Modified oligonucleotides

Abstract

Disclosed herein are compositions comprising a modified oligonucleotide. In some embodiments, the oligonucleotides include hydrophobic moieties. In some embodiments, the oligonucleotide comprises a vinyl phosphonate. In some embodiments, the oligonucleotides include modified oligonucleotide sugars, such as 2 '-modified ribose molecules. In some embodiments, the oligonucleotides include a modified inter-nucleoside linkage, such as a thiophosphate linkage. In some embodiments, the oligonucleotide comprises a siRNA. In some embodiments, the siRNA comprises a duplex region having a protruding end at either end.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 324,487, filed on March 28, 2022, and U.S. Provisional Application No. 63 / 429,756, filed on December 2, 2022, both of which are incorporated herein by reference.

[0003] Sequence Listing

[0004] This application is submitted with a sequence listing in electronic format. The sequence listing is provided as a file named 54462-739601_PCT.xml, created on March 20, 2023, and is 541 kilobytes in size. The information in the sequence listing in electronic format is incorporated herein by reference in its entirety. Background Art

[0005] There is a need in the art for improved oligonucleotides for reducing gene expression. Summary of the invention

[0006] In some embodiments, compositions comprising oligonucleotides are described herein, which target mRNA and reduce target mRNA or protein levels when administered in an effective amount. In some embodiments, compositions comprising small interfering RNA (siRNA) are described herein, which siRNA comprises a sense strand, an antisense strand, and a lipid portion connected to the end of the sense strand or antisense strand; wherein the lipid portion comprises a phenyl or cyclohexyl linker, wherein the linker is connected to the lipid and connected to the end of the sense strand or antisense strand. The lipid portion can be connected to the 5' end or 3' end of the sense strand or antisense strand. In some embodiments, the ends of the lipid and sense strand or antisense strand are connected to a phenyl or cyclohexyl linker with a 1,4, 1,3, or 1,2 substitution pattern (e.g., para, meta or ortho phenyl configuration). In some embodiments, the ends of the lipid and sense strand or antisense strand are connected to a phenyl or cyclohexyl linker with 1,4 (para phenyl). In some embodiments, the lipid portion comprises the following structure:

[0007] Wherein the dotted line indicates a connection (e.g., covalent connection) to the end of the sense strand or the antisense strand, and R is an alkyl group containing 4-18 carbons. In some embodiments, R is not octane. In some embodiments, R is an alkyl group containing 4-7 or 9-18 carbons. In some embodiments, the lipid portion comprises the following structure:

[0008] Wherein the dotted line indicates the connection (e.g., covalent connection) to the end of the sense strand or the antisense strand, n is 1-3, and R is an alkyl group containing 4-18 carbons. In some embodiments, the lipid portion comprises the following structure:

[0009] Wherein the dotted line indicates the connection (e.g., covalent connection) to the end of the sense strand or the antisense strand, n is 0-3, and R is an alkyl group containing 4-18 carbons. In some embodiments, the lipid portion comprises the following structure: Wherein the dotted line indicates the connection (e.g., covalent connection) to the end of the sense strand or the antisense strand, and R is an alkyl group containing 4-18 carbons. In some embodiments, the lipid portion comprises any one of the following structures:

[0010]

[0011] Wherein the dotted line indicates the connection (e.g., covalent connection) to the end of the sense strand or antisense strand, n is 0-3, and R is an alkyl group containing 4-18 carbons. In some embodiments, the lipid portion comprises a lipid portion depicted in Table 1. In some embodiments, the lipid portion is connected to the 5' end of the sense strand or antisense strand. In some embodiments, the lipid portion is connected to the 5' end of the sense strand. In some embodiments, the lipid portion is connected to the 5' end of the sense strand or antisense strand through a phosphate. In some embodiments, the lipid portion is connected to the end of the sense strand. In some embodiments, the antisense strand comprises vinyl phosphonate. In some embodiments, the antisense strand comprises 5' vinyl phosphonate. In some embodiments, the sense strand or antisense strand comprises one or two thiophosphate linkages at the 5' end or 3' end of the sense strand or antisense strand. In some embodiments, the antisense strand comprises one or two 5' thiophosphate linkages. For example, there may be a phosphorothioate between the first and second nucleotides at the 5' end of the antisense strand, or there may be a phosphorothioate between the first, second and third nucleotides at the 5' end of the antisense strand. In some embodiments, the antisense strand comprises one or two 3' phosphorothioate linkages. For example, there may be a phosphorothioate between the first and second nucleotides at the 3' end of the antisense strand, or there may be a phosphorothioate between the first, second and third nucleotides at the 3' end of the antisense strand. In some embodiments, the sense strand comprises one or two 5' phosphorothioate linkages. For example, there may be a phosphorothioate between the first and second nucleotides at the 5' end of the sense strand, or there may be a phosphorothioate between the first, second and third nucleotides at the 5' end of the sense strand. In some embodiments, the sense strand does not comprise one or two 5' phosphorothioate linkages. For example, in some embodiments, there is no phosphorothioate between the last 3 nucleotides at the 5' end of the sense strand. In some embodiments, the sense strand comprises a 5' phosphate linkage. In some embodiments, the sense strand comprises one or two 3' phosphorothioate linkages. For example, a phosphorothioate may be present between the first and second nucleotides at the 3' end of the sense strand, or a phosphorothioate may be present between the first, second, and third nucleotides at the 3' end of the sense strand.In some embodiments, with respect to the sense strand, any of the following is true: all purine nucleosides contain 2' fluorine and all pyrimidine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl; all purine nucleosides contain 2'-O-methyl and all pyrimidine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl; all purine nucleosides contain 2' fluorine and all pyrimidine nucleosides contain 2' fluorine and 2'-O-methyl; all pyrimidine nucleosides contain 2' fluorine and all purine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl; all pyrimidine nucleosides contain 2' fluorine and all purine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl; or all pyrimidine nucleosides contain 2' fluorine and all purine nucleosides contain 2'-O-methyl; provided 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 of the following is true: all purine nucleosides contain 2' fluorine, and all pyrimidine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl; all purine nucleosides contain 2'-O-methyl, and all pyrimidine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl; all purine nucleosides contain 2'-O-methyl, and all pyrimidine nucleosides contain 2' fluorine; all pyrimidine nucleosides contain 2' fluorine, and all purine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl; all pyrimidine nucleosides contain 2'-O-methyl, and all purine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl; or all pyrimidine nucleosides contain 2'-O-methyl, and all purine nucleosides contain 2' fluorine. In some embodiments, with respect to the sense strand or the antisense strand (provided that the sense strand or the antisense strand may include deoxynucleosides), any of the following is true: all purines comprise 2' fluorine-modified purines, and all pyrimidines comprise a mixture of 2' fluorine and 2'-O-methyl modified pyrimidines; all purines comprise 2'-O-methyl modified purines, and all pyrimidines comprise a mixture of 2' fluorine and 2'-O-methyl modified pyrimidines; all purines comprise 2' fluorine-modified purines, and all pyrimidines comprise 2'-O-methyl modified pyrimidines; all pyrimidines comprise 2' fluorine-modified pyrimidines, and all purines comprise a mixture of 2' fluorine and 2'-O-methyl modified purines; all pyrimidines comprise 2'-O-methyl modified pyrimidines, and all purines comprise a mixture of 2' fluorine and 2'-O-methyl modified purines; or all pyrimidines comprise 2' fluorine-modified pyrimidines, and all purines comprise 2'-O-methyl modified purines.In some embodiments, with respect to the sense strand, any of the following is true: all purines comprise 2' fluorine-modified purines, and all pyrimidines comprise a mixture of 2' fluorine and 2'-O-methyl modified pyrimidines; all purines comprise 2'-O-methyl modified purines, and all pyrimidines comprise a mixture of 2' fluorine and 2'-O-methyl modified pyrimidines; all purines comprise 2' fluorine-modified purines, and all pyrimidines comprise 2'-O-methyl modified pyrimidines; all pyrimidines comprise 2' fluorine-modified pyrimidines, and all purines comprise a mixture of 2' fluorine and 2'-O-methyl modified purines; all pyrimidines comprise 2'-O-methyl modified pyrimidines, and all purines comprise a mixture of 2' fluorine and 2'-O-methyl modified purines; or all pyrimidines comprise 2' fluorine-modified pyrimidines, and all purines comprise 2'-O-methyl modified purines; provided that in any of the above, the sense strand may include deoxynucleosides. In some embodiments, the sense strand does not include deoxynucleosides. In some embodiments, with respect to the sense strand, any 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 purines, 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 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'-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 include a 2-deoxy modification at position 9.

[0012] In some embodiments, described herein are compositions comprising: a small interfering RNA (siRNA) comprising a sense strand and an antisense strand; wherein the antisense strand comprises a 5' end comprising a vinylphosphonate and two phosphorothioate linkages and a 3' end comprising two phosphorothioate linkages; wherein the sense strand comprises a 5' end comprising a hydrophobic portion and a 3' end comprising two phosphorothioate linkages; wherein with respect to the sense strand (provided that the sense strand may comprise a 2' deoxynucleoside), any of the following is true: all purine nucleosides comprise 2' fluorine, and all pyrimidine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl, all purine nucleosides comprise 2' fluorine, and all pyrimidine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl, all purine nucleosides comprise 2' fluorine, and all pyrimidine nucleosides comprise 2'-O-methyl, all pyrimidine nucleosides comprise 2' fluorine, and all purine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl, all pyrimidine nucleosides comprise 2' fluorine, and all purine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl, wherein the antisense strand comprises a 2'-O-methyl group, wherein the purine nucleosides comprise a 2'-O-methyl group, and all purine nucleosides are modified with a mixture of 2'-fluoro and 2'-O-methyl groups, or the pyrimidine nucleosides comprise a 2'-fluoro group, and all purine nucleosides comprise a 2'-O-methyl group; and wherein with respect to the antisense strand, any of the following is true: all purine nucleosides comprise a 2'-fluoro group, and all pyrimidine nucleosides are modified with a mixture of 2'-fluoro and 2'-O-methyl groups, all purine nucleosides comprise a 2'-O-methyl group, and all pyrimidine nucleosides are modified with a mixture of 2'-fluoro and 2'-O-methyl groups; In some embodiments, the sense strand comprises a 2' deoxynucleoside. In some embodiments, the sense strand comprises a 2' deoxynucleoside. In some embodiments, the sense strand comprises a 2' deoxynucleoside. In some embodiments, the sense strand comprises a 2' deoxynucleoside.In some embodiments, described herein are compositions comprising a small interfering RNA (siRNA) comprising a sense strand and an antisense strand; wherein the antisense strand comprises a 5' end comprising a vinylphosphonate and two phosphorothioate linkages and a 3' end comprising two phosphorothioate linkages; wherein the sense strand comprises a 5' end comprising a hydrophobic portion and a 3' end comprising two phosphorothioate linkages; wherein with respect to the sense strand, any of the following is true: all purines comprise 2' fluorine-modified purines, and all pyrimidines comprise A mixture of 2'-fluoro and 2'-O-methyl modified pyrimidines, all purines contain 2'-O-methyl modified purines, and all pyrimidine nucleosides contain 2'-fluoro and 2'-O-methyl modified pyrimidines. A mixture of 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 2'-fluoro and 2'-O-methyl modified purines. A mixture of all pyrimidines contain 2'-O-methyl modified pyrimidines, and all purines contain 2'-fluoro and 2'-O-methyl modified purines. All pyrimidines contain 2'-O-methyl modified pyrimidines, and all purines contain 2 wherein the antisense strand comprises 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, any 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 purines, 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 In some embodiments, the sense strand comprises a 2'-O-methyl modified purine and all pyrimidines comprise a 2'-fluorine modified pyrimidine, all pyrimidines comprise a 2'-fluorine modified pyrimidine, and all purines comprise a mixture of 2'-fluorine and 2'-O-methyl modified purines, all pyrimidines comprise a 2'-O-methyl modified pyrimidine, and all purines comprise a mixture of 2'-fluorine and 2'-O-methyl modified purines, or all pyrimidines comprise a 2'-O-methyl modified pyrimidine, and all purines comprise a 2'-fluorine modified purine, all of which have the condition that the sense strand may comprise a deoxynucleoside. In some embodiments, the sense strand does not comprise a deoxynucleoside.In some embodiments, described herein are compositions comprising a small interfering RNA (siRNA) comprising a sense strand and an antisense strand; wherein the antisense strand comprises a 5' end comprising a vinylphosphonate and two phosphorothioate linkages and a 3' end comprising two phosphorothioate linkages; wherein the sense strand comprises a 5' end comprising a hydrophobic portion and a 3' end comprising two phosphorothioate linkages; wherein with respect to the sense strand, any of the following is true: all purines comprise 2' fluorine-modified purines, and all There is a mixture of pyrimidines containing 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 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, any 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 2'-fluoro and 2'-O-methyl modified pyrimidines; In some embodiments, the 5' end of the sense strand comprises two nucleotides connected via a phosphate linkage rather than a thiophosphate linkage. In some embodiments, the 5' end of the sense strand comprises two nucleosides connected via a phosphate linkage rather than a thiophosphate linkage, and the 3' end of the sense strand comprises two nucleosides connected via a thiophosphate linkage. In some embodiments, the 5' end of the sense strand further comprises one or two thiophosphate linkages. In some embodiments, the hydrophobic moiety comprises a lipid connected to the 5' end of the sense strand via a phenyl or cyclohexyl linker. In some embodiments, the 5' end of the lipid and sense strand is connected 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 5' end of the lipid and sense strand is connected to the phenyl or cyclohexyl linker in a 1,4 (para-phenyl) configuration.In some embodiments, the hydrophobic portion comprises any of the following structures:.

[0013] Wherein the dotted line indicates the connection to the end of the sense strand or the antisense strand, n is 1-3, and R is an alkyl group containing 4-18 carbons. In some embodiments, the hydrophobic portion comprises the following structure:

[0014] Wherein the dotted line indicates a connection (e.g., covalent connection) to the end of the sense strand or antisense strand, and R is an alkyl group containing 4-18 carbons. In some embodiments, R is not octane. In some embodiments, R is an alkyl group containing 4-7 or 9-18 carbons. In some embodiments, the hydrophobic portion comprises the following structure:

[0015] Wherein the dotted line indicates the connection (e.g., covalent connection) to the end of the sense strand or the antisense strand, n is 1-3, and R is an alkyl group containing 4-18 carbons. In some embodiments, the hydrophobic portion comprises the following structure:

[0016] Wherein the dotted line indicates the connection (e.g., covalent connection) to the end of the sense strand or the antisense strand, n is 1-3, and R is an alkyl group containing 4-18 carbons. In some embodiments, the hydrophobic portion comprises the following structure:

[0017] Wherein the dotted line indicates a connection (eg, covalent connection) to the terminus of the sense strand or antisense strand, and R is an alkyl group containing 4-18 carbons. In some embodiments, the hydrophobic moiety comprises a lipid moiety depicted in Table 1. The sense strand may include a 2-deoxy modification at position 9.

[0018] 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 nucleotides. In some embodiments, the sense strand overhang comprises 2 nucleotides. 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 nucleotides. In some embodiments, the antisense strand overhang comprises 2 nucleotides. 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 comprise a pharmaceutically acceptable carrier. In some embodiments, methods of treatment are described herein, comprising administering an effective amount of a composition to an object in need thereof. In some embodiments, methods of reducing the amount of RNA or protein in a cell are described herein, comprising administering a composition to a cell. In some embodiments, administering a composition to a cell comprises administering a composition to an object comprising a cell.

[0019] In some embodiments, methods of treatment are described herein, comprising administering an effective amount of a composition or siRNA as described herein to a subject in need thereof. In some embodiments, administration comprises subcutaneous, intravitreal, intrathecal, or intraventricular administration. In some embodiments, the siRNA targets an mRNA encoding a protein that mediates a subject's disease. In some embodiments, the mRNA is located in a subject's tissue, including the eye, liver, fat, brain, or spinal cord.

[0020] In some embodiments, described herein is a method for reducing the amount of a target RNA or a protein encoded by RNA in a cell, comprising administering a composition or siRNA as described herein to a cell, wherein the antisense strand comprises a sequence complementary to and combined with RNA. In some embodiments, administering a composition to a cell comprises administering a composition to an object comprising a cell. In some embodiments, administration comprises subcutaneous, intravitreal, intrathecal or intraventricular administration. In some embodiments, administration reduces the measured value of a target RNA or a protein encoded by RNA by at least 10% relative to a baseline measured value or a control. In some embodiments, cells include eye, liver, fat, brain or spinal cord cells. DETAILED DESCRIPTION

[0021] In some embodiments, modified oligonucleotides are disclosed herein. The modified oligonucleotide can be siRNA, which comprises modifications to the ribose ring and phosphate linkages. The modification can be a specific pattern that maximizes cell delivery, stability, and efficiency. The siRNA can also comprise vinylphosphonates and hydrophobic groups. These modifications can help deliver to cells or tissues in the subject. The modified oligonucleotide can be used in methods such as methods of treatment or methods of reducing gene expression.

[0022] In some embodiments, the oligonucleotide comprises a duplex consisting of 21 nucleotide single strands, with base pairing between 19 base pairs. In some embodiments, the duplex comprises 2 nucleotide overhangs of a single strand at the 3' end of each strand. One strand (antisense strand) is complementary to the target mRNA. Each end of the antisense strand has one to two thiophosphate bonds. The 5' end has an optional phosphate mimic, such as vinylphosphonate. 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, 1-2 thiophosphates are present at the 3' end. In some embodiments, 1 or no thiophosphates are present at the 5' end. In some embodiments, a hydrophobic conjugate of 12 to 25 carbons is attached via a phosphodiester bond at the 5' end.

[0023] I. Composition

[0024] In some embodiments, compositions comprising oligonucleotides are disclosed herein. In some embodiments, the composition comprises oligonucleotides targeting target oligonucleotides. The target oligonucleotide may comprise a target RNA. In some embodiments, the composition consists of oligonucleotides targeting 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 for treating a disorder of a subject in need. Some embodiments relate to compositions comprising oligonucleotides, which are used in a method for treating a disorder. Some embodiments relate to the use of compositions comprising oligonucleotides in a method for treating a disorder.

[0025] Targets can be identified in a variety of ways. In some cases, the target oligonucleotide comprises an mRNA with an expression level associated with the incidence of a disease (e.g., a fat-related or eye-related disease). In some cases, the target oligonucleotide comprises an mRNA encoded by a gene with a specific genotype associated with the disease. Large-scale human genetics data can improve the success rate of drug discovery and development. Genome-wide association studies (GWAS) can detect the association between genetic variants and traits in population samples. GWAS can enable a better understanding of the biology of the disease and provide applicable treatment methods. GWAS can utilize genotyping and / or sequencing data, and generally involves the evaluation of millions of relatively evenly distributed genetic variants on the genome. The most common GWAS design is a case-control study, which involves comparing the variant frequencies of cases relative to controls. If the variant has a significantly different frequency in the case relative to the control, the variant is said to be associated with the disease. The association statistics that can be used in GWAS are p-values ​​as a measure of statistical significance; odds ratios (OR) as a measure of effect size; or beta coefficients (β) as a measure of effect size. Researchers typically assume an additive genetic model and calculate the allelic advantage ratio, which is the increased (or decreased) disease risk conferred by each additional copy of the allele (compared to not carrying this allele copy). Another concept in GWAS design and interpretation is linkage disequilibrium, which is the non-random association of alleles. The presence of linkage disequilibrium may confuse which variant is "causal".

[0026] Functional annotation of variants and / or wet lab experiments are used to identify causal genetic variants identified via GWAS, and in many cases lead to the identification of disease-causing genes. In particular, understanding the functional effects of causal genetic variants (e.g., loss of protein function, gain of protein function, increase in gene expression, or reduction in gene expression) allows this variant to be used as a representative for therapeutic regulation of the target gene, or to gain insight into the potential therapeutic efficacy and safety of therapeutic agents that regulate this target.

[0027] The identification of such gene-disease associations has provided insights into disease biology and has been used to identify novel therapeutic targets for the pharmaceutical industry. In order to transform therapeutic insights derived from human genetics, the disease biology in patients is exogenously "programmed" to replicate observations from human genetics. There are several therapeutic options that can be utilized in converting therapeutic targets identified via human genetics into novel drugs. These include well-established therapeutic modalities, such as small molecules and monoclonal antibodies; mature methods, such as oligonucleotides; and emerging methods, 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), related tissues (e.g., lungs or eyes), and related indications. Such studies can be performed to identify targets associated with specific disorders for siRNA inhibition by compositions or compounds described herein. In some cases, the target may be associated with a specific tissue.

[0028] Disclosed herein are compositions comprising oligonucleotides targeting target RNA. In the case of disclosing the suppression or targeting of target RNA, it is envisioned that some embodiments may include suppression or targeting of target protein or target RNA. For example, RNA (e.g., mRNA) encoded by a target gene is suppressed or targeted by using oligonucleotides as described herein, because the target protein produced by the translation of the target RNA is less, the target protein can be suppressed or targeted; or the target protein can be combined with or interacted with the target RNA and reduce the oligonucleotide targeting or suppression of the target protein produced by the target RNA. Therefore, targeting can refer to combining the target RNA and reducing the target RNA or protein level. The oligonucleotide may include small interfering RNA (siRNA) or antisense oligonucleotide (ASO). Also provided herein is a method for treating a target disease by providing an oligonucleotide targeting a target to an object in need thereof.

[0029] Some embodiments include a composition comprising an oligonucleotide, which targets mRNA and reduces the target mRNA or protein level in a cell, fluid or tissue when applied to an object in an effective amount. In some embodiments, the composition comprises an oligonucleotide, which targets RNA and reduces the target mRNA level in a cell or tissue when applied to an object in an effective amount. In some embodiments, as compared to before application, the target mRNA level is reduced by about 2.5% or more, about 5% or more, or about 7.5% or more. In some embodiments, as compared to before application, the target mRNA level is reduced by about 10% or more. In some embodiments, as compared to before application, 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%. In some embodiments, as compared to before application, the target mRNA level is reduced by no more than about 2.5%, no more than about 5% or no more than about 7.5%. In some embodiments, as compared with before administration, target mRNA level has reduced by no more than about 10%. In some embodiments, as compared with before administration, target mRNA level has reduced by no more than about 20%, no more than about 30%, no more than about 40%, no more than about 50%, no more than about 60%, no more than about 70%, no more than about 80% or no more than about 90%. In some embodiments, target mRNA level has 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 the scope defined by any two aforementioned percentages.

[0030] In some embodiments, the composition comprises an oligonucleotide that targets a target mRNA and reduces the target protein level in a cell, fluid, or tissue when administered to a subject in an effective amount. In some embodiments, the target protein level is reduced by about 2.5% or more, about 5% or more, or about 7.5% or more, as compared to before administration. In some embodiments, the target protein level is reduced by about 10% or more, as compared to before administration. In some embodiments, the target protein 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%, as compared to before administration. In some embodiments, the target protein level is reduced by no more than about 2.5%, no more than about 5%, or no more than about 7.5%, as compared to before administration. In some embodiments, the target protein level is reduced by no more than about 10%, as compared to before administration. In some embodiments, the target protein level is reduced by no more than about 20%, no more than about 30%, no more than about 40%, no more than about 50%, no more than about 60%, no more than about 70%, no more than about 80%, or no more than about 90% as 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.

[0031] In some embodiments, the composition comprises an oligonucleotide that targets mRNA and weakens the disease phenotype when applied 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, as compared to before administration. In some embodiments, the disease phenotype is reduced by about 10% or more, as 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%, as compared to before administration. In some embodiments, the disease phenotype is reduced by no more than about 2.5%, no more than about 5%, or no more than about 7.5%, as compared to before administration. In some embodiments, the disease phenotype is reduced by no more than about 10%, as compared to before administration. In some embodiments, the disease phenotype is reduced by no more than about 20%, no more than about 30%, no more than about 40%, no more than about 50%, no more than about 60%, no more than about 70%, no more than about 80%, or no more than about 90% as 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.

[0032] In some embodiments, the composition comprises an oligonucleotide that targets mRNA and enhances the protective phenotype of the disease of the object when applied to the object 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, as compared to before administration. In some embodiments, the protective phenotype is increased by about 10% or more, as 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, as 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, as compared to before administration. In some embodiments, the protective phenotype is increased by no more than about 2.5%, no more than about 5%, or no more than about 7.5% as compared to before administration. In some embodiments, the protective phenotype is increased by no more than about 10% as compared to before administration. In some embodiments, the protective phenotype is increased by no more than about 20%, no more than about 30%, no more than about 40%, no more than about 50%, no more than about 60%, no more than about 70%, no more than about 80%, no more than about 90%, or no more than about 100% as compared to before administration. In some embodiments, the protective phenotype is increased by no more than about 200%, no more than about 300%, no more than about 400%, no more than about 500%, no more than about 600%, no more than about 700%, no more than about 800%, no more than about 900%, or no more than about 1000% as 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%, 1000%, or a range defined by any two of the foregoing percentages.

[0033] A.siRNA

[0034] In some embodiments, the composition comprises an oligonucleotide targeting a target RNA such as an mRNA, wherein the oligonucleotide comprises a small interfering RNA (siRNA). In some embodiments, the composition comprises an oligonucleotide targeting a target mRNA, wherein the oligonucleotide comprises a small interfering RNA (siRNA) comprising a sense strand and an antisense strand.

[0035] In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of a target mRNA, wherein the oligonucleotide comprises an siRNA comprising a sense strand and an antisense strand, wherein the sense strand is 12-30 nucleosides in length. In some embodiments, the composition comprises a sense strand of 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 sense strand of a range defined by any two of the foregoing numbers. The length of the sense strand can be 14-30 nucleosides. In some embodiments, the composition comprises an antisense strand of 12-30 nucleosides in length. In some embodiments, the composition comprises an antisense strand having a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleosides, or a range defined by any two of the foregoing numbers. The antisense strand may be 14-30 nucleosides in length.

[0036] In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of a target mRNA, wherein the oligonucleotide comprises an siRNA comprising a sense strand and an antisense strand, the length of each strand being independently about 12-30 nucleosides, and at least one of the sense strand and the antisense strand comprises a nucleoside sequence of about 12-30 consecutive nucleosides comprising 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 of 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 consecutive nucleosides comprising a target RNA.

[0037] In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of a target mRNA, wherein the oligonucleotide comprises an siRNA comprising a sense strand and an antisense strand, wherein 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.

[0038] 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 foregoing 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 foregoing numbers. In some embodiments, the 5' overhang comprises 1, 2, or more nucleosides. In some embodiments, the 5' overhang comprises 2 nucleosides.

[0039] 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 nucleotide range defined by any two of the aforementioned 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 nucleotide range defined by any two of the aforementioned numbers. In some embodiments, the 5' overhang comprises 1, 2 or more nucleosides. In some embodiments, the 5' overhang comprises 2 nucleosides.

[0040] In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of a target mRNA, wherein the oligonucleotide comprises an siRNA comprising a sense strand and an antisense strand, wherein the siRNA binds to a 19-mer in a human target mRNA. In some embodiments, the siRNA binds to a 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 a human target mRNA.

[0041] In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of a target mRNA, wherein the oligonucleotide comprises an siRNA comprising a sense strand and an antisense strand, wherein the siRNA binds to a 17-mer in a non-human primate target mRNA. In some embodiments, the siRNA binds to a 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 a non-human primate target mRNA.

[0042] In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of a target mRNA, wherein the oligonucleotide comprises an siRNA comprising a sense strand and an antisense strand, wherein the siRNA binds to a human target mRNA and less than or equal to 20 human off-targets, wherein there are no more than 2 mismatches in the antisense strand. In some embodiments, the siRNA binds to a human target mRNA and less than or equal to 10 human off-targets, with no more than 2 mismatches in the antisense strand. In some embodiments, the siRNA binds to a human target mRNA and less than or equal to 30 human off-targets, with no more than 2 mismatches in the antisense strand. In some embodiments, the siRNA binds to a human target mRNA and less than or equal to 40 human off-targets, with no more than 2 mismatches in the antisense strand. In some embodiments, the siRNA binds to a human target mRNA and less than or equal to 50 human off-targets, with no more than 2 mismatches in the antisense strand. In some embodiments, the siRNA binds to a human target mRNA and less than or equal to 10 human off-targets, with no more than 3 mismatches in the antisense strand. In some embodiments, the siRNA binds to a human target mRNA and less than or equal to 20 human off-targets, with no more than 3 mismatches in the antisense strand. In some embodiments, the siRNA binds to a human target mRNA and less than or equal to 30 human off-targets, with no more than 3 mismatches in the antisense strand. In some embodiments, the siRNA binds to a human target mRNA and less than or equal to 40 human off-targets, with no more than 3 mismatches in the antisense strand. In some embodiments, the siRNA binds to a human target mRNA and less than or equal to 50 human off-targets, with no more than 3 mismatches in the antisense strand.

[0043] In some embodiments, the compositions include oligonucleotides that inhibit the expression of target mRNA, wherein the oligonucleotides include siRNAs that include sense strands and antisense strands, and the siRNAs bind to human target mRNA target sites that do not have a minor allele frequency (MAF) greater than or equal to 1% of a SNP (position 2-18). In some embodiments, the MAF is greater than or equal to about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19% or about 20%.

[0044] B.ASO

[0045] In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of a target mRNA, wherein the oligonucleotide comprises an antisense oligonucleotide (ASO). In some embodiments, the length of the ASO is 12-30 nucleosides. In some embodiments, the length of the ASO is 14-30 nucleosides. In some embodiments, the length of 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, or the scope defined by any two aforementioned numbers. In some embodiments, the length of the ASO is 15-25 nucleosides. In some embodiments, the length of the ASO is 20 nucleosides.

[0046] In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of a target mRNA, wherein the oligonucleotide comprises an ASO having a length of about 12-30 nucleosides and comprising a nucleoside sequence complementary to about 12-30 consecutive nucleosides of a full-length human target mRNA sequence; wherein (i) the oligonucleotide comprises a modification comprising a modified nucleoside and / or a modified internucleoside linkage, and / or (ii) the composition comprises a pharmaceutically 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 consecutive nucleosides of a target mRNA.

[0047] C. Modification

[0048] In some embodiments, the composition comprises an oligonucleotide that inhibits the 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 pharmaceutically 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 alkylphosphonate, a thiophosphate, a methylphosphonate, a dithiophosphate, an alkylthiophosphonate, an aminophosphate, a carbamate, a carbonate, a phosphotriester, an acetimidate or a carboxymethyl ester, or a combination thereof. In some embodiments, the modified internucleoside linkage comprises one or more thiophosphate linkages. Phosphorothioates can include non-bridging oxygen atoms substituted by sulfur in the phosphate backbone of an oligonucleotide. The modified internucleoside linkage can be included in siRNA or ASO. Benefits of modified internucleoside linkages may include reduced toxicity or improved pharmacokinetics.

[0049] In some embodiments, the composition comprises an oligonucleotide that inhibits expression of a target mRNA, wherein the oligonucleotide comprises a modified internucleoside linkage, wherein 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 of modified internucleoside linkages defined by any two of the foregoing numbers. In some embodiments, the oligonucleotide comprises no more than 18 modified internucleoside linkages. In some embodiments, the oligonucleotide comprises no more than 20 modified internucleoside linkages. In some embodiments, the oligonucleotide comprises 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, a plurality of 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.

[0050] In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of a target mRNA, wherein 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. In some embodiments, the modified nucleoside comprises a 2'-O-alkyl. 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 a 2'-methoxyethyl thymine. In some embodiments, the modified nucleoside includes a 2'-O-methyl group. 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-methylacetylamino (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 includes a 2'-deoxyfluoro nucleoside. 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. The benefits of the modified nucleoside may include reduced toxicity or improved pharmacokinetics.

[0051] 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 of nucleosides defined by any two of the aforementioned numbers. In some embodiments, the oligonucleotide comprises no more than 19 modified nucleosides. In some embodiments, the oligonucleotide comprises no more than 21 modified nucleosides. In some embodiments, the 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.

[0052] In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of a target mRNA, wherein the oligonucleotide comprises a portion attached to the 3' or 5' end of the oligonucleotide. Examples of portions include hydrophobic portions or sugar portions, or a combination thereof. In some embodiments, the oligonucleotide is an siRNA with a sense strand, and the portion is attached to the 5' end of the sense strand. In some embodiments, the oligonucleotide is an siRNA with a sense strand, and the portion is attached to the 3' end of the sense strand. In some embodiments, the oligonucleotide is an siRNA with an antisense strand, and the portion is attached to the 5' end of the antisense strand. In some embodiments, the oligonucleotide is an siRNA with an antisense strand, and the portion is attached to the 3' end of the antisense strand. In some embodiments, the oligonucleotide is an ASO, and the portion is attached to the 5' end of the ASO. In some embodiments, the oligonucleotide is an ASO, and the portion is attached to the 3' end of the ASO.

[0053] In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of a target mRNA, wherein the oligonucleotide comprises a hydrophobic portion. An oligonucleotide comprising a hydrophobic portion may include or be referred to as a hydrophobic conjugate. The hydrophobic portion may be used to enhance cellular uptake. The hydrophobic portion may be attached to the 3' or 5' end of the oligonucleotide. The hydrophobic portion may include a lipid, such as a fatty acid. The hydrophobic portion may include a hydrocarbon. The hydrophobic portion may be linear. The hydrophobic portion may be non-linear. The hydrophobic portion may include a lipid portion or a cholesterol portion, or a combination thereof. In some embodiments, the hydrophobic portion includes a cyclohexyl group. In some embodiments, the hydrophobic portion includes a lipid. In some embodiments, the hydrophobic portion is used in a specific format as described herein. In some embodiments, the hydrophobic portion is attached to the 5' end of the sense strand without any phosphorothioate group or linkage at the 5' end. The hydrophobic portion may include an esterified lipid.

[0054] The hydrophobic part can be or include a lipid part. In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of a target mRNA, wherein the oligonucleotide comprises a lipid attached to the 3' or 5' end of the oligonucleotide. In some embodiments, the lipid comprises cholesterol, myristyl, palmityl, stearyl, lithocholyl, behenyl, docosahexaenyl, myristyl, palmityl stearyl or alpha-tocopherol, or a combination thereof. In some embodiments, the lipid comprises stearyl, tert-butylphenol, n-butylphenol, octylphenyl, dodecylphenyl, n-dodecylphenyl, octadecylbenzamide, hexadecylbenzamide or octadecylcyclohexyl. In some embodiments, the lipid comprises p-C12 phenyl. The lipid part can be esterified.

[0055] In some embodiments, the oligonucleotide includes any aspect of the following structures: In some embodiments, R is not octane. In some embodiments, R is not octane. In some embodiments, R is an alkyl group containing 4-7 or 9-18 carbons. In some embodiments, the oligonucleotide includes any aspect of the following structures: In some embodiments, the oligonucleotide includes any aspect of the following structures: In some embodiments, the oligonucleotide includes any aspect of the following structures: Aspects included in the oligonucleotide may include the entire structure of any structure shown, or may include a lipid portion. 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 defined by any two of the foregoing carbon numbers. In some embodiments, the alkyl group contains 4-18 carbons. In some embodiments, R is not octane (C8). In some embodiments, R is C 4-7 In some embodiments, R is C 9-18 In some embodiments, R is C 4-7 Alkyl and C 9-18 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 includes a branched carbon chain. In some embodiments, R includes an unbranched carbon chain. In some embodiments, the lipid portion comprises an alcohol or an ether. In some embodiments, the lipid portion has at least one degree of unsaturation. In some embodiments, the lipid portion is an ω-fatty acid, such as an ω-3, ω-5, ω-6, ω-7, or ω-9 fatty acid.

[0056] In some embodiments, the lipid comprises a fatty acid. In some embodiments, the lipid comprises a lipid depicted in Table 1. The exemplary lipid moieties in Table 1 are shown attached to the 5' end of the oligonucleotide, wherein the 5' terminal phosphate of the oligonucleotide is shown with a lipid moiety. In some embodiments, the lipid moieties in Table 1 can be attached to a different attachment point than shown. For example, the attachment point of any lipid moiety in the table can be at the 3' oligonucleotide end. In some embodiments, the lipid is used to target the oligonucleotide to a non-hepatic cell or tissue.

[0057] Table 1: Examples of lipid fractions

[0058]

[0059]

[0060] In some embodiments, the lipid or lipid moiety comprises 16 to 18 carbons. In some embodiments, the lipid comprises 16 carbons. In some embodiments, the lipid comprises 17 carbons. In some embodiments, the lipid comprises 18 carbons. In some embodiments, the lipid moiety comprises 16 carbons. In some embodiments, the lipid moiety comprises 17 carbons. In some embodiments, the lipid moiety comprises 18 carbons. In some embodiments, the lipid moiety comprises 19 carbons. In some embodiments, the lipid moiety comprises 20 carbons.

[0061] The hydrophobic portion may include a linker comprising a carbocycle. The carbocycle may be hexacyclic. 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 in turn may be attached to the phosphate (e.g., 5' or 3' phosphate) of the oligonucleotide. In some embodiments, the lipid or hydrocarbon and the end of the sense are connected 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 are connected 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 can be attached to the carbocyclic ring in a 1,2 orientation relative to the oligonucleotide.

[0062] The lipid portion may comprise or consist of the following structures:

[0063] In some embodiments, the lipid portion comprises or consists of the following structure: In some embodiments, the lipid portion comprises or consists of the following structure: In some embodiments, the lipid portion comprises or consists of the following structure: In some embodiments, the dotted line indicates a covalent connection. The covalent connection can be between the ends of the sense strand or the antisense strand. For example, the connection can 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 includes a branched carbon chain. In some embodiments, R includes 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 foregoing carbon 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 4-18 carbons. In some embodiments, the lipid moiety is not phenyloctyl. In some embodiments, R is not octane. In some embodiments, R is C 4-7 In some embodiments, R is C 9-18 In certain embodiments, R is C 4-7 Alkyl and C 9-18 In some embodiments, the lipid moiety is not phenyloctyl.

[0064] In some embodiments, the 5' hydrophobic portion includes any of the following structures:

[0065]

[0066] Wherein the dotted line indicates the covalent attachment 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 moiety comprises a hydrophobic moiety in Table 1. In some embodiments, the 5' hydrophobic moiety comprises a p-C12 phenyl group. 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 ω-3 position, ω-4 position, ω-5 position, ω-6 position, ω-7 position, ω-8 position, ω-9 position, or a combination thereof. In some embodiments, the 5' hydrophobic moiety is not phenyloctyl.

[0067] The hydrophobic portion may include a linker comprising a carbocycle. The carbocycle may be hexacyclic. 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 in turn may be attached to the phosphate (e.g., 5' or 3' phosphate) of the oligonucleotide. In some embodiments, the lipid or hydrocarbon and the end of the sense are connected 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 are connected 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 can be attached to the carbocyclic ring in a 1,2 orientation relative to the oligonucleotide.

[0068] The lipid moiety can be attached to the 5' end of the oligonucleotide. The 5' end can have a phosphate that connects the lipid moiety to the 5' carbon of the sugar of the oligonucleotide. The 5' end can have two phosphates that connect the lipid moiety to the 5' carbon of the sugar of the oligonucleotide. The 5' end can have three phosphates that connect the lipid moiety to the 5' carbon of the sugar of the oligonucleotide. The 5' end can have a phosphate that connects the lipid moiety to the 5' carbon of the sugar of the oligonucleotide, wherein one of the phosphates is connected to the lipid moiety. The 5' end can have two phosphates that connect to the 5' carbon of the sugar of the oligonucleotide, wherein one of the two phosphates is connected to the lipid moiety. The 5' end can have three phosphates that connect to the 5' carbon of the sugar of the oligonucleotide, wherein one of the three phosphates is connected to the lipid moiety. The sugar can include ribose. The sugar can include deoxyribose. The sugar can be modified, such as 2' modified sugars (e.g., 2'O-methyl or 2' fluororibose). A phosphate at the 5' end can include modifications, such as replacing oxygen with sulfur. The two phosphates at the 5' end can include modifications, such as replacing oxygen with sulfur. The three phosphates at the 5' end may contain modifications, such as replacing oxygen with sulfur. In some embodiments, the oligonucleotide is not d(pT-GGGGG).

[0069] In some embodiments, the oligonucleotide comprises 1 lipid moiety. In some embodiments, the oligonucleotide comprises 2 lipid moieties. In some embodiments, the oligonucleotide comprises 3 lipid moieties. In some embodiments, the oligonucleotide comprises 4 lipid moieties.

[0070] Some embodiments relate to a method for preparing an oligonucleotide comprising a hydrophobic conjugate. Strategies for preparing hydrophobic conjugates can include the use of a phosphoramidite reagent based on a 6-membered cyclic alcohol (such as phenol or cyclohexanol). The phosphoramidite can react with a nucleotide to connect the nucleotide to a hydrophobic moiety, thereby producing a hydrophobic conjugate. Some examples of phosphoramidite reagents that can be used to produce hydrophobic conjugates are provided below: 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 defined by any two of the aforementioned carbon numbers. In some embodiments, R comprises or consists of an alkyl group containing 4-18 carbons. Any of the phosphoramidite reagents can react with the 5' end of the oligonucleotide to produce an oligonucleotide comprising a hydrophobic portion. In some embodiments, the phosphoramidite reagent reacts with the 5' end of the sense strand of the siRNA. The sense strand can then be hybridized with the antisense strand to form a duplex. Hybridization can be performed by incubating the sense strand and the antisense strand in a solution at a given temperature. The temperature can be gradually reduced. The temperature can include or comprise a temperature that is an annealing temperature for the sense strand and the antisense strand. The temperature can be lower than or comprise a temperature that is lower than the annealing temperature for the sense strand and the antisense strand. The temperature can be lower than the melting temperature of the sense strand and the antisense strand.

[0071] In some embodiments, oligonucleotides include negatively charged groups. Negatively charged groups can contribute to cell or tissue penetration. Negatively charged groups can be attached to the 5' or 3' end (e.g., 5' end) of oligonucleotides. This can be referred to as end groups. End groups can be or include thiophosphate, dithiophosphate, vinylphosphonate, methylphosphonate, cyclopropylphosphonate or deoxy-C-malonyl. End groups can include additional 5' phosphates, such as additional 5' phosphates. Combinations of end groups can be used.

[0072] In some embodiments, the oligonucleotide comprises a phosphate mimetic. In some embodiments, the phosphate mimetic comprises vinyl phosphonate. In some embodiments, the vinyl phosphonate comprises trans-vinyl phosphonate. In some embodiments, the vinyl phosphonate comprises cis-vinyl phosphonate. Examples of nucleotides comprising vinyl phosphonate are as follows.

[0073]

[0074] 5' vinylphosphonate 2'O-methyluridine

[0075] In some embodiments, vinyl phosphonates increase the stability of oligonucleotides. In some embodiments, vinyl phosphonates increase the accumulation of oligonucleotides in tissues. In some embodiments, vinyl phosphonates protect oligonucleotides from exonucleases or phosphatases. In some embodiments, vinyl phosphonates increase the binding affinity of oligonucleotides to siRNA processing systems.

[0076] In some embodiments, the oligonucleotide comprises 1 vinyl phosphonate. In some embodiments, the oligonucleotide comprises 2 vinyl phosphonates. In some embodiments, the oligonucleotide comprises 3 vinyl phosphonates. In some embodiments, the oligonucleotide comprises 4 vinyl phosphonates. In some embodiments, the antisense strand of the oligonucleotide comprises vinyl phosphonate at the 5' end. In some embodiments, the antisense strand of the oligonucleotide comprises vinyl phosphonate at the 3' end. In some embodiments, the sense strand of the oligonucleotide comprises vinyl phosphonate at the 5' end. In some embodiments, the sense strand of the oligonucleotide comprises vinyl phosphonate at the 3' end.

[0077] The oligonucleotide may include a purine. Examples of purines include adenine (A) or guanine (G) or modified forms thereof. The oligonucleotide may include a pyrimidine. Examples of pyrimidines include cytosine (C), thymine (T) or uracil (U) or modified forms thereof.

[0078] In some embodiments, the purines of the oligonucleotides comprise 2' fluorine-modified purines. In some embodiments, the purines of the oligonucleotides comprise 2'-O-methyl-modified purines. In some embodiments, the purines of the oligonucleotides comprise a mixture of 2'-fluorine and 2'-O-methyl-modified purines. In some embodiments, all purines of the oligonucleotides comprise 2'-fluorine-modified purines. In some embodiments, all purines of the oligonucleotides comprise 2'-O-methyl-modified purines. In some embodiments, all purines of the oligonucleotides comprise a mixture of 2'-fluorine and 2'-O-methyl-modified purines. In some embodiments, 2'-O-methyl comprises 2'O-methyl.

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

[0080] In some embodiments, the purines of the oligonucleotides comprise 2' fluorine-modified purines, and the pyrimidines of the oligonucleotides comprise a mixture of 2' fluorine and 2'-O-methyl modified pyrimidines. In some embodiments, the purines of the oligonucleotides comprise 2'-O-methyl modified purines, and the pyrimidines of the oligonucleotides comprise a mixture of 2' fluorine and 2'-O-methyl modified pyrimidines. In some embodiments, the purines of the oligonucleotides comprise 2' fluorine-modified purines, and the pyrimidines of the oligonucleotides comprise 2'-O-methyl modified pyrimidines. In some embodiments, the purines of the oligonucleotides comprise 2' fluorine-modified purines, and the pyrimidines of the oligonucleotides comprise 2' fluorine-modified pyrimidines. In some embodiments, the pyrimidines of the oligonucleotides comprise 2' fluorine-modified pyrimidines, and the purines of the oligonucleotides comprise a mixture of 2' fluorine and 2'-O-methyl modified purines. In some embodiments, the pyrimidines of the oligonucleotides comprise 2'-O-methyl modified pyrimidines, and the purines of the oligonucleotides comprise a mixture of 2' fluorine 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.

[0081] In some embodiments, all purines of the oligonucleotide comprise 2' fluorine-modified purines, and all pyrimidines of the oligonucleotide comprise a mixture of 2' fluorine 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' fluorine and 2'-O-methyl modified pyrimidines. In some embodiments, all purines of the oligonucleotide comprise 2' fluorine-modified purines, and all pyrimidines of the oligonucleotide comprise 2'-O-methyl modified pyrimidines. In some embodiments, all purines of the oligonucleotide comprise 2' fluorine-modified purines, and all pyrimidines of the oligonucleotide comprise 2' fluorine-modified pyrimidines. In some embodiments, all pyrimidines of the oligonucleotide comprise 2' fluorine-modified pyrimidines, and all purines of the oligonucleotide comprise a mixture of 2' fluorine 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' fluorine and 2'-O-methyl modified purines. In some embodiments, all pyrimidines of the oligonucleotide comprise 2' fluoro-modified pyrimidines, and all purines of the oligonucleotide comprise 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 2' fluoro-modified purines.

[0082] 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 consecutive 2' fluoro-modified pyrimidines. In some embodiments, the odd positions of the antisense strand comprise 2'-O-methyl-modified nucleotides. In some embodiments, the even 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' fluorine-modified pyrimidine; all purines of the sense strand comprise 2'-O-methyl-modified purines; 1, 2, 3, 4, or 5 pyrimidines between positions 5 and 11 comprise 2' fluorine-modified pyrimidines, provided that there are not three consecutive 2' fluorine-modified pyrimidines; the odd-numbered positions of the antisense strand comprise 2'-O-methyl-modified nucleotides; and the even-numbered positions of the antisense strand comprise 2' fluorine-modified nucleotides and unmodified deoxyribonucleotides.

[0083] In some embodiments, position 9 of the sense strand comprises a 2' fluorine-modified purine. In some embodiments, all pyrimidines of the sense strand comprise 2'-O-methyl-modified purines. In some embodiments, 1, 2, 3, 4, or 5 purines between positions 5 and 11 comprise 2' fluorine-modified purines, provided that there are no three consecutive 2' fluorine-modified purines. In some embodiments, the odd positions of the antisense strand comprise 2'-O-methyl-modified nucleotides. In some embodiments, the even positions of the antisense strand comprise 2' fluorine-modified nucleotides and unmodified deoxyribonucleotides. In some embodiments, position 9 of the sense strand comprises a 2' fluorine-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' fluorine-modified purines, provided that there are not three consecutive 2' fluorine-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' fluorine-modified nucleotides and unmodified deoxyribonucleotides. In some embodiments, there are not three consecutive 2' fluorine-modified purines. In some embodiments, there are not three consecutive 2' fluorine-modified pyrimidines.

[0084] In some embodiments, position 9 of the sense strand comprises an unmodified deoxyribonucleotide. 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 to 21 of the sense strand comprise 2'-O-methyl-modified pyrimidines, and all purines at positions 10 to 21 of the sense strand comprise 2'-O-methyl-modified purines or 2' fluoro-modified purines. In some embodiments, the odd positions of the antisense strand comprise 2'-O-methyl-modified nucleotides. In some embodiments, the even positions of the antisense strand comprise 2' fluoro-modified nucleotides and unmodified deoxyribonucleotides. In some embodiments, position 9 of the sense strand comprises an unmodified deoxyribonucleotide; positions 5, 7, and 8 of the sense strand comprise 2' fluorine-modified nucleotides; all pyrimidines at positions 10 to 21 of the sense strand comprise 2'-O-methyl-modified pyrimidines, and all purines at positions 10 to 21 comprise 2'-O-methyl-modified purines or 2' fluorine-modified purines; the odd-numbered positions of the antisense strand comprise 2'-O-methyl-modified nucleotides; and the even-numbered positions of the antisense strand comprise 2' fluorine-modified nucleotides and unmodified deoxyribonucleotides.

[0085] In some embodiments, position 9 of the sense strand comprises an unmodified deoxyribonucleotide. In some embodiments, positions 5, 7, and 8 of the sense strand comprise 2' fluorine-modified nucleotides. In some embodiments, all purines at positions 10 to 21 of the sense strand comprise 2'-O-methyl-modified purines, and all pyrimidines at positions 10 to 21 of the sense strand comprise 2'-O-methyl-modified pyrimidines or 2' fluorine-modified pyrimidines. In some embodiments, the odd positions of the antisense strand comprise 2'-O-methyl-modified nucleotides. In some embodiments, the even positions of the antisense strand comprise 2' fluorine-modified nucleotides and unmodified deoxyribonucleotides. In some embodiments, position 9 of the sense strand comprises an unmodified deoxyribonucleotide; positions 5, 7, and 8 of the sense strand comprise 2' fluorine-modified nucleotides; all purines at positions 10 to 21 of the sense strand comprise 2'-O-methyl-modified purines, and all pyrimidines at positions 10 to 21 comprise 2'-O-methyl-modified pyrimidines or 2' fluorine-modified pyrimidines; the odd-numbered positions of the antisense strand comprise 2'-O-methyl-modified nucleotides; and the even-numbered positions of the antisense strand comprise 2' fluorine-modified nucleotides and unmodified deoxyribonucleotides.

[0086] The modifications described herein can be used for delivery to cells or tissues, such as extrahepatic delivery or targeting of oligonucleotide compositions. The modifications described herein can be used to target oligonucleotide compositions to cells or tissues.

[0087] 1. Modified siRNA

[0088] In some embodiments, a composition comprises an oligonucleotide that inhibits expression of a target nucleic acid, wherein the oligonucleotide comprises a siRNA comprising a sense strand and an antisense strand, wherein the sense strand comprises modification pattern 1S: 5'-NfsnNfnNfnNfnNfnNfnNfnNfnNfsnsn-3', wherein "Nf" is a 2' fluorine-modified nucleoside, "n" is a 2' O-methyl-modified nucleoside, and "s" is a phosphorothioate linkage. In some embodiments, the sense strand comprises modification pattern 2S: 5'-NfsnNfnNfnNfnNfnNfnNfnNfnNfsnsn-3', wherein "Nf" is a 2' fluorine-modified nucleoside, "n" is a 2' O-methyl-modified nucleoside, and "s" is a phosphorothioate linkage. In some embodiments, the sense strand comprises modification pattern 3S: 5'-NfnNfnNfnNfnNfnNfnNfnNfnNfsnsn-3', wherein "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 modification pattern 4S: 5'-NfsnsNfnNfnNfnNfnNfnNfnNfnNfnNfsnsn-3', wherein "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 a modification pattern 5S: 5'-nnnnnnNfnNfnnnnnnnnnnsnsn-3', wherein "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 a modification pattern 6S: 5'-nnnnnnnNfNfNfNfnnnnnnnnsnsn-3', wherein "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 a modification pattern 7S: 5'-nnnnNfnnnNfnNfnnnnnnnnsnsn-3', wherein "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', wherein "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 a modification pattern 9S: 5'-nnnnnnNfNfNfNfNfnnnnnnnnsnsn-3', wherein "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 a modification pattern 10S: 5'-nnnnNfnNfNfNfNfnnnnnnnnnsnsn-3', wherein "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 a modification pattern 11S: 5'-nnnnnNfNfNfNfnNfnnnnnnnnsnsn-3', wherein "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 a modification pattern 12S: 5'-nnnnnNfNfNfNfnnnnnnnnnnsnsn-3', wherein "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 modification pattern 13S: 5'-nnnnNfNfNfNfNfnnnnnnnnsnsn-3', wherein "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 modification pattern 14S: 5'-nnnnnnnnNfNfnnnnnnnnnsnsn-3', wherein "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 modification pattern 15S: 5'-nnnnnnNfNfNfNfnnnnnnnnnsn-3', wherein "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 modification pattern 16S: 5'-nnnnNfnnNfnnNfnNfnnnnnnnnnsn-3', wherein "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 modification pattern 17S: 5'-nnnnNfnNfnNfnNfnnnnnnnnnsn-3', wherein "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 modification pattern 18S: 5'-nnnnnNfnnNfnNfnnnnnnnnsnsn-3', wherein "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 modification pattern 19S: 5'-nnnnnNfNfnNfnnnnnnnnnnsnsn-3', wherein "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 modification pattern 20S: 5'-nnnnnnNfnNfNfnnnnnnnnnsnsn-3', wherein "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 a modification pattern 21S: 5'-nNfnNfnNfnNfnnnnnNfnNfNfnsnsn-3', wherein "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 a modification pattern 22S: 5'-snnnnnNfNfNfNfnNfnnnnnnnnsnsn-3', wherein "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 a modification pattern 23S: 5'-snnnnnNfNfNfNfnnnnnnnnsnsn-3', wherein "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 a modification pattern 24S: 5'-snnnnNfNfNfNfNfnnnnnnnnnnnsn-3', wherein "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 a modification pattern 25S: 5'-snnnnnnnnNfNfnnnnnnnnnsnsn-3', wherein "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 a modification pattern 26S: 5'-snnnnNfNfnnNfnNfnnnnnnnnsnsn-3', wherein "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'-snNfnNfnNfnNfnnnnnNfnNfNfnsnsn-3', wherein "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'-NfnNfnNfnNfNfnNfnNfnNfnNfnNfsnsn-3', wherein "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 modification pattern 29S: 5'-nnNfnNfnNfnNfnNfnNfnNfnNfsnsn-3', wherein "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 modification pattern 30S: 5'-nnnnnNfNfNfNfNfnnnnnnnnnsnsn-3', wherein "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.

[0089] In some embodiments, the composition comprises an oligonucleotide that inhibits expression of a target mRNA, wherein the oligonucleotide comprises a siRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises modification pattern 1AS: 5'-nsNfsnNfnNfnNfnNfnNfnNfnNfnsnsn-3', wherein "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 modification pattern 2AS: 5'-nsNfsnNfnNfnNfnNfnnnNfnNfnNfnsnsn-3', wherein "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 modification pattern 3AS: 5'-nsnsnNfnNfnNfnNfnNfnNfnNfnNfnsnsn-3', wherein "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 modification pattern 4AS: 5'-nsNfsnnnNfnNfnnnnnNfnNfnnnsnsn-3', wherein "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', wherein "Nf" is a 2' fluoro modified nucleoside, "n" is a 2' O-methyl modified nucleoside, and "s" is a phosphorothioate linkage.

[0090] In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of a target nucleic acid, wherein the oligonucleotide comprises an siRNA comprising a sense strand and an antisense strand, wherein the sense strand comprises a pattern 1S and the antisense strand comprises a pattern 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises a pattern 2S and the antisense strand comprises a pattern 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises a pattern 3S and the antisense strand comprises a pattern 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises a pattern 4S and the antisense strand comprises a pattern 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises a pattern 5S and the antisense strand comprises a pattern 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises a pattern 6S and the antisense strand comprises a pattern 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 7S and the antisense strand comprises pattern 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 8S and the antisense strand comprises pattern 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 9S and the antisense strand comprises pattern 1AS, 2AS, 3AS, 4AS, or 5AS. In some embodiments, the sense strand comprises pattern 10S and the antisense strand comprises pattern 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 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 the 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.

[0091] In some embodiments, the sense strand comprises the 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 the pattern 1AS. In some embodiments, the sense strand comprises the 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 the pattern 2AS. In some embodiments, the sense strand comprises the 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 the pattern 3AS. In some embodiments, the sense strand comprises the 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 the pattern 4AS. In some embodiments, the sense strand comprises the 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 the pattern 5AS.

[0092] In some embodiments, the sense strand comprises the modification pattern 1AS, 2AS, 3AS, or 4AS. In some embodiments, the sense strand or antisense strand comprises the modification pattern 1AS, 2AS, 3AS, 4AS, or 5S. In some embodiments, the antisense strand comprises the 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 strand or the 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.

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

[0094] 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.

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

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

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

[0098] 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.

[0099] In some embodiments, the purines of the antisense strand comprise 2' fluorine-modified purines, and the pyrimidines of the antisense strand comprise a mixture of 2' fluorine and 2'-O-methyl modified pyrimidines. In some embodiments, the purines of the antisense strand comprise 2'-O-methyl modified purines, and the pyrimidines of the antisense strand comprise a mixture of 2' fluorine and 2'-O-methyl modified pyrimidines. In some embodiments, the purines of the antisense strand comprise 2' fluorine-modified purines, and the pyrimidines of the antisense strand comprise 2'-O-methyl modified pyrimidines. In some embodiments, the purines of the antisense strand comprise 2'-O-methyl modified purines, and the pyrimidines of the antisense strand comprise 2' fluorine-modified pyrimidines. In some embodiments, the pyrimidines of the antisense strand comprise 2' fluorine-modified pyrimidines, and the purines of the antisense strand comprise a mixture of 2' fluorine and 2'-O-methyl modified purines. In some embodiments, the pyrimidines of the antisense strand comprise 2'-O-methyl modified pyrimidines, and the purines of the antisense strand comprise a mixture of 2' fluoro and 2'-O-methyl modified purines. In some embodiments, the pyrimidines of the antisense strand comprise 2' fluoro modified pyrimidines, and the purines of the antisense strand comprise 2'-O-methyl modified purines. In some embodiments, the pyrimidines of the antisense strand comprise 2'-O-methyl modified pyrimidines, and the purines of the antisense strand comprise 2' fluoro modified purines.

[0100] In some embodiments, all purines of the antisense strand comprise 2' fluoro-modified purines, and all pyrimidines of the antisense strand comprise a mixture of 2' fluoro- and 2'-O-methyl-modified pyrimidines. In some embodiments, all purines of the antisense strand comprise 2'-O-methyl-modified purines, and all pyrimidines of the antisense strand comprise a mixture of 2' fluoro- and 2'-O-methyl-modified pyrimidines. In some embodiments, all purines of the antisense strand comprise 2' fluoro-modified purines, and all pyrimidines of the antisense strand comprise 2'-O-methyl-modified purines. In some embodiments, all purines of the antisense strand comprise 2'-O-methyl-modified purines, and all pyrimidines of the antisense strand comprise 2' fluoro-modified pyrimidines. In some embodiments, all pyrimidines of the antisense strand comprise 2' fluoro-modified pyrimidines, and all purines of the antisense strand comprise a mixture of 2' fluoro- and 2'-O-methyl-modified purines. In some embodiments, all pyrimidines of the antisense strand comprise 2'-O-methyl modified pyrimidines, and all purines of the antisense strand comprise a mixture of 2' fluoro and 2'-O-methyl modified purines. In some embodiments, all pyrimidines of the antisense strand comprise 2' fluoro modified pyrimidines, and all purines of the antisense strand comprise 2'-O-methyl modified purines. In some embodiments, all pyrimidines of the antisense strand comprise 2'-O-methyl modified pyrimidines, and all purines of the antisense strand comprise 2' fluoro modified purines.

[0101] In some embodiments, the siRNA comprises a sense strand, an antisense strand, and a lipid moiety attached to a terminus of the sense strand or the antisense strand; wherein the lipid moiety comprises a phenyl or cyclohexyl linker, wherein the linker is attached to the lipid and to the terminus of the sense strand or the antisense strand. In some embodiments, with respect to the sense strand, any 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 purines, 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 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'-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 vinylphosphonate and two phosphorothioate linkages and a 3' end comprising two phosphorothioate linkages; wherein the sense strand comprises a 5' end comprising a hydrophobic portion and a 3' end comprising two phosphorothioate linkages; wherein with respect to the sense strand, any of the following is true: all purines comprise 2' fluorine-modified purines, and all pyrimidines comprise a mixture of 2' fluorine- 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'-fluoro modified purines, and all pyrimidines include 2'-O-methyl modified pyrimidines, all pyrimidines include 2'-fluoro modified pyrimidines, 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 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, any 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 The purine comprises a 2'-O-methyl modified purine and all pyrimidine nucleosides comprise a 2' fluorine modified pyrimidine, all pyrimidines comprise a 2' fluorine modified pyrimidine and all purines comprise a mixture of 2' fluorine and 2'-O-methyl modified purines, all pyrimidines comprise a 2'-O-methyl modified pyrimidine and all purines comprise a mixture of 2' fluorine and 2'-O-methyl modified purines, or all pyrimidines comprise a 2'-O-methyl modified pyrimidine and all purines comprise a 2' fluorine modified purine.

[0102] In some embodiments, with respect to the sense strand, any 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'-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; with the proviso that in any of the above, the sense strand may include deoxynucleosides. In some embodiments, all purines comprise 2' fluorine-modified purines, and all pyrimidines comprise a mixture of 2' fluorine and 2'-O-methyl modified pyrimidines; provided that deoxynucleosides 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' fluorine and 2'-O-methyl modified pyrimidines; provided that deoxynucleosides may be included in the sense strand. In some embodiments, in the sense strand, all purines comprise 2' fluorine-modified purines, and all pyrimidines comprise 2'-O-methyl modified pyrimidines; provided that deoxynucleosides may be included in the sense strand. In some embodiments, all pyrimidines comprise 2' fluorine-modified pyrimidines, and all purines comprise 2' fluorine and 2'-O-methyl modified purines; provided that deoxynucleosides 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; provided that deoxynucleosides 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; provided that deoxynucleosides may be included in the sense strand. In some embodiments, the sense strand comprises deoxynucleosides. Deoxynucleosides may be at nucleoside position 9 of the sense strand. In some embodiments, the sense strand does not comprise deoxynucleosides. The deoxynucleosides of the sense strand may otherwise be unmodified.

[0103] In some embodiments, with respect to the antisense strand, any 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'-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 include deoxynucleosides. In some embodiments, all purines comprise 2' fluorine-modified purines, and all pyrimidines comprise a mixture of 2' fluorine and 2'-O-methyl modified pyrimidines; provided that deoxynucleosides 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' fluorine and 2'-O-methyl modified pyrimidines; provided that deoxynucleosides 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' fluorine-modified pyrimidines; provided that deoxynucleosides may be included in the antisense strand. In some embodiments, in the antisense strand, all pyrimidines comprise 2' fluorine-modified pyrimidines, and all purines comprise a mixture of 2' fluorine and 2'-O-methyl modified purines; provided that deoxynucleosides may be included in the antisense strand. In some embodiments, in the antisense strand, all pyrimidines comprise 2'-O-methyl modified pyrimidines, and all purines comprise a mixture of 2'-fluoro and 2'-O-methyl modified purines; provided that deoxynucleosides may be included in the antisense strand. In some embodiments, in the antisense strand, all pyrimidines comprise 2'-O-methyl modified pyrimidines, and all purines comprise 2'-fluoro modified purines; provided that deoxynucleosides may be included in the antisense strand. In some embodiments, the antisense strand comprises deoxynucleosides. Deoxynucleosides may be at nucleoside position 9 of the antisense strand. In some embodiments, the antisense strand does not include deoxynucleosides. The deoxynucleosides of the antisense strand may be unmodified in other aspects.

[0104] In some embodiments, with respect to the sense strand, any 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'-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; provided that in any of the above, the sense strand may include 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; provided 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; provided 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; provided that deoxynucleosides or 2'-O-methoxyethyl nucleosides may be included in the sense strand. In some embodiments, all pyrimidines comprise 2'fluoro-modified pyrimidines, and all purines comprise a mixture of 2'fluoro and 2'-O-methyl modified purines; provided that deoxynucleosides or 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; provided that deoxynucleosides or 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; provided that deoxynucleosides or 2'-O-methoxyethyl nucleosides may be included in the sense strand. In some embodiments, the sense strand comprises deoxynucleosides. Deoxynucleosides may be at nucleoside position 9 of the sense strand. In some embodiments, the sense strand does not comprise deoxynucleosides. The deoxynucleosides of the sense strand may be otherwise unmodified. In some embodiments, the sense strand includes 2'-O-methoxyethyl nucleosides. The 2'-O-methoxyethyl nucleosides may be at nucleoside position 4 of the sense strand. The 2'-O-methoxyethyl nucleosides may include 2'-O-methoxyethyl thymidine.In some embodiments, the sense strand does not include 2'-O-methoxyethyl nucleosides. Additionally, the 2'-O-methoxyethyl nucleosides of the sense strand can be otherwise unmodified.

[0105] In some embodiments, with respect to the antisense strand, any 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'-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; provided that in any of the above, the sense strand may include 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; provided 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; provided 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; provided that deoxynucleosides or 2'-O-methoxyethyl nucleosides may be included in the antisense strand. In some embodiments, in the antisense strand, all pyrimidines comprise 2'-fluoro-modified pyrimidines, and all purines comprise a mixture of 2'-fluoro and 2'-O-methyl-modified purines; provided that deoxynucleosides or 2'-O-methoxyethyl nucleosides may be included in the antisense strand. In some embodiments, in the antisense strand, all pyrimidines comprise 2'-O-methyl-modified pyrimidines, and all purines comprise a mixture of 2'-fluoro and 2'-O-methyl-modified purines; provided that deoxynucleosides or 2'-O-methoxyethyl nucleosides may be included in the antisense strand. In some embodiments, in the antisense strand, all pyrimidines comprise 2'-O-methyl-modified pyrimidines, and all purines comprise 2'-fluoro-modified purines; provided that deoxynucleosides or 2'-O-methoxyethyl nucleosides may be included in the antisense strand. In some embodiments, the antisense strand comprises deoxynucleosides. Deoxynucleosides may be at nucleoside position 9 of the antisense strand. In some embodiments, the antisense strand does not comprise deoxynucleosides. The deoxynucleosides of the antisense strand may otherwise be unmodified. In some embodiments, the antisense strand includes 2'-O-methoxyethyl nucleosides. The 2'-O-methoxyethyl nucleosides may be at nucleoside position 4 of the sense strand.The 2'-O-methoxyethyl nucleosides may include 2'-O-methoxyethyl thymidine. In some embodiments, the antisense strand does not include 2'-O-methoxyethyl nucleosides. The 2'-O-methoxyethyl nucleosides of the antisense strand may be unmodified in other aspects.

[0106] In some embodiments, with respect to the sense strand, any 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'-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; with the proviso that in any of the above, the sense strand may include 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; provided that 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; provided that 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; provided that 2'-O-methoxyethyl nucleosides may be included in the sense strand. In some embodiments, all pyrimidines comprise 2' fluoro-modified pyrimidines, and all purines comprise a mixture of 2' fluoro- and 2'-O-methyl-modified purines; provided 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; provided 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; provided that 2'-O-methoxyethyl nucleosides may be included in the sense strand. In some embodiments, the sense strand comprises 2'-O-methoxyethyl nucleosides. 2'-O-methoxyethyl nucleosides may be at nucleoside position 4 of the sense strand. 2'-O-methoxyethyl nucleosides may include 2'-O-methoxyethyl thymidine. In some embodiments, the sense strand does not include 2'-O-methoxyethyl nucleosides. The 2'-O-methoxyethyl nucleosides of the sense strand may be unmodified in other aspects.

[0107] In some embodiments, with respect to the antisense strand, any 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'-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; provided that in any of the above, the sense strand may include deoxynucleosides or 2'-O-methoxyethyl nucleosides. In some embodiments, all purines comprise 2' fluorine-modified purines, and all pyrimidines comprise a mixture of 2' fluorine and 2'-O-methyl modified pyrimidines; provided 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' fluorine and 2'-O-methyl modified pyrimidines; provided 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' fluorine-modified pyrimidines; provided that 2'-O-methoxyethyl nucleosides may be included in the antisense strand. In some embodiments, in the antisense strand, all pyrimidines comprise 2' fluorine-modified pyrimidines, and all purines comprise a mixture of 2' fluorine and 2'-O-methyl modified purines; provided 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'-fluorine and 2'-O-methyl modified purines; provided that 2'-O-methoxyethyl nucleosides can 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'-fluorine modified purines; provided that 2'-O-methoxyethyl nucleosides can be included in the antisense strand. In some embodiments, the antisense strand includes 2'-O-methoxyethyl nucleosides. 2'-O-methoxyethyl nucleosides can be at nucleoside position 4 of the sense strand. 2'-O-methoxyethyl nucleosides can include 2'-O-methoxyethyl thymidine. In some embodiments, the antisense strand does not include 2'-O-methoxyethyl nucleosides. The 2'-O-methoxyethyl nucleosides of the antisense strand can be unmodified in other aspects.

[0108] In some embodiments, with respect to the sense strand (provided that the sense strand may include 2' deoxynucleosides), any of the following is true: all purine nucleosides contain 2' fluorine and all pyrimidine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl, all purine nucleosides contain 2'-O-methyl and all pyrimidine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl, all purine nucleosides contain 2' fluorine and all pyrimidine nucleosides contain 2'-O-methyl, all pyrimidine nucleosides contain 2' fluorine and all purine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl, all pyrimidine nucleosides contain 2' fluorine and all purine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl; or all pyrimidine nucleosides contain 2' fluorine and all purine nucleosides contain 2'-O-methyl. In some embodiments, in the sense strand, all purine nucleosides contain 2' fluorine, and all pyrimidine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl, provided that the sense strand may include 2' deoxynucleosides. In some embodiments, in the sense strand, all purine nucleosides contain 2'-O-methyl, and all pyrimidine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl, provided that the sense strand may include 2' deoxynucleosides. In some embodiments, in the sense strand, all purine nucleosides contain 2' fluorine, and all pyrimidine nucleosides contain 2'-O-methyl, provided that the sense strand may include 2' deoxynucleosides. In some embodiments, in the sense strand, all pyrimidine nucleosides contain 2' fluorine, and all purine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl, provided that the sense strand may include 2' deoxynucleosides. In some embodiments, in the sense strand, all pyrimidine nucleosides contain 2'-O-methyl, and all purine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl, provided that the sense strand may include 2' deoxynucleosides. In some embodiments, in the sense strand, all pyrimidine nucleosides contain 2' fluorine, and all purine nucleosides contain 2'-O-methyl, provided 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 a condition that the sense strand may include 2'-O-methoxyethyl nucleosides (e.g., at position 4, 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.

[0109] In some embodiments, with respect to the sense strand, any of the following is true: all purine nucleosides contain 2' fluorine, and all pyrimidine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl; all purine nucleosides contain 2'-O-methyl, and all pyrimidine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl; all purine nucleosides contain 2' fluorine, and all pyrimidine nucleosides contain 2' fluorine, and all purine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl; all pyrimidine nucleosides contain 2' fluorine, and all purine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl; or all pyrimidine nucleosides contain 2' fluorine, and all purine nucleosides contain 2' fluorine. In some embodiments, in the sense strand, all purine nucleosides contain 2' fluorine, and all pyrimidine nucleosides are modified with a mixture of 2' fluorine 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'-O-methyl and 2'-O-methyl. In some embodiments, in the sense strand, all purine nucleosides include 2'-fluorine, and all pyrimidine nucleosides include 2'-O-methyl. In some embodiments, in the sense strand, all pyrimidine nucleosides include 2'-fluorine, and all purine nucleosides are modified with a mixture of 2'-fluorine 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'-fluorine and 2'-O-methyl. In some embodiments, in the sense strand, all pyrimidine nucleosides include 2'-fluorine, and all purine nucleosides include 2'-O-methyl. Some embodiments include a condition that the sense strand may include 2'-O-methoxyethyl nucleosides (e.g., at position 4, 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.

[0110] In some embodiments, with respect to the antisense strand (provided that the sense strand may include 2' deoxynucleosides), any of the following is true: all purine nucleosides contain 2' fluorine and all pyrimidine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl, all purine nucleosides contain 2'-O-methyl and all pyrimidine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl, all purine nucleosides contain 2'-O-methyl and all pyrimidine nucleosides contain 2' fluorine, all pyrimidine nucleosides contain 2' fluorine and all purine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl, all pyrimidine nucleosides contain 2'-O-methyl and all purine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl; or all pyrimidine nucleosides contain 2'-O-methyl and all purine nucleosides contain 2' fluorine. In some embodiments, in the antisense strand, all purine nucleosides include 2' fluorine, and all pyrimidine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl, provided that the antisense strand may include 2' deoxynucleosides. In some embodiments, in the antisense strand, all purine nucleosides include 2' fluorine, and all pyrimidine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl, provided that the antisense strand may include 2' deoxynucleosides. In some embodiments, in the antisense strand, all purine nucleosides include 2' fluorine, and all pyrimidine nucleosides include 2'-O-methyl, provided that the antisense strand may include 2' deoxynucleosides. In some embodiments, in the antisense strand, all pyrimidine nucleosides include 2' fluorine, and all purine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl, provided that the antisense strand may 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' fluorine and 2'-O-methyl, provided that the antisense strand may include 2' deoxynucleosides. In some embodiments, in the antisense strand, all pyrimidine nucleosides include 2' fluorine, and all purine nucleosides include 2'-O-methyl, provided 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 a condition that the antisense strand may include 2'-O-methoxyethyl nucleosides (e.g., at position 4, 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.

[0111] In some embodiments, with respect to the antisense strand, any of the following is true: all purine nucleosides contain 2' fluorine, and all pyrimidine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl; all purine nucleosides contain 2'-O-methyl, and all pyrimidine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl; all purine nucleosides contain 2'-O-methyl, all pyrimidine nucleosides contain 2' fluorine, and all purine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl; all pyrimidine nucleosides contain 2'-O-methyl, and all purine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl; or all pyrimidine nucleosides contain 2'-O-methyl, and all purine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl. In some embodiments, in the antisense strand, all purine nucleosides contain 2' fluorine, and all pyrimidine nucleosides are modified with a mixture of 2' fluorine 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' fluorine and 2'-O-methyl. In some embodiments, in the antisense strand, all purine nucleosides include 2' fluorine, and all pyrimidine nucleosides include 2'-O-methyl. In some embodiments, in the antisense strand, all pyrimidine nucleosides include 2' fluorine, and all purine nucleosides are modified with a mixture of 2' fluorine 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' fluorine and 2'-O-methyl. In some embodiments, in the antisense strand, all pyrimidine nucleosides include 2' fluorine, and all purine nucleosides include 2'-O-methyl. Some embodiments include a condition that the antisense strand may include 2'-O-methoxyethyl nucleosides (e.g., at position 4, 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.

[0112] In some embodiments, the antisense strand comprises one or two 3' phosphorothioate linkages. For example, there may be a phosphorothioate between the first and second nucleotides at the 3' end of the antisense strand, or there may be a phosphorothioate between the first, second, and third nucleotides at the 3' end of the antisense strand. In some embodiments, the sense strand comprises one or two 5' phosphorothioate linkages. For example, there may be a phosphorothioate linkage between the first and second nucleotides at the 5' end of the sense strand, or there may be a phosphorothioate linkage between the first, second, and third nucleotides at the 5' end of the sense strand. In some embodiments, the sense strand does not comprise one or two 5' phosphorothioate linkages. For example, in some embodiments, there is no phosphorothioate linkage between the last 3 nucleotides at the 5' end of the sense strand. In some embodiments, the sense strand comprises a 5' phosphate linkage. In some embodiments, the sense strand comprises one or two 3' phosphorothioate linkages. For example, phosphorothioate may be present between the first and second nucleotides at the 3' end of the sense strand, or phosphorothioate may be present between the first, second, and third nucleotides at the 3' end of the sense strand.

[0113] In some embodiments, the antisense strand includes a 5' end comprising two phosphorothioate linkages. The 5' end may include three nucleosides separated by two phosphorothioate linkages. In some embodiments, the antisense strand includes a 3' end comprising two phosphorothioate linkages. The 3' end may include three nucleosides separated by two phosphorothioate linkages.

[0114] In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of a target nucleic acid, wherein the oligonucleotide comprises an siRNA comprising a sense strand and an antisense strand, wherein the oligonucleotide comprises a hydrophobic portion. In some embodiments, the hydrophobic portion may be attached to the 5' end of the sense strand. In some embodiments, the hydrophobic portion may be attached to the 3' end of the sense strand. In some embodiments, the hydrophobic portion may be attached to the 5' end of the antisense strand. In some embodiments, the hydrophobic portion may be attached to the 3' end of the antisense strand.

[0115] In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of a target nucleic acid, wherein the oligonucleotide comprises an siRNA comprising a sense strand and an antisense strand, wherein the oligonucleotide comprises one or more vinyl phosphonates. In some embodiments, the one or more vinyl phosphonates may be attached to the 5' end of the sense strand. In some embodiments, the one or more vinyl phosphonates may be attached to the 3' end of the sense strand. In some embodiments, the one or more vinyl phosphonates may be attached to the 5' end of the antisense strand. In some embodiments, the one or more vinyl phosphonates may be attached to the 3' end of the antisense strand.

[0116] In some embodiments, the sense strand comprises RNA or modified RNA nucleotides or consists of them. In some embodiments, the sense strand comprises deoxynucleosides. Deoxynucleosides may include DNA nucleosides. In some embodiments, deoxynucleosides comprise 2' deoxynucleosides or consist of them. Deoxynucleosides may be located at positions (5' to 3', wherein the 5' position is 1) within the sense strand. The position within the sense strand may be or include positions 2, 4, 6, 8, 9, 10, 12, 14, 16 or 18, or a combination of said positions. The position within the sense strand may be or include positions 2, 4, 6, 8, 10, 12, 14, 16 or 18, or a combination of said positions. The position within the sense strand may be or include positions 2, 6, 9, 10, 14 or 18, or a combination of said positions. The position within the sense strand may be or include positions 2, 6, 10, 14 or 18, or a combination of said positions. The position within the sense strand can be or include position 4, 8, 9, 12 or 16, or a combination of the positions. The position within the sense strand can be or include position 4, 8, 12 or 16, or a combination of the positions. The position within the sense strand can include position 9. The position within the sense strand can be position 9. The sense strand can 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 includes 1 deoxynucleoside. The sense strand can include 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 deoxynucleoside numbers. The sense strand can include deoxynucleosides at all even positions. The sense strand may include deoxynucleosides at some even-numbered positions. The sense strand may include deoxynucleosides at every other even-numbered position. The sense strand may include 1 deoxynucleoside. The sense strand may include at least 1 deoxynucleoside. The sense strand may include at least 2 deoxynucleosides. The sense strand may include at least 3 deoxynucleosides. The sense strand may include at least 4 deoxynucleosides. The sense strand may include at least 5 deoxynucleosides. The sense strand may include at least 6 deoxynucleosides. The sense strand may include at least 7 deoxynucleosides. The sense strand may include at least 8 deoxynucleosides. The sense strand may include at least 9 deoxynucleosides. The sense strand may include at least 10 deoxynucleosides. The sense strand may include no more than 2 deoxynucleosides. The sense strand may include no more than 3 deoxynucleosides. The sense strand may include no more than 4 deoxynucleosides. The sense strand may include no more than 5 deoxynucleosides. The sense strand may include no more than 6 deoxynucleosides. The sense strand may include no more than 7 deoxynucleosides. The sense strand may include no more than 8 deoxynucleosides. The sense strand may include no more than 9 deoxynucleosides. The sense strand may include no more than 10 deoxynucleosides.

[0117] In some embodiments, the antisense strand comprises RNA or modified RNA nucleotides or consists of them. In some embodiments, the antisense strand comprises deoxynucleosides. Deoxynucleosides can include DNA nucleosides. In some embodiments, deoxynucleosides comprise 2' deoxynucleosides or consist of them. The antisense strand can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 deoxynucleosides, or a deoxynucleoside range defined by any two of the aforementioned deoxynucleoside numbers.

[0118] In some embodiments where deoxynucleosides are included in the sense strand (e.g., at the 9th nucleotide counted from the 5' end), the nucleosides at positions 1-8 include a mixture of 2'-fluoro and 2'-O-methyl modified nucleosides. In some embodiments where deoxynucleosides are included in the sense strand, the purines at positions 1-8 include a mixture of 2'-fluoro and 2'-O-methyl modified nucleosides. In some embodiments where deoxynucleosides are included in the sense strand, the pyrimidines at positions 1-8 include a mixture of 2'-fluoro and 2'-O-methyl modified nucleosides. In some embodiments where deoxynucleosides are included in the sense strand, the nucleosides at positions 1-8 all include 2'-O-methyl modified nucleosides. In some embodiments where deoxynucleosides are included in the sense strand, the purines at positions 1-8 all include 2'-O-methyl modified nucleosides. In some embodiments where deoxynucleosides are included in the sense strand, the pyrimidines at positions 1-8 all include 2'-O-methyl modified nucleosides. In some embodiments wherein deoxynucleosides are included in the sense strand, the purines at positions 1-8 include 2'-fluoro and 2'-O-methyl modified nucleosides, and the pyrimidines at positions 1-8 all include 2'-O-methyl modified nucleosides. In some embodiments wherein deoxynucleosides are included in the sense strand, the pyrimidines at positions 1-8 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.

[0119] The antisense strand can start in an alternating pattern where even-numbered nucleosides include 2' fluorine-modified nucleosides and odd-numbered nucleosides include 2'-O-methyl-modified nucleosides. The sense strand can then be optimized by replacing some of the 2' fluorine modifications with 2'-O-methyl modifications. The antisense strand can include a mixture of 2' fluorine modifications and 2'-O-methyl modifications.

[0120] 2. Modified ASO

[0121] In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of a target nucleic acid, wherein the oligonucleotide comprises an antisense oligonucleotide (ASO). In some embodiments, the ASO comprises a modification pattern of 1S, 2S, 3S, 4S, 1AS or 2AS, or any other combination of modifications described herein. In some embodiments, the ASO comprises a modification pattern of 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.

[0122] D. Preparation

[0123] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition is sterile. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0124] In some embodiments, the pharmaceutically acceptable carrier comprises water. In some embodiments, the pharmaceutically acceptable carrier comprises a buffer. In some embodiments, the pharmaceutically acceptable carrier comprises a saline solution. In some embodiments, the pharmaceutically acceptable carrier comprises water, a buffer, or a saline solution. In some embodiments, the composition comprises liposomes. In some embodiments, the pharmaceutically acceptable carrier comprises liposomes, lipids, nanoparticles, proteins, protein-antibody complexes, peptides, cellulose, nanogels, or combinations thereof.

[0125] In some embodiments, the composition is formulated to pass through the blood-brain barrier. In some embodiments, the composition is formulated for central nervous system (CNS) delivery. In some embodiments, the composition includes a lipophilic compound. The lipophilic compound can be used to pass through the blood-brain barrier or for CNS delivery.

[0126] In some embodiments, the composition is formulated for administration. Administration can be systemic. In some embodiments, administration is intravenous administration. In some embodiments, administration is by injection. In some embodiments, injection is subcutaneous injection. In some embodiments, injection is subcutaneous injection. In some embodiments, injection is intraperitoneal injection. In some embodiments, injection is intramuscular injection. In some embodiments, injection is intrathecal injection. Administration can be applied to the eye (e.g., in the vitreous body). Administration can be applied to neural tissue. Administration can be applied to the brain. Administration can be intraventricular administration. Administration can be intrathecal administration. Administration can be applied to the spinal cord or spinal canal. In some embodiments, the formulation allows compounds such as oligonucleotides to be delivered to eye cells. In some embodiments, the formulation allows compounds such as oligonucleotides to be delivered to neural cells.

[0127] E. Kit

[0128] In some embodiments, kits are described herein. The kit may include oligonucleotides, such as siRNAs described herein. The oligonucleotides may be conjugated to lipid moieties. The kit may include lipid moieties described herein. The oligonucleotides may include nucleoside modifications or modified internucleoside linkages. The oligonucleotides may include any modification described herein, such as modifications from base sequences. The kit may include a delivery agent, such as an injection. The kit may include instructions for use, such as methods for use in the methods described herein.

[0129] II. Methods and Uses

[0130] Disclosed herein, in some embodiments, are methods of administering a composition described herein to a subject.Some embodiments relate to using a composition described herein, such as administering the composition to a subject.

[0131] Some embodiments relate to a method of treating a condition in a subject in need thereof. Some embodiments relate to the use of a composition as described herein in a method of treatment. Some embodiments include administering a composition as described herein to a subject suffering from a condition. In some embodiments, administering treats a condition in a subject. In some embodiments, the composition treats a condition in a subject.

[0132] In some embodiments, treatment includes preventing, inhibiting, or reversing a condition in a subject. Some embodiments relate to the use of a composition as described herein in a method for preventing, inhibiting, or reversing a condition. Some embodiments relate to a method for preventing, inhibiting, or reversing a condition in a subject in need thereof. Some embodiments include administering a composition as described herein to a subject having a condition. In some embodiments, administration prevents, inhibits, or reverses a condition in a subject. In some embodiments, the composition prevents, inhibits, or reverses a condition in a subject.

[0133] Some embodiments relate to a method of preventing a condition in a subject in need thereof. Some embodiments relate to the use of a composition described herein in a method of preventing a condition. Some embodiments include administering a composition described herein to a subject having a condition. In some embodiments, administration prevents the condition in a subject. In some embodiments, the composition prevents the condition in a subject.

[0134] Some embodiments relate to a method of inhibiting a condition in a subject in need thereof. Some embodiments relate to the use of a composition described herein in a method of inhibiting a condition. Some embodiments include administering a composition described herein to a subject having a condition. In some embodiments, administration inhibits the condition of the subject. In some embodiments, the composition inhibits the condition of the subject.

[0135] Some embodiments relate to a method of reversing a condition in a subject in need thereof. Some embodiments relate to the use of a composition as described herein in a method of reversing a condition. Some embodiments include administering a composition as described herein to a subject having a condition. In some embodiments, administration reverses the condition of the subject. In some embodiments, the composition reverses the condition of the subject.

[0136] In some embodiments, administration is systemic. In some embodiments, administration is intravenous administration. In some embodiments, administration is by injection. In some embodiments, injection is subcutaneous injection. In some embodiments, injection is subcutaneous injection. In some embodiments, injection is intraperitoneal injection. In some embodiments, injection is intramuscular injection. Administration can be administered to the eye (e.g., in the vitreous body). Administration can be administered to neural tissue. Administration can be administered to the brain. Administration can be intraventricular administration. In some embodiments, injection is intrathecal injection. Administration can be intrathecal administration. Administration can be administered to the spinal cord or spinal canal.

[0137] In some embodiments, disclosed herein is a method of targeting siRNA to a cell or tissue. The method may include conjugating the siRNA (e.g., the sense strand of the siRNA) to a lipid moiety as described herein. The method may include delivering the siRNA conjugated to the lipid moiety to a tissue or cell. The method may include contacting a tissue or cell with the siRNA conjugated to the lipid moiety.

[0138] In some embodiments, disclosed herein is a method for reducing the amount of a target RNA or protein in a cell or tissue, comprising administering or delivering a composition described herein to a cell or tissue. The composition may include an siRNA having an antisense strand having a sequence complementary to an RNA. The antisense strand may bind to the RNA.

[0139] In some embodiments, methods for preparing siRNA compositions are disclosed herein. The method may include conjugating the siRNA to a lipid moiety. The siRNA and lipid moiety may include any siRNA or lipid moiety described herein. The method may include synthesizing the siRNA. The method may include synthesizing the lipid moiety.

[0140] A. Symptoms

[0141] 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 lung disorder.

[0142] B. Object

[0143] 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, a mammal, a dog, a cat, a cow, a rodent, a mouse, a rat, a primate, or a monkey. In some embodiments, the subject is a human.

[0144] 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 old).

[0145] C. Baseline Measurements

[0146] Some embodiments of the methods described herein include obtaining a baseline measurement from a subject. For example, in some embodiments, a baseline measurement is obtained from a subject prior to treating the subject.

[0147] In some embodiments, the baseline measurements are obtained directly from the subject. In some embodiments, the baseline measurements are obtained by observation, such as by observing the subject or the subject's tissue. In some embodiments, the baseline measurements are obtained in a non-invasive manner using an imaging device.

[0148] In some embodiments, the baseline measurement value is obtained in a sample from an object. In some embodiments, the baseline measurement value is obtained in one or more histological tissue sections. In some embodiments, the baseline measurement value is obtained by performing an assay such as an immunoassay, a colorimetric assay, or a fluorometric assay on a sample obtained from an object. In some embodiments, the baseline measurement value is obtained by an assay such as an immunoassay, a colorimetric assay, a fluorometric assay, or a chromatography (e.g., HPLC) assay. In some embodiments, the baseline measurement value is obtained by PCR.

[0149] 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 indicated as the mass or percentage / sample weight of the target protein. In some embodiments, the baseline target protein level is indicated as the mass or percentage / sample volume of the target protein. In some embodiments, the baseline target protein level is indicated as the mass or percentage / total protein of the target protein in the sample. In some embodiments, the baseline target protein measurement is a baseline circulation / 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 fluorometric assay.

[0150] In some embodiments, the baseline measurement is a baseline target mRNA measurement. In some embodiments, the baseline target mRNA measurement includes a baseline target mRNA level. In some embodiments, the baseline target mRNA level is indicated as the amount or percentage / sample weight of the target mRNA. In some embodiments, the baseline target mRNA level is indicated as the amount or percentage / sample volume of the target mRNA. In some embodiments, the baseline target mRNA level is indicated as the amount or percentage / total mRNA of the target mRNA in the sample. In some embodiments, the baseline target mRNA level is indicated as the amount or percentage / total nucleic acid of the target mRNA in the sample. In some embodiments, the baseline target mRNA level is relative to another mRNA level in the sample, such as the mRNA level of a housekeeping gene. 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 a determination such as a polymerase chain reaction (PCR) determination. In some embodiments, PCR includes quantitative PCR (qPCR). In some embodiments, PCR includes reverse transcription of the target mRNA.

[0151] Some embodiments of the methods described herein include obtaining a sample from a subject. In some embodiments, the baseline measurement is obtained from a sample from a subject. In some embodiments, the sample is obtained from the subject before administering a composition as described herein or treating the subject with a composition as described herein. In some embodiments, the baseline measurement is obtained from a sample obtained from the subject before administering the composition to the subject. In some embodiments, the sample is obtained from a subject in a fasting state. In some embodiments, the sample is obtained from a subject after an overnight fast. In some embodiments, the sample is obtained from a subject in a fed state.

[0152] In some embodiments, the sample comprises a fluid. In some embodiments, the sample is a fluid sample. In some embodiments, the sample is a blood, plasma or serum sample. 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 is a plasma sample. The blood sample can be a plasma sample. In some embodiments, the sample comprises serum. In some embodiments, the sample is a serum sample. The blood sample can be a serum sample. In some embodiments, the fluid comprises cerebrospinal fluid (CSF). For example, the fluid can be obtained by spinal puncture. In some embodiments, the fluid comprises spinal fluid. In some embodiments, the fluid comprises brain fluid. In some embodiments, the fluid comprises brain fluid.

[0153] In some embodiments, the sample includes tissue. In some embodiments, the sample is a tissue sample. In some embodiments, the tissue includes eye, kidney, muscle, fat, brain, lung or vascular tissue. For example, baseline target mRNA measurements or baseline target protein measurements can be obtained in kidney, fat, brain, lung, or vascular samples obtained from patients. In some embodiments, the tissue includes kidney tissue. Kidney tissue may include renal medullary cells or renal proximal tubule cells. In some embodiments, the tissue includes adipose tissue. In some embodiments, adipose tissue includes white adipose tissue. Adipose tissue may include adipocytes. In some embodiments, the tissue includes liver tissue. The liver may include hepatocytes. In some embodiments, the tissue is non-liver tissue. In some embodiments, the tissue includes eye tissue. In some embodiments, eye tissue includes retinal tissue. In some embodiments, the tissue includes brain tissue. Brain tissue may include neurons or glial cells. Brain tissue may include cerebellum. Brain tissue may include frontal cortex. Brain tissue may include temporal cortex. Brain tissue may include tissue from the right hemisphere of the brain (e.g., right frontal cortex or right temporal cortex). Brain tissue can include tissue from the left hemisphere of the brain (e.g., left frontal cortex or left temporal cortex). Brain tissue can include hippocampal tissue. In some embodiments, tissue includes neural tissue. In some embodiments, tissue includes nerves. In some embodiments, tissue includes spinal tissue. In some embodiments, tissue includes tissue of the spinal cord or spinal canal. In some embodiments, tissue includes lung tissue, and in some embodiments, tissue includes vascular tissue. Vascular tissue can include vascular endothelial cells.

[0154] In some embodiments, the sample includes cells. In some embodiments, the sample includes cells. In some embodiments, the cells include eye cells, kidney cells, muscle cells, fat cells, brain cells or vascular system cells. In some embodiments, the cells are kidney cells. In some embodiments, the kidney cells are renal medullary cells. In some embodiments, the kidney cells are renal proximal tubule cells. In some embodiments, the cells are fat cells. In some embodiments, the fat cells are fat cells. In some embodiments, the cells are liver cells. In some embodiments, the liver cells are hepatocytes. In some embodiments, the cells are non-liver cells, or are not liver cells. In some embodiments, the cells are eye cells. In some embodiments, the eye cells are retinal cells. In some embodiments, the eye cells are rod cells. In some embodiments, the eye cells are cone cells. In some embodiments, the cells are brain cells. In some embodiments, the brain cells are from brain tissues, such as cerebellum, cortex, hippocampus tissue. In some embodiments, the brain cells are neurons. In some embodiments, the brain cells are glial cells. In some embodiments, the cells are spinal cells. In some embodiments, the cells are spinal cord cells. 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.

[0155] D. Effect

[0156] In some embodiments, the composition or administration of the composition affects a measurement, such as a target protein measurement or a target mRNA measurement relative to a baseline measurement.

[0157] Some embodiments of the methods described herein include obtaining a measurement from a subject. For example, the measurement can be obtained from the subject after the subject is treated. In some embodiments, after the composition is applied to the subject, the measurement is obtained in a second sample (such as a fluid or tissue sample described herein) obtained from the subject. In some embodiments, the measurement is an indication that the condition has been treated.

[0158] In some embodiments, the measured value is obtained directly from the object. In some embodiments, the measured value is obtained using an imaging device in a non-invasive manner. In some embodiments, the measured value is obtained in a second sample from the object. In some embodiments, the measured value is obtained in one or more histological tissue sections. In some embodiments, the measured value is obtained by measuring the second sample obtained from the object. In some embodiments, the measured value is obtained by measuring (such as the measurement described herein). In some embodiments, the determination is an immunoassay, a colorimetric determination, a fluorometric determination, a chromatography (e.g., HPLC) determination or a PCR determination. In some embodiments, the measured value is obtained by a determination such as an immunoassay, a colorimetric determination, a fluorometric determination or a chromatography (e.g., HPLC) determination. In some embodiments, the measured value is obtained by PCR. In some embodiments, the measured value is obtained by histology. In some embodiments, the measured value is obtained by observation. In some embodiments, additional measurements are performed, such as in the 3rd sample, the 4th sample or the 5th sample.

[0159] In some embodiments, the measured value is obtained within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, or 24 hours after applying the composition. In some embodiments, the measured value is obtained within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after applying the composition. In some embodiments, the measured value is 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 applying the composition. In some embodiments, the measured value is obtained after 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, or 24 hours after applying the composition. In some embodiments, the measured value is obtained after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after applying the composition. In some embodiments, the measurements are obtained after 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.

[0160] In some embodiments, the composition reduces the measured value relative to the baseline measured value. In some embodiments, after the composition is applied to the object, a reduction is measured in the second sample obtained from the object. In some embodiments, after the composition is applied to the object, a reduction is measured directly in the object. In some embodiments, relative to the baseline measured value, the measured value is reduced by about 2.5% or more, about 5% or more, or about 7.5% or more. In some embodiments, relative to the baseline measured value, the measured value is reduced by about 10% or more. In some embodiments, relative to the baseline measured value, the measured value 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. In some embodiments, relative to the baseline measured value, the measured value is reduced by no more than about 2.5%, no more than about 5% or no more than about 7.5%. In some embodiments, relative to the baseline measured value, the measured value is reduced by no more than about 10%. In some embodiments, the measured value is reduced by no more than about 20%, no more than about 30%, no more than about 40%, no more than about 50%, no more than about 60%, no more than about 70%, no more than about 80%, no more than about 90%, or no more than about 100% relative to the baseline measured value. In some embodiments, the measured value is reduced 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.

[0161] In some embodiments, the composition increases the measured value relative to the baseline measured value. In some embodiments, after the composition is applied to the object, an increase is measured in the second sample obtained from the object. In some embodiments, after the composition is applied to the object, an increase is measured directly in the object. In some embodiments, relative to the baseline measured value, the measured value increases by about 2.5% or more, about 5% or more, or about 7.5% or more. In some embodiments, relative to the baseline measured value, the measured value increases by about 10% or more. In some embodiments, relative to the baseline measured value, the measured value 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. In some embodiments, relative to the baseline measured value, the measured value increases by about 100% or more, increases by about 250% or more, increases by about 500% or more, increases by about 750% or more, or increases by about 1000% or more. In some embodiments, the measured value increases by no more than about 2.5%, no more than about 5%, or no more than about 7.5% relative to the baseline measurement value. In some embodiments, the measured value increases by no more than about 10% relative to the baseline measurement value. In some embodiments, the measured value increases by no more than about 20%, no more than about 30%, no more than about 40%, no more than about 50%, no more than about 60%, no more than about 70%, no more than about 80%, no more than about 90%, or no more than about 100% relative to the baseline measurement value. In some embodiments, the measured value increases by no more than about 100%, no more than about 250%, no more than about 500%, no more than about 750%, or no more than about 1000% relative to the baseline measurement value. In some embodiments, the measured value 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.

[0162] In some embodiments, the measured value is a target protein measured value. In some embodiments, the target protein measured value comprises a target protein level. In some embodiments, the target protein level is indicated as the mass or percentage / sample weight of the target protein. In some embodiments, the target protein level is indicated as the mass or percentage / sample volume of the target protein. In some embodiments, the target protein level is indicated as the mass or percentage / total protein of the target protein in the sample. In some embodiments, the target protein measured value is a circulating / tissue target protein measured value. In some embodiments, the target protein measured value is obtained by an assay such as an immunoassay, a colorimetric assay, or a fluorometric assay.

[0163] In some embodiments, the composition reduces the target protein measurement relative to the baseline target protein measurement. In some embodiments, the composition reduces the circulating target protein level relative to the baseline target protein measurement. In some embodiments, the composition reduces the tissue target protein level relative to the baseline target protein measurement. In some embodiments, after the composition is applied to the object, the reduced target protein level is measured in the second sample obtained from the object. In some embodiments, relative to the baseline target protein measurement, the target protein measurement is reduced by about 2.5% or more, about 5% or more, or about 7.5% or more. In some embodiments, relative to the baseline target protein measurement, the target protein measurement is reduced by about 10% or more. In some embodiments, relative to the baseline target protein measurement, 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%. In some embodiments, relative to the baseline target protein measurement, the target protein measurement is reduced by no more than about 2.5%, no more than about 5%, or no more than about 7.5%. In some embodiments, relative to the baseline target protein measurement value, the target protein measurement value is reduced by no more than about 10%. In some embodiments, relative to the baseline target protein measurement value, the target protein measurement value is reduced by no more than about 20%, no more than about 30%, no more than about 40%, no more than about 50%, no more than about 60%, no more than about 70%, no more than about 80%, no more than about 90% or no more than about 100%. In some embodiments, the target protein measurement value is reduced by 2.5%, 5%, 7.5%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, or the range defined by any two of the aforementioned percentages.

[0164] In some embodiments, the measured value is a target mRNA measured value. In some embodiments, the target mRNA measured value includes a target mRNA level. In some embodiments, the target mRNA level is indicated as the amount or percentage / sample weight of the target mRNA. In some embodiments, the target mRNA level is indicated as the amount or percentage / sample volume of the target mRNA. In some embodiments, the target mRNA level is indicated as the amount or percentage / total mRNA of the target mRNA in the sample. In some embodiments, the target mRNA level is indicated as the amount or percentage / total nucleic acid of the target mRNA in the sample. In some embodiments, the target mRNA level is indicated relative to another mRNA level (such as the mRNA level of a housekeeping gene) in the sample. In some embodiments, the target mRNA measured value is a circulating / tissue target mRNA measured value. In some embodiments, the target mRNA measured value is obtained by a determination such as a PCR determination. In some embodiments, PCR includes qPCR. In some embodiments, PCR includes reverse transcription of the target mRNA.

[0165] In some embodiments, the composition reduces the target mRNA measurement value relative to the baseline target mRNA measurement value. In some embodiments, after the composition is applied to the object, the target mRNA measurement value is obtained in the second sample obtained from the object. In some embodiments, the composition reduces the target mRNA level relative to the baseline target mRNA level. In some embodiments, after the composition is applied to the object, the reduced target mRNA level is measured in the second sample obtained from the object. In some embodiments, the second sample is a liver sample. In some embodiments, the sample is a fat sample. In some embodiments, relative to the baseline target mRNA measurement value, the target mRNA measurement value is reduced by about 2.5% or more, about 5% or more or about 7.5% or more. In some embodiments, relative to the baseline target mRNA measurement value, the target mRNA measurement value is reduced by about 10% or more. In some embodiments, relative to the baseline target mRNA measurement value, the target mRNA measurement value 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%. In some embodiments, relative to baseline target mRNA measured value, target mRNA measured value reduces by no more than about 2.5%, no more than about 5% or no more than about 7.5%. In some embodiments, relative to baseline target mRNA measured value, target mRNA measured value reduces by no more than about 10%. In some embodiments, relative to baseline target mRNA measured value, target mRNA measured value reduces by no more than about 20%, no more than about 30%, no more than about 40%, no more than about 50%, no more than about 60%, no more than about 70%, no more than about 80%, no more than about 90% or no more than about 100%. In some embodiments, target mRNA measured value reduces by 2.5%, 5%, 7.5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, or the scope defined by any two aforementioned percentages.

[0166] III. Definitions

[0167] Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by those of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ease of reference, and such definitions contained herein should not necessarily be construed as indicating a substantial difference from what is generally understood in the art.

[0168] Throughout this application, various embodiments can be presented in the form of ranges. It should be understood that the description of the range form is only for convenience and brevity, and should not be interpreted as a hard limit to the scope of the present disclosure. Therefore, the description of the range should be regarded as having all possible sub-ranges disclosed and the individual numerical values ​​within this range. For example, the description of a range such as 1 to 6 should be regarded as having the sub-ranges disclosed, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the width of the scope, this applies.

[0169] As used in the specification and claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a sample" includes a plurality of samples, including mixtures thereof.

[0170] The terms "determine," "measure," "evaluate," "assess," "measure," and "analyze" are generally used interchangeably herein to refer to forms of measurement. The terms include determining whether an element is present (e.g., detecting). These terms may include quantitative, qualitative, or quantitative and qualitative determinations. Assessments may be relative or absolute. In addition to determining whether something is present or absent based on context, "detecting the presence of" may also include determining the amount of something that is present.

[0171] The terms "subject" and "patient" are used interchangeably herein. A "subject" can be a biological entity containing expressed genetic material. A biological entity can be a plant, an animal, or a microorganism, including, for example, bacteria, viruses, fungi, and protozoa. A subject can be a mammal. A mammal can be a human. A subject may be diagnosed or suspected of being at high risk for a disease. In some cases, a subject is not necessarily diagnosed or suspected of being at high risk for a disease.

[0172] As used herein, the term "about" a number refers to the number plus or minus 10% of the number. The term "about" a range refers to the range minus 10% of its lower value and plus 10% of its upper value.

[0173] As used herein, the term "treatment" is used to refer to a drug or other intervention regimen for obtaining a beneficial or desired result in a recipient. Beneficial or desired results include, but are not limited to, therapeutic benefits and / or preventive benefits. Therapeutic benefits may refer to eradicating or improving the underlying condition or its symptoms being treated. In addition, therapeutic benefits may be achieved by eradicating or improving one or more physiological symptoms associated with the underlying condition so that improvements are observed in the subject, although the subject may still be troubled by the underlying condition. Preventive effects include delaying, preventing or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing down, stopping or reversing the progress of a disease or condition, or any combination thereof. For preventive benefits, treatment may be performed on an object at risk of a specific disease or on an object reporting one or more physiological symptoms of a disease (even if the diagnosis of the disease may not yet have been made).

[0174] "Treatment" or "treating" may include methods for obtaining beneficial or desired results (including but not limited to therapeutic benefits and / or preventive benefits) associated with a disease, disorder, or medical condition. Therapeutic benefits may include, for example, eradication or improvement of the underlying disorder being treated. In addition, therapeutic benefits may include, for example, eradication or improvement of one or more physiological symptoms associated with the underlying disorder so that improvement is observed in the subject, although the subject may still be troubled by the underlying disorder. In certain embodiments, for preventive benefits, 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 though a diagnosis of the disease may not have been made. Treatment via administration of the compounds described herein does not require the involvement of a medical professional.

[0175] Some embodiments relate to nucleic acid sequence information. It is envisioned that in some embodiments, thymine (T) can be interchanged with uracil (U), or vice versa. For example, some sequences in the sequence table can list T, but in some embodiments, these can be replaced by U. In some oligonucleotides with a nucleic acid sequence comprising uracil, uracil can be replaced by thymine. Similarly, in some oligonucleotides with a nucleic acid sequence comprising thymine, thymine can be replaced by uracil. In some embodiments, oligonucleotides such as siRNA include RNA or are composed of RNA. In some embodiments, oligonucleotides can include DNA. For example, oligonucleotides can include 2' deoxyribonucleotides. ASOs can include DNA or are composed of DNA. To any extent that the disclosure of the sequence table and the specification sheet contradicts, the specification sheet takes precedence.

[0176] Some aspects include sequences with nucleotide modifications or modified internucleoside linkages. Typically and 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' deoxy-modified nucleosides, n (e.g., a, c, g, t, or u) refers to 2' O-methyl-modified nucleosides, and "s" refers to phosphorothioate linkages.

[0177] Pyrimidine may include cytosine (C), thymine (T), or uracil (U). Pyrimidine may include C or U. Pyrimidine may include C or T. Reference to pyrimidine may include nucleosides or nucleotides comprising pyrimidine. Purine may include guanine (G) or adenine (A). Reference to purine may include nucleosides or nucleotides comprising purine.

[0178] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0179] The term "C x-y " or "C x -C y " when used in conjunction with a chemical moiety such as an alkyl, alkenyl, or alkynyl group, is intended to include groups containing from x to y carbons in the chain. For example, the term "C 1-6 "Alkyl" refers to a substituted or unsubstituted saturated hydrocarbon group, including straight-chain alkyl and branched-chain alkyl groups containing 1 to 6 carbon atoms.

[0180] The term "C x-y "Alkenyl" and "C x-y "Alkynyl" refers to a substituted or unsubstituted unsaturated aliphatic group analogous in length and possible substitution to the alkyl groups described above, but containing at least one double or triple bond, respectively.

[0181] As used herein, the term "carbocycle" refers to a saturated ring, an unsaturated ring or an aromatic ring, wherein each atom of the ring is carbon.Carbocycles include 3 to 10-membered monocycles, 5 to 12-membered bicyclic rings, 5 to 12-membered spiral bicyclic rings and 5 to 12-membered bridged rings.Each ring of the bicyclic carbocycle can be selected from saturated rings, unsaturated rings and aromatic rings.In an exemplary embodiment, an aromatic ring, such as phenyl, can be fused with a saturated or unsaturated ring, such as cyclohexane, cyclopentane or cyclohexene.Where valence permits, the bicyclic carbocycle includes any combination of saturated bicyclic rings, unsaturated bicyclic rings and aromatic bicyclic rings.Bicyclic carbocycles also include spiral bicyclic rings, such as spiropentane. Bicyclic carbocycles include any combination of ring sizes, such as 3-3 spiro ring systems, 4-4 spiro ring systems, 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. Exemplary carbocycles include cyclopentyl, cyclohexyl, cyclohexenyl, adamantyl, phenyl, indenyl, naphthyl, and bicyclo[1.1.1]pentanyl.

[0182] The term "aryl" refers to an aromatic monocyclic or polycyclic hydrocarbon ring system. An aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon and contains from 5 to 18 carbon atoms, wherein at least one ring in the ring system is aromatic, i.e., it 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.

[0183] The term "cycloalkyl" refers to a saturated ring in which each atom of the ring is carbon. Cycloalkyl can include monocycles and polycycles, such as 3-10 monocycles, 5-12 bicycles, 5-12 spiro bicycles, and 5-12 bridged rings. In certain embodiments, cycloalkyl comprises 3 to 10 carbon atoms. In other embodiments, cycloalkyl comprises 5 to 7 carbon atoms. Cycloalkyl 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 groups include, for example, adamantyl, spiropentane, bornyl (i.e., bicyclo [2.2.1] heptyl), decahydronaphthyl, 7,7 dimethylbicyclo [2.2.1] heptyl, bicyclo [1.1.1] pentyl, etc.

[0184] The term "cycloalkenyl" refers to a saturated ring, wherein each atom of the ring is carbon, and there is at least one double bond between two ring carbons. Cycloalkenyl can include monocycles and polycycles, such as 3 to 10 monocycles, 6 to 12 bicycles and 5 to 12 bridged rings. In other embodiments, cycloalkenyl includes 5 to 7 carbon atoms. Cycloalkenyl can be attached to the remainder of the molecule by a single bond. The example of monocyclic cycloalkenyl includes, for example, cyclopentenyl, cyclohexenyl, cycloheptenyl and cyclooctenyl.

[0185] The term "halo" or alternatively "halogen" or "halide" means fluoro, chloro, bromo or iodo. In some embodiments, halo is fluoro, chloro or bromo.

[0186] The term "haloalkyl" refers to an alkyl group as defined above, substituted with one or more halo groups, for example, trifluoromethyl, dichloromethyl, bromomethyl, 2,2,2-trifluoroethyl, 1-chloromethyl-2-fluoroethyl, etc. In some embodiments, the alkyl portion of the haloalkyl is optionally further substituted as described herein.

[0187] As used herein, the term "heterocycle" refers to a saturated ring, an unsaturated ring or an aromatic ring containing one or more heteroatoms. Exemplary heteroatoms include N, O, Si, P, B and S atoms. Heterocycles include 3-10-membered monocycles, 6-12-membered bicyclic rings, 5-12-membered spiral bicyclic rings and 5-12-membered bridged rings. Where valence permits, bicyclic heterocycles include any combination of saturated bicyclic rings, unsaturated bicyclic rings and aromatic bicyclic rings. In an exemplary embodiment, an aromatic ring, such as a pyridyl group, can be fused with a saturated or unsaturated ring, such as 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 also include spiro bicycles, for example 5- to 12-membered spiro bicycles, such as 2-oxa-6-azaspiro[3.3]heptane.

[0188] The term "heteroaryl" refers to a group derived from a 5- to 18-membered aromatic ring group containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur. As used herein, a heteroaryl group is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one ring in the ring system is aromatic, i.e., according to the Hückel theory, it contains a cyclic, delocalized (4n+2) π-electron system. Heteroaryl includes fused or bridged ring systems. One or more heteroatoms in the heteroaryl are optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. The heteroaryl is attached to the remainder of the molecule through any atom of the ring.Examples of heteroaryl groups include, but are not limited to, azepine, 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, benzodioxadienyl, benzothienyl / benzothiophenyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl, 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]cyclopenta[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, furano[3,2-c]pyridinyl, 5,6,7,8,9,10-hexahydrocyclooctano[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocyclooctano[d]pyridazinyl, 5,6,7,8,9,10-hexahydrocyclooctano[d] pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolyl, isoindolyl, isoquinolyl, indolizinyl, isoxazolyl, 5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl, 1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinetrienyl, 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[3,4-d]pyrimidinyl , pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolyl, isoquinolyl, tetrahydroquinolyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cyclopenta[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).

[0189] The term "heterocycloalkyl" refers to a saturated ring with carbon atoms and at least one heteroatom. Exemplary heteroatoms include N, O, Si, P, B and S atoms. Heterocycloalkyl can include monocycles and polycycles, such as 3 to 10 monocycles, 6 to 12 bicycles, 5 to 12 spiral bicycles and 5 to 12 bridged rings. The heteroatoms in the heterocycloalkyl group are optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. Where valence permits, heterocycloalkyl is attached to the remainder of the molecule by any atom of heterocycloalkyl (such as any carbon or nitrogen atom of heterocycloalkyl). Examples of heterocycloalkyl groups include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, 2-oxa-6-azaspiro[3.3]heptane, and 1,1-dioxo-thiomorpholinyl.

[0190] The term "heterocycloalkenyl" refers to an unsaturated ring having carbon atoms and at least one heteroatom, and at least one double bond between two ring carbons. Heterocycloalkenyl does not include heteroaryl rings. Exemplary heteroatoms include N, O, Si, P, B and S atoms. Heterocycloalkenyl can 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, the heterocycloalkenyl includes 5 to 7 ring atoms. The heterocycloalkenyl can be attached to the remainder of the molecule by a single bond. Examples of monocyclic cycloalkenyl groups include, for example, pyrroline (dihydropyrrole), pyrazoline (dihydropyrazole), imidazoline (dihydroimidazole), triazoline (dihydrotriazole), dihydrofuran, dihydrothiophene, oxazoline (dihydrooxazole), isoxazoline (dihydroisoxazole), thiazoline (dihydrothiazole), isothiazolin (dihydroisothiazole), oxadiazolin (dihydrooxadiazole), thiadiazoline (dihydrothiadiazole), dihydropyridine, tetrahydropyridine, dihydropyridazine, tetrahydropyridazine, dihydropyrimidine, tetrahydropyrimidine, dihydropyrazine, tetrahydropyrazine, pyran, dihydropyran, thiopyran, dihydrothiopyran, dioxadiene, dihydrodioxine, oxazine, dihydrooxazine, thiazine, and dihydrothiazine.

[0191] The term "substituted" refers to a substituent having a hydrogen or substitutable heteroatom (e.g., NH or NH2 of a compound) that replaces one or more carbons. It should be understood that "substituted" or "substituted by..." includes implicit prerequisites, i.e., such substitutions meet the allowed valences of the substituted atom and the substituent, and the substitution produces a stable compound, i.e., a compound that does not spontaneously undergo transformations such as rearrangement, cyclization, elimination, etc. In certain embodiments, substituted refers to a substituent having a substituent that replaces two hydrogen atoms on the same carbon atom, such as replacing two hydrogen atoms on a single carbon with an oxo group, an imino group, or a thio group. As used herein, it is contemplated that the term "substituted" includes all allowed substituents of an organic compound. In a broad sense, allowed substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of an organic compound. For appropriate organic compounds, allowed substituents may be one or more and may be identical or different.

[0192] In some embodiments, the substituents may include any of the substituents described herein, for example, halogen, hydroxyl, oxo (=O), thio (=S), cyano (-CN), nitro (-NO2), imino (=NH), oxime (=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)OR a , -R b -N(R a )C(O)R a , -R b -N(R a )S(O) t R a (where t is 1 or 2), -R b -S(O)t R a (where t is 1 or 2), -R b -S(O) t OR a (where t is 1 or 2) and -R b -S(O) t N(R a )2 (wherein t is 1 or 2); and alkyl, alkenyl, alkynyl, aryl, aralkyl, aralkenyl, aralkynyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl and heteroarylalkyl, any of which may be optionally replaced by alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, oxo (=O), thio (=S), cyano (-CN), nitro (-NO2), imino (=NH), oxime (=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)OR a , -R b -N(R a )C(O)R a , -R b -N(R a )S(O) t R a (where t is 1 or 2), -R b -S(O) t R a (where t is 1 or 2), -R b -S(O) t OR a (where t is 1 or 2) and -R b -S(O) t N(Ra )2 (wherein t is 1 or 2); wherein each R a is independently selected from hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, wherein each R a The alkyl radical may be optionally substituted with an alkyl radical, an alkenyl radical, an alkynyl radical, a halogen radical, a haloalkyl radical, a haloalkenyl radical, a haloalkynyl radical, an oxo radical (═O), a thio radical (═S), a cyano radical (—CN), a nitro radical (—NO2), an imino radical (═NH), an oxime radical (═N—OH), a hydrazine radical (═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 (where t is 1 or 2), -R b -S(O) t R a (where t is 1 or 2), -R b -S(O) t OR a (where t is 1 or 2) and -R b -S(O) t N(R a )2 (wherein t is 1 or 2); and wherein each R b are independently selected from a direct bond or a straight or branched alkylene, alkenylene or alkynylene chain, and each R cis a straight or branched alkylene, alkenylene or alkynylene chain.

[0193] Double bonds to oxygen atoms, such as oxo groups, are represented herein as both "=O" and "(O)". Double bonds to nitrogen atoms are represented as both "=NR" and "(NR)". Double bonds to sulfur atoms are represented as both "=S" and "(S)".

[0194] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to 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, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0195] As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0196] As used herein, the phrase "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to a pharmaceutically 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 injurious to the patient. Some examples of materials that can be used as pharmaceutically 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, ethyl cellulose, and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn starch, etc. Rice oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerol, 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) ethanol; (20) phosphate buffered saline; and (21) other nontoxic compatible substances used in pharmaceutical preparations.

[0197] The term "salt" or "pharmaceutically acceptable salt" refers to salts derived from various organic and inorganic counterions 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 base 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 can be derived include, for example, primary, secondary and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine and ethanolamine. In some embodiments, pharmaceutically acceptable base addition salts are selected from ammonium, potassium, sodium, calcium and magnesium salts.

[0198] VI. Examples

[0199] Example 1: Intravitreal injection of siRNA duplexes:

[0200] For intravitreal injections in mice (4-7 week old ICR, Envigo), a 33 gauge needle and glass microinjector (10 μL volume; Hamilton) were used. The eyeball was extruded and the needle was passed through the equatorial sclera and inserted into the vitreous cavity at an angle of approximately 45 degrees, taking care to avoid contact with the posterior lens or retina. 5 μg of siRNA duplexes in 1 μL phosphate buffered saline vehicle were injected into the vitreous. The injected siRNA duplexes are depicted in Table 2.

[0201] Table 2: siRNA duplexes

[0202]

[0203] Mice were euthanized on the 14th day after injection. Both eyes of each animal were harvested and dissected along the equator, and the anterior and posterior hemispheres were placed in RNAlater. Total RNA was extracted from homogenized tissue and reverse transcribed into cDNA using the first chain III cDNA synthesis kit. Fluorescently labeled TaqMan probe / primer sets of selected genes (ANGPTL7, MYOC, COL1A1, COL5A1, VCAN, FN1, and PPIA) were used to perform normalized cDNA quantification by real-time TaqMan PCR. Reactions were performed in 20 μL aliquots using TaqManUniversa1 PCR Master Mix No AmpErase UNG (running on an ABI Prism 7500 fast real-time PCR system sequence detection system) and analyzed by 7500 system software. Relative quantification (RQ) values ​​between treated and untreated samples were calculated by the formula 2-ΔΔCT, where CT is the cycle at threshold (automatically measured), 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 the treated sample minus the ΔCT of the same gene in the untreated sample (calibrator).

[0204] The results are depicted in Table 3. ETD1208 was more effective than ETD1207, which was more effective than EDT 1206. This suggests that having fewer (or no) phosphorothioates at the 5' end of the sense strand is more effective than having 2 phosphorothioates at the end where the hydrophobic group is attached.

[0205] ETD1588 is more active than ETD1589, which means that some sequences with a 5' vinyl phosphonate on the antisense strand show greater activity than those without a 5' vinyl phosphonate on the antisense strand.

[0206] The ETD 1716-ETD 1787 series enables different hydrophobic groups containing 12-25 carbons, all conjugated to phosphorothioates at the 5' end of the sense strand.

[0207] Table 3: mRNA expression levels

[0208] <![CDATA[ s iRNA]]> Relative to the mean mRNA of untreated control animals ETD01206 026 ETD01207 0.20 ETD01208 0.14 ETD01588 0.43 ETD01589 0.89 ETD01716 0.44 ETD01736 0.09 ETD01778 0.34 ETD01779 0.40 ETD01786 0.29 ETD01787 0.23

[0209] Example 2: Intravitreal injection with mRNA and siRNA quantification

[0210] As described above, 20 μg siRNA was injected intravitreally with 1 μL. 14 days after injection, the eyes were harvested and mRNA was quantified as shown in the previous example. siRNA was quantified using a stem-loop assay according to published procedures (Cheng A, LiM, Liang Y, Wang Y, Wong L, Chen C, Vlassov AV, Magdaleno S2009. Stem-loop RT-PCR quantification of siRNAs in vitro and in vivo. Oligonucleotides 19: 203–208).

[0211] The results are depicted in Table 4. There is a correlation between the size of the hydrophobic group, the percent knockdown, and the siRNA levels observed in the tissue. In general, hydrophobic groups with more carbons result in increased percent knockdown and more siRNA observed in the tissue.

[0212] Table 4: mRNA quantification

[0213]

[0214] Example 3: siRNA-mediated knockdown of PLIN1 in mouse adipose tissue

[0215] 4-7 week old ICR mice (Envigo Labs) mice in the 1st group (n=4) were given 100uL phosphate buffered saline (PBS) or 500μg siRNA targeting mouse PLIN1 in 100uL PBS by subcutaneous injection. On the 14th day, mice were euthanized, and abdominal white fat samples from each mouse were collected and placed in RNAlater (ThermoFisher catalog number AM7020). Total liver RNA was prepared by homogenizing liver tissue in homogenization buffer (Maxwell RSC simplyRNA tissue kit) for two 10 second cycles using a Percellys 24 tissue homogenizer (Bertin Instruments) set to 5000rpm. Total RNA from lysate was purified on a Maxwell RSC 48 platform (Promega Corporation) according to the manufacturer's recommendation. The levels of PLIN1 mRNA in the liver 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 animals receiving PBS.

[0216] The results are depicted in Table 5. The addition of 5' vinyl phosphonate resulted in increased potency and enabled delivery into adipose tissue.

[0217] Table 5: mRNA expression levels

[0218]

[0219] Example 4: Delivery to the Central Nervous System Injection

[0220] siRNA with the modifications and hydrophobic conjugates were injected intraventricularly or intrathecally according to published procedures (Alterman, JF, Godinho, BMDC, Hassler, MR et al. Adivalent siRNA chemicalscaffold 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)). Fourteen days after injection, mice were euthanized, and brain hemispheres were harvested, frozen, and subsequently homogenized and assayed for target mRNA and protein expression.

[0221] Example 5: Key points description of siRNA

[0222] Example siRNAs are described below:

[0223] 21-mer:

[0224] a.19 base pairs

[0225] b. 2 nucleotide (nt) overhang

[0226] Antisense strand:

[0227] a. Vinylphosphonate (VP) at the 5′ end

[0228] b. 2 phosphorothioate bonds (PS) at each end

[0229] Sense strand:

[0230] a. Hydrophobic group (C 16-C18) at the 5′ end

[0231] b. Optional 0-2 PS at the 5′ end

[0232] c. 2 PS at the 3′ end

[0233] · Modification patterns of 2′ fluoro and 2′ methyl.

[0234] Example 6: Modification of Motif 1

[0235] Example siRNAs contain a combination of the following modifications:

[0236] • Position 9 (from 5' to 3') of the sense strand is 2'F.

[0237] If position 9 is a pyrimidine, all purines in the sense strand are 2'OMe and 1-5 pyrimidines between positions 5 and 11 are 2'F, provided that three 2'F modifications do not occur consecutively.

[0238] If position 9 is a purine, all pyrimidines in the sense strand are 2'OMe and 1-5 purines between positions 5 and 11 are 2'F, provided that three 2'F modifications do not occur consecutively.

[0239] The antisense strand has 2'OMe at odd positions and a mixture of 2'F and 2'Deoxy at even positions.

[0240] Example 7: Modification Motif 2

[0241] Example siRNAs contain a combination of the following modifications:

[0242] • Position 9 (from 5' to 3') of the sense strand is 2' deoxy.

[0243] • Sense strand positions 5, 7 and 8 are 2'F.

[0244] All pyrimidines at positions 10-21 are 2'OMe, and purines are 2'OMe and 2'F

[0245] 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.

[0246] The antisense strand has 2'OMe at odd positions and a mixture of 2'F and 2'Deoxy at even positions.

[0247] Example 8: Synthesis of ETL phosphoramidite

[0248]

[0249] Example 8A: Synthesis of ETL20 phosphoramidite

[0250] Synthesis of N-(4-hydroxyphenylethyl) palmitic acid amide (5):

[0251] 12.82 grams of 1 (palmitic acid) were weighed and dissolved in 450 mL of CH2Cl2. 16.3 mL of diisopropylethylamine (DIEA) was added to the solution of 1. Then, 12.88 mL of 2 (2,2,2-perfluorophenyl trifluoroacetate, "PFP") was added dropwise, and the reaction was stirred for 10 minutes after the addition was complete. 8.26 grams of 4 (4-(2-aminoethyl)phenol) were added to the solution of PFP activated acid via an addition funnel, and the addition funnel was rinsed with 50 mL of CH2Cl2. The reaction was placed under argon and stirred overnight. After stirring overnight, 5 formed a precipitate. The precipitate was collected via filtration and washed with 75 mL of MTBE, which had been cooled to -20°C. The white to off-white solid was dried overnight under high vacuum.

[0252] The product was used in the next step without further purification.

[0253]

[0254] Synthesis of ETL20 phosphoramidite (6): 100 mL of anhydrous ethyl acetate was added to N-(4-hydroxyphenylethyl) palmitic acid amide 5 (5.2 grams), 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, and the solution became turbid. 3-((chloro(diisopropylamino)phosphino)oxy)propionitrile (3.5 mL) was slowly added to the turbid solution. After the addition was complete, the reaction mixture was taken out of the ice bath and stirred overnight at room temperature under Ar. The reaction mixture was then diluted with ethyl acetate (200 mL), washed with saturated NaHCO solution (2x50 mL), and then washed with brine (50 mL). The solution was dried over NaSO, filtered and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography and eluted with 5%-30% ethyl acetate in hexane containing 2% triethylamine.

[0255] Using hydroxybenzylamine and lauric acid (C 12 ) or myristic acid (C 14 )Procedure for the synthesis of ETL20 phosphoramidite ETL18 (C 14 ) and ETL19(C 12)phosphoramidites.

[0256] Example 9: Intravitreal injection of siRNA duplexes:

[0257] For intravitreal injections in mice (4-7 week old ICR, Envigo), a 33 gauge needle and glass microinjector (10 μL volume; Hamilton) were used. The eyeball was extruded and the needle was passed through the equatorial sclera and inserted into the vitreous cavity at an angle of approximately 45 degrees, taking care to avoid contact with the posterior lens or retina. 2.5 or 1 μg of siRNA duplexes in 1 μL of phosphate buffered saline vehicle were injected into the vitreous. The injected siRNA duplexes are described in Table 1.

[0258] Table 6: siRNA duplexes

[0259]

[0260] Mice were euthanized on the 14th day after injection. Both eyes of each animal were harvested and dissected along the equator, and the anterior and posterior hemispheres were placed in RNAlater. Total RNA was extracted from homogenized tissue and reverse transcribed into cDNA using a first-strand III cDNA synthesis kit. Normalized cDNA quantification was performed by real-time TaqMan PCR using fluorescently labeled TaqMan probe / primer sets of 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 (run 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 at threshold (automatically measured), 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 the treated sample minus the ΔCT of the same gene in the untreated sample (calibrator).

[0261] Furthermore, the ETD1928-1930 series showed the usefulness of different hydrophobic groups containing 12-16 carbons attached to tyramine. The hydrophobic groups were conjugated without phosphorothioate at the 5' end of the sense strand.

[0262] Table 7: mRNA expression levels

[0263]

[0264] Example 10: Intraventricular Injection

[0265] Mice were induced to anesthesia in an induction chamber with 1.2% isoflurane vaporized by 1.0 L / min of oxygen and then transferred and fixed to a stereotaxic frame while maintaining anesthesia with 0.8% isoflurane through a mask. After exposing the skull and placing a needle at a coordinate below the bregma, a standard U-shaped mouse stereotaxic instrument (Harvard Apparatus 75-1808) and a stereotaxic anesthesia adapter with an anesthesia mask (Harvard Apparatus 75-1856) were used at 500 nlmin. -1 A single intraventricular injection was performed (5 μl, artificial cerebrospinal fluid as vehicle): -0.2 mm anterior-posterior, 0.8 mm mediolateral, and -2.5 mm dorsoventral.

[0266] Table 8: siRNA duplexes

[0267]

[0268] ETL3 was stearyl conjugated to the 5' end of the sense strand using stearyl phosphoramidite (Glen Research, 10-1979-90).

[0269] Mice were euthanized on day 14 after injection. The brain of each animal was harvested and dissected into right and left hemispheres.

[0270] Total RNA was extracted from homogenized tissue and reverse transcribed into cDNA using the first strand III cDNA synthesis kit. Fluorescently labeled TaqMan probe / primer sets of selected genes (MTRES1, MYOC, COL1A1, COL5A1, VCAN, FN1, and PPIA) were used to perform normalized cDNA quantification by real-time TaqMan PCR. TaqMan Universal PCR Master Mix No AmpErase UNG (run on an ABI Prism 7500 fast real-time PCR system sequence detection system) was used to react in 20 μL aliquots and analyzed by the 7500 system software. The relative quantification (RQ) values ​​between treated and untreated samples were calculated by the formula 2-ΔΔCT, where CT is the cycle at the threshold (automatic measurement), ΔCT is the CT of the measured gene minus the CT of the endogenous control (PPIA), and ΔΔCT is the ΔCT of the normalized measured gene in the treated sample minus the ΔCT of the same gene in the untreated sample (calibrator).

[0271] Table 9: mRNA expression levels

[0272]

[0273] Example 11: Intrathecal injection in rats

[0274] Male Sprague Dawley rats (n=2) were used for intrathecal (IT) injection of siRNA. Duplex ETD02273 was injected at 30 mg ml in artificial cerebrospinal fluid (aCSF). -1 was formulated and administered to rats as a 30 μl IT injection via lumbar puncture in the dorsal region of the spine between the L3-L5 intervertebral spaces. Duplex ETD02210 was diluted in artificial cerebrospinal fluid (aCSF) at 9 mg ml -1 It was formulated and administered to rats as a 100 μl IT injection via lumbar puncture in the dorsal region of the spine between the L3-L5 intervertebral spaces.

[0275] After anesthetizing the rats with isoflurane, the rats were placed on a warm heating pad. The IT injection site was shaved and disinfected. An incision was made to expose the spine. siRNA was administered with a 30-gauge insulin syringe. Correct placement of the needle was confirmed by CSF reflux through the center of the needle and tail flicks. Once siRNA administration was complete, gentle constant pressure was maintained on the plunger for 30 seconds. The incision was sutured and fixed with tissue glue. The rats were placed on a heating pad in sternal recumbency until recovery.

[0276] Rats were euthanized on day 14 after injection. Samples of liver, right and left (R / L) frontal cortex, R / L temporal cortex, hippocampus, cerebellum, and spinal cord were collected from each rat and placed in RNAlater (ThermoFisher Cat. No. AM7020) until processing.

[0277] Total liver RNA was prepared by homogenizing liver tissue in homogenization buffer (Maxwell RSC simplyRNA tissue kit) for two 10 second cycles using a Percellys 24 tissue homogenizer (Bertin Instruments) set to 5000 rpm. Total RNA from lysate was purified on a Maxwell RSC 48 platform (Promega Corporation) according to the manufacturer's recommendations. cDNA was prepared using Quanta qScript cDNA SuperMix (VWR, catalog number 95048-500) according to the manufacturer's instructions.

[0278] TaqMan assays for rat MTRES1 (ThermoFisher, assay number Rn01441122_m1) and rat housekeeping gene PPIA (ThermoFisher, assay number Rn03302269_gH) were used as well as Low ROX TM (VWR, catalog number 101419-222) and RT-qPCR in QuantStudio TM The relative levels of hepatic MTRES1 mRNA were assessed in triplicate on the 6Pro Real-Time PCR System.

[0279] Data were normalized to mean MTRES1 mRNA levels in animals that did not receive treatment. The results are shown in Tables 10 and 11 below.

[0280] Table 10: mRNA expression levels

[0281]

[0282] Table 11: siRNA duplexes

[0283]

[0284] Although preferred embodiments of the present invention have been shown and described herein, it is obvious to those skilled in the art that such embodiments are provided only by way of example. Without departing from the present invention, those skilled in the art will now expect multiple modifications, changes and substitutions. It should be understood that the various alternatives of the embodiments of the present invention described herein may be used to implement the present invention. The appended claims are intended to limit the scope of the present invention and are intended to cover methods and compositions and their equivalents within the scope of these claims.

Claims

1. A composition comprising: Small interfering RNA (siRNA) comprising a sense strand, an antisense strand, and a lipid moiety attached to a terminus of the sense strand or the antisense strand; wherein the lipid moiety comprises a phenyl or cyclohexyl linker, wherein the linker is attached to the lipid and to the terminus of the sense strand or the antisense strand.

2. The composition of claim 1, wherein the lipid and the termini of the sense strand or the antisense strand are linked to the phenyl or cyclohexyl linker in a 1,4, 1,3, or 1,2 substitution pattern.

3. The composition of claim 1, wherein the lipid and the termini of the sense strand or the antisense strand are linked to the phenyl or cyclohexyl linker in a 1,4 substitution pattern.

4. The composition of claim 1, wherein the lipid portion comprises the following structure: wherein the dashed line indicates a covalent linkage to the terminus of the sense strand or the antisense strand, provided that R is not octane.

5. The composition of claim 1, wherein the lipid portion comprises the following structure: wherein the dashed line indicates a covalent attachment to the terminus of the sense strand or the antisense strand, n is 1-3, and R is an alkyl group containing 4-18 carbons.

6. The composition of claim 1, wherein the lipid portion comprises the following structure: wherein the dashed line indicates a covalent attachment to the terminus of the sense strand or the antisense strand, n is 0-3, and R is an alkyl group containing 4-18 carbons.

7. The composition of claim 1, wherein the lipid portion comprises the following structure: Wherein the dashed line indicates a covalent linkage to the terminus of the sense strand or the antisense strand.

8. The composition of claim 1, wherein the lipid fraction comprises the lipid fraction depicted in Table 1.

9. The composition of claim 1, wherein the lipid moiety is attached to the 5' end of the sense strand or the antisense strand.

10. The composition of claim 1, wherein the lipid moiety is linked to the 5' end of the sense strand or the antisense strand via a phosphate.

11. The composition of claim 1, wherein the lipid moiety is attached to the end of the sense strand.

12. The composition of claim 1, wherein the antisense strand comprises a 5' vinyl phosphonate.

13. The composition of claim 1, wherein the antisense strand comprises one or two 5' phosphorothioate linkages.

14. The composition of claim 1, wherein the antisense strand comprises one or two 3' phosphorothioate linkages.

15. The composition of claim 1, wherein the sense strand comprises one or two 5' phosphorothioate linkages.

16. The composition of claim 1, wherein the sense strand does not comprise one or two 5' phosphorothioate linkages.

17. The composition of claim 1, wherein the sense strand comprises a 5' phosphonate linkage.

18. The composition of claim 1, wherein the sense strand comprises one or two 3' phosphorothioate linkages.

19. The composition of claim 1, wherein with respect to the sense strand, any of the following is true: All purine nucleosides contain a 2' fluorine, and all pyrimidine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl; All purine nucleosides contain a 2'-O-methyl group, and all pyrimidine nucleosides are modified with a mixture of 2'-fluoro and 2'-O-methyl groups; All purine nucleosides contain a 2' fluorine, and all pyrimidine nucleosides contain a 2'-O-methyl group; All pyrimidine nucleosides contain a 2' fluorine, and all purine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl; All pyrimidine nucleosides contain 2'-O-methyl groups and all purine nucleosides are modified with a mixture of 2'-fluoro and 2'-O-methyl groups; or All pyrimidine nucleosides contain a 2' fluorine, and all purine nucleosides contain a 2'-O-methyl group; Provided that in any of the above, the sense strand may include 2' deoxynucleosides.

20. The composition of claim 19, wherein the sense strand comprises the 2' deoxynucleoside.

21. The composition of claim 19, wherein the sense strand does not include the 2' deoxynucleoside.

22. The composition of claim 1, wherein with respect to the antisense strand, any of the following is true: All purine nucleosides contain a 2' fluorine, and all pyrimidine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl; All purine nucleosides contain a 2'-O-methyl group, and all pyrimidine nucleosides are modified with a mixture of 2'-fluoro and 2'-O-methyl groups; All purine nucleosides contain a 2'-O-methyl group, and all pyrimidine nucleosides contain a 2' fluorine; All pyrimidine nucleosides contain a 2' fluorine, and all purine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl; All pyrimidine nucleosides contain 2'-O-methyl groups and all purine nucleosides are modified with a mixture of 2'-fluoro and 2'-O-methyl groups; or All pyrimidine nucleosides contain a 2'-O-methyl group, and all purine nucleosides contain a 2' fluorine group.

23. A composition comprising: Small interfering RNA (siRNA), which contains a sense strand and an antisense strand; wherein the antisense strand comprises a 5' end comprising vinylphosphonate and two phosphorothioate linkages and a 3' end comprising two phosphorothioate linkages; wherein the sense strand comprises a 5' end comprising a hydrophobic portion and a 3' end comprising two phosphorothioate linkages; Wherein any of the following is true with respect to the sense strand, provided that the sense strand may include 2' deoxynucleosides: All purine nucleosides contain a 2' fluorine, and all pyrimidine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl, All purine nucleosides contain a 2'-O-methyl group, and all pyrimidine nucleosides are modified with a mixture of 2'-fluoro and 2'-O-methyl groups, All purine nucleosides contain a 2' fluorine, and all pyrimidine nucleosides contain a 2'-O-methyl group, All pyrimidine nucleosides contain a 2' fluorine, and all purine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl, All pyrimidine nucleosides contain 2'-O-methyl groups, and all purine nucleosides are modified with a mixture of 2'-fluoro and 2'-O-methyl groups, or All pyrimidine nucleosides contain a 2' fluorine, and all purine nucleosides contain a 2'-O-methyl group; and wherein with respect to the antisense strand, any of the following is true: All purine nucleosides contain a 2' fluorine, and all pyrimidine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl, All purine nucleosides contain a 2'-O-methyl group, and all pyrimidine nucleosides are modified with a mixture of 2'-fluoro and 2'-O-methyl groups, All purine nucleosides contain a 2'-O-methyl group, and all pyrimidine nucleosides contain a 2' fluoro group. All pyrimidine nucleosides contain a 2' fluorine, and all purine nucleosides are modified with a mixture of 2' fluorine and 2'-O-methyl, All pyrimidine nucleosides contain 2'-O-methyl groups, and all purine nucleosides are modified with a mixture of 2'-fluoro and 2'-O-methyl groups, or All pyrimidine nucleosides contain a 2'-O-methyl group, and all purine nucleosides contain a 2' fluorine group.

24. The composition of claim 23, wherein the sense strand comprises the 2' deoxynucleoside.

25. The composition of claim 23, wherein the sense strand does not include the 2' deoxynucleoside.

26. The composition of claim 23, wherein the 5' end of the sense strand comprises two nucleotides linked via a phosphate linkage rather than by a phosphorothioate linkage.

27. The composition of claim 23, wherein the 5' end of the sense strand further comprises 1 or 2 phosphorothioate linkages.

28. The composition of claim 23, wherein the hydrophobic moiety comprises a lipid attached to the 5' end of the sense strand via a phenyl or cyclohexyl linker.

29. The composition of claim 28, wherein the lipid and the 5' end of the sense strand are linked to the phenyl or cyclohexyl linker in the 1,4, 1,3, or 1,2 substitution pattern.

30. The composition of claim 28, wherein the lipid and the 5' end of the sense strand are linked to the phenyl or cyclohexyl linker in the 1,4 substitution pattern.

31. The composition of claim 28, wherein the hydrophobic portion comprises the structure: wherein the dashed line indicates a covalent attachment to the terminus of the sense strand or the antisense strand, and R is an alkyl group containing 4-18 carbons, with the proviso that R is not octane.

32. The composition of claim 28, wherein the hydrophobic portion comprises the structure: wherein the dashed line indicates a covalent attachment to the terminus of the sense strand or the antisense strand, n is 1-3, and R is an alkyl group containing 4-18 carbons.

33. The composition of claim 28, wherein the hydrophobic portion comprises the structure: wherein the dashed line indicates a covalent attachment to the terminus of the sense strand or the antisense strand, n is 1-3, and R is an alkyl group containing 4-18 carbons.

34. The composition of claim 28, wherein the hydrophobic portion comprises the structure: wherein the dashed line indicates a covalent attachment to the terminus of the sense strand or the antisense strand, and R is an alkyl group containing 4-18 carbons.

35. The composition of claim 28, wherein the hydrophobic portion comprises a lipid portion described in Table 1.

36. The composition of claim 1 or 23, wherein the sense strand and the antisense strand form a duplex.

37. The composition of claim 36, wherein 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.

38. The composition of claim 36, wherein the duplex comprises 19 base pairs.

39. The composition of claim 36, wherein the duplex comprises a sense strand overhang.

40. The composition of claim 39, wherein the sense strand overhang comprises 1-4 nucleotides.

41. The composition of claim 39, wherein the sense strand overhang comprises 2 nucleotides.

42. The composition of claim 39, wherein the sense strand overhang comprises uracil.

43. The composition of claim 39, wherein the duplex comprises an antisense strand overhang.

44. The composition of claim 43, wherein the antisense strand overhang comprises 1-4 nucleotides.

45. The composition of claim 43, wherein the antisense strand overhang comprises 2 nucleotides.

46. ​​The composition of claim 43, wherein the antisense strand overhang comprises uracil.

47. The composition of claim 23, wherein the 5' end of the antisense strand comprises 3 nucleosides separated by the 2 phosphorothioate linkages.

48. The composition of claim 23, wherein the 3' end of the antisense strand comprises three nucleosides separated by the two phosphorothioate linkages.

49. The composition of claim 1 or 23, further comprising a pharmaceutically acceptable carrier.

50. A method of treatment, comprising administering an effective amount of the composition of claim 49 to a subject in need thereof.

51. The method of claim 50, wherein the administering comprises subcutaneous, intravitreal, intrathecal, or intracerebroventricular administration.

52. The method of claim 50, wherein the siRNA targets an mRNA encoding a protein that mediates the disease in the subject.

53. The method of claim 51, wherein the mRNA is located in a tissue of the subject, the tissue comprising eye, liver, fat, brain, or spinal cord.

54. A method of reducing the amount of a target RNA or a protein encoded by the RNA in a cell, comprising administering to the cell a composition as described in claim 49, wherein the antisense strand comprises a sequence that is complementary to and binds to the RNA.

55. The method of claim 54, wherein administering the composition to the cell comprises administering the composition to a subject comprising the cell.

56. The method of claim 55, wherein the administering comprises subcutaneous, intravitreal, intrathecal, or intracerebroventricular administration.

57. The method of claim 54, wherein the administration reduces a measurement of the target RNA or a protein encoded by the RNA by at least 10% relative to a baseline measurement or a control.

58. The method of claim 54, wherein the cells comprise eye, liver, fat, brain, or spinal cord cells.

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