Ethylene glycol-modified oligonucleotides

Abstract

This disclosure relates to compositions, systems, and methods comprising ethylene glycol chemical modification of oligonucleotides for regulating silencing activity and improving stability.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Patent Application No. 63 / 591,271, filed on 18 October 2023. The entire contents of the aforementioned patent application are incorporated herein by reference.

[0002] This disclosure relates to the use of ethylene glycol chemical modification of modified oligonucleotides to modulate silencing activity and improve stability. [Background technology]

[0003] Chemically modified siRNAs represent a cutting-edge form of oligonucleotide therapy. Exemplary chemical modifications include, but are not limited to, 2'-fluoroRNA (2'FRNA), 2'-O-methylRNA (2'OMeRNA), phosphorothioate, vinyl phosphonate, alkyl, locked nucleic acid (LNA), unlocked nucleic acid (UNA), 2'-O-methoxyethyl (2'-MOE), and DNA modifications. These modifications can improve the efficacy, stability, and duration of treatment of oligonucleotides. However, existing modification patterns can result in non-silencing or over-silencing of target mRNA. Over-silencing of the target can lead to undesirable side effects, while under-silencing may not provide therapeutic benefit. Existing modification patterns may also hinder long-term in vivo silencing of mRNA by reducing the duration for which therapeutic oligonucleotides remain active.

[0004] Therefore, in this field, optimized chemical modifications of oligonucleotides are needed to regulate silencing activity and improve stability. [Overview of the Initiative]

[0005] Provided herein are modified oligonucleotides having ethylene glycol chemical modification, which can be used to adjust the level of activity of the modified oligonucleotide to enable strong silencing (e.g., silencing of about 75% or more) or moderate silencing (e.g., silencing of about 25% to about 50%) over a short period (i.e., less than 6 months) or a long period (i.e., 6 months or more), and to improve the stability of the oligonucleotide.

[0006] In one embodiment, the Disclosure provides an RNA molecule having a 5' end and a 3' end, wherein the RNA molecule has at least one ethylene glycol modification at one or both of the 5' and 3' ends, and the at least one ethylene glycol modification comprises 1 to 20 ethylene glycol units.

[0007] In certain embodiments, at least one ethylene glycol modification comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ethylene glycol units.

[0008] In certain embodiments, the RNA molecule contains 2 to 5 ethylene glycol modifications.

[0009] In certain embodiments, the RNA molecule contains two ethylene glycol modifications.

[0010] In certain embodiments, the RNA molecule contains three ethylene glycol modifications.

[0011] In certain embodiments, the RNA molecule contains four ethylene glycol modifications.

[0012] In certain embodiments, the RNA molecule contains five ethylene glycol modifications.

[0013] In certain embodiments, the at least one ethylene glycol modification is located between two adjacent nucleotides.

[0014] In certain embodiments, the at least one ethylene glycol modification does not substitute a nucleotide at the same position in the RNA molecule, relative to an RNA molecule that does not comprise the at least one ethylene glycol modification at the position in the RNA molecule.

[0015] In certain embodiments, the at least one ethylene glycol modification substitutes a nucleotide at the same position in the RNA molecule, relative to an RNA molecule that does not comprise the at least one ethylene glycol modification at the position in the RNA molecule.

[0016] In certain embodiments, the RNA molecule further comprises at least one alkyl modification to one or both of the 5' end and the 3' end.

[0017] In certain embodiments, the at least one alkyl modification is C1~C 10 comprises alkyl.

[0018] In certain embodiments, the at least one alkyl modification comprises C4 alkyl.

[0019] In certain embodiments, the RNA molecule comprises 2 to 5 alkyl modifications.

[0020] In certain embodiments, the RNA molecule comprises 2 alkyl modifications.

[0021] In certain embodiments, the RNA molecule comprises 3 alkyl modifications.

[0022] In certain embodiments, the RNA molecule comprises 4 alkyl modifications.

[0023] In certain embodiments, the RNA molecule comprises 5 alkyl modifications.

[0024] In certain embodiments, at least one alkyl modification is located between two adjacent nucleotides.

[0025] In certain embodiments, at least one alkyl modification does not substitute a nucleotide at the same position within the RNA molecule for RNA molecules that do not contain at least one alkyl modification at that position within the RNA molecule.

[0026] In certain embodiments, at least one alkyl modification replaces a nucleotide at the same position within the RNA molecule in an RNA molecule that does not contain at least one alkyl modification at that position within the RNA molecule.

[0027] In certain embodiments, the RNA molecule includes single-stranded (ss)RNA or double-stranded (ds)RNA.

[0028] In certain embodiments, the RNA molecule includes a dsRNA having an antisense strand with a 5' end and a 3' end, and a sense strand with a 5' end and a 3' end.

[0029] In certain embodiments, the antisense chain includes at least one ethylene glycol modification at one or both of its 5' and 3' ends, and the at least one ethylene glycol modification includes 1 to 20 ethylene glycol units.

[0030] In certain embodiments, at least one ethylene glycol modification comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ethylene glycol units.

[0031] In certain embodiments, the antisense chain contains 2 to 5 ethylene glycol modifications.

[0032] In certain embodiments, the antisense chain includes two ethylene glycol modifications.

[0033] In certain embodiments, the antisense chain includes three ethylene glycol modifications.

[0034] In certain embodiments, the antisense chain includes four ethylene glycol modifications.

[0035] In certain embodiments, the antisense chain includes five ethylene glycol modifications.

[0036] In certain embodiments, at least one ethylene glycol modification is located between two adjacent nucleotides.

[0037] In certain embodiments, at least one ethylene glycol modification does not substitute a nucleotide at the same position within the RNA molecule for RNA molecules that do not contain at least one ethylene glycol modification at that position within the RNA molecule.

[0038] In certain embodiments, at least one ethylene glycol modification replaces a nucleotide at the same position within the RNA molecule in an RNA molecule that does not contain at least one ethylene glycol modification at that position within the RNA molecule.

[0039] In certain embodiments, the antisense chain further comprises at least one alkyl modification to one or both of its 5' and 3' ends.

[0040] In certain embodiments, at least one alkyl modification is C1-C 10 Contains alkyl.

[0041] In certain embodiments, at least one alkyl modification includes a C4 alkyl group.

[0042] In certain embodiments, the antisense chain includes 2 to 5 alkyl modifications.

[0043] In certain embodiments, the antisense chain includes two alkyl modifications.

[0044] In certain embodiments, the antisense chain includes three alkyl modifications.

[0045] In certain embodiments, the antisense chain includes four alkyl modifications.

[0046] In certain embodiments, the antisense chain includes five alkyl modifications.

[0047] In certain embodiments, at least one alkyl modification is located between two adjacent nucleotides.

[0048] In certain embodiments, at least one alkyl modification does not substitute a nucleotide at the same position within the RNA molecule for RNA molecules that do not contain at least one alkyl modification at that position within the RNA molecule.

[0049] In certain embodiments, at least one alkyl modification replaces a nucleotide at the same position within the RNA molecule in an RNA molecule that does not contain at least one alkyl modification at that position within the RNA molecule.

[0050] In certain embodiments, the antisense strand is 15 to 25 nucleotides long.

[0051] In certain embodiments, the antisense chain is 18, 19, 20, 21, 22, or 23 nucleotides long.

[0052] In certain embodiments, the sense strand is 15 to 25 nucleotides long.

[0053] In certain embodiments, the sense strand is 14, 15, 16, or 17 nucleotides long.

[0054] In certain embodiments, at least one ethylene glycol modification is located at one of positions 1 to 25 from the 5' end of the antisense chain.

[0055] In certain embodiments, at least one alkyl modification is located at one of positions 1 to 25 from the 5' end of the antisense chain.

[0056] In certain embodiments, the RNA molecule comprises at least one chemically modified nucleotide, which is distinct from at least one alkyl modification or at least one ethylene glycol modification.

[0057] In certain embodiments, at least one chemically modified nucleotide includes 2'-O-methyl modified nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, debasalized nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, or mixtures thereof.

[0058] In certain embodiments, the dsRNA includes at least one modified nucleotide bond.

[0059] In certain embodiments, the modified nucleotide bond includes a phosphorothioate nucleotide bond.

[0060] In certain embodiments, the RNA molecule contains 4 to 16 phosphorothioate nucleotide interlinks.

[0061] In certain embodiments, the RNA molecule contains 8 to 13 phosphorothioate nucleotide interlinks.

[0062] In certain embodiments, the dsRNA contains blunt ends.

[0063] In certain embodiments, the dsRNA includes at least one single-stranded nucleotide overhang.

[0064] In certain embodiments, the dsRNA contains single-stranded nucleotide overhangs of approximately 2 to 5 nucleotides.

[0065] In certain embodiments, the dsRNA contains a single-stranded nucleotide overhang of two nucleotides.

[0066] In certain embodiments, the dsRNA includes a single-stranded nucleotide overhang of three nucleotides.

[0067] In certain embodiments, the dsRNA includes a single-stranded nucleotide overhang of four nucleotides.

[0068] In certain embodiments, the dsRNA contains a single-stranded nucleotide overhang of 5 nucleotides.

[0069] In certain embodiments, the single-stranded nucleotide overhang includes at least two alkyl modifications.

[0070] In certain embodiments, the single-stranded nucleotide overhang includes two, three, four, or five alkyl modifications.

[0071] In certain embodiments, the RNA molecule comprises an antisense strand having one of the chemical modification patterns provided in Tables 2-6.

[0072] In certain embodiments, the RNA molecule comprises a sense strand having one of the chemical modification patterns provided in Tables 2-3.

[0073] In another embodiment, the Disclosure provides a double-stranded (ds)RNA comprising an antisense strand having a 5' end and a 3' end, and a sense strand having a 5' end and a 3' end, wherein the antisense strand comprises at least one ethylene glycol modification at one or both of the 5' and 3' ends, and the at least one ethylene glycol modification comprises 1 to 20 ethylene glycol units.

[0074] In certain embodiments, the antisense strand is 15 to 25 nucleotides long.

[0075] In certain embodiments, the antisense chain is 18, 19, 20, 21, 22, or 23 nucleotides long.

[0076] In certain embodiments, the sense strand is 15 to 25 nucleotides long.

[0077] In certain embodiments, the sense strand is 14, 15, 16, or 17 nucleotides long.

[0078] In certain embodiments, at least one ethylene glycol modification is located at one of positions 1 to 25 from the 5' end of the antisense chain.

[0079] In certain embodiments, the dsRNA includes at least one alkyl modification.

[0080] In certain embodiments, at least one alkyl modification is located at one of positions 1 to 25 from the 5' end of the antisense chain.

[0081] In certain embodiments, the dsRNA comprises at least one chemically modified nucleotide that is different from at least one alkyl modification or at least one ethylene glycol modification.

[0082] In certain embodiments, at least one chemically modified nucleotide includes 2'-O-methyl modified nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, debasalized nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, or mixtures thereof.

[0083] In certain embodiments, the dsRNA includes at least one modified nucleotide bond.

[0084] In certain embodiments, the modified nucleotide bond includes a phosphorothioate nucleotide bond.

[0085] In certain embodiments, the dsRNA contains 4 to 16 phosphorothioate nucleotide interlinks.

[0086] In certain embodiments, the dsRNA contains 8 to 13 phosphorothioate nucleotide interlinks.

[0087] In certain embodiments, the dsRNA contains blunt ends.

[0088] In certain embodiments, the dsRNA includes at least one single-stranded nucleotide overhang.

[0089] In certain embodiments, the dsRNA contains single-stranded nucleotide overhangs of approximately 2 to 5 nucleotides.

[0090] In certain embodiments, the dsRNA contains a single-stranded nucleotide overhang of two nucleotides.

[0091] In certain embodiments, the dsRNA includes a single-stranded nucleotide overhang of three nucleotides.

[0092] In certain embodiments, the dsRNA includes a single-stranded nucleotide overhang of four nucleotides.

[0093] In certain embodiments, the dsRNA contains a single-stranded nucleotide overhang of 5 nucleotides.

[0094] In certain embodiments, the single-stranded nucleotide overhang includes at least two alkyl modifications.

[0095] In certain embodiments, the single-stranded nucleotide overhang includes two, three, four, or five alkyl modifications.

[0096] In certain embodiments, the dsRNA comprises an antisense strand having one of the chemical modification patterns provided in Tables 2-6.

[0097] In certain embodiments, the dsRNA comprises a sense strand having one of the chemical modification patterns provided in Tables 2-3.

[0098] In another embodiment, the Disclosure provides a double-stranded (ds)RNA comprising an antisense strand and a sense strand, each having a 5' end and a 3' end, and at least one single-stranded nucleotide overhang of 2 to 5 nucleotides, wherein the single-stranded nucleotide overhang comprises at least one ethylene glycol modification.

[0099] In certain embodiments, the single-stranded nucleotide overhang includes two, three, four, or five nucleotide modifications consisting of ethylene glycol modifications.

[0100] In certain embodiments, each nucleotide in a single-stranded nucleotide overhang contains the same nucleotide modification.

[0101] In certain embodiments, the single-stranded nucleotide overhang further includes alkyl modification.

[0102] In another embodiment, the Disclosure provides a double-stranded (ds)RNA comprising an antisense strand and a sense strand, each having a 5' end and a 3' end, wherein the antisense strand comprises one of the chemical modification patterns provided in Tables 2-6.

[0103] In another embodiment, the Disclosure provides a double-stranded (ds)RNA comprising an antisense strand and a sense strand, each having a 5' end and a 3' end, wherein the sense strand comprises one of the chemical modification patterns provided in Tables 2-3.

[0104] In another aspect, the Disclosure provides a method for reducing the expression of a target mRNA in a subject, the method comprising administering an RNA molecule or dsRNA to the subject to reduce the expression of the target mRNA.

[0105] In certain embodiments, the expression of the target mRNA is reduced by at least about 20%, at least about 30%, at least about 40%, or at least about 50% compared to the expression level before administration of the RNA molecule or dsRNA.

[0106] In certain embodiments, the expression of the target mRNA is reduced for at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months after administration of the RNA molecule or dsRNA.

[0107] The aforementioned and other features and advantages of this disclosure will be better understood by reading the following detailed description of exemplary embodiments in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0108] [Figure 1] The chemical modifications used as baseline modification patterns, in which one or more ethylene glycol-containing modifications are applied, and four siRNA patterns (patterns 1, 1T, 2, and 2T) are shown. [Figure 2] This disclosure shows one or more exemplary embodiments of chemical modification. [Figure 3]This shows the relative HTT mRNA levels in cells incubated with various chemically modified siRNAs at different doses. The siRNAs include the following 3'-terminal modifications: 3× butane modification, 5× butane modification, 2× butane modification with 1×6 ethylene glycol unit modification (hexaethylene glycol (HEG)), or 2×6 ethylene glycol unit modification (HEG). Specific chemical modification patterns are listed in Table 6. [Modes for carrying out the invention]

[0109] Unless otherwise specified, the nomenclature used herein in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein, as well as nucleic acid chemistry and hybridization, is well known and commonly used in the art. Furthermore, the methods and techniques provided herein are carried out in accordance with conventional methods well known in the art, and unless otherwise specified, as described in the various general and more specific references cited and discussed throughout this specification. Enzyme reactions and purification techniques are carried out in accordance with the manufacturer's specifications, as commonly achieved in the art, or as described herein. The terminology, laboratory procedures, and techniques used herein in relation to analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, delivery, and patient treatment.

[0110] Unless otherwise defined herein, scientific and technical terms used herein have the meanings generally understood by those skilled in the art. In the event of any potential ambiguity, the definitions provided herein shall prevail over any dictionary or external definitions. Unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms. The use of "or" shall mean "and / or" unless otherwise stated. The use of the term "including," and other forms such as "include," "includes," and "included," is not limited to these.

[0111] To make this disclosure easier to understand, certain terms are defined first.

[0112] The term "nucleoside" refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine, and thymidine. Further exemplary nucleosides include inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine, and N2,N2-dimethylguanosine (also known as a “rare” nucleoside). The term "nucleotide" refers to a nucleoside having one or more phosphate groups linked to the sugar moiety by ester linkage. Exemplary nucleotides include nucleoside monophosphates, diphosphates, and triphosphates. The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein and refer to polymers of nucleotides linked together by unmodified phosphodiester bonds between 5' and 3' carbon atoms or chemically modified intersubunit bonds.

[0113] The terms “RNA,” “RNA molecule,” or “ribonucleic acid molecule” refer to polymers of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, or more ribonucleotides). The terms “DNA,” “DNA molecule,” or “deoxyribonucleic acid molecule” refer to polymers of deoxyribonucleotides. DNA and RNA can be synthesized spontaneously (e.g., by DNA replication or DNA transcription, respectively). RNA can be modified after transcription. DNA and RNA can also be synthesized chemically. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double-stranded, i.e., dsRNA and dsDNA, respectively). “mRNA” or “messenger RNA” is single-stranded RNA that identifies the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis when ribosomes bind to mRNA.

[0114] As used herein, the term “small interfering RNA” (“siRNA”) (also known in the art as “short interfering RNA”) refers to RNA (or RNA analogues) containing approximately 10 to 50 nucleotides (or nucleotide analogues) that can direct or mediate RNA interference. The siRNAs of this disclosure may be single-stranded (i.e., a single antisense strand) or double-stranded (i.e., the antisense strand and sense strand are annealed together to form a double helix). The double-stranded siRNAs of this disclosure include an antisense strand that is sufficiently complementary to a target mRNA to mediate the silencing of said mRNA, and a sense strand that is sufficiently complementary to the antisense strand to form a double helix. In certain embodiments, an siRNA comprises about 15–30 nucleotides or nucleotide analogs, or about 16–25 nucleotides (or nucleotide analogs), or about 18–23 nucleotides (or nucleotide analogs), or about 19–22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21, or 22 nucleotides or nucleotide analogs). The term "short" siRNA refers to an siRNA containing about 21 nucleotides (or nucleotide analogs), e.g., 19, 20, 21, or 22 nucleotides. The term "long" siRNA refers to an siRNA containing about 24–25 nucleotides, e.g., 23, 24, 25, or 26 nucleotides. Short siRNA may, in some cases, contain fewer than 19 nucleotides, e.g., 16, 17, or 18 nucleotides, provided that the short siRNA retains its ability to mediate RNAi. Similarly, long siRNA may, in some cases, contain more than 26 nucleotides, provided that the longer siRNA retains its ability to mediate RNAi without further processing of the short siRNA, e.g., enzymatic processing.

[0115] The terms “nucleotide analog,” “modified nucleotide,” or “modified nucleotide” refer to non-standard nucleotides, such as ribonucleotides or deoxyribonucleotides, which do not exist in nature. Exemplary nucleotide analogs are modified at any position to alter the specific chemical properties of a nucleotide, while retaining the ability of the nucleotide analog to perform its intended function. Examples of nucleotide positions that can be derivatized include the 5-position, e.g., 5-(2-amino)propyluridine, 5-bromouridine, 5-propyneuridine, 5-propenyluridine; the 6-position, e.g., 6-(2-amino)propyluridine; and the 8-position of adenosine and / or guanosine, e.g., 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine. Furthermore, examples of nucleotide analogs include deazanucleotides, such as 7-deaza-adenosine, O- and N-modified (e.g., alkylated, e.g., N6-methyladenosine, or those known in the art) nucleotides, and other heterocyclic modified nucleotide analogs, such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug.10(4):297-310.

[0116] Nucleotide analogs may also include modifications to the sugar moiety of the nucleotide. For example, the 2'OH group may be substituted with a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH2, NHR, NR2, or COOR, where R is a substituted or unsubstituted C1-C6 alkyl, alkenyl, alkynyl, aryl, etc. Other possible modifications are described in U.S. Patents No. 5,858,988 and No. 6,291,438.

[0117] The phosphate group of a nucleotide can also be modified, for example, by substituting one or more oxygen atoms of the phosphate group with sulfur (e.g., phosphorothioate), or by making other substitutions so that the nucleotide can perform its intended function, as described in Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr. 10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000 Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct. 11(5):317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001 Apr. 11(2):77-85, and U.S. Patent No. 5,684,143. The specific modifications referenced above (e.g., phosphate group modifications) reduce the hydrolysis rate of polynucleotides, including their analogs, for example, in vivo or in vitro.

[0118] The term "oligonucleotide" refers to polymers of nucleotides and / or nucleotide analogs. Oligonucleotides include, but are not limited to, siRNA, antisense oligonucleotides, miRNA, ribozymes, and mRNA.

[0119] The term “RNA analog” refers to a polynucleotide (e.g., a chemically synthesized polynucleotide) that has at least one modified or altered nucleotide compared to the corresponding unmodified or unaltered RNA, but retains the same or similar properties or functions as the corresponding unmodified or unaltered RNA. As described above, oligonucleotides may be linked by phosphodiester links that, compared to RNA molecules with phosphodiester links, result in a reduced hydrolysis rate of the RNA analog. For example, the nucleotides of an analog may include methylenediol, ethylenediol, oxymethylthio, oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phosphoramidate, and / or phosphorothioate links. Examples of RNA analogs include, but are not limited to, sugar and / or skeletal-modified ribonucleotides and / or deoxyribonucleotides. Such alterations or alterations may further include the addition of non-nucleotide substances, such as to the terminal(s) or internal(s) (one or more nucleotides) of the RNA. RNA analogs only need to be sufficiently similar to native RNA that has the ability to mediate RNA interference.

[0120] As used herein, the term “RNA interference” (“RNAi”) refers to the selective intracellular degradation of RNA. RNAi occurs naturally within cells and removes foreign RNA (e.g., viral RNA). Natural RNAi proceeds via fragments cleaved from free dsRNA, directing the degradation mechanism to other similar RNA sequences. Alternatively, RNAi can be initiated by humans, for example, to silencing the expression of a target gene.

[0121] RNAi agents, such as RNA silencing agents, have a chain containing a sequence sufficiently complementary to the target mRNA sequence to direct target-specific RNA interference (RNAi), meaning that the chain contains a sequence sufficient to cause the destruction of the target mRNA by the RNAi mechanism or process.

[0122] As used herein, “isolated RNA” (e.g., “isolated siRNA” or “isolated siRNA precursor”) refers to an RNA molecule that, if produced by recombinant technology, substantially does not contain other cell material or culture medium, and to an RNA molecule that, if chemically synthesized, substantially does not contain chemical precursors or other chemical substances.

[0123] As used herein, the term “RNA silencing” refers to a group of sequence-specific regulatory mechanisms mediated by RNA molecules (e.g., RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), querring, co-repression, and translational repression) that result in inhibition or “silencing” of the expression of the corresponding protein-coding gene. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

[0124] The term “discriminative RNA silencing” refers to the ability of an RNA molecule to substantially inhibit the expression of a “first” or “target” polynucleotide sequence while not substantially inhibiting the expression of a “second” or “non-target” polynucleotide sequence, for example, when both polynucleotide sequences are present in the same cell. In certain embodiments, the target polynucleotide sequence corresponds to a target gene, and the non-target polynucleotide sequence corresponds to a non-target gene. In other embodiments, the target polynucleotide sequence corresponds to a target allele, and the non-target polynucleotide sequence corresponds to a non-target allele. In certain embodiments, the target polynucleotide sequence is a DNA sequence encoding a regulatory region (e.g., a promoter or enhancer element) of the target gene. In other embodiments, the target polynucleotide sequence is the target mRNA encoded by the target gene.

[0125] The term "in vitro" has the meaning recognized in the art, including, for example, purified reagents or extracts, such as cell extracts. The term "in vivo" also has the meaning recognized by those skilled in the art, including, for example, living cells, such as immortalized cells, primary cells, cell lines, and / or cells within an organism.

[0126] As used herein, the term “target gene” refers to a gene whose expression is substantially inhibited or “silenced.” This silencing can be achieved, for example, by cleaving the mRNA of the target gene or by silencing the RNA by translational repression of the target gene. The term “non-target gene” refers to a gene whose expression is not substantially silenced. In one embodiment, the polynucleotide sequences of the target gene and the non-target gene (e.g., the mRNA encoded by the target gene and the non-target gene) may differ by one or more nucleotides. In another embodiment, the target and non-target genes may differ by one or more polymorphisms (e.g., single nucleotide polymorphisms or SNPs). In another embodiment, the target and non-target genes may share less than 100% sequence identity. In another embodiment, the non-target gene may be a homolog (e.g., an ortholog or paralog) of the target gene.

[0127] As used herein, the term “RNA silencing agent” refers to RNA capable of inhibiting or “silencing” the expression of a target gene. In certain embodiments, RNA silencing agents can prevent the complete processing of mRNA molecules (e.g., complete translation and / or expression) through a post-transcriptional silencing mechanism. Examples of RNA silencing agents include small (<50 b.p.) non-coding RNA molecules, such as double-stranded RNAs containing paired strands, and precursor RNAs capable of generating such small non-coding RNAs. Exemplary RNA silencing agents include siRNA, miRNA, siRNA-like double-stranded RNAs, antisense oligonucleotides, GAPMER molecules, and dual-function oligonucleotides, as well as their precursors. In one embodiment, RNA silencing agents can induce RNA interference. In another embodiment, RNA silencing agents can mediate translational repression.

[0128] As used herein, the term “rare nucleotide” refers to naturally occurring nucleotides that occur infrequently, such as naturally occurring deoxyribonucleotides or ribonucleotides that do not occur infrequently, such as guanosine, adenosine, cytosine, or uridine. Examples of rare nucleotides, but not limited to, include inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine, and 2,2N,N-dimethylguanosine.

[0129] The term "engineered" indicates that the precursor or molecule is not found in nature, in that all or part of the nucleic acid sequence of the precursor or molecule is created or selected by humans, as in the case of an engineered RNA precursor or engineered nucleic acid molecule. Once created or selected, the sequence is replicated, translated, transcribed, or otherwise processed by intracellular mechanisms. Therefore, an RNA precursor produced intracellularly from a transgene containing an engineered nucleic acid molecule is an engineered RNA precursor.

[0130] As used herein, the term “microRNA” (“miRNA”) is also referred to in the art as “small temporal RNA” (“stRNA”) and refers to a small (e.g., 10 to 50 nucleotides) RNA that is genetically encoded (e.g., by the genome of a virus, mammal, or plant) and can direct or mediate RNA silencing. “miRNA disorder” refers to a disease or disorder characterized by abnormal expression or activity of miRNA.

[0131] As used herein, the term “dual-functional oligonucleotide” refers to an RNA silencing agent having the formula TL-μ, where T is the mRNA targeting portion, L is the linking portion, and μ is the miRNA recruitment portion. As used herein, the terms “mRNA targeting portion,” “targeting portion,” “mRNA targeting portion,” or “targeting portion” refer to a domain, portion, or region of a dual-functional oligonucleotide that is sufficiently sized and sufficiently complementary to a portion or region of mRNA selected or targeted for silencing (i.e., the portion has a sequence sufficient to capture the target mRNA). As used herein, the term “linking portion” or “linking portion” refers to a domain, portion, or region of an RNA silencing agent that covalently joins or links mRNA.

[0132] As used herein, the term “antisense strand” of an RNA silencing agent, such as siRNA or RNA silencing agent, refers to a strand substantially complementary to a section of approximately 10–50 nucleotides, e.g., approximately 15–30, 16–25, 18–23, or 19–22 nucleotides, of the mRNA of the targeted gene for silencing. The antisense strand, or first strand, has a sequence that is sufficiently complementary to the desired target mRNA sequence to direct target-specific silencing, e.g., sufficient complementarity to cause the destruction of the desired target mRNA by an RNAi mechanism or process (RNAi interference), or sufficient complementarity to induce translational repression of the desired target mRNA.

[0133] The terms “sense strand” or “second strand” of an RNA silencing agent, such as siRNA, refer to a strand complementary to the antisense strand or first strand. The antisense strand and sense strand can also be referred to as the first strand or second strand, where the first strand or second strand is complementary to the target sequence, and each second strand or first strand is complementary to the first strand or second strand. A miRNA double-stranded intermediate or siRNA-like double-stranded complex includes a miRNA strand that is sufficiently complementary to a section of approximately 10–50 nucleotides of the mRNA of the gene targeted for silencing, and a miRNA* strand that is sufficiently complementary to form a double-stranded complex with the miRNA strand.

[0134] As used herein, the term “guide strand” refers to the strand of RNA silencing agent, such as a double-stranded siRNA or antisense strand of an siRNA sequence, that enters the RISC complex and directs the cleavage of the target mRNA.

[0135] As used herein, the term “asymmetry” refers to an unevenness in the binding strength or base pair strength between the ends of an RNA silencing agent (e.g., between the terminal nucleotides on the first strand or stem portion and the terminal nucleotides on the opposing second strand or stem portion), such as in the asymmetry of the double-stranded region of the RNA silencing agent (e.g., the stem of shRNA). This results in the 5' end of one strand of the double helix being more frequently in a transient unpaired state, e.g., a single-stranded state, than the 5' end of the complementary strand. This structural difference determines that one strand of the double helix is ​​preferentially incorporated into the RISC complex. The strand whose 5' end is less firmly paired with the complementary strand will preferentially be incorporated into RISC and mediate RNAi.

[0136] As used herein, the terms “bond strength” or “base pair strength” primarily refer to the strength of interactions between pairs of nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide double-strand (e.g., an siRNA double-strand), and between these nucleotides (or nucleotide analogs), such as through H bonds and van der Waals interactions.

[0137] As used herein, the "5' end" (e.g., the antisense or sense strand of an oligonucleotide) refers to the 5' terminal nucleotide, e.g., the 1 to about 5 nucleotides at the 5' end of an oligonucleotide. In certain embodiments, the 5' end of an oligonucleotide corresponds to the first 5 nucleotides of the oligonucleotide. In certain embodiments, the 5' end of an oligonucleotide is the first nucleotide. In certain embodiments, the 5' end of an oligonucleotide is the first two consecutive nucleotides. In certain embodiments, the 5' end of an oligonucleotide is the first three consecutive nucleotides. In certain embodiments, the 5' end of an oligonucleotide is the first four consecutive nucleotides. In certain embodiments, the 5' end of an oligonucleotide is the first five consecutive nucleotides.

[0138] As used herein, the "3' end" (e.g., the antisense or sense strand of an siRNA), such as the 3' end of an oligonucleotide, refers to the 3' terminal nucleotides, e.g., the 1 to about 5 nucleotides at the 3' end of an oligonucleotide. In certain embodiments, the 3' end of an oligonucleotide corresponds to the last 5 nucleotides of the oligonucleotide. In certain embodiments, the 3' end of an oligonucleotide is the last nucleotide. In certain embodiments, the 3' end of an oligonucleotide is the last 2 consecutive nucleotides. In certain embodiments, the 3' end of an oligonucleotide is the last 3 consecutive nucleotides. In certain embodiments, the 3' end of an oligonucleotide is the last 4 consecutive nucleotides. In certain embodiments, the 3' end of an oligonucleotide is the last 5 consecutive nucleotides.

[0139] As used herein, the term “destabilized nucleotide” refers to a first nucleotide or nucleotide analog that can form a base pair with a second nucleotide or nucleotide analog such that the base pair has a lower binding strength than a conventional base pair (i.e., a Watson-Crick base pair). In certain embodiments, the destabilized nucleotide can form a mismatch base pair with the second nucleotide. In other embodiments, the destabilized nucleotide can form a fluctuation base pair with the second nucleotide. In yet another embodiment, the destabilized nucleotide can form an ambiguous base pair with the second nucleotide.

[0140] As used herein, the term “base pair” refers to the interaction between pairs of nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide double helix (e.g., the strands of an RNA silencing agent and the double helix formed by a target mRNA sequence), primarily through H bonds between said nucleotides (or nucleotide analogs), van der Waals interactions, and the like. As used herein, the terms “bond strength” or “base pair strength” refer to the strength of a base pair.

[0141] As used herein, the term "mismatched base pair" refers to a base pair consisting of a non-complementary or non-Watson-Crick base pair that is not a normal complementary G:C, A:T, or A:U base pair. As used herein, the term "ambiguous base pair" (also known as a non-recognized base pair) refers to a base pair formed by a universal nucleotide.

[0142] As used herein, the term “universal nucleotide” (also known as “neutral nucleotide”) includes nucleotides (e.g., certain destabilized nucleotides) that have bases that do not significantly distinguish between bases on a complementary polynucleotide when forming base pairs (“universal bases” or “neutral bases”). Universal nucleotides are primarily hydrophobic molecules and can efficiently pack into antiparallel double-stranded nucleic acids (e.g., double-stranded DNA or RNA) through stacking interactions. The base portion of a universal nucleotide typically contains a nitrogen-containing aromatic heterocyclic moiety.

[0143] As used herein, the terms “sufficient complementarity” or “sufficient degree of complementarity” mean that the RNA silencing agent has sequences (e.g., in the antisense strand, mRNA targeting portion, or miRNA recruiting portion) sufficient to bind to the desired target and induce RNA silencing of the target mRNA, respectively.

[0144] As used herein, the term “translational repression” refers to the selective inhibition of mRNA translation. Spontaneous translational repression proceeds via miRNA cleaved from shRNA precursors. Both RNAi and translational repression are mediated by RISC. Both RNAi and translational repression occur spontaneously or can be initiated by humans, for example, to silence the expression of a target gene.

[0145] As used herein, the term "alkyl" refers to a saturated hydrocarbon group that may be linear or branched. n~m"Alkyl" refers to an alkyl group having n to m carbon atoms. An alkyl group formally corresponds to an alkane in which one C-H bond is replaced by a point of attachment to the rest of the compound of said alkyl group. In some embodiments, the alkyl group contains 1 to 10 carbon atoms, 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; higher homologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, and the like.

[0146] The term "heteroalkyl" refers to an optionally substituted alkyl radical having one or more backbone chain atoms selected from atoms other than carbon, for example, oxygen, nitrogen (e.g., NH or N-alkyl), sulfur, phosphorus, silicon, or combinations thereof. In some embodiments, heteroalkyl refers to an alkyl group in which one of the backbone atoms of the alkyl is oxygen. In some embodiments, heteroalkyl refers to an alkyl group in which one of the backbone atoms of the alkyl is NH or N-alkyl. In some embodiments, heteroalkyl refers to an alkyl group in which one of the backbone atoms of the alkyl is O or S. Exemplary heteroalkyl groups include -(CH₂) n O-CH₃, -(CH₂) n OCH(CH₃)₂, -CH(CH₃)O-(CH₂)ₙ-CH₃, -C(CH₃)₂O-CH₃, -(CH₂) n S-CH₃, -(CH₂) n SCH(CH₃)₂, -CH(CH₃)S-(CH₂) n -CH₃, -CH(CH₃)SO₂-(CH₂) nExamples include, but are not limited to, -CH3, -C(CH3)2SO2-CH3, -CH2NH-(C1-C6 alkyl), -C(CH3)2NH-(C1-C6 alkyl), and -CH(CH3)NH-(C1-C6 alkyl)2. In certain embodiments, the heteroatom(s) are positioned at any internal position of the heteroalkyl group. Examples include, but are not limited to, -CH2-O-CH3, -CH2-CH2-O-CH3, -CH2-NH-CH3, -CH2-CH2-NH-CH3, -CH2-N(CH3)-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -O-CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -O-CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, and -CH=CH-N(CH3)-CH3. In certain embodiments, if the heteroalkyl group contains a CH3 group, the heteroalkyl group is located at the 5' and / or 3' ends of the oligonucleotide.

[0147] As used herein, the term "alkoxy" refers to an -O-alkyl group (alkyl is as defined herein). Examples of alkoxys include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, and t-butoxy. In one embodiment, C1-C6 alkoxy groups are provided herein.

[0148] As used herein, the term “halo” or “halogen” means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably fluorine, chlorine, or bromine, more preferably fluorine or chlorine, either alone or as part of another substituent.

[0149] As used herein, the term "hydroxy," either alone or as part of another substituent, means the alcohol moiety having the formula -OH unless otherwise specified.

[0150] As used herein, the term “ethylene glycol,” either alone or as part of another substituent, means the alcohol moiety having the formula HO(CH2)(CH2)OH unless otherwise specified. The term “n to m ethylene glycol units” means the alcohol moiety having n to m ethylene glycol units. In some embodiments, ethylene glycol includes 1 to 20 ethylene glycol units, 1 to 18 ethylene glycol units, 1 to 18 ethylene glycol units, 1 to 16 ethylene glycol units, 1 to 14 ethylene glycol units, 1 to 12 ethylene glycol units, 1 to 10 ethylene glycol units, 1 to 8 ethylene glycol units, 1 to 6 ethylene glycol units, 1 to 4 ethylene glycol units, or 1 to 2 ethylene glycol units. In some embodiments, ethylene glycol includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ethylene glycol units. "Ethylene glycol modification" may contain n to m ethylene glycol units, as defined above.

[0151] The preparation of linkers may involve the protection and deprotection of various chemical groups. The need for protection and deprotection, as well as the selection of appropriate protecting groups, can be readily determined by those skilled in the art. The chemistry of protecting groups can be found, for example, in Greene, et al., Protective Groups in Organic Synthesis, 4d. Ed., Wiley & Sons, 2007 (which is incorporated herein by reference in its entirety). The protecting groups described herein, as well as the adjustments to the methods of formation and cleavage, can be adjusted as necessary, taking into account various substituents.

[0152] Various methodologies of this disclosure include a step of comparing values, levels, features, properties, etc., with a “preferred control,” which is interchangeably referred to herein as “appropriate control.” A “preferred control” or “appropriate control” is any control or standard well known to those skilled in the art and useful for comparison purposes. In one embodiment, a “preferred control” or “appropriate control” is a value, level, feature, property, etc., determined before performing the RNAi methodology as described herein. For example, transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristics or properties, genotype, phenotype, etc., can be determined before introducing the RNA silencing agent of this disclosure into cells or organisms. In another embodiment, a “preferred control” or “appropriate control” is a value, level, feature, property, etc., determined in cells or organisms, e.g., a control or, e.g., normal cells or organisms exhibiting normal traits. In yet another embodiment, a “preferred control” or “appropriate control” is a predefined value, level, feature, property, etc.

[0153] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure pertains. Similar or equivalent methods and materials may be used in the implementation or testing of this disclosure, but suitable methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. In case of any conflict, this specification shall prevail, including definitions. In addition, materials, methods, and examples are illustrative and not intended to be limiting.

[0154] Various aspects of this disclosure are described in further detail in the following subsections.

[0155] Nucleotide modification and chemical modification patterns Provided herein are a series of nucleotide modifications, any one or more of which can be applied to RNA molecules (e.g., dsRNA) to improve efficacy and, for example, extend in vivo silencing activity after a single dose. Also provided are RNA chemical modification patterns (e.g., chemical modification patterns of the antisense and sense strands) that achieve improved efficacy and extended in vivo silencing. In non-limiting examples, RNA chemical modification patterns (e.g., patterns 1, 1T, 2, and 2T) as shown in Figure 1 can be applied to RNA molecules of this disclosure.

[0156] In certain embodiments, the RNA molecules of this disclosure include one or more nucleotide modifications selected from the group consisting of ethylene glycol modifications, alkyl modifications, locked nucleic acid (LNA) modifications, unlocked nucleic acid (UNA) modifications, 2'-deoxy modifications, and 2'-MOE modifications. In non-limiting examples, the RNA molecules of this disclosure may include nucleotide modifications as shown in Figure 2.

[0157] In certain embodiments, the RNA molecule contains at least one ethylene glycol modification, at least two ethylene glycol modifications, at least three ethylene glycol modifications, at least four ethylene glycol modifications, at least five ethylene glycol modifications, at least six ethylene glycol modifications, at least seven ethylene glycol modifications, at least eight ethylene glycol modifications, at least nine ethylene glycol modifications, or at least ten ethylene glycol modifications. In certain embodiments, the RNA molecule contains two to five ethylene glycol modifications. In certain embodiments, the RNA molecule contains two ethylene glycol modifications. In certain embodiments, the RNA molecule contains three ethylene glycol modifications. In certain embodiments, the RNA molecule contains four ethylene glycol modifications. In certain embodiments, the RNA molecule contains five ethylene glycol modifications. In certain embodiments, the ethylene glycol modification may contain n to m ethylene glycol units as defined above. In certain embodiments, the RNA molecule containing two or more ethylene glycol modifications may contain the same number of ethylene glycol units in each modification. In some embodiments, the RNA molecule containing two or more ethylene glycol modifications may contain a different number of ethylene glycol units in each modification. In some embodiments, an RNA molecule containing two or more ethylene glycol modifications may contain at least two modifications having different numbers of ethylene glycol units. For example, an RNA molecule containing two or more ethylene glycol modifications may contain at least one modification having m ethylene glycol units and at least one modification having n ethylene glycol units, where n ≠ m.

[0158] In certain embodiments, the RNA molecule contains at least one ethylene glycol modification within nucleotide positions 1-5 at either or both of its 5' and 3' ends.

[0159] In certain embodiments, the RNA molecule includes an ethylene glycol modification at nucleotide position 1 at the 5' end. In certain embodiments, the RNA molecule includes an ethylene glycol modification at nucleotide position 2 at the 5' end. In certain embodiments, the RNA molecule includes an ethylene glycol modification at nucleotide position 3 at the 5' end. In certain embodiments, the RNA molecule includes an ethylene glycol modification at nucleotide position 4 at the 5' end. In certain embodiments, the RNA molecule includes an ethylene glycol modification at nucleotide position 5 at the 5' end. In certain embodiments, the RNA molecule includes ethylene glycol modifications at nucleotide positions 1 and 2 at the 5' end. In certain embodiments, the RNA molecule includes ethylene glycol modifications at nucleotide positions 1-3 at the 5' end. In certain embodiments, the RNA molecule includes ethylene glycol modifications at nucleotide positions 1-4 at the 5' end. In certain embodiments, the RNA molecule includes ethylene glycol modifications at nucleotide positions 1-5 at the 5' end.

[0160] In certain embodiments, the RNA molecule includes an ethylene glycol modification at nucleotide position 1 at the 3' end. In certain embodiments, the RNA molecule includes an ethylene glycol modification at nucleotide position 2 at the 3' end. In certain embodiments, the RNA molecule includes an ethylene glycol modification at nucleotide position 3 at the 3' end. In certain embodiments, the RNA molecule includes an ethylene glycol modification at nucleotide position 4 at the 3' end. In certain embodiments, the RNA molecule includes an ethylene glycol modification at nucleotide position 5 at the 3' end. In certain embodiments, the RNA molecule includes ethylene glycol modifications at nucleotide positions 1 and 2 at the 3' end. In certain embodiments, the RNA molecule includes ethylene glycol modifications at nucleotide positions 1-3 at the 3' end. In certain embodiments, the RNA molecule includes ethylene glycol modifications at nucleotide positions 1-4 at the 3' end. In certain embodiments, the RNA molecule includes ethylene glycol modifications at nucleotide positions 1-5 at the 3' end.

[0161] In certain embodiments, the RNA molecule contains 1 to 20 ethylene glycol units. In certain embodiments, the RNA molecule contains 1 ethylene glycol unit. In certain embodiments, the RNA molecule contains 2 ethylene glycol units. In certain embodiments, the RNA molecule contains 3 ethylene glycol units. In certain embodiments, the RNA molecule contains 4 ethylene glycol units. In certain embodiments, the RNA molecule contains 5 ethylene glycol units. In certain embodiments, the RNA molecule contains 6 ethylene glycol units. In certain embodiments, the RNA molecule contains 7 ethylene glycol units. In certain embodiments, the RNA molecule contains 8 ethylene glycol units. In certain embodiments, the RNA molecule contains 9 ethylene glycol units. In certain embodiments, the RNA molecule contains 10 ethylene glycol units. In certain embodiments, the RNA molecule contains 11 ethylene glycol units. In certain embodiments, the RNA molecule contains 12 ethylene glycol units. In certain embodiments, the RNA molecule contains 13 ethylene glycol units. In certain embodiments, the RNA molecule contains 14 ethylene glycol units. In certain embodiments, the RNA molecule contains 15 ethylene glycol units. In certain embodiments, the RNA molecule contains 16 ethylene glycol units. In certain embodiments, the RNA molecule contains 17 ethylene glycol units. In certain embodiments, the RNA molecule contains 18 ethylene glycol units. In certain embodiments, the RNA molecule contains 19 ethylene glycol units. In certain embodiments, the RNA molecule contains 20 ethylene glycol units.

[0162] Ethylene glycol modification, if adopted, can be inserted between two adjacent nucleotides or inserted in place of a nucleotide (i.e., can substitute for a nucleotide). In a non-limiting example, the RNA molecules of this disclosure may include ethylene glycol substitutions of nucleotides (e.g., hexaethylene glycol (HEG) substituted nucleotides) as shown in Figure 2. To illustrate the location of ethylene glycol modification, the RNA molecule with the sequence ATGC is used. When the ethylene glycol modification is inserted between two adjacent nucleotides, the exemplary sequence is AT(EG)GC, where "EG" corresponds to the internal ethylene glycol modification. When the ethylene glycol modification is inserted in place of a nucleotide, the exemplary sequence is AT(EG)C, where "EG" corresponds to the ethylene glycol substitution modification.

[0163] In certain embodiments, at least one ethylene glycol modification is located between two adjacent nucleotides. In non-limiting examples, the RNA molecules of this disclosure may include at least one ethylene glycol modification located between two adjacent nucleotides, as shown in Figure 2 (e.g., hexaethylene glycol (HEG) bonded between two nucleotides).

[0164] In certain embodiments, at least one ethylene glycol modification located between two adjacent nucleotides does not substitute a nucleotide at the same position within the RNA molecule for RNA molecules that do not contain at least one ethylene glycol modification at that position within the RNA molecule.

[0165] In certain embodiments, at least one ethylene glycol modification replaces a nucleotide at the same position within the RNA molecule in an RNA molecule that does not contain at least one ethylene glycol modification at that position within the RNA molecule.

[0166] In certain embodiments, the RNA molecule further includes at least one alkyl modification, at least two alkyl modifications, at least three alkyl modifications, at least four alkyl modifications, at least five alkyl modifications, at least six alkyl modifications, at least seven alkyl modifications, at least eight alkyl modifications, at least nine alkyl modifications, or at least ten alkyl modifications. In certain embodiments, the RNA molecule includes two to five alkyl modifications. In certain embodiments, the RNA molecule includes two alkyl modifications. In certain embodiments, the RNA molecule includes three alkyl modifications. In certain embodiments, the RNA molecule includes four alkyl modifications. In certain embodiments, the RNA molecule includes five alkyl modifications.

[0167] In certain embodiments, the RNA molecule contains at least one alkyl modification within nucleotide positions 1-5 at either or both of its 5' and 3' ends.

[0168] In certain embodiments, the RNA molecule includes an alkyl modification at nucleotide position 1 at the 5' end. In certain embodiments, the RNA molecule includes an alkyl modification at nucleotide position 2 at the 5' end. In certain embodiments, the RNA molecule includes an alkyl modification at nucleotide position 3 at the 5' end. In certain embodiments, the RNA molecule includes an alkyl modification at nucleotide position 4 at the 5' end. In certain embodiments, the RNA molecule includes an alkyl modification at nucleotide position 5 at the 5' end. In certain embodiments, the RNA molecule includes alkyl modifications at nucleotide positions 1 and 2 at the 5' end. In certain embodiments, the RNA molecule includes alkyl modifications at nucleotide positions 1-3 at the 5' end. In certain embodiments, the RNA molecule includes alkyl modifications at nucleotide positions 1-4 at the 5' end. In certain embodiments, the RNA molecule includes alkyl modifications at nucleotide positions 1-5 at the 5' end.

[0169] In certain embodiments, the RNA molecule includes an alkyl modification at nucleotide position 1 at the 3' end. In certain embodiments, the RNA molecule includes an alkyl modification at nucleotide position 2 at the 3' end. In certain embodiments, the RNA molecule includes an alkyl modification at nucleotide position 3 at the 3' end. In certain embodiments, the RNA molecule includes an alkyl modification at nucleotide position 4 at the 3' end. In certain embodiments, the RNA molecule includes an alkyl modification at nucleotide position 5 at the 3' end. In certain embodiments, the RNA molecule includes alkyl modifications at nucleotide positions 1 and 2 at the 3' end. In certain embodiments, the RNA molecule includes alkyl modifications at nucleotide positions 1-3 at the 3' end. In certain embodiments, the RNA molecule includes alkyl modifications at nucleotide positions 1-4 at the 3' end. In certain embodiments, the RNA molecule includes alkyl modifications at nucleotide positions 1-5 at the 3' end.

[0170] In certain embodiments, alkyl modification is C1-C 10 Contains alkyl. In certain embodiments, the alkyl modification includes C1 alkyl. In certain embodiments, the alkyl modification includes C2 alkyl. In certain embodiments, the alkyl modification includes C3 alkyl. In certain embodiments, the alkyl modification includes C4 alkyl (i.e., butyl). In certain embodiments, the alkyl modification includes C5 alkyl. In certain embodiments, the alkyl modification includes C6 alkyl. In certain embodiments, the alkyl modification includes C7 alkyl. In certain embodiments, the alkyl modification includes C8 alkyl. In certain embodiments, the alkyl modification includes C9 alkyl. In certain embodiments, the alkyl modification includes C 10 Contains alkyl.

[0171] In certain embodiments, alkyl modification is branched C3~C 10Contains alkyl. In certain embodiments, the alkyl modification includes a branched C3 alkyl. In certain embodiments, the alkyl modification includes a branched C4 alkyl. In certain embodiments, the alkyl modification includes a branched C5 alkyl. In certain embodiments, the alkyl modification includes a branched C6 alkyl. In certain embodiments, the alkyl modification includes a branched C7 alkyl. In certain embodiments, the alkyl modification includes a branched C8 alkyl. In certain embodiments, the alkyl modification includes a branched C9 alkyl. In certain embodiments, the alkyl modification includes a branched C 10 Contains alkyl.

[0172] In certain embodiments, the branched alkyl is isopropyl. In certain embodiments, the branched alkyl is isobutyl. In certain embodiments, the branched alkyl is sec-butyl. In certain embodiments, the branched alkyl is tert-butyl.

[0173] In certain embodiments, branching from branched alkyl is C3~C 10 Contains alkyl. In certain embodiments, the branch from the branched alkyl contains a C3 alkyl. In certain embodiments, the branch from the branched alkyl contains a C4 alkyl. In certain embodiments, the branch from the branched alkyl contains a C5 alkyl. In certain embodiments, the branch from the branched alkyl contains a C6 alkyl. In certain embodiments, the branch from the branched alkyl contains a C7 alkyl. In certain embodiments, the branch from the branched alkyl contains a C8 alkyl. In certain embodiments, the branch from the branched alkyl contains a C9 alkyl. In certain embodiments, the branch from the branched alkyl contains a C 10 Contains alkyl.

[0174] Alkyl modifications, when adopted, can be inserted between two adjacent nucleotides or inserted in place of a nucleotide (i.e., can substitute for a nucleotide). To illustrate the locations of alkyl modifications, an RNA molecule of the sequence ATGC is used. When the alkyl modification is inserted between two adjacent nucleotides, the example sequence is AT(ibut)GC, where "ibut" corresponds to an internal butyl modification. When the alkyl modification is inserted in place of a nucleotide, the example sequence is AT(but)C, where "but" corresponds to a butyl substitution modification.

[0175] In certain embodiments, at least one alkyl modification is located between two adjacent nucleotides.

[0176] In certain embodiments, at least one alkyl modification located between two adjacent nucleotides does not substitute a nucleotide at the same position within the RNA molecule for an RNA molecule that does not contain at least one alkyl modification at that position within the RNA molecule.

[0177] In certain embodiments, at least one alkyl modification replaces a nucleotide at the same position within the RNA molecule in an RNA molecule that does not contain at least one alkyl modification at that position within the RNA molecule.

[0178] In certain embodiments, the alkyl modification is linear (i.e., unbranched).

[0179] In other embodiments, the alkyl modification is branched.

[0180] In certain embodiments, the RNA molecule includes single-stranded (ss)RNA or double-stranded (ds)RNA. The dsRNA of this disclosure includes an antisense strand complementary to the target mRNA and a sense strand complementary to the antisense strand, each strand having a 5' end and a 3' end.

[0181] In certain embodiments, the antisense strand is 15 to 25 nucleotides long. In certain embodiments, the antisense strand is 18, 19, 20, 21, 22, or 23 nucleotides long. In certain embodiments, the sense strand is 15 to 25 nucleotides long. In certain embodiments, the sense strand is 14, 15, 16, or 17 nucleotides long.

[0182] In certain embodiments, the dsRNA includes a double-stranded region of 15 to 20 base pairs. In certain embodiments, the dsRNA includes a double-stranded region of 15 base pairs. In certain embodiments, the dsRNA includes a double-stranded region of 16 base pairs. In certain embodiments, the dsRNA includes a double-stranded region of 18 base pairs. In certain embodiments, the dsRNA includes a double-stranded region of 20 base pairs.

[0183] In certain embodiments, the dsRNA includes a blunt end. In certain embodiments, the dsRNA includes at least one single-stranded nucleotide overhang. In certain embodiments, the dsRNA includes a single-stranded nucleotide overhang of approximately 2 to 5 nucleotides. In certain embodiments, the dsRNA includes a single-stranded nucleotide overhang of 2 nucleotides. In certain embodiments, the dsRNA includes a single-stranded nucleotide overhang of 5 nucleotides.

[0184] Nucleotide modifications selected from the group consisting of ethylene glycol modification, alkyl modification, locked nucleic acid (LNA) modification, unlocked nucleic acid (UNA) modification, 2'-deoxy modification, and 2'-MOE modification can be applied to any one or more nucleotide positions in the antisense or sense strand.

[0185] In certain embodiments, the antisense strand includes an ethylene glycol modification at one or more nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, counted from the 5' end.

[0186] In certain embodiments, the antisense strand includes an ethylene glycol modification at one or more nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, counted from the 5' end.

[0187] In certain embodiments, the antisense chain includes alkyl modifications at one or more nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, counted from the 5' end.

[0188] In certain embodiments, the antisense strand includes an LNA modification at one or more nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, counted from the 5' end.

[0189] In certain embodiments, the antisense strand contains a UNA modification at one or more of the nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, counted from the 5' end.

[0190] In certain embodiments, the antisense strand contains a 2'-deoxy modification at one or more nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, counted from the 5' end.

[0191] In certain embodiments, the antisense strand contains a 2'-MOE modification at one or more of the nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, counted from the 5' end.

[0192] In certain embodiments, the antisense strand contains one or more unmodified RNA nucleotides at nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, counted from the 5' end.

[0193] In certain embodiments, the sense strand includes an ethylene glycol modification at one or more nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, counted from the 5' end.

[0194] In certain embodiments, the sense strand includes alkyl modifications at one or more nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, counted from the 5' end.

[0195] In certain embodiments, the sense strand contains an LNA modification at one or more nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, counted from the 5' end.

[0196] In certain embodiments, the sense strand contains a UNA modification at one or more of the nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, counted from the 5' end.

[0197] In certain embodiments, the sense strand contains a 2'-deoxy modification at one or more nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, counted from the 5' end.

[0198] In certain embodiments, the sense strand contains a 2'-MOE modification at one or more nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, counted from the 5' end.

[0199] In certain embodiments, the sense strand contains one or more unmodified RNA nucleotides at nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, counted from the 5' end.

[0200] In one embodiment, the disclosure provides a double-stranded (ds)RNA comprising an antisense strand having a 5' end and a 3' end, and a sense strand having a 5' end and a 3' end, wherein the antisense strand comprises at least one ethylene glycol modification.

[0201] In certain embodiments, the antisense chain includes at least one alkyl glycol modification.

[0202] In certain embodiments of the RNA molecule or dsRNA of this disclosure, the RNA molecule or dsRNA comprises at least one modified nucleotide bond.

[0203] In certain embodiments, the modified nucleotide bonds include phosphorothioate nucleotide bonds. In certain embodiments, the RNA molecule or dsRNA contains 4 to 16 phosphorothioate nucleotide bonds. In certain embodiments, the RNA molecule or dsRNA contains 8 to 13 phosphorothioate nucleotide bonds.

[0204] Antisense single-chain overhang modifier This disclosure further provides antisense single-stranded overhang (i.e., tail) nucleotide modifications. Antisense single-stranded overhangs are formed when the antisense strand is longer than the sense strand of the dsRNA. Single-stranded overhangs can be 1 to 6 nucleotides long.

[0205] In certain embodiments, the single-stranded overhang is 2 nucleotides long, 3 nucleotides long, 4 nucleotides long, or 5 nucleotides long.

[0206] In certain embodiments, the single-stranded overhang includes an ethylene glycol modification at one or more nucleotide positions 1, 2, 3, 4, or 5, counted from the 5' end.

[0207] In certain embodiments, the single-stranded overhang includes alkyl modification at one or more nucleotide positions 1, 2, 3, 4, or 5, counted from the 5' end.

[0208] In certain embodiments, the single-stranded overhang includes an LNA modification at one or more nucleotide positions 1, 2, 3, 4, or 5, counted from the 5' end.

[0209] In certain embodiments, the single-stranded overhang includes a UNA modification at one or more nucleotide positions 1, 2, 3, 4, or 5, counted from the 5' end.

[0210] In certain embodiments, the single-stranded overhang includes a 2'-deoxy modification at one or more nucleotide positions 1, 2, 3, 4, or 5, counted from the 5' end.

[0211] In certain embodiments, the single-stranded overhang includes a 2'-MOE modification at one or more nucleotide positions 1, 2, 3, 4, or 5, counted from the 5' end.

[0212] In certain embodiments, the single-stranded overhang contains one or more unmodified RNA nucleotides at nucleotide positions 1, 2, 3, 4, or 5, counted from the 5' end.

[0213] Exemplary chemical modification patterns Tables 1-5 below provide examples of antisense and sense chains with chemical modifications. [Table 1] [Table 2-1] Table 2-2 Table 2-3 Table 2-4 Table 2-5 Table 2-6 Table 3-1 Table 3-2 Table 3-3 Table 3-4 Table 3-5 Table 3-6 Table 3-7 Table 4-1 Table 4-2 Table 4-3 Table 4-4 Table 5-1 [Table 5-2] [Table 5-3] [Table 5-4]

[0214] For the chemical modification patterns listed in Tables 1-5 above, the following abbreviations are used: "as" corresponds to the siRNA antisense strand, "s" to the siRNA sense strand, "m" to the 2'-O-methyl (2'-OMe) chemical modification, "f" to the 2'-fluoro (2'-F) chemical modification, "EG" to the ethylene glycol chemical modification (substitution of a nucleotide with ethylene glycol), "iEG" to the internal ethylene glycol chemical modification (ethylene glycol bonded between two nucleotides), "r" to the unmodified ribonucleotide, and "#" to the phosphorothioate nucleotide bond.

[0215] siRNA design In some embodiments, the siRNA is designed as follows: First, a portion of the target gene is selected. The cleavage of mRNA at these sites eliminates the need for translation of the corresponding protein. The antisense strand is designed based on the target sequence, and the sense strand is designed to be complementary to the antisense strand. Hybridization of the antisense and sense strands forms the siRNA double helix. The antisense strand contains approximately 19–25 nucleotides, e.g., 19, 20, 21, 22, 23, 24, or 25 nucleotides. In other embodiments, the antisense strand contains 20, 21, 22, or 23 nucleotides. The sense strand contains approximately 14–25 nucleotides, e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In other embodiments, the sense strand is 15 nucleotides. In other embodiments, the sense strand is 16 nucleotides. In other embodiments, the sense strand is 17 nucleotides. In other embodiments, the sense strand is 18 nucleotides. In other embodiments, the sense strand is 19 nucleotides. In other embodiments, the sense strand is 20 nucleotides. However, those skilled in the art will understand that siRNAs having antisense strands of less than 19 nucleotides or more than 25 nucleotides in length may also function to mediate RNAi. Thus, siRNAs of such lengths are also within the scope of this disclosure, insofar as they retain the ability to mediate RNAi. Longer RNAi agents have been demonstrated to induce interferon or PKR responses in certain mammalian cells, which may be undesirable. In certain embodiments, the RNAi agents of this disclosure do not induce a PKR response (i.e., are sufficiently short in length). However, longer RNAi agents may be useful, for example, in cell types that cannot produce a PKR response, or in situations where the PKR response is downregulated or suppressed by alternative means.

[0216] Sense strand sequences can be designed so that the target sequence is essentially located in the center of the strand. By shifting the target sequence to an off-center position, the efficiency of siRNA cleavage may, in some cases, be reduced. Such compositions, i.e., less efficient compositions, may be desirable for use when off-silencing of wild-type mRNA is detected.

[0217] The antisense strand may be the same length as the sense strand and may contain complementary nucleotides. In one embodiment, the strands are perfectly complementary; that is, they have blunt ends when aligned or annealed. In another embodiment, the strands are aligned or annealed such that a 1, 2, 3, 4, 5, 6, 7, or 8 nucleotide overhang is generated; that is, the 3' end of the sense strand is 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides longer than the 5' end of the antisense strand, and / or the 3' end of the antisense strand is 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides longer than the 5' end of the sense strand. The overhang may contain (or consist of) nucleotides corresponding to the target gene sequence (or its complement). Alternatively, the overhang may contain (or consist of) deoxyribonucleotides, e.g., dT, or nucleotide analogs, or other suitable non-nucleotide substances.

[0218] To facilitate the entry of the antisense strand into RISC (and thus increase or improve the efficiency of target cleavage and silencing), the base pair strength between the 5' end of the sense strand and the 3' end of the antisense strand may be modified (e.g., reduced or decreased) (these are described in detail below in U.S. Patents No. 7,459,547, No. 7,772,203 and No. 7,732,593, titled "Methods and Compositions for Controlling Efficacy of RNA Silencing" (filed June 2, 2003) and U.S. Patents No. 8,309,704, No. 7,750,144, No. 8,304,530, No. 8,329,892 and No. 8,309,705, titled "Methods and Compositions for Enhancing the Efficacy and Specificity of (RNAi (filed June 2, 2003), the entirety of which is incorporated by reference). In one embodiment of these aspects of the present disclosure, the base pair strength is low because there are fewer G:C base pairs between the 5' end of the first or antisense strand and the 3' end of the second or sense strand than there are between the 3' end of the first or antisense strand and the 5' end of the second or sense strand. In another embodiment, the base pair strength is low due to at least one mismatched base pair between the 5' end of the first or antisense strand and the 3' end of the second or sense strand. In certain exemplary embodiments, the mismatched base pair is selected from the group consisting of: G:A, C:A, C:U, G:G, A:A, C:C, and U:U. In another embodiment, the base pair strength is low due to at least one fluctuation base pair, e.g., G:U, between the 5' end of the first or antisense strand and the 3' end of the second or sense strand. In another embodiment, the base pair strength is lower due to at least one base pair containing a rare nucleotide, such as inosine (I). In certain exemplary embodiments, the base pair is selected from the group consisting of I:A, I:U, and I:C. In yet another embodiment, the base pair strength is lower due to at least one base pair containing a modified nucleotide.In certain exemplary embodiments, the modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

[0219] To verify the effectiveness of siRNA in disrupting mRNA (e.g., mRNA expressed from a target gene), siRNA can be incubated with cDNA (e.g., cDNA derived from a target gene) in a Drosophila-based in vitro mRNA expression system. 32 Newly synthesized mRNA (e.g., target mRNA) radiolabeled with 3P is detected by autoradiography on an agarose gel. The presence of cleaved mRNA indicates mRNA nuclease activity. Suitable controls include the omission of siRNA. Alternatively, a control siRNA is selected that has the same nucleotide composition as the selected siRNA but does not have significant sequence complementarity to the appropriate target gene. Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected siRNA. A homology search can be performed to confirm that the negative control lacks homology to any other gene in the appropriate genome. Furthermore, negative control siRNA can be designed by introducing one or more base mismatches into the sequence. The siRNA-mRNA complementation site is selected to yield optimal mRNA specificity and maximum mRNA cleavage.

[0220] Characteristics of RNA silencing agents In certain embodiments, the RNA silencing agent comprises at least 80% chemically modified nucleotides. In certain embodiments, the RNA silencing agent is fully chemically modified, i.e., 100% of the nucleotides are chemically modified.

[0221] In certain embodiments, the RNA silencing agent is 2'-O-methyl rich, i.e., contains more than 50% 2'-O-methyl. In certain embodiments, the RNA silencing agent contains at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% 2'-O-methyl nucleotide content. In certain embodiments, the RNA silencing agent contains at least about 70% 2'-O-methyl nucleotide modification. In certain embodiments, the RNA silencing agent contains about 70% to about 90% 2'-O-methyl nucleotide modification. In certain embodiments, the RNA silencing agent is dsRNA including an antisense strand and a sense strand. In certain embodiments, the antisense strand contains at least about 70% 2'-O-methyl nucleotide modification. In certain embodiments, the antisense strand contains about 70% to about 90% 2'-O-methyl nucleotide modification. In certain embodiments, the sense strand contains at least about 70% 2'-O-methylnucleotide modifications. In certain embodiments, the sense strand contains about 70% to about 90% 2'-O-methylnucleotide modifications. In certain embodiments, the sense strand contains 100% 2'-O-methylnucleotide modifications. 2'-O-methyl-rich RNA silencing agents and specific chemical modification patterns are further described in USSN 16 / 550,076 (filed on 23 August 2019, issued as US 11,279,930 on 22 March 2022) and USSN 16 / 999,759 (filed on 21 August 2020, claiming priority over 62 / 891,185 (filed on 23 August 2019)), all of which are incorporated herein by reference.

[0222] Conjugated functional parts In other embodiments, the RNA silencing agent may be modified with one or more functional moieties. The functional moieties are molecules that confer one or more additional activities to the RNA silencing agent. In certain embodiments, the functional moieties enhance cellular uptake by target cells (e.g., nerve cells). Accordingly, the disclosure includes RNA silencing agents that are conjugated to other moieties (e.g., non-nucleic acid moieties such as peptides), organic compounds (e.g., dyes), etc. (e.g., at the 5' and / or 3' ends), or not conjugated. Binding can be achieved using methods known in the art, for example, Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001) (described on nucleic acids loaded onto polyalkylcyanoacrylate (PACA) nanoparticles), Fattal et al., J. Control Release 53(1-3): 137-43 (1998) (described on nucleic acids bound to nanoparticles), Schwab et al., Ann. Oncol. 5 Suppl. 4: 55-8 (1994) (described on nucleic acids linked to inserts, hydrophobic groups, polycations, or PACA nanoparticles), and Godard et al., Eur. J. Biochem. 232(2): 404-10 (1995) (described on nucleic acids linked to nanoparticles).

[0223] In certain embodiments, the functional portion is a hydrophobic portion. In certain embodiments, the hydrophobic portion is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides and nucleoside analogs, endocannabinoids, and vitamins. In certain embodiments, the steroid is selected from the group consisting of cholesterol and lithocholic acid (LCA). In certain embodiments, the fatty acid is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanic acid (DCA). In certain embodiments, the vitamin is selected from the group consisting of choline, vitamin A, vitamin E, and their derivatives or metabolites. In certain embodiments, the vitamin is selected from the group consisting of retinoic acid and alpha-succinate tocopherol.

[0224] In certain embodiments, the RNA silencing agent of the disclosure is bound to a lipophilic moiety. In one embodiment, the lipophilic moiety is a ligand containing a cationic group. In another embodiment, the lipophilic moiety is attached to one or both strands of the siRNA. In an exemplary embodiment, the lipophilic moiety is attached to one end of the sense strand of the siRNA. In another exemplary embodiment, the lipophilic moiety is attached to the 3' end of the sense strand. In certain embodiments, the lipophilic moiety is selected from the group consisting of cholesterol, vitamin E, vitamin K, vitamin A, folic acid, and cationic dyes (e.g., Cy3). In an exemplary embodiment, the lipophilic moiety is cholesterol. Other lipophilic components include cholic acid, adamantane acetate, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

[0225] In certain embodiments, the functional portion may include one or more ligands tethered to the RNA silencing agent to improve stability, hybridization thermodynamics with target nucleic acids, targeting to specific tissues or cell types, or cell permeability, for example, by endocytosis-dependent or independent mechanisms. The ligands and associated modifications may also enhance sequence specificity, thereby reducing off-site targeting. The tethering ligand may include one or more modified bases or sugars that can function as intercalators. These may also be located within internal regions, such as within the bulge of the RNA silencing agent / target double helix. The intercalators may be aromatic, e.g., polycyclic aromatic or heterocyclic aromatic compounds. Polycyclic intercalators may have stacking capabilities and may include systems having two, three, or four fused rings. The universal bases described herein may be included in the ligand. In one embodiment, the ligand may include a cleavage group that contributes to the inhibition of the target gene by cleaving the target nucleic acid. The cleavage group may be, for example, bleomycin (e.g., bleomycin-A5, bleomycin-A2, or bleomycin-B2), pyrene, phenanthroline (e.g., O-phenanthroline), polyamine, tripeptide (e.g., lys-tyr-lys tripeptide), or a metal ion chelating group. Examples of metal ion chelating groups include Lu(III) or EU(III) macrocyclic complexes, Zn(II) 2,9-dimethylphenanthroline derivatives, Cu(II) terpyridine, or acridine, which can promote the selective cleavage of target RNA at the bulge region by free metal ions such as Lu(III). In some embodiments, a peptide ligand can be tethered to an RNA silencing agent to promote cleavage of target RNA at the bulge region, for example. For example, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (Cycram) can be conjugated to a peptide (e.g., via an amino acid derivative) to promote target RNA cleavage.The tethering ligand can be an aminoglycoside ligand, which can impart improved hybridization properties or improved sequence specificity to the RNA silencing agent. Exemplary aminoglycosides include glycosylated polylysine, galactosylated polylysine, neomycin B, tobramycin, kanamycin A, and acridine-binding aminoglycosides, such as Neo-N-acridine, Neo-S-acridine, Neo-C-acridine, Tobra-N-acridine, and KanaA-N-acridine. The use of acridine analogs can enhance sequence specificity. For example, neomycin B has high affinity for RNA compared to DNA, but lower sequence specificity. The acridine analog neo-5-acridine has high affinity for the HIV Rev-response element (RRE). In some embodiments, guanidine analogs (guanidinoglycosides) of aminoglycoside ligands are tethered to the RNA silencing agent. Within guanidinoglycosides, the amine group on the amino acid is replaced with a guanidine group. The attachment of a guanidine analog can increase the cellular permeability of RNA silencing agents. The tethering ligand may be a polyarginine peptide, peptoid, or peptide mimetic that can increase the cellular uptake of oligonucleotide agents.

[0226] Exemplary ligands are coupled to ligand-binding carriers, either directly or indirectly via intervening tethering factors. In certain embodiments, the coupling is via covalent bonding. In certain embodiments, the ligand is attached to the carrier via intervening tethering factors. In certain embodiments, the ligand alters the distribution, targeting, or lifetime of the RNA silencing agent into which it is incorporated. In certain embodiments, the ligand provides, for example, higher affinity to selected targets, such as molecules, cells or cell types, compartments, such as cell or organ compartments, tissues, organs, or regions of the body, compared to species in which such ligands are absent.

[0227] Exemplary ligands can improve transport, hybridization, and specificity properties, and may also improve the nuclease resistance of polymer molecules and / or natural or modified ribonucleotides, including the resulting natural or modified RNA silencing agents or any combination of monomers described herein. Ligands generally include, for example, therapeutic modifiers to increase uptake, such as diagnostic compounds or reporter groups to monitor distribution, crosslinkers, nuclease resistance-constituting moieties, and natural or aberrant nucleic acid bases. Common examples include lipophilic substances, lipids, steroids (e.g., ubaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, friederin, epifriederanol-deranolic acid-derived lithocholic acid), vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins, protein binders, integrin target molecules, polycations, peptides, polyamines, and peptide mimetic compounds. Ligands can be naturally occurring substances (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), amino acids, or lipids. Ligands may also be recombinant or synthetic molecules, such as synthetic polymers, such as synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-coglycolated) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphatidine.Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudo-peptide polyamines, peptide-mimicking polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helical peptides.

[0228] The ligand may also include a target group that binds to a specific cell type, such as kidney cells, e.g., cell or tissue targeting agents, e.g., lectins, glycoproteins, lipids or proteins, e.g., antibodies. The target group may also be thyroid-stimulating hormone, melanotropin, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetylgalactosamine (GalNAc) or its derivatives, N-acetylglucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, biotin, or RGD peptide or RGD peptide mimetic. Other examples of ligands include dyes, inserts (e.g., acridine and substituted acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphylline, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine, phenanthroline, pyrene), lys-tyr-lys tripeptides, aminoglycosides, guanidiaminaminoguanidiaminaminoglycozides, artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol and its thio analogues), cholic acid, cholanic acid, lithocholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, glycerol (e.g., its esters (e.g., mono, bis, or tris fatty acid esters, e.g., C) 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C18 , C 19 or C 20 fatty acids) and ethers, for example, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , or C 20 Alkyl compounds (e.g., 1,3-bis-O(hexadecyl)glycerol, 1,3-bis-O(octadecyl)glycerol), geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, stearic acid (e.g., glyceryl distearate), oleic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., ante Napedia peptides, Tat peptides), alkylating agents, phosphates, amino acids, mercaptos, PEGs (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, naproxen, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu tetraazama macrocycles) 3+ Examples include complexes, dinitrophenyl, HRP, or AP. In certain embodiments, the ligand is GalNAc or a derivative thereof.

[0229] Ligands can be proteins such as glycoproteins, peptides such as molecules with specific affinity for colligands, or antibodies such as antibodies that bind to specific cell types such as cancer cells, endothelial cells, or osteocytes. Examples of ligands include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, or polyvalent fucose. Ligands can be, for example, lipopolysaccharides, p38 MAP kinase activators, or NF-κB activators.

[0230] A ligand can be a substance, such as a drug, that can increase the uptake of RNA silencing agents into cells by, for example, disrupting the cytoskeleton of a cell, for example, by disrupting the microtubules, microfilaments, and / or intermediate filaments of a cell. Drugs may be, for example, taxone, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latranculine A, phalloidin, swinholide A, indanosine, or myoservin. Ligands can increase the uptake of RNA silencing agents into cells by, for example, activating an inflammatory response. Exemplary ligands having such effects include tumor necrosis factor alpha (TNFα), interleukin-1 beta, or gamma interferon. In one embodiment, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule can bind to a serum protein, for example, human serum albumin (HSA). HSA-binding ligands enable the distribution of the conjugate to target tissues, for example, non-renal target tissues of the body. For example, the target tissue may be the liver, including the parenchymal cells of the liver. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipid or lipid-based ligands can be used to (a) improve resistance to the degradation of the conjugate, (b) increase targeting or transport to target cells or cell membranes, and / or (c) modulate binding to serum proteins, e.g., HSA. Lipid-based ligands can be used to modulate, e.g., control the binding of the conjugate to target tissues. For example, a lipid or lipid-based ligand that binds strongly to HSA is less likely to target the kidney and therefore less likely to be removed from the body. A lipid or lipid-based ligand that does not bind very strongly to HSA can be used to target the conjugate to the kidney. In certain embodiments, the lipid-based ligand binds to HSA. The lipid-based ligand can bind to HSA with sufficient affinity so that the conjugate is distributed to non-renal tissues.However, HSA-ligand binding is intended to be reversible, meaning that the affinity for the lipid-based ligand is not so strong that it cannot reverse the HSA-ligand binding. In another embodiment, the lipid-based ligand binds weakly to HSA or not at all, so that the conjugate is distributed in the kidney. Other parts targeting kidney cells can also be used instead of, or in addition to, the lipid-based ligand.

[0231] In another embodiment, the ligand is a portion taken up by target cells, e.g., proliferating cells, e.g., a vitamin. These may be particularly useful in treating undesirable cell proliferation, e.g., disorders characterized by malignant or non-malignant forms, e.g., cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include vitamin B (e.g., folic acid, B12, riboflavin, biotin, pyridoxal) or other vitamins or nutrients taken up by cancer cells. HSA and low-density lipoprotein (LDL) are also included.

[0232] In another embodiment, the ligand is a cell permeabilizer, such as a helical cell permeabilizer. In certain embodiments, the agent is amphiphilic. Exemplary agents are peptides such as tat or antennopedia. If the agent is a peptide, it may be modified, including the use of peptidyl mimes, invertomers, non-peptide or pseudo-peptide bonds, and D-amino acids. The helical agent may be an alpha-helical agent that may have a lipophilic phase and an oleophobic phase.

[0233] The ligand may be a peptide or a peptide mimetic. A peptide mimetic (also referred to herein as an oligopeptide mimetic) is a molecule that can fold into a defined three-dimensional structure similar to that of a natural peptide. The attachment of peptides and peptide mimetics to oligonucleotide agents can affect the pharmacokinetic distribution of RNA silencing agents, for example, by improving cellular recognition and absorption. The peptide or peptide mimetic moiety may be about 5 to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. The peptide or peptide mimetic may be, for example, a cell-permeable peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (e.g., mainly consisting of Tyr, Trp, or Phe). The peptide moiety may be a dendrimeric peptide, a restrictive peptide, or a cross-linked peptide. The peptide moiety may be an L-peptide or a D-peptide. Alternatively, the peptide moiety may contain a hydrophobic membrane translocation sequence (MTS). Peptides or peptide mimetic compounds can be encoded by random DNA sequences, such as peptides identified from phage presentation libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature 354:82-84, 1991). In exemplary embodiments, the peptide or peptide mimetic compound tethered to the RNA silencing agent via an incorporated monomer unit is a cell-targeted peptide, such as an arginine-glycine-aspartate (RGD) peptide or RGD mimic. The peptide moiety may range in length from approximately 5 to approximately 40 amino acids. The peptide moiety can undergo structural modifications, for example, to improve stability or induce conformational properties. Any of the structural modifications described below may be used.

[0234] In certain embodiments, the functional portion is ligated to the 5' and / or 3' ends of the RNA silencing agent of the disclosure. In certain embodiments, the functional portion is ligated to the 5' and / or 3' ends of the antisense strand of the RNA silencing agent of the disclosure. In certain embodiments, the functional portion is ligated to the 5' and / or 3' ends of the sense strand of the RNA silencing agent of the disclosure. In certain embodiments, the functional portion is ligated to the 3' end of the sense strand of the RNA silencing agent of the disclosure.

[0235] In certain embodiments, the functional moiety is linked to the RNA silencing agent by a linker. In certain embodiments, the functional moiety is linked to the antisense strand and / or sense strand by a linker. In certain embodiments, the functional moiety is linked to the 3' end of the sense strand by a linker. In certain embodiments, the linker includes a divalent or trivalent linker. In certain embodiments, the linker includes an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof. In certain embodiments, the divalent or trivalent linker is selected from the following: [ka] (wherein n is 1, 2, 3, 4, or 5).

[0236] In certain embodiments, the linker further comprises a phosphodiester or a phosphodiester derivative. In certain embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of: [ka] In the formula, X is O, S, or BH3.

[0237] Various functional portions of this disclosure and means for conjugating them to RNA silencing agents are described in further detail in WO2017 / 030973A1 and WO2018 / 031933A2, which are incorporated herein by reference.

[0238] Branched oligonucleotides Two or more RNA silencing agents disclosed above, such as oligonucleotide constructs including siRNA, can be linked together by one or more moieties independently selected from linkers, spacers, and branching points to form a branched oligonucleotide RNA silencing agent. In certain embodiments, the branched oligonucleotide RNA silencing agent consists of two siRNAs to form a di-siRNA ("di-siRNA") scaffold for delivering two siRNAs. In typical embodiments, the nucleic acids of the branched oligonucleotide each include an antisense strand (or a portion thereof), the antisense strand having sufficient complementarity to the target mRNA and mediating an RNA-mediated silencing mechanism (e.g., RNAi).

[0239] In exemplary embodiments, the branched oligonucleotide may have 2 to 8 RNA silencing agents attached via a linker. The linker may be hydrophobic. In one embodiment, the branched oligonucleotide of the present application has 2 to 3 oligonucleotides. In one embodiment, the oligonucleotides independently have substantial chemical stability (e.g., at least 40% of the constituent bases are chemically modified). In exemplary embodiments, the oligonucleotides have complete chemical stability (i.e., all constituent bases are chemically modified). In some embodiments, the branched oligonucleotide comprises one or more single-stranded phosphorothioate tails, each independently having 2 to 20 nucleotides. In non-limiting embodiments, each single-stranded tail has 2 to 10 nucleotides.

[0240] In certain embodiments, branched oligonucleotides are characterized by three properties: (1) a branched structure, (2) complete metabolic stabilization, and (3) the presence of a single-stranded tail containing a phosphorothioate linker. In certain embodiments, branched oligonucleotides have two or three branches. It is thought that an increase in the overall size of the branched structure promotes increased uptake. Also, although not bound by any particular theory regarding activity, it is thought that multiple adjacent branches (e.g., two or three) allow each branch to act in coordination, and thus dramatically improve the rates of internalization, transport, and release.

[0241] Branched oligonucleotides are provided in a variety of structurally diverse embodiments. In some embodiments, the nucleic acid attached at the branching point is single-stranded or double-stranded and consists of a miRNA inhibitor, gapmer, micmer, SSO, PMO, or PNA. These single strands can be attached to the 3' or 5' end. Combinations of siRNA and single-stranded oligonucleotides can also be used for dual function. In another embodiment, short nucleic acids complementary to gapmers, micmers, miRNA inhibitors, SSO, PMO, and PNA are used to carry these active single-stranded nucleic acids and enhance their distribution and intracellular integration. The short double-stranded region has a low melting temperature (Tm approximately 37°C) to rapidly dissociate when the branched structure is taken up into the cell.

[0242] Di-siRNA branched oligonucleotides may contain chemically diverse conjugates, such as the functional moieties described above. The conjugate bioactive ligands may be used to enhance cell specificity and promote membrane binding, internalization, and serum protein binding. Examples of bioactive moieties used for conjugation include DHA, GalNAc, and cholesterol. These moieties can be attached to the Di-siRNA via a connecting linker or spacer, or added via an additional linker or spacer attached to another free siRNA end.

[0243] Due to the presence of the branched structure, the tissue retention level in the brain is improved by more than 100 times compared with unbranched compounds of the same chemical composition, which suggests a new mechanism of cell retention and distribution. Branched oligonucleotides are unexpectedly uniformly distributed throughout the spinal cord and the entire brain. Furthermore, branched oligonucleotides exhibit unexpectedly efficient systemic delivery to various tissues and very high levels of tissue accumulation.

[0244] Branched oligonucleotides include various therapeutic nucleic acids such as siRNA, ASO, miRNA, miRNA inhibitors, splice switching, PMO, and PNA. In some embodiments, the branched oligonucleotide further comprises a conjugated hydrophobic moiety and exhibits unprecedented silencing and effects in vitro and in vivo.

[0245] Linker In one embodiment of the branched oligonucleotide, each linker is independently selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, phosphate, phosphonate, phosphoramidate, ester, amide, triazole, and combinations thereof; wherein any carbon or oxygen atom of the linker is optionally substituted with a nitrogen atom, bears a hydroxyl substituent, or bears an oxo substituent. In one embodiment, each linker is an ethylene glycol chain. In another embodiment, each linker is an alkyl chain. In another embodiment, each linker is a peptide. In another embodiment, each linker is RNA. In another embodiment, each linker is DNA. In another embodiment, each linker is phosphate. In another embodiment, each linker is phosphonate. In another embodiment, each linker is phosphoramidate. In another embodiment, each linker is an ester. In another embodiment, each linker is an amide. In another embodiment, each linker is triazole.

[0246] Branched oligonucleotides, including their synthesis and use, are described in further detail in WO2017 / 132669, which is incorporated herein by reference.

[0247] Method for introducing RNA silencing agents The RNA silencing agents of this disclosure can be introduced directly into cells (e.g., nerve cells) (i.e., intracellularly), or they can be introduced from outside the cell into cavities, spaces, or the circulation of an organism, or they can be introduced orally, or by immersing cells or organisms in a solution containing nucleic acids. Blood vessels or extravascular circulation, the blood or lymphatic system, and cerebrospinal fluid are sites into which nucleic acids can be introduced.

[0248] The RNA silencing agents of this disclosure may be introduced using nucleic acid delivery methods known in the art, such as injection of a nucleic acid-containing solution, impact with nucleic acid-coated particles, immersion of cells or organisms in a nucleic acid solution, or electroporation of cell membranes in the presence of nucleic acid. Other methods known in the art for introducing nucleic acids into cells may also be used, such as lipid-mediated transport, chemical-mediated transport, and cationic liposome transfection, such as calcium phosphate. The nucleic acids may be introduced together with other components that perform one or more of the following activities: enhancing nucleic acid uptake by cells, or increasing inhibition of target genes.

[0249] Physical methods for introducing nucleic acids include injection of an RNA-containing solution, impact with RNA-coated particles, immersion of cells or organisms in an RNA solution, or electroporation of cell membranes in the presence of RNA. Other methods known in the art for introducing nucleic acids into cells, such as lipid-mediated transport, chemical-mediated transport, such as calcium phosphate, may also be used. Thus, RNA may be introduced with components that perform one or more activities, such as enhancing RNA uptake by cells, inhibiting single-strand annealing, stabilizing single strands, or otherwise increasing inhibition of target genes.

[0250] RNA can be introduced directly into cells (i.e., into cells), or it can be introduced from outside cells into cavities, spaces, or the circulation of organisms, orally, or by immersing cells or organisms in a solution containing RNA. Blood vessels or extravascular circulation, the blood or lymphatic system, and cerebrospinal fluid are sites into which RNA can be introduced.

[0251] Cells containing the target gene may originate from germline or somatic cells, totipotent or pluripotent, mitotic or non-mitotic, parenchymal or epithelial tissue, immortalized or transformed cells, etc. The cells may be stem cells or differentiated cells. Differentiated cell types include adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelial cells, neurons, glial cells, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells of endocrine or exocrine glands.

[0252] Depending on the specific target gene and the dose of the delivered double-stranded RNA material, this process can result in partial or complete loss of function of the target gene. Typical results include a reduction or loss of gene expression in at least 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or more, of target cells. Inhibition of gene expression refers to the absence (or observable reduction) of levels of protein and / or mRNA products from the target gene. Specificity refers to the ability to inhibit the target gene without apparent effect on other genes in the cell. The results of inhibition can be confirmed by examining the external characteristics of the cell or organism (as shown in the examples below) or by biochemical methods, such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring by microarray, antibody binding, enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence-activated cell sorting (FACS).

[0253] In the case of RNA-mediated inhibition in cell lines or whole organisms, gene expression can be conveniently assayed by using reporters or drug resistance genes whose protein products are readily assayable. Examples of such reporter genes include acetohydroxy acid synthase (AHAS), alkaline phosphatase (AP), beta-galactosidase (LacZ), beta-glucolonidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopalin synthase (NOS), octopine synthase (OCS), and their derivatives. Multiple select markers conferring resistance to ampicillin, bleomycin, chloramphenicol, gentamicin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracycline are available. Depending on the assay, quantification of gene expression levels can determine inhibition levels greater than 10%, 33%, 50%, 90%, 95%, or 99% compared to cells not treated with this disclosure. If the dose of injected material is small and the time elapsed since RNAi agent administration is long, inhibition may occur in a smaller percentage of cells (e.g., at least 10%, 20%, 50%, 75%, 90%, or 95% of target cells). Quantification of intracellular gene expression may indicate similar levels of inhibition at the level of target mRNA accumulation or target protein translation. For example, the efficiency of inhibition may be determined by evaluating the amount of intracellular gene products. mRNA can be detected by a hybridization probe having a nucleotide sequence outside the region used for the inhibitory double-stranded RNA, or the translated polypeptide can be detected by an antibody produced against the polypeptide sequence in that region.

[0254] RNA can be introduced in amounts that allow for delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500, or 1000 copies per cell) may result in more effective inhibition, while lower doses may be useful for specific applications.

[0255] In exemplary embodiments, the efficacy of the RNAi agents of this disclosure (e.g., siRNA targeting a target sequence) is tested by their ability to specifically degrade mutant mRNA (e.g., production of target mRNA and / or target protein) intracellularly, particularly in neurons (e.g., striatal or cortical neuron clones, and / or primary neurons). Other readily transfectable cells, such as HeLa cells or COS cells, are also suitable for cell-based validation assays. Cells are transfected with human wild-type or mutant cDNA (e.g., human wild-type or mutant target cDNA). Standard siRNA, modified siRNA, or a vector capable of producing siRNA from U-loop mRNA is co-transfected. Selective reduction in target mRNA and / or target protein is measured. The reduction in target mRNA or protein can be compared to the level of target mRNA or protein in the absence of the RNAi agent or in the presence of an RNAi agent that does not target the target mRNA. Exogenously introduced mRNA or protein (or endogenous mRNA or protein) can be assayed for comparison. When using nerve cells that are known to have some degree of resistance to standard transfection techniques, it may be desirable to introduce RNAi agents (e.g., siRNA) by passive uptake.

[0256] Treatment method As used herein, “treatment” or “to treat” is defined as the application or administration of a therapeutic agent (e.g., an RNA agent) to a patient having a disease or disorder, symptoms of a disease or disorder, or a predisposition to a disease or disorder, for the purpose of curing, resolving, mitigating, alleviating, altering, treating, improving, enhancing, or influencing the disease or disorder, symptoms of a disease or disorder, or a predisposition to a disease or disorder, or the application or administration of a therapeutic agent to tissue or cell lines isolated from such patient.

[0257] In one embodiment, the Disclosure provides a method for preventing the aforementioned disease or disorder in a subject by administering a therapeutic agent (e.g., an RNAi agent or vector or a transgene encoding such agent) to the subject. Subjects at risk of the disease can be identified, for example, by one or a combination of the diagnostic assays or prognostic assays described herein. Administration of the prophylactic agent can be performed before the appearance of symptoms characteristic of the disease or disorder, thereby preventing the disease or disorder or slowing its progression.

[0258] Another aspect of this disclosure relates to methods for therapeutically treating an object, i.e., methods for altering the onset of symptoms of a disease or disorder.

[0259] With respect to both prophylactic and therapeutic methods of treatment, such treatments may be specifically adapted or modified based on knowledge derived from the field of pharmacogenomics. As used herein, “pharmacogenomics” refers to the application of genomics techniques, such as gene sequencing, statistical genetics, and gene expression analysis, to drugs in clinical development and on the market. More specifically, the term refers to the study of how a patient’s genes determine their response to a drug (e.g., a patient’s “drug response phenotype” or “drug response genotype”). Thus, another aspect of this disclosure provides a method for tailoring prophylactic or therapeutic treatments to an individual using either a target gene molecule or a target gene regulator of this disclosure according to the individual’s drug response genotype. Pharmacogenomics enables clinicians or physicians to target prophylactic or therapeutic treatments to patients who would benefit most from them, and to avoid treatments for patients who experience toxic drug-related side effects.

[0260] Therapeutic agents can be tested in appropriate animal models. For example, the efficacy, toxicity, or side effects of treatment with RNAi agents can be determined using animal models with RNAi agents (or expression vectors or transgenes encoding them) as described herein. Alternatively, the mechanism of action of such agents can be determined using animal models with therapeutic agents. For example, the efficacy, toxicity, or side effects of treatment with such agents can be determined using animal models with agents. Alternatively, the mechanism of action of such agents can be determined using animal models with agents.

[0261] Pharmaceutical composition and administration method This disclosure relates to the use of the above agents for prophylactic and / or therapeutic treatments as described below. Accordingly, the modifiers of this disclosure (e.g., RNAi agents) can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include nucleic acid molecules, proteins, antibodies, or modifier compounds and pharmaceutically acceptable carriers. In this specification, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic and absorption retarders, etc., that are suitable for drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is not suitable for the active compound, its use in a composition is intended. Complementary active ingredients can also be incorporated into the composition.

[0262] The pharmaceutical compositions of this disclosure are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, intraperitoneal, intramuscular, oral (e.g., inhalation), transdermal (topical), and transmucosal administration. In certain exemplary embodiments, the pharmaceutical compositions of this disclosure are administered intravenously and can cross the blood-brain barrier to enter the central nervous system. In certain exemplary embodiments, the pharmaceutical compositions of this disclosure are delivered to the cerebrospinal fluid (CSF) by routes of administration including, but not limited to, intrastriatal (IS), intraventricular (ICV), and intrathecal (IT) administration (e.g., via pump, infusion, etc.).

[0263] In certain embodiments, compositions comprising the compounds of the present disclosure can be delivered to the nervous system of a subject by various routes. Exemplary routes include intrathecal, parenchymal (e.g., intracerebral), nasal, and ocular delivery. The composition can also be delivered systemically, for example, by intravenous, subcutaneous or intramuscular injection. One route of delivery is directly to the brain, e.g., to the ventricles or the hypothalamus of the brain, or to the lateral or dorsal regions of the brain. The compounds for neuronal delivery can be incorporated into pharmaceutical compositions that are suitable for administration.

[0264] For example, the composition can comprise one or more species of the compounds of the present disclosure and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the present disclosure can be administered in multiple ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration can be topical (ocular, intranasal, transdermal, etc.), oral, or parenteral. Parenteral administration includes intravenous infusion, subcutaneous, intraperitoneal or intramuscular injection, intrathecal, or intracerebroventricular (e.g., intracerebral·intracerebroventricular) administration. In certain exemplary embodiments, the RNA silencing agents of the present disclosure are delivered across the blood-brain barrier (BBB) using various suitable compositions and methods described herein.

[0265] The delivery route may depend on the disorder of the patient. In addition to the compounds of the present disclosure, the patient can be administered a second therapy, such as a palliative therapy and / or a disease-specific therapy. The second therapy can be, for example, symptomatic therapy (e.g., to alleviate symptoms), neuroprotection (e.g., to delay or stop the progression of the disease), or recovery (e.g., to reverse the disease process). Other therapies can include psychotherapy, physical therapy, speech therapy, communication and memory aids, social support services, as well as dietary advice.

[0266] The compounds of this disclosure can be delivered to nerve cells in the brain. In certain embodiments, the compounds of this disclosure can be delivered to the brain without direct administration to the central nervous system; that is, the compounds can be delivered intravenously and enter the brain by crossing the blood-brain barrier. Delivery methods that do not require the composition to cross the blood-brain barrier can be utilized. For example, a pharmaceutical composition containing the compounds of this disclosure can be delivered to a patient by direct injection into an area containing cells affected by the disease. For example, a pharmaceutical composition can be delivered by direct injection into the brain. The injection may be by stereotactic injection into a specific area of ​​the brain (e.g., the substantia nigra, cortex, hippocampus, striatum, or globus pallidus). The compounds can be delivered to multiple areas of the central nervous system (e.g., multiple areas of the brain, and / or the spinal cord). The compounds can be delivered to diffuse areas of the brain (e.g., diffuse delivery to the cerebral cortex).

[0267] In one embodiment, the compound may be delivered by a cannula or other delivery device having one end implanted in a tissue, such as the brain, such as the substantia nigra, cortex, hippocampus, striatum, or globus pallidus. The cannula may be connected to a reservoir containing the compound. Flow or delivery may be mediated by a pump, such as an osmotic pump or minipump, such as an Alzet pump (Durect, Cupertino, CA). In one embodiment, the pump and reservoir are implanted in a region away from the tissue, such as the abdomen, and delivery is carried out by a conduit leading from the pump or reservoir to a release site. Devices for delivery to the brain are described, for example, in U.S. Patents 6,093,180 and 5,814,014.

[0268] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein can be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. While specific embodiments have been described in detail so far, the same will be more clearly understood by referring to the following examples, which are included for illustrative purposes only and are not intended to limit the scope of the embodiments disclosed herein. [Examples]

[0269] Example 1. Testing of ethylene glycol modification in siRNA. Ethylene glycol modification was tested with siRNA. In particular, Figure 3 shows the relative HTT mRNA levels in cells incubated with various chemically modified siRNAs at different doses, each containing one or two ethylene glycol modifications at the 3' end of the antisense strand. These results indicate that siRNAs containing one or two ethylene glycol modifications (referred to as "HEG-1," "HEG-2," and "HEG-3," corresponding to oligo IDs 30912, 30913, and 30914 in Table 6) showed improved targeted silencing compared to chemically modified siRNAs without ethylene glycol modification, such as alkyl-modified siRNAs (e.g., 5x or 3x butane-modified siRNAs).

[0270] Table 6 below shows exemplary siRNAs with various chemical modifications. [Table 6] In Table 6, "P" corresponds to the 5' phosphate, "m" corresponds to the 2'-OMe modification, "f" corresponds to the 2'-fluoro modification, "#" corresponds to the phosphorothioate internucleotide bond, "HEG" corresponds to the ethylene glycol modification (hexaethylene glycol) with six ethylene glycol units, and "but" corresponds to the alkyl modification with butyl.

[0271] Embedding by reference The content of all references (including references to documents, patents, patent applications, and websites) that may be cited throughout this application, as well as the references described herein, are expressly incorporated by reference in their entirety for any purpose. This disclosure uses immunological, molecular biological, and cell biological techniques that are well known as prior art unless otherwise specified.

[0272] This disclosure also incorporates, by reference, a whole range of techniques well known in the fields of molecular biology and drug delivery. These techniques include, but are not limited to, those described in the following publications: Atwell et al.J.Mol.Biol.1997,270:26-35; Ausubel et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &Sons, NY (1993); Ausubel,FMet al.eds.,SHORT PROTOCOLS IN MOLECULAR BIOLOGY(4th Ed.1999) John Wiley & Sons,NY.(ISBN 0-471-32938-X); CONTROLLED DRUG BIOAVAILABILITY, DRUG PRODUCT DESIGN AND PERFORMANCE, Smolen and Ball(eds.), Wiley, New York(1984); Giege, R. and Ducruix, A. Barrett, CRYSTALLIZATION OF NUCLEIC ACIDS AND PROTEINS, a Practical Approach, 2nd ea., pp.20 1-16, Oxford University Press, New York, New York, (1999); Goodson,in MEDICAL APPLICATIONS OF CONTROLLED RELEASE,vol.2,pp.115-138(1984); Hammerling, et al., in: MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS 563-681 (Elsevier, NY, 1981; Harlow et al., ANTIBODIES:A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Kabat et al.,SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST(National Institutes of Health,Bethesda,Md.(1987) and (1991); Kabat,E.A.,et al.(1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242; Kontermann and Dubel eds.,ANTIBODY ENGINEERING(2001) Springer-Verlag.New York.790 pp.(ISBN 3-540-41354-5). Kriegler,Gene Transfer and Expression,A Laboratory Manual,Stockton Press,NY(1990); Lu and Weiner eds.,CLONING AND EXPRESSION VECTORS FOR GENE FUNCTION ANALYSIS(2001) BioTechniques Press.Westborough,MA.298 pp.(ISBN 1-881299-21-X). MEDICAL APPLICATIONS OF CONTROLLED RELEASE,Langer and Wise(eds.),CRC Pres.,Boca Raton,Fla.(1974); Old,R.W. & S.B.Primrose,PRINCIPLES OF GENE MANIPULATION:AN INTRODUCTION TO GENETIC ENGINEERING (3d Ed.1985) Blackwell Scientific Publications,Boston.Studies in Microbiology;V.2:409 pp.(ISBN 0-632-01318-4). Sambrook, J. et al. eds., MOLECULAR CLONING: A LABORATORY MANUAL (2d Ed.1989) Cold Spring Harbor Laboratory Press, NY. Vols. 1-3. (ISBN 0-87969-309-6). SUSTAINED AND CONTROLLED RELEASE DRUG DELIVERY SYSTEMS, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978 Winnacker, ELFROM GENES TO CLONES:INTRODUCTION TO GENE TECHNOLOGY(1987) VCH Publishers, NY(translated by Horst Ibelgaufts), 634 pp.(ISBN 0-89573-614-4).

[0273] Equal portions This disclosure may be embodied in other specific forms without departing from its spirit or essential features. Therefore, the embodiments described above should be considered illustrative in all respects and not limiting to this disclosure. Accordingly, the scope of this disclosure is indicated not by the foregoing description but by the appended claims, and all modifications that fall within the meaning and equivalents of the claims are intended to be incorporated herein.

Claims

1. An RNA molecule having a 5' end and a 3' end, wherein the RNA molecule has at least one ethylene glycol modification on one or both of the 5' end and the 3' end, and the at least one ethylene glycol modification contains 1 to 20 ethylene glycol units.

2. The RNA molecule according to claim 1, wherein the at least one ethylene glycol modification comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ethylene glycol units.

3. The RNA molecule according to claim 1 or 2, wherein the RNA molecule contains 2 to 5 ethylene glycol modifications.

4. The RNA molecule according to claim 3, wherein the RNA molecule contains two ethylene glycol modifications.

5. The RNA molecule according to claim 3, wherein the RNA molecule contains three ethylene glycol modifications.

6. The RNA molecule according to claim 3, wherein the RNA molecule contains four ethylene glycol modifications.

7. The RNA molecule according to claim 3, wherein the RNA molecule contains five ethylene glycol modifications.

8. The RNA molecule according to any one of claims 1 to 7, wherein the at least one ethylene glycol modification is located between two adjacent nucleotides.

9. The RNA molecule according to any one of claims 1 to 8, wherein the at least one ethylene glycol modification does not substitute a nucleotide at the same position in the RNA molecule for an RNA molecule that does not contain the at least one ethylene glycol modification at that position in the RNA molecule.

10. The RNA molecule according to any one of claims 1 to 9, wherein the at least one ethylene glycol modification replaces a nucleotide at the same position in the RNA molecule as in an RNA molecule that does not contain the at least one ethylene glycol modification at that position in the RNA molecule.

11. The RNA molecule according to any one of claims 1 to 10, wherein the RNA molecule further comprises at least one alkyl modification to one or both of the 5' and 3' ends.

12. The aforementioned at least one alkyl modification is C １ ~C １０ The RNA molecule according to claim 11, comprising an alkyl group.

13. The aforementioned at least one alkyl modification is C ４ An RNA molecule according to claim 11 or 12, comprising an alkyl group.

14. The RNA molecule according to any one of claims 11 to 13, wherein the RNA molecule comprises 2 to 5 alkyl modifications.

15. The RNA molecule according to claim 14, wherein the RNA molecule comprises two alkyl modifications.

16. The RNA molecule according to claim 14, wherein the RNA molecule comprises three alkyl modifications.

17. The RNA molecule according to claim 14, wherein the RNA molecule comprises four alkyl modifications.

18. The RNA molecule according to claim 14, wherein the RNA molecule comprises five alkyl modifications.

19. The RNA molecule according to any one of claims 11 to 18, wherein the at least one alkyl modification is located between two adjacent nucleotides.

20. The RNA molecule according to any one of claims 11 to 19, wherein the at least one alkyl modification does not substitute a nucleotide at the same position in the RNA molecule for an RNA molecule that does not contain the at least one alkyl modification at that position in the RNA molecule.

21. The RNA molecule according to any one of claims 11 to 19, wherein the at least one alkyl modification replaces a nucleotide at the same position in the RNA molecule with respect to an RNA molecule that does not contain the at least one alkyl modification at that position in the RNA molecule.

22. The RNA molecule according to any one of claims 1 to 21, wherein the RNA molecule comprises single-stranded (ss) RNA or double-stranded (ds) RNA.

23. The RNA molecule according to claim 22, comprising an antisense strand having a 5' end and a 3' end, and a dsRNA having a sense strand having a 5' end and a 3' end.

24. The RNA molecule according to claim 23, wherein the antisense strand comprises at least one ethylene glycol modification at one or both of the 5' and 3' ends, and the at least one ethylene glycol modification comprises 1 to 20 ethylene glycol units.

25. The RNA molecule according to claim 24, wherein the at least one ethylene glycol modification comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ethylene glycol units.

26. The RNA molecule according to claim 25, wherein the antisense strand contains 2 to 5 ethylene glycol modifications.

27. The RNA molecule according to claim 26, wherein the antisense strand comprises two ethylene glycol modifications.

28. The RNA molecule according to claim 26, wherein the antisense strand contains three ethylene glycol modifications.

29. The RNA molecule according to claim 26, wherein the antisense strand contains four ethylene glycol modifications.

30. The RNA molecule according to claim 26, wherein the antisense strand contains five ethylene glycol modifications.

31. The RNA molecule according to any one of claims 24 to 30, wherein the at least one ethylene glycol modification is located between two adjacent nucleotides.

32. The RNA molecule according to any one of claims 24 to 31, wherein the at least one ethylene glycol modification does not substitute a nucleotide at the same position in the RNA molecule for an RNA molecule that does not contain the at least one ethylene glycol modification at that position in the RNA molecule.

33. The RNA molecule according to any one of claims 24 to 31, wherein the at least one ethylene glycol modification replaces a nucleotide at the same position in the RNA molecule as in an RNA molecule that does not contain the at least one ethylene glycol modification at that position in the RNA molecule.

34. The RNA molecule according to any one of claims 24 to 33, wherein the antisense strand further comprises at least one alkyl modification to one or both of the 5' and 3' ends.

35. The aforementioned at least one alkyl modification is C １ ~C １０ The RNA molecule according to claim 34, comprising an alkyl group.

36. The aforementioned at least one alkyl modification is C ４ An RNA molecule comprising an alkyl group, according to claim 34 or 35.

37. The RNA molecule according to any one of claims 34 to 36, wherein the antisense strand comprises 2 to 5 alkyl modifications.

38. The RNA molecule according to claim 37, wherein the antisense strand comprises two alkyl modifications.

39. The RNA molecule according to claim 37, wherein the antisense strand comprises three alkyl modifications.

40. The RNA molecule according to claim 37, wherein the antisense strand comprises four alkyl modifications.

41. The RNA molecule according to claim 37, wherein the antisense strand comprises five alkyl modifications.

42. The RNA molecule according to any one of claims 34 to 41, wherein the at least one alkyl modification is located between two adjacent nucleotides.

43. The RNA molecule according to any one of claims 34 to 42, wherein the at least one alkyl modification does not substitute a nucleotide at the same position in the RNA molecule for an RNA molecule that does not contain the at least one alkyl modification at that position in the RNA molecule.

44. The RNA molecule according to any one of claims 34 to 42, wherein the at least one alkyl modification replaces a nucleotide at the same position in the RNA molecule with respect to an RNA molecule that does not contain the at least one alkyl modification at that position in the RNA molecule.

45. The RNA molecule according to any one of claims 23 to 44, wherein the antisense strand is 15 to 25 nucleotides long.

46. The RNA molecule according to claim 45, wherein the antisense strand has a length of 18, 19, 20, 21, 22, or 23 nucleotides.

47. The RNA molecule according to any one of claims 23 to 46, wherein the sense strand is 15 to 25 nucleotides long.

48. The RNA molecule according to claim 47, wherein the sense strand is 14, 15, 16, or 17 nucleotides long.

49. The RNA molecule according to any one of claims 23 to 48, wherein the at least one ethylene glycol modification is located at any one of positions 1 to 25 from the 5' end of the antisense strand.

50. The RNA molecule according to any one of claims 34 to 49, wherein the at least one alkyl modification is located at any one of positions 1 to 25 from the 5' end of the antisense strand.

51. The RNA molecule according to any one of claims 22 to 50, further comprising at least one chemically modified nucleotide, which is different from the at least one alkyl modification or the at least one ethylene glycol modification.

52. The RNA molecule according to claim 51, wherein the at least one chemically modified nucleotide comprises a 2'-O-methyl modified nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a debasalized nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, or a mixture thereof.

53. The RNA molecule according to any one of claims 22 to 52, wherein the dsRNA comprises at least one modified internucleotide bond.

54. The RNA molecule according to claim 53, wherein the modified nucleotide bond includes a phosphorothioate nucleotide bond.

55. An RNA molecule according to any one of claims 22 to 54, comprising 4 to 16 phosphorothioate nucleotide interbonds.

56. An RNA molecule according to any one of claims 22 to 55, comprising 8 to 13 phosphorothioate nucleotide interlinks.

57. The RNA molecule according to any one of claims 22 to 56, wherein the dsRNA includes a blunt end.

58. The RNA molecule according to any one of claims 22 to 57, wherein the dsRNA comprises at least one single-stranded nucleotide overhang.

59. The RNA molecule according to claim 58, wherein the dsRNA includes a single-stranded nucleotide overhang of about 2 to 5 nucleotides.

60. The RNA molecule according to claim 58, wherein the dsRNA includes a single-stranded nucleotide overhang of two nucleotides.

61. The RNA molecule according to claim 58, wherein the dsRNA includes a single-stranded nucleotide overhang of three nucleotides.

62. The RNA molecule according to claim 58, wherein the dsRNA includes a single-stranded nucleotide overhang of four nucleotides.

63. The RNA molecule according to claim 58, wherein the dsRNA includes a single-stranded nucleotide overhang of 5 nucleotides.

64. The RNA molecule according to any one of claims 58 to 63, wherein the single-stranded nucleotide overhang comprises at least two alkyl modifications.

65. The RNA molecule according to claims 58 to 64, wherein the single-stranded nucleotide overhang comprises two, three, four, or five alkyl modifications.

66. An RNA molecule according to any one of claims 1 to 65, comprising an antisense strand having one of the chemical modification patterns provided in Tables 2 to 6.

67. An RNA molecule according to any one of claims 22 to 66, comprising a sense strand having one of the chemical modification patterns provided in Tables 2 to 3.

68. A double-stranded (ds) RNA comprising an antisense strand having a 5' end and a 3' end, and a sense strand having a 5' end and a 3' end, wherein the antisense strand comprises at least one ethylene glycol modification at one or both of the 5' and 3' ends, and the at least one ethylene glycol modification comprises 1 to 20 ethylene glycol units.

69. The dsRNA according to claim 68, wherein the antisense strand is 15 to 25 nucleotides long.

70. The dsRNA according to claim 69, wherein the antisense strand is 18, 19, 20, 21, 22, or 23 nucleotides long.

71. The dsRNA according to any one of claims 68 to 70, wherein the sense strand is 15 to 25 nucleotides long.

72. The dsRNA according to claim 71, wherein the sense strand is 14, 15, 16, or 17 nucleotides long.

73. The dsRNA according to any one of claims 68 to 72, wherein the at least one ethylene glycol modification is located at any one of positions 1 to 25 from the 5' end of the antisense strand.

74. The dsRNA according to any one of claims 68 to 73, further comprising at least one alkyl modification.

75. The dsRNA according to any one of claims 74, wherein the at least one alkyl modification is located at any one of positions 1 to 25 from the 5' end of the antisense strand.

76. The dsRNA according to any one of claims 68 to 75, further comprising at least one chemically modified nucleotide, which is different from the at least one alkyl modification or the at least one ethylene glycol modification.

77. The dsRNA according to claim 76, wherein the at least one chemically modified nucleotide comprises a 2'-O-methyl modified nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a debasalized nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, or a mixture thereof.

78. The dsRNA according to any one of claims 68 to 77, wherein the dsRNA comprises at least one modified internucleotide bond.

79. The dsRNA according to claim 78, wherein the modified nucleotide bond includes a phosphorothioate nucleotide bond.

80. The dsRNA according to any one of claims 68 to 79, comprising 4 to 16 phosphorothioate nucleotide interlinks.

81. The dsRNA according to any one of claims 68 to 80, comprising 8 to 13 phosphorothioate nucleotide interlinks.

82. The dsRNA according to any one of claims 68 to 81, wherein the dsRNA includes a blunt end.

83. The dsRNA according to any one of claims 68 to 82, wherein the dsRNA comprises at least one single-stranded nucleotide overhang.

84. The dsRNA according to claim 83, wherein the dsRNA includes a single-stranded nucleotide overhang of about 2 to 5 nucleotides.

85. The dsRNA according to claim 83, wherein the dsRNA includes a single-stranded nucleotide overhang of two nucleotides.

86. The dsRNA according to claim 83, wherein the dsRNA includes a single-stranded nucleotide overhang of three nucleotides.

87. The dsRNA according to claim 83, wherein the dsRNA includes a single-stranded nucleotide overhang of four nucleotides.

88. The dsRNA according to claim 83, wherein the dsRNA includes a single-stranded nucleotide overhang of 5 nucleotides.

89. The dsRNA according to any one of claims 83 to 88, wherein the single-stranded nucleotide overhang comprises at least two alkyl modifications.

90. The dsRNA according to any one of claims 83 to 89, wherein the single-stranded nucleotide overhang comprises two, three, four, or five alkyl modifications.

91. The dsRNA according to any one of claims 68 to 90, comprising an antisense strand having one of the chemical modification patterns provided in Tables 2 to 6.

92. The dsRNA according to any one of claims 68 to 91, comprising a sense strand having one of the chemical modification patterns provided in Tables 2 to 3.

93. A double-stranded (ds)RNA comprising an antisense strand and a sense strand, each having a 5' end and a 3' end, and at least one single-stranded nucleotide overhang of 2 to 5 nucleotides, wherein the single-stranded nucleotide overhang comprises at least one ethylene glycol modification.

94. The dsRNA according to claim 93, wherein the single-stranded nucleotide overhang comprises two, three, four, or five nucleotide modifications consisting of ethylene glycol modifications.

95. The dsRNA according to claim 93 or 94, wherein each nucleotide in the single-stranded nucleotide overhang contains the same nucleotide modification.

96. The dsRNA according to any one of claims 93 to 95, wherein the single-stranded nucleotide overhang further comprises alkyl modification.

97. A double-stranded (ds) RNA comprising an antisense strand and a sense strand, each having a 5' end and a 3' end, wherein the antisense strand contains one of the chemical modification patterns provided in Tables 2 to 6.

98. A double-stranded (ds) RNA comprising an antisense strand and a sense strand, each having a 5' end and a 3' end, wherein the sense strand contains one of the chemical modification patterns provided in Tables 2-3.

99. A method for reducing the expression of a target mRNA in a subject, comprising administering to the subject an RNA molecule according to any one of claims 1 to 67, or a dsRNA according to any one of claims 68 to 98, thereby reducing the expression of the target mRNA.

100. The method according to claim 99, wherein the expression of the target mRNA is reduced by at least about 20%, at least about 30%, at least about 40%, or at least about 50% compared to the expression level before administration of the RNA molecule or dsRNA.

101. The method according to claim 99 or 100, wherein the expression of the target mRNA decreases for at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months after administration of the RNA molecule or dsRNA.

Images