Oligonucleotides with ethylene glycol modification

Abstract

The present disclosure relates to compositions, systems and methods comprising ethylene glycol chemical modifications to oligonucleotides to modulate silence activity and improve stability.

Description

Cross-reference to related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 591,271, filed October 18, 2023. The entire contents of the aforementioned patent application are incorporated herein by reference. Technical Field

[0002] This disclosure relates to the use of ethylene glycol to chemically modify modified oligonucleotides to modulate silencing activity and improve stability. Background Technology

[0003] Chemically modified siRNAs are at the forefront of oligonucleotide therapeutics. Exemplary chemical modifications include, but are not limited to, 2'-fluoroRNA (2'FRNA), 2'-O-methylRNA (2'OMeRNA), phosphate thioesters, vinylphosphonates, alkyl groups, locked nucleic acids (LNA), unlocked nucleic acids (UNA), 2'-O-methoxy-ethyl (2'-MOE), and DNA modifications, and these modifications can enhance the efficacy, stability, and duration of treatment of oligonucleotides. However, existing modification modalities may result in non-silencing or over-silencing of target mRNAs. Over-silencing of the target may lead to unwanted side effects, while under-silencing may result in no therapeutic benefit. Existing modification modalities may also hinder long-term in vivo silencing of mRNAs by reducing the time during which therapeutic oligonucleotides remain active.

[0004] Therefore, there is a need in the art to optimize the chemical modification of oligonucleotides to modulate their silencing activity and improve their stability. Summary of the Invention

[0005] This article provides modified oligonucleotides with ethylene glycol chemical modifications that can be used to modulate the activity level of the modified oligonucleotides to achieve robust silencing (e.g., silencing about 75% or more) or moderate silencing (e.g., silencing about 25% to about 50%) over a short period of time (i.e., less than 6 months) or a long period of time (i.e., 6 months or longer), and to improve the stability of the oligonucleotides.

[0006] In one aspect, this disclosure provides an RNA molecule comprising a 5' end and a 3' end, wherein the RNA molecule comprises at least one ethylene glycol modification of one or both of the 5' end and the 3' end, wherein the at least one ethylene glycol modification comprises 1-20 ethylene glycol units.

[0007] In some embodiments, 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.

[0008] In some implementations, the RNA molecule contains 2-5 ethylene glycol modifications.

[0009] In some implementations, the RNA molecule contains two ethylene glycol modifications.

[0010] In some implementations, the RNA molecule contains three ethylene glycol modifications.

[0011] In some implementations, the RNA molecule contains four ethylene glycol modifications.

[0012] In some implementations, the RNA molecule contains five ethylene glycol modifications.

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

[0014] In some embodiments, the at least one ethylene glycol modification does not replace the nucleotide at that position within the RNA molecule, as opposed to an RNA molecule that does not contain the at least one ethylene glycol modification at the same position within the RNA molecule.

[0015] In some embodiments, the at least one ethylene glycol modification replaces a nucleotide at that position within the RNA molecule, relative to an RNA molecule that does not contain at least one ethylene glycol modification at the same position within the RNA molecule.

[0016] In some embodiments, the RNA molecule also includes at least one alkyl modification of one or both of the 5' and 3' ends.

[0017] In some embodiments, the at least one alkyl modification includes C1-C 10 alkyl.

[0018] In some embodiments, the at least one alkyl modification includes a C4 alkyl group.

[0019] In some implementations, the RNA molecule contains 2-5 alkyl modifications.

[0020] In some implementations, the RNA molecule contains two alkyl modifications.

[0021] In some implementations, the RNA molecule contains three alkyl modifications.

[0022] In some implementations, the RNA molecule contains four alkyl modifications.

[0023] In some implementations, the RNA molecule contains five alkyl modifications.

[0024] In some embodiments, the at least one alkyl modification is positioned between two adjacent nucleotides.

[0025] In some embodiments, the at least one alkyl modification does not replace the nucleotide at that position within the RNA molecule, as opposed to an RNA molecule that does not contain the at least one alkyl modification at the same position within the RNA molecule.

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

[0027] In some implementations, the RNA molecule comprises single-stranded (ss) RNA or double-stranded (ds) RNA.

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

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

[0030] In some embodiments, 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.

[0031] In some implementations, the antisense chain contains 2-5 ethylene glycol modifications.

[0032] In some implementations, the antisense chain contains two ethylene glycol modifications.

[0033] In some implementations, the antisense chain contains three ethylene glycol modifications.

[0034] In some implementations, the antisense chain contains four ethylene glycol modifications.

[0035] In some implementations, the antisense chain contains five ethylene glycol modifications.

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

[0037] In some embodiments, the at least one ethylene glycol modification does not replace the nucleotide at that position within the RNA molecule, as opposed to an RNA molecule that does not contain the at least one ethylene glycol modification at the same position within the RNA molecule.

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

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

[0040] In some embodiments, the at least one alkyl modification includes C1-C 10 alkyl.

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

[0042] In some implementations, the antisense chain contains 2-5 alkyl modifications.

[0043] In some implementations, the antisense chain contains two alkyl modifications.

[0044] In some implementations, the antisense chain contains three alkyl modifications.

[0045] In some implementations, the antisense chain contains four alkyl modifications.

[0046] In some implementations, the antisense chain contains five alkyl modifications.

[0047] In some embodiments, the at least one alkyl modification is positioned between two adjacent nucleotides.

[0048] In some embodiments, the at least one alkyl modification does not replace the nucleotide at that position within the RNA molecule, as opposed to an RNA molecule that does not contain the at least one alkyl modification at the same position within the RNA molecule.

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

[0050] In some implementations, the length of the antisense strand is between 15 and 25 nucleotides.

[0051] In some implementations, the antisense strand is 18, 19, 20, 21, 22, or 23 nucleotides in length.

[0052] In some implementations, the length of the sense strand is between 15 and 25 nucleotides.

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

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

[0055] In some embodiments, the at least one alkyl modification is at any of positions 1-25 starting from the 5' end of the antisense chain.

[0056] In some embodiments, the RNA molecule contains at least one chemically modified nucleotide that is different from the at least one alkyl modification or the at least one glycol modification.

[0057] In some embodiments, the at least one chemically modified nucleotide includes 2'-O-methyl modified nucleotides, 2'-deoxy-2'-fluorine modified nucleotides, 2'-deoxy modified nucleotides, lock nucleotides, debased nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholinonucleotides, aminophosphates, nucleotides containing non-natural bases, or mixtures thereof.

[0058] In some implementations, dsRNA contains at least one modified nucleotide inter-bond.

[0059] In some embodiments, the modified nucleotide inter-bonds include phosphate thioester nucleotide inter-bonds.

[0060] In some implementations, the RNA molecule contains 4-16 phosphate thioester nucleotide bonds.

[0061] In some implementations, the RNA molecule contains 8-13 phosphate thioester nucleotide bonds.

[0062] In some implementations, the dsRNA contains blunt ends.

[0063] In some implementations, the dsRNA contains at least one single-stranded nucleotide overhang.

[0064] In some implementations, dsRNA contains a single-stranded nucleotide overhang of about 2 to 5 nucleotides.

[0065] In some implementations, dsRNA contains a single-stranded nucleotide overhang of 2 nucleotides.

[0066] In some implementations, dsRNA contains a single-stranded nucleotide overhang of 3 nucleotides.

[0067] In some implementations, dsRNA contains a single-stranded nucleotide overhang of 4 nucleotides.

[0068] In some implementations, dsRNA contains a 5-nucleotide single-stranded nucleotide overhang.

[0069] In some implementations, the overhang of a single-stranded nucleotide contains at least two alkyl modifications.

[0070] In some implementations, the overhangs of a single-stranded nucleotide contain 2, 3, 4, or 5 alkyl modifications.

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

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

[0073] In another aspect, this 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 of one or both of the 5' end and the 3' end, wherein the at least one ethylene glycol modification comprises 1-20 ethylene glycol units.

[0074] In some implementations, the length of the antisense strand is between 15 and 25 nucleotides.

[0075] In some implementations, the antisense strand is 18, 19, 20, 21, 22, or 23 nucleotides in length.

[0076] In some implementations, the length of the sense strand is between 15 and 25 nucleotides.

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

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

[0079] In some implementations, the dsRNA contains at least one alkyl modification.

[0080] In some embodiments, the at least one alkyl modification is at any of positions 1-25 starting from the 5' end of the antisense chain.

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

[0082] In some embodiments, the at least one chemically modified nucleotide includes 2'-O-methyl modified nucleotides, 2'-deoxy-2'-fluorine modified nucleotides, 2'-deoxy modified nucleotides, lock nucleotides, debased nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholinonucleotides, aminophosphates, nucleotides containing non-natural bases, or mixtures thereof.

[0083] In some implementations, dsRNA contains at least one modified nucleotide inter-bond.

[0084] In some embodiments, the modified nucleotide inter-bonds include phosphate thioester nucleotide inter-bonds.

[0085] In some implementations, dsRNA contains 4-16 phosphate thionucleotide bonds.

[0086] In some implementations, dsRNA contains 8-13 phosphate thionucleotide bonds.

[0087] In some implementations, the dsRNA contains blunt ends.

[0088] In some implementations, the dsRNA contains at least one single-stranded nucleotide overhang.

[0089] In some implementations, dsRNA contains a single-stranded nucleotide overhang of about 2 to 5 nucleotides.

[0090] In some implementations, dsRNA contains a single-stranded nucleotide overhang of 2 nucleotides.

[0091] In some implementations, dsRNA contains a single-stranded nucleotide overhang of 3 nucleotides.

[0092] In some implementations, dsRNA contains a single-stranded nucleotide overhang of 4 nucleotides.

[0093] In some implementations, dsRNA contains a 5-nucleotide single-stranded nucleotide overhang.

[0094] In some implementations, the overhang of a single-stranded nucleotide contains at least two alkyl modifications.

[0095] In some implementations, the overhangs of a single-stranded nucleotide contain 2, 3, 4, or 5 alkyl modifications.

[0096] In some implementations, the dsRNA comprises an antisense strand having any of the chemical modification patterns provided in Tables 2-6.

[0097] In some implementations, the dsRNA comprises a sense strand having any of the chemical modification patterns provided in Tables 2-3.

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

[0099] In some implementations, the single-stranded nucleotide overhang contains 2, 3, 4, or 5 nucleotide modifications consisting of ethylene glycol modifications.

[0100] In some implementations, each nucleotide in the overhang of a single-stranded nucleotide contains the same nucleotide modification.

[0101] In some implementations, the overhangs of the single-stranded nucleotide also contain alkyl modifications.

[0102] On the other hand, this disclosure provides a double-stranded (ds) RNA comprising an antisense strand and a sense strand, each strand having a 5' end and a 3' end, wherein the antisense strand comprises any of the chemical modification patterns provided in Tables 2-6.

[0103] On the other hand, this disclosure provides a double-stranded (ds) RNA comprising an antisense strand and a sense strand, each strand having a 5' end and a 3' end, wherein the sense strand comprises any of the chemical modification patterns provided in Tables 2-3.

[0104] In another aspect, this 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 some implementations, 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 prior to the administration of the RNA molecule or dsRNA.

[0106] In some implementations, the reduction in target mRNA expression persists 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. Attached Figure Description

[0107] The above and other features and advantages of this disclosure will be more fully understood in conjunction with the accompanying drawings and the following detailed description of illustrative embodiments.

[0108] Figure 1 Chemical modifications and four siRNA patterns (patterns 1, 1T, 2, and 2T) used as baseline modification patterns were described, with one or more ethylene glycol-containing modifications applied to these patterns.

[0109] Figure 2 Chemical modifications according to one or more exemplary embodiments of the present disclosure are described.

[0110] Figure 3 The relative HTT mRNA levels in cells incubated with different doses of various chemically modified siRNAs were depicted. The siRNAs contained the following 3' end modifications: 3x butane modification, 5x butane modification, 2x butane modification, and 1x 6-ethylene glycol unit modification (hexaethylene glycol, HEG) or 2x 6-ethylene glycol unit modification (HEG). Specific chemical modification patterns are listed in Table 6. Detailed Implementation

[0111] Unless otherwise specified, the nomenclature described herein in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, as well as hybridization, is that which is well known and commonly used in the art. Furthermore, unless otherwise stated, the methods and techniques provided herein are performed according to conventional methods well known in the art and described in the various general and more specific references cited and discussed throughout this specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as is commonly done in the art, or as described herein. Unless precisely defined, the nomenclature used in conjunction with the nomenclature and laboratory procedures and techniques of these chemical fields described herein are those well known and commonly used in the art. Standard techniques are used in chemical synthesis, chemical analysis, drug preparation, formulation, delivery, and patient treatment.

[0112] Unless otherwise defined herein, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. In the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. Unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. Unless otherwise stated, the use of “or” means “and / or”. The use of the term “including” and other forms such as “includes” and “included” is not restrictive.

[0113] To make this disclosure easier to understand, some terms are first defined.

[0114] The term "nucleoside" refers to a molecule having a purine or pyrimidine base covalently linked to ribose or deoxyribose. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine, and thymidine. Other exemplary nucleosides include inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, nucleothymidine, 2N-methylguanosine, and N2,N2-dimethylguanosine (also known as "rare" nucleosides). The term "nucleotide" refers to a nucleoside having one or more phosphate groups linked to a sugar moiety by an ester bond. Exemplary nucleotides include nucleoside monophosphate, nucleoside diphosphate, and nucleoside triphosphate. The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein and refer to a polymer of nucleotides linked together by unmodified phosphodiester or chemically modified subunit bonds between the 5' and 3' carbon atoms.

[0115] The term "RNA" or "RNA molecule" or "ribonucleic acid molecule" refers to a polymer of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, or more ribonucleotides). The term "DNA" or "DNA molecule" or "deoxyribonucleic acid molecule" refers to a polymer of deoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., through DNA replication or DNA transcription, respectively). RNA can be post-transcriptionally modified. DNA and RNA can also be chemically synthesized. 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 a single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. This information is translated when the ribosome binds to the mRNA during protein synthesis.

[0116] As used herein, the term “small interfering RNA” (“siRNA”) (also referred to in the art as “short interfering RNA”) refers to an RNA (or RNA analog) comprising about 10-50 nucleotides (or nucleotide analogs) capable of directing or mediating RNA interference. The siRNA of this disclosure may be single-stranded (i.e., a single antisense strand) or double-stranded (i.e., an antisense strand and a sense strand annealed together to form a duplex). The double-stranded siRNA of this disclosure comprises an antisense strand sufficiently complementary to the target mRNA to mediate the silencing of said mRNA, and a sense strand sufficiently complementary to the antisense strand to form a duplex. In some embodiments, the 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 siRNA containing approximately 21 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21, or 22 nucleotides). The term "long" siRNA refers to siRNA containing approximately 24-25 nucleotides, such as 23, 24, 25, or 26 nucleotides. In some cases, short siRNA may include fewer than 19 nucleotides, such as 16, 17, or 18 nucleotides, as long as the shorter siRNA retains the ability to mediate RNAi. Similarly, in some cases, long siRNA may include more than 26 nucleotides, as long as the longer siRNA retains the ability to mediate RNAi without further processing (e.g., enzymatic processing) into short siRNA.

[0117] The terms "nucleotide analog," "altered nucleotide," or "modified nucleotide" refer to non-standard nucleotides, including ribonucleotides or deoxyribonucleotides that are not naturally occurring. Exemplary nucleotide analogs are modified at any position to alter certain chemical properties of the nucleotide while retaining the ability of the nucleotide analog to perform its intended function. Examples of nucleotide positions that can be derivatized include: position 5, such as 5-(2-amino)propyluridine, 5-bromouridine, 5-propynyluridine, 5-propenyluridine, etc.; position 6, such as 6-(2-amino)propyluridine; and position 8 of adenosine and / or guanosine, such as 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine, etc. Nucleotide analogs also include denitronucleotides, such as 7-denitro-adenosine; O- and N-modified (e.g., alkylated, such as N6-methyladenosine, or as otherwise known in the art) nucleotides; and other heterocyclic modified nucleotide analogs, such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., August 2000, 10(4):297-310.

[0118] Nucleotide analogs may also include modifications to the sugar moiety of the nucleotide. For example, the 2' OH- group may be replaced by 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 include those described in U.S. Patent Nos. 5,858,988 and 6,291,438.

[0119] The phosphate group of a nucleotide can also be modified, for example, by replacing one or more oxygen atoms in the phosphate group with sulfur (e.g., thiophosphate), or by making other substitutions that allow the nucleotide to perform its intended function, such as those described in the following literature: Eckstein, Antisense Nucleic Acid Drug Dev. April 2000, 10(2):117-21; Rusckowski et al., Antisense Nucleic Acid Drug Dev. October 2000, 10(5):333-45; Stein, Antisense Nucleic Acid Drug Dev. October 2001, 11(5):317-25; Vorobjev et al., Antisense Nucleic Acid Drug Dev. April 2001, 11(2):77-85; and U.S. Patent No. 5,684,143. Some of the modifications mentioned above (such as phosphate group modifications) reduce the rate of hydrolysis of polynucleotides, such as those containing the like, in vivo or in vitro.

[0120] The term "oligonucleotide" refers to a polymer of nucleotides and / or nucleotide analogs. Oligonucleotides include, but are not limited to, siRNA, antisense oligonucleotides, miRNA, ribozymes, and mRNA.

[0121] The term "RNA analog" refers to a polynucleotide (e.g., a chemically synthesized polynucleotide) that has at least one altered or modified nucleotide compared to its corresponding unaltered or unmodified RNA, but retains the same or similar properties or functions as the corresponding unaltered or unmodified RNA. As discussed above, oligonucleotides can be linked by bonds, which results in a lower hydrolysis rate for RNA analogs compared to RNA molecules with phosphodiester bonds. For example, the nucleotides of the analog may contain methylene glycol, ethylene glycol, oxymethylthio, oxyethio, oxycarbonyloxy, phosphoryldiamine, phosphoramide, and / or thiophosphate bonds. Examples of RNA analogs include, but are not limited to, sugar and / or backbone-modified ribonucleotides and / or deoxyribonucleotides. Such alterations or modifications may also include the addition of non-nucleotide materials, such as addition to the ends or interior of the RNA (at one or more nucleotides of the RNA). An RNA analog only needs to be sufficiently similar to native RNA to have the ability to mediate RNA interference.

[0122] As used herein, the term “RNA interference” (“RNAi”) refers to the selective intracellular degradation of RNA. RNAi occurs naturally in cells to remove exogenous RNA (such as viral RNA). Natural RNAi proceeds through fragments cleaved from free dsRNA, which direct the degradation mechanism to other similar RNA sequences. Alternatively, RNAi can be artificially initiated, for example, by silencing the expression of a target gene.

[0123] An RNAi agent (e.g., an RNA silencer) is one in which the strand contains a sequence that is sufficiently complementary to the target mRNA sequence to guide target-specific RNA interference (RNAi).

[0124] As used herein, the term “isolated RNA” (e.g., “isolated siRNA” or “isolated siRNA precursor”) refers to an RNA molecule that is substantially free from other cellular material or culture medium produced by recombinant technology, or substantially free from chemical precursors or other compounds produced by chemical synthesis.

[0125] As used herein, the term “RNA silencing” refers to a set of sequence-specific regulatory mechanisms mediated by RNA molecules (e.g., RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), suppression, co-repression, and translational repression) that result in the suppression or “silencing” of the expression of the corresponding protein-coding genes. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

[0126] The term "discriminative RNA silencing" refers to the ability of an RNA molecule to substantially suppress the expression of a "first" or "target" polynucleotide sequence while substantially not suppressing the expression of a "second" or "non-target" polynucleotide sequence, for example, when both polynucleotide sequences are present in the same cell. In some 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 some 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.

[0127] The term "in vitro" has its generally accepted meaning in the art, for example, referring to purified reagents or extracts, such as cell extracts. The term "in vivo" also has its generally accepted meaning in the art, for example, referring to living cells, such as immortalized cells, primary cells, cell lines, and / or cells within an organism.

[0128] As used herein, the term "target gene" is a gene whose expression will be substantially suppressed or "silenced." This silencing can be achieved through RNA silencing, such as by cleaving the mRNA of the target gene or by translational repression of the target gene. The term "non-target gene" is a gene whose expression will not be substantially silenced. In one embodiment, the polynucleotide sequences of the target gene and non-target genes (e.g., the mRNA encoded by the target gene and non-target genes) may differ by one or more nucleotides. In another embodiment, the target gene and non-target genes may differ due to one or more polymorphisms (e.g., single nucleotide polymorphisms or SNPs). In yet another embodiment, the target gene and non-target genes may share less than 100% sequence identity. In yet another embodiment, the non-target gene may be a homolog of the target gene (e.g., an ortholog or paralog).

[0129] As used herein, the term "RNA silencer" refers to RNA capable of inhibiting or "silencing" the expression of a target gene. In some embodiments, RNA silencers are capable of preventing the complete processing (e.g., complete translation and / or expression) of mRNA molecules through a post-transcriptional silencing mechanism. RNA silencers include small (<50 bp), non-coding RNA molecules, such as RNA duplexes containing paired strands, and precursor RNAs from which such small non-coding RNAs can be generated. Exemplary RNA silencers include siRNA, miRNA, siRNA-like duplexes, antisense oligonucleotides, GAPMER molecules, and bifunctional oligonucleotides, and their precursors. In one embodiment, the RNA silencer is capable of inducing RNA interference. In another embodiment, the RNA silencer is capable of mediating translational repression.

[0130] As used herein, the term "rare nucleotide" refers to naturally occurring nucleotides that are not frequently found, including naturally occurring deoxyribonucleotides or ribonucleotides that are not naturally occurring ribonucleotides such as guanosine, adenosine, cytosine, or uridine. Examples of rare nucleotides include, but are not limited to, inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, nucleothymidine, 2N-methylguanosine, and 2,2N,N-dimethylguanosine.

[0131] The term "engineered" in engineered RNA precursors or engineered nucleic acid molecules indicates that the precursor or molecule does not exist in nature because all or part of its nucleic acid sequence was generated or selected by humans. Once the sequence is generated or selected, it can be replicated, translated, transcribed, or otherwise processed through intracellular mechanisms. Therefore, an RNA precursor produced intracellularly by a transgene including an engineered nucleic acid molecule is an engineered RNA precursor.

[0132] As used herein, the term “microRNA” (“miRNA”), also known in the art as “hour sequence RNA” (“stRNA”), refers to a small (10–50 nucleotides) RNA that is genetically encoded (e.g., through the genomes of viruses, mammals, or plants) and capable of directing or mediating RNA silencing. “miRNA syndrome” should refer to a disease or condition characterized by the abnormal expression or activity of miRNAs.

[0133] As used herein, the term "bifunctional oligonucleotide" refers to an RNA silencing agent having the formula TL-μ, where T is the mRNA targeting moiety, L is the linking moiety, and μ is the miRNA recruitment moiety. As used herein, the terms "mRNA targeting moiety," "targeting moiety," "mRNA targeting portion," or "targeting portion" refer to a domain, portion, or region of a bifunctional oligonucleotide that is of sufficient size and sufficiently complementary to the selected or targeted mRNA portion or region for silencing (i.e., the portion has a sequence sufficient to capture the target mRNA). As used herein, the term "linking moiety" or "linking portion" refers to a domain, portion, or region of an RNA silencing agent that is covalently linked or connected to mRNA.

[0134] As used herein, the “antisense strand” of the term RNA silencing agent (e.g., siRNA or RNA silencing agent) refers to a strand substantially complementary to approximately 10–50 nucleotides (e.g., approximately 15–30, 16–25, 18–23, or 19–22 nucleotides) of the mRNA of the gene targeted for silencing. The antisense strand, or first strand, has a sequence sufficiently complementary to the desired target mRNA sequence to guide target-specific silencing, such as complementarity sufficient to trigger an RNAi mechanism or process (RNAi interference) to disrupt the complementarity of the desired target mRNA or sufficient to trigger translational repression of the desired target mRNA.

[0135] The term RNA silencing agent, such as siRNA, or the "sense strand" or "second strand" of an RNA silencing agent, refers to the strand complementary to the antisense strand or the first strand. Antisense and sense strands can also be referred to as the first or second strand, where the first or second strand is complementary to the target sequence, and the corresponding second or first strand is complementary to the first or second strand. miRNA duplex intermediates or siRNA-like duplexes consist of a miRNA strand sufficiently complementary to approximately 10-50 nucleotides of the mRNA targeting the silenced gene, and a miRNA* strand sufficiently complementary to form a duplex with the miRNA.

[0136] As used herein, the term “guide strand” refers to the strand of an RNA silencing agent, such as a double-stranded siRNA or an antisense strand of an siRNA sequence, which enters the RISC complex and guides the cleavage of the target mRNA.

[0137] As used herein, the term "asymmetry," such as in the asymmetry of RNA silencing duplex regions (e.g., the stem of shRNA), refers to an unequal bond strength or base pairing strength between the ends of the RNA silencing agent (e.g., between the terminal nucleotides of the first strand or stem and the terminal nucleotides of the opposite second strand or stem), such that the 5' end of one strand of the duplex is in a transiently unpaired state, such as a single-stranded state, more frequently than the 5' end of the complementary strand. This structural difference determines that one strand of the duplex is preferentially incorporated into the RISC complex. The strand with a less tight 5' pairing with the complementary strand will preferentially be incorporated into the RISC and mediate RNAi.

[0138] As used herein, the term “bond strength” or “base pair strength” refers to the strength of the interaction between pairs of nucleotides (or nucleotide analogs) on opposite strands of an oligonucleotide duplex (e.g., siRNA duplex), primarily due to H-bonding, van der Waals interactions, etc., between the nucleotides (or nucleotide analogs).

[0139] As used herein, "5' end," such as in the 5' end of an oligonucleotide (e.g., the antisense or sense strand of siRNA), refers to the 5' terminal nucleotide, for example, the 5' end of an oligonucleotide consisting of between one and about five nucleotides. In some embodiments, the 5' end of an oligonucleotide corresponds to the first five nucleotides of the oligonucleotide. In some embodiments, the 5' end of an oligonucleotide is the first nucleotide. In some embodiments, the 5' end of an oligonucleotide is the first two consecutive nucleotides. In some embodiments, the 5' end of an oligonucleotide is the first three consecutive nucleotides. In some embodiments, the 5' end of an oligonucleotide is the first four consecutive nucleotides. In some embodiments, the 5' end of an oligonucleotide is the first five consecutive nucleotides.

[0140] As used herein, "3' end," such as in the 3' end of an oligonucleotide (e.g., the antisense or sense strand of siRNA), refers to the 3' terminal nucleotide, for example, between one and about five nucleotides at the 3' end of an oligonucleotide. In some embodiments, the 3' end of an oligonucleotide corresponds to the last five nucleotides of the oligonucleotide. In some embodiments, the 3' end of an oligonucleotide is the last nucleotide. In some embodiments, the 3' end of an oligonucleotide is the last two consecutive nucleotides. In some embodiments, the 3' end of an oligonucleotide is the last three consecutive nucleotides. In some embodiments, the 3' end of an oligonucleotide is the last four consecutive nucleotides. In some embodiments, the 3' end of an oligonucleotide is the last five consecutive nucleotides.

[0141] As used herein, the term "destabilized nucleotide" refers to a first nucleotide or nucleotide analog capable of forming a base pair with a second nucleotide or nucleotide analog such that the bond strength of that base pair is lower than that of a conventional base pair (i.e., a Watson-Crick base pair). In some embodiments, the destabilized nucleotide is capable of forming a mismatched base pair with the second nucleotide. In other embodiments, the destabilized nucleotide is capable of forming a wobbly base pair with the second nucleotide. In still other embodiments, the destabilized nucleotide is capable of forming a fuzzy base pair with the second nucleotide.

[0142] As used herein, the term "base pair" refers to the interaction between pairs of nucleotides (or nucleotide analogs) on opposite strands of an oligonucleotide duplex (e.g., a duplex formed by a strand of an RNA silencer and a target mRNA sequence), primarily due to H-bonding, van der Waals interactions, etc., between said nucleotides (or nucleotide analogs). As used herein, the term "bond strength" or "base pair strength" refers to the strength of a base pair.

[0143] As used herein, the term “mismatched base pair” refers to a base pair consisting of non-complementary or non-Watson-Crick base pairs, such as non-normally complementary G:C, A:T, or A:U base pairs. As used herein, the term “fuzzy base pair” (also known as an undiscriminate base pair) refers to a base pair formed from universal nucleotides.

[0144] 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 the bases on complementary polynucleotides when forming base pairs ("universal bases" or "neutral bases"). Universal nucleotides are primarily hydrophobic molecules that can efficiently assemble into antiparallel double-stranded nucleic acids (e.g., double-stranded DNA or RNA) due to stacking interactions. The base portion of a universal nucleotide typically contains a nitrogen-containing aromatic heterocyclic moiety.

[0145] As used herein, the terms “sufficient complementarity” or “sufficient complementarity” mean that the RNA silencing agent has a sequence sufficient to bind to the desired target RNA and trigger RNA silencing of the target mRNA (e.g., in the antisense strand, the mRNA targeting portion, or the miRNA recruitment portion).

[0146] As used herein, the term "translation repression" refers to the selective repression of mRNA translation. Natural translation repression occurs via miRNA cleaved from shRNA precursors. Both RNAi and translation repression are RISC-mediated. Both RNAi and translation repression can occur naturally or can be artificially initiated, for example, by silencing the expression of target genes.

[0147] As used herein, the term "alkyl" refers to a saturated hydrocarbon group that can be straight-chain or branched. The term "C"... n-m "Alkyl" refers to an alkyl group having n to m carbon atoms. Alkyl groups formally correspond to alkane groups, where one CH bond is replaced by the alkyl group at the junction with the rest of the compound. 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 groups include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; and higher homologues such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, etc.

[0148] The term "heteroalkyl" refers to an optionally substituted alkyl group having one or more skeletal chain atoms selected from atoms other than carbon, such as oxygen, nitrogen (e.g., NH or N-alkyl), sulfur, phosphorus, silicon, or combinations thereof. In some embodiments, a heteroalkyl group is an alkyl group in which one of the skeletal atoms is oxygen. In some embodiments, a heteroalkyl group is an alkyl group in which one of the skeletal atoms is NH or N-alkyl. In some embodiments, a heteroalkyl group is an alkyl group in which one of the skeletal atoms is O or S. Exemplary heteroalkyl groups include, but are not limited to, -(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) n -CH3, -C(CH3)2SO2-CH3, -CH2NH-(C1-C6alkyl), -C(CH3)2NH-(C1-C6alkyl), -CH(CH3)NH-(C1-C6alkyl)2. In some embodiments, the heteroatom is located 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 some embodiments, where the heteroalkyl group comprises a CH3 group, the heteroalkyl group is present at the 5' and / or 3' end of the oligonucleotide.

[0149] As used herein, the term "alkoxy" refers to an -O-alkyl group, wherein the alkyl group is as defined herein. Alkoxy groups include, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, etc. In one embodiment, C1-C6 alkoxy groups are provided herein.

[0150] As used herein, unless otherwise stated, the term "halo / halogen" alone or as part of another substituent means a fluorine, chlorine, bromine or iodine atom, preferably fluorine, chlorine or bromine, more preferably fluorine or chlorine.

[0151] As used herein, unless otherwise stated, the term "hydroxyl" alone or as part of another substituent means an alcohol moiety having the formula -OH.

[0152] As used herein, unless otherwise stated, the term "ethylene glycol," alone or as part of another substituent, means an alcohol moiety having the formula HO(CH2)(CH2)OH. The term nm ethylene glycol units refers to an alcohol moiety having n to m ethylene glycol units. In some embodiments, the ethylene glycol contains 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, the ethylene glycol contains 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" can contain nm ethylene glycol units, as defined above.

[0153] The preparation of linkers can 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, 4th ed., Wiley & Sons, 2007, which is incorporated herein by reference in its entirety. The modification of protecting groups and the formation and cleavage methods described herein can be adapted to various substituents as needed.

[0154] The various methods disclosed herein include steps involving comparing values, levels, characteristics, properties, etc., with a “suitable control” (which may be interchangeably referred to herein as a “appropriate control”). A “suitable control” or “appropriate control” is any control or standard familiar to those skilled in the art for comparative purposes. In one embodiment, a “suitable control” or “appropriate control” is a value, level, characteristic, property, etc., determined prior to performing the RNAi method as described herein. For example, transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristics or properties, genotype, phenotype, etc., may be determined prior to introducing the RNA silencing agent of this disclosure into cells or organisms. In another embodiment, a “suitable control” or “appropriate control” is a value, level, characteristic, property, etc., determined in cells or organisms exhibiting, for example, normal characteristics (such as controls or normal cells or organisms). In yet another embodiment, a “suitable control” or “appropriate control” is a predefined value, level, characteristic, property, etc.

[0155] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although similar or equivalent methods and materials may be used in practice or testing of the methods and materials described herein, the following description is of suitable methods and materials. All patents, patent applications, and other references cited herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, these materials, methods, and examples are illustrative only and not intended to be limiting.

[0156] Various aspects of this disclosure are described in more detail in the following subsections.

[0157] Nucleotide modification and chemical modification patterns This document provides a series of nucleotide modifications, any one or more of which can be applied to RNA molecules (e.g., dsRNA) to improve efficacy and prolong in vivo silencing activity, such as after a single administration. RNA chemical modification patterns (e.g., antisense and sense strand chemical modification patterns) for achieving improved efficacy and prolonged in vivo silencing are also provided. In non-limiting examples, such as... Figure 1 The RNA chemical modification patterns shown (e.g., patterns 1, 1T, 2, and 2T) can be applied to the RNA molecules of this disclosure.

[0158] In some embodiments, the RNA molecule of this disclosure comprises one or more 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. In a non-limiting example, the RNA molecule of this disclosure may comprise, as shown below... Figure 2The nucleotide modifications shown.

[0159] In some 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 some embodiments, the RNA molecule contains 2-5 ethylene glycol modifications. In some embodiments, the RNA molecule contains 2 ethylene glycol modifications. In some embodiments, the RNA molecule contains 3 ethylene glycol modifications. In some embodiments, the RNA molecule contains 4 ethylene glycol modifications. In some embodiments, the RNA molecule contains 5 ethylene glycol modifications. In some embodiments, the ethylene glycol modification may contain nm ethylene glycol units, as defined above. In some embodiments, RNA molecules containing 2 or more ethylene glycol modifications may include the same number of ethylene glycol units in each modification. In some embodiments, RNA molecules containing 2 or more ethylene glycol modifications may include different numbers of ethylene glycol units in each modification. In some embodiments, an RNA molecule containing two or more ethylene glycol modifications may include at least two modifications with different numbers of ethylene glycol units. For example, an RNA molecule containing two or more ethylene glycol modifications may include at least one modification having m ethylene glycol units and at least one modification having n ethylene glycol units, where n ≠ m.

[0160] In some embodiments, the RNA molecule contains at least one ethylene glycol modification at nucleotide positions 1-5 of one or both of the 5' and 3' ends.

[0161] In some embodiments, the RNA molecule contains an ethylene glycol modification at nucleotide position 1 at the 5' end. In some embodiments, the RNA molecule contains an ethylene glycol modification at nucleotide position 2 at the 5' end. In some embodiments, the RNA molecule contains an ethylene glycol modification at nucleotide position 3 at the 5' end. In some embodiments, the RNA molecule contains an ethylene glycol modification at nucleotide position 4 at the 5' end. In some embodiments, the RNA molecule contains an ethylene glycol modification at nucleotide position 5 at the 5' end. In some embodiments, the RNA molecule contains ethylene glycol modifications at nucleotide positions 1 and 2 at the 5' end. In some embodiments, the RNA molecule contains ethylene glycol modifications at nucleotide positions 1-3 at the 5' end. In some embodiments, the RNA molecule contains ethylene glycol modifications at nucleotide positions 1-4 at the 5' end. In some embodiments, the RNA molecule contains ethylene glycol modifications at nucleotide positions 1-5 at the 5' end.

[0162] In some embodiments, the RNA molecule contains an ethylene glycol modification at nucleotide position 1 at the 3' end. In some embodiments, the RNA molecule contains an ethylene glycol modification at nucleotide position 2 at the 3' end. In some embodiments, the RNA molecule contains an ethylene glycol modification at nucleotide position 3 at the 3' end. In some embodiments, the RNA molecule contains an ethylene glycol modification at nucleotide position 4 at the 3' end. In some embodiments, the RNA molecule contains an ethylene glycol modification at nucleotide position 5 at the 3' end. In some embodiments, the RNA molecule contains ethylene glycol modifications at nucleotide positions 1 and 2 at the 3' end. In some embodiments, the RNA molecule contains ethylene glycol modifications at nucleotide positions 1-3 at the 3' end. In some embodiments, the RNA molecule contains ethylene glycol modifications at nucleotide positions 1-4 at the 3' end. In some embodiments, the RNA molecule contains ethylene glycol modifications at nucleotide positions 1-5 at the 3' end.

[0163] In some embodiments, the RNA molecule contains 1-20 ethylene glycol units. In some embodiments, the RNA molecule contains 1 ethylene glycol unit. In some embodiments, the RNA molecule contains 2 ethylene glycol units. In some embodiments, the RNA molecule contains 3 ethylene glycol units. In some embodiments, the RNA molecule contains 4 ethylene glycol units. In some embodiments, the RNA molecule contains 5 ethylene glycol units. In some embodiments, the RNA molecule contains 6 ethylene glycol units. In some embodiments, the RNA molecule contains 7 ethylene glycol units. In some embodiments, the RNA molecule contains 8 ethylene glycol units. In some embodiments, the RNA molecule contains 9 ethylene glycol units. In some embodiments, the RNA molecule contains 10 ethylene glycol units. In some embodiments, the RNA molecule contains 11 ethylene glycol units. In some embodiments, the RNA molecule contains 12 ethylene glycol units. In some embodiments, the RNA molecule contains 13 ethylene glycol units. In some embodiments, the RNA molecule contains 14 ethylene glycol units. In some embodiments, the RNA molecule contains 15 ethylene glycol units. In some embodiments, the RNA molecule contains 16 ethylene glycol units. In some embodiments, the RNA molecule contains 17 ethylene glycol units. In some embodiments, the RNA molecule contains 18 ethylene glycol units. In some embodiments, the RNA molecule contains 19 ethylene glycol units. In some embodiments, the RNA molecule contains 20 ethylene glycol units.

[0164] When modified with ethylene glycol, it can be used to insert or replace a nucleotide between two adjacent nucleotides (i.e., to replace the nucleotide). In a non-limiting example, the RNA molecule of this disclosure may contain, as... Figure 2The nucleotides shown are ethylene glycol substitutions (e.g., hexaethylene glycol (HEG) substitutions). The RNA molecule with the sequence ATGC will be used to illustrate the location of ethylene glycol modifications. An exemplary sequence would be AT(EG)GC when an ethylene glycol modification is inserted between two adjacent nucleotides, where “EG” corresponds to an internal ethylene glycol modification. An exemplary sequence would be AT(EG)C when a substituted nucleotide is inserted for an ethylene glycol modification, where “EG” corresponds to an ethylene glycol substitution modification.

[0165] In some embodiments, the at least one ethylene glycol modification is located between two adjacent nucleotides. In a non-limiting example, the RNA molecule of this disclosure may include at least one ethylene glycol modification located between two adjacent nucleotides, such as... Figure 2 As shown (e.g., hexaethylene glycol (HEG) linked between two nucleotides).

[0166] In some embodiments, the at least one ethylene glycol modification located between two adjacent nucleotides does not replace the nucleotide at that position in the RNA molecule, relative to an RNA molecule that does not contain the at least one ethylene glycol modification at the same position within the RNA molecule.

[0167] In some embodiments, the at least one ethylene glycol modification replaces a nucleotide at that position within the RNA molecule, relative to an RNA molecule that does not contain the at least one ethylene glycol modification at the same position within the RNA molecule.

[0168] In some embodiments, the RNA molecule further comprises 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 some embodiments, the RNA molecule comprises 2-5 alkyl modifications. In some embodiments, the RNA molecule comprises two alkyl modifications. In some embodiments, the RNA molecule comprises three alkyl modifications. In some embodiments, the RNA molecule comprises four alkyl modifications. In some embodiments, the RNA molecule comprises five alkyl modifications.

[0169] In some embodiments, the RNA molecule contains at least one alkyl modification at nucleotide positions 1-5 of one or both of the 5' and 3' ends.

[0170] In some embodiments, the RNA molecule contains an alkyl modification at nucleotide position 1 at the 5' end. In some embodiments, the RNA molecule contains an alkyl modification at nucleotide position 2 at the 5' end. In some embodiments, the RNA molecule contains an alkyl modification at nucleotide position 3 at the 5' end. In some embodiments, the RNA molecule contains an alkyl modification at nucleotide position 4 at the 5' end. In some embodiments, the RNA molecule contains an alkyl modification at nucleotide position 5 at the 5' end. In some embodiments, the RNA molecule contains alkyl modifications at nucleotide positions 1 and 2 at the 5' end. In some embodiments, the RNA molecule contains alkyl modifications at nucleotide positions 1-3 at the 5' end. In some embodiments, the RNA molecule contains alkyl modifications at nucleotide positions 1-4 at the 5' end. In some embodiments, the RNA molecule contains alkyl modifications at nucleotide positions 1-5 at the 5' end.

[0171] In some embodiments, the RNA molecule contains an alkyl modification at nucleotide position 1 at the 3' end. In some embodiments, the RNA molecule contains an alkyl modification at nucleotide position 2 at the 3' end. In some embodiments, the RNA molecule contains an alkyl modification at nucleotide position 3 at the 3' end. In some embodiments, the RNA molecule contains an alkyl modification at nucleotide position 4 at the 3' end. In some embodiments, the RNA molecule contains an alkyl modification at nucleotide position 5 at the 3' end. In some embodiments, the RNA molecule contains alkyl modifications at nucleotide positions 1 and 2 at the 3' end. In some embodiments, the RNA molecule contains alkyl modifications at nucleotide positions 1-3 at the 3' end. In some embodiments, the RNA molecule contains alkyl modifications at nucleotide positions 1-4 at the 3' end. In some embodiments, the RNA molecule contains alkyl modifications at nucleotide positions 1-5 at the 3' end.

[0172] In some embodiments, alkyl modifications include C1-C 10 Alkyl groups. In some embodiments, the alkyl modification includes C1 alkyl. In some embodiments, the alkyl modification includes C2 alkyl. In some embodiments, the alkyl modification includes C3 alkyl. In some embodiments, the alkyl modification includes C4 alkyl (i.e., butyl). In some embodiments, the alkyl modification includes C5 alkyl. In some embodiments, the alkyl modification includes C6 alkyl. In some embodiments, the alkyl modification includes C7 alkyl. In some embodiments, the alkyl modification includes C8 alkyl. In some embodiments, the alkyl modification includes C9 alkyl. In some embodiments, the alkyl modification includes C... 10 alkyl.

[0173] In some embodiments, alkyl modification includes branched C3-C... 10Alkyl group. In some embodiments, the alkyl modification branch includes branched C3 alkyl. In some embodiments, the alkyl modification branch includes branched C4 alkyl. In some embodiments, the alkyl modification branch includes branched C5 alkyl. In some embodiments, the alkyl modification branch includes branched C6 alkyl. In some embodiments, the alkyl modification branch includes branched C7 alkyl. In some embodiments, the alkyl modification branch includes branched C8 alkyl. In some embodiments, the alkyl modification branch includes branched C9 alkyl. In some embodiments, the alkyl modification branch includes branched C... 10 alkyl.

[0174] In some embodiments, the branched alkyl group is isopropyl. In some embodiments, the branched alkyl group is isobutyl. In some embodiments, the branched alkyl group is sec-butyl. In some embodiments, the branched alkyl group is tert-butyl.

[0175] In some embodiments, the branches derived from branched alkyl groups contain C3-C... 10 Alkyl group. In some embodiments, the branch from the branched alkyl group comprises C3 alkyl. In some embodiments, the branch from the branched alkyl group comprises C4 alkyl. In some embodiments, the branch from the branched alkyl group comprises C5 alkyl. In some embodiments, the branch from the branched alkyl group comprises C6 alkyl. In some embodiments, the branch from the branched alkyl group comprises C7 alkyl. In some embodiments, the branch from the branched alkyl group comprises C8 alkyl. In some embodiments, the branch from the branched alkyl group comprises C9 alkyl. In some embodiments, the branch from the branched alkyl group comprises C... 10 alkyl.

[0176] When an alkyl modification is used, it can be inserted between two adjacent nucleotides or substituted for a nucleotide (i.e., the nucleotide is replaced). An RNA molecule with the sequence ATGC will be used to illustrate the location of alkyl modifications. An exemplary sequence would be AT(ibut)GC when an alkyl modification is inserted between two adjacent nucleotides, where "ibut" corresponds to an internal butyl modification. An exemplary sequence would be AT(but)C when an alkyl modification is inserted substituted for a nucleotide, where "but" corresponds to a butyl substitution modification.

[0177] In some embodiments, the at least one alkyl modification is positioned between two adjacent nucleotides.

[0178] In some embodiments, the at least one alkyl modification located between two adjacent nucleotides does not replace the nucleotide at that position within the RNA molecule, relative to an RNA molecule that does not contain the at least one alkyl modification at the same position within the RNA molecule.

[0179] In some embodiments, the at least one alkyl modification replaces the nucleotide at that position within the RNA molecule, relative to an RNA molecule that does not contain the at least one alkyl modification at the same position within the RNA molecule.

[0180] In some embodiments, the alkyl modification is linear (i.e., unbranched).

[0181] In other embodiments, the alkyl modification is branched.

[0182] In some embodiments, the RNA molecule comprises single-stranded (ss) RNA or double-stranded (ds) RNA. The dsRNA disclosed herein comprises an antisense strand complementary to the target mRNA and a sense strand complementary to the antisense strand, each strand comprising a 5' end and a 3' end.

[0183] In some embodiments, the antisense strand is between 15 and 25 nucleotides in length. In some embodiments, the antisense strand is 18, 19, 20, 21, 22, or 23 nucleotides in length. In some embodiments, the sense strand is between 15 and 25 nucleotides in length. In some embodiments, the sense strand is 14, 15, 16, or 17 nucleotides in length.

[0184] In some embodiments, the dsRNA comprises a double-stranded region of 15 to 20 base pairs. In some embodiments, the dsRNA comprises a double-stranded region of 15 base pairs. In some embodiments, the dsRNA comprises a double-stranded region of 16 base pairs. In some embodiments, the dsRNA comprises a double-stranded region of 18 base pairs. In some embodiments, the dsRNA comprises a double-stranded region of 20 base pairs.

[0185] In some embodiments, the dsRNA includes blunt ends. In some embodiments, the dsRNA includes at least one single-stranded nucleotide overhang. In some embodiments, the dsRNA includes a single-stranded nucleotide overhang of about 2 to 5 nucleotides. In some embodiments, the dsRNA includes a single-stranded nucleotide overhang of 2 nucleotides. In some embodiments, the dsRNA includes a single-stranded nucleotide overhang of 5 nucleotides.

[0186] 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 within the antisense or sense strand.

[0187] In some implementations, the antisense strand contains ethylene glycol modification at one or more of the following nucleotide positions, counted from the 5' end: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23.

[0188] In some implementations, the antisense strand contains ethylene glycol modification at one or more of the following nucleotide positions, counted from the 5' end: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23.

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

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

[0192] In some implementations, the antisense strand contains a 2'-deoxy modification at one or more of the following nucleotide positions, counted from the 5' end: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23.

[0193] In some implementations, the antisense strand contains a 2'-MOE modification at one or more of the following nucleotide positions, counted from the 5' end: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23.

[0194] In some implementations, the antisense strand contains unmodified RNA nucleotides at one or more of the following nucleotide positions, counted from the 5' end: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23.

[0195] In some embodiments, the sense strand contains ethylene glycol 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.

[0196] In some embodiments, the sense strand contains an alkyl 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 some implementations, the sense strand contains an LNA 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.

[0198] In some implementations, the sense strand contains an 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.

[0199] In some embodiments, the sense strand contains a 2'-deoxy 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.

[0200] In some implementations, the sense 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, or 20, counted from the 5' end.

[0201] In some implementations, the sense strand contains unmodified RNA nucleotides at one or more of the following nucleotide positions, counted from the 5' end: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23.

[0202] In one aspect, this 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.

[0203] In some embodiments, the antisense chain comprises at least one alkyl diol modification.

[0204] In some embodiments of the RNA molecule or dsRNA disclosed herein, the RNA molecule or dsRNA contains at least one modified nucleotide inter-bond.

[0205] In some embodiments, the modified internucleotide bonds include phosphate-thioester nucleotide bonds. In some embodiments, the RNA molecule or dsRNA contains 4-16 phosphate-thioester nucleotide bonds. In some embodiments, the RNA molecule or dsRNA contains 8-13 phosphate-thioester nucleotide bonds.

[0206] Antonym single-chain protrusion modification This disclosure also provides antisense single-stranded overhang (i.e., tail) nucleotide modifications. An antisense single-stranded overhang is formed when the antisense strand is longer than the sense strand of the dsRNA. The length of the single-stranded overhang can be between 1 and 6 nucleotides.

[0207] In some implementations, the single-stranded overhang is 2 nucleotides long, 3 nucleotides long, 4 nucleotides long, or 5 nucleotides long.

[0208] In some implementations, the single-stranded overhang contains ethylene glycol modification at one or more of nucleotide positions 1, 2, 3, 4, or 5, counted from the 5' end.

[0209] In some implementations, the single-stranded overhang contains an alkyl modification at one or more of nucleotide positions 1, 2, 3, 4, or 5, counted from the 5' end.

[0210] In some implementations, the single-stranded overhang contains an LNA modification at one or more of the nucleotide positions 1, 2, 3, 4, or 5, counted from the 5' end.

[0211] In some implementations, the single-stranded overhang contains an UNA modification at one or more of the nucleotide positions 1, 2, 3, 4, or 5, counted from the 5' end.

[0212] In some implementations, the single-stranded overhang contains a 2'-deoxy modification at one or more of the nucleotide positions 1, 2, 3, 4, or 5, counted from the 5' end.

[0213] In some implementations, the single-stranded overhang contains a 2'-MOE modification at one or more of the nucleotide positions 1, 2, 3, 4, or 5, counted from the 5' end.

[0214] In some implementations, the single-stranded overhang contains unmodified RNA nucleotides at one or more of nucleotide positions 1, 2, 3, 4, or 5, counted from the 5' end.

[0215] Exemplary chemical modification patterns Examples of antisense and sense chains with chemical modifications are provided in Tables 1-5 below.

[0216] Table 1 – Antisense and sense chains of chemical modifications used as baseline modification patterns for applying one or more alternative modifications.

[0217] Table 2 – Ethylene glycol (EG)-containing chemical modifications for baseline modification patterns 1, 2 and 5 (P1, P2 and P5).

[0218] Table 3 – Chemical modifications of baseline modification patterns 1, 2 and 5 (P1, P2 and P5) containing internal ethylene glycol (iEG).

[0219] Table 4 – Chemical modifications of the baseline modification pattern 3 (P3) at the 3' end, including ethylene glycol (EG) and alkyl-containing modifications.

[0220] Table 5 – Chemical modifications of the baseline modification pattern 4 (P4) at the 3' end, including ethylene glycol (EG) and alkyl-containing modifications.

[0221] For the chemical modification patterns listed in Tables 1-5 above, the following abbreviations are used: "as" corresponds to the antisense strand of siRNA; "s" corresponds to the sense strand of siRNA; "m" corresponds to 2'-O-methyl (2'-OMe) chemical modification; "F" corresponds to 2'-fluorine (2'-F) chemical modification; "EG" corresponds to ethylene glycol chemical modification (replacing the nucleotide with ethylene glycol); "iEG" corresponds to internal ethylene glycol chemical modification (ethylene glycol linked between two nucleotides); "r" corresponds to unmodified ribonucleotide; and "#" corresponds to the internucleotide bond of phosphate thioester.

[0222] siRNA design In some embodiments, siRNA is designed as follows. First, a portion of a target gene is selected. Cutting the mRNA at these sites should eliminate the translation of the corresponding protein. An antisense strand is designed based on the target sequence, and a sense strand is designed to be complementary to the antisense strand. The hybridization of the antisense and sense strands forms an siRNA duplex. The antisense strand comprises about 19 to 25 nucleotides, for example, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In other embodiments, the antisense strand comprises 20, 21, 22, or 23 nucleotides. The sense strand comprises about 14 to 25 nucleotides, for example, 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 with antisense strands shorter than 19 nucleotides or longer than 25 nucleotides can also mediate RNAi. Therefore, siRNAs of this length are also within the scope of this disclosure, as long as they retain the ability to mediate RNAi. Longer RNAi agents have been shown to elicit interferon or PKR responses in some mammalian cells, which may be undesirable. In some embodiments, the RNAi agents of this disclosure do not elicit a PKR response (i.e., have a sufficiently short length). However, longer RNAi agents may be useful, for example, in cell types that do not produce a PKR response, or where the PKR response has been downregulated or inhibited by alternative methods.

[0223] A sense strand sequence can be designed so that the target sequence is essentially located in the middle of the strand. In some cases, moving the target sequence off-center can reduce the cleavage efficiency of the siRNA. Such compositions, i.e., less efficient compositions, may be necessary if wild-type mRNA silencing is detected.

[0224] The antisense strand may be the same length as the sense strand and include complementary nucleotides. In one embodiment, the strands are perfectly complementary, i.e., the strands are blunt-ended during alignment or annealing. In another embodiment, the strands are aligned or annealed to produce a 1, 2, 3, 4, 5, 6, 7, or 8 nucleotide overhang, i.e., the 3' end of the sense strand extends 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides beyond the 5' end of the antisense strand and / or the 3' end of the antisense strand extends 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides beyond the 5' end of the sense strand. The overhang may contain (or consist of) nucleotides corresponding to the target gene sequence (or its complementary sequence). Alternatively, the overhang may contain deoxyribonucleotides, such as dT, or nucleotide analogs, or other suitable non-nucleotide material (or consist of).

[0225] To facilitate antisense strand entry into RISC (thereby increasing or enhancing 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 can be altered, for example, weakened or reduced, as detailed in U.S. Patent Nos. 7,459,547, 7,772,203, and 7,732,593 (filed June 2, 2003), entitled "Methods and Compositions for Controlling Efficacy of RNA Silencing," and U.S. Patent Nos. 8,309,704, 7,750,144, 8,304,530, 8,329,892, and 8,309,705 (filed June 2, 2003), entitled "Methods and Compositions for Enhancing the Efficacy and Specificity of RNAi," the contents of which are incorporated herein by reference in their entirety. In one embodiment of these aspects of the present disclosure, the base pair strength is lower 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 compared to the G:C base pair between the 3' end of the first or antisense strand and the 5' end of the second or sense strand. In another embodiment, the lower base pair strength is 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 some 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 lower due to at least one wobbly base pair, such as 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 lower base pair strength is due to at least one base pair containing a rare nucleotide, such as inosine (I). In some exemplary embodiments, the base pair is selected from the group consisting of I:A, I:U, and I:C. In yet another embodiment, the lower base pair strength is due to at least one base pair containing a modified nucleotide. In some 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.

[0226] To verify the effectiveness of siRNA in disrupting mRNA (e.g., mRNA expressed from the target gene), siRNA can be combined with cDNA (e.g., cDNA derived from the target gene) in a assay based on... fruit flies It will be incubated in an in vitro mRNA expression system. Using 32Newly synthesized mRNAs (e.g., target mRNAs) radiolabeled with P are detected by autoradiography on agarose gels. The presence of cleaved mRNA indicates mRNA nuclease activity. Suitable controls include omitting the siRNA. Alternatively, a control siRNA with the same nucleotide composition as the selected siRNA but without significant sequence complementarity to the appropriate target gene is chosen. This negative control can be designed by randomly scrambling the nucleotide sequence of the selected siRNA; homology searches can be performed to ensure that the negative control lacks homology with any other gene in the appropriate genome. Furthermore, a negative control siRNA can be designed by introducing one or more base mismatches into the sequence. The siRNA-mRNA complement site that results in optimal mRNA specificity and maximum mRNA cleavage is selected.

[0227] RNA silencing agent characteristics In some embodiments, the RNA silencer contains at least 80% chemically modified nucleotides. In some embodiments, the RNA silencer is completely chemically modified, i.e., 100% of the nucleotides are chemically modified.

[0228] In some embodiments, the RNA silencer is rich in 2'-O-methyl, i.e., contains more than 50% 2'-O-methyl. In some embodiments, the RNA silencer contains at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% 2'-O-methyl nucleotide content. In some embodiments, the RNA silencer contains at least about 70% 2'-O-methyl nucleotide modification. In some embodiments, the RNA silencer contains between about 70% and about 90% 2'-O-methyl nucleotide modification. In some embodiments, the RNA silencer is a dsRNA comprising an antisense strand and a sense strand. In some embodiments, the antisense strand contains at least about 70% 2'-O-methyl nucleotide modification. In some embodiments, the antisense strand contains between about 70% and about 90% 2'-O-methyl nucleotide modification. In some embodiments, the sense strand contains at least about 70% 2'-O-methyl nucleotide modification. In some embodiments, the sense strand comprises between about 70% and about 90% 2'-O-methyl nucleotide modification. In some embodiments, the sense strand comprises 100% 2'-O-methyl nucleotide modification.

[0229] RNA silencing agents rich in 2'-O-methyl and specific chemical modification patterns are further described in USSN 16 / 550,076 (filed August 23, 2019, and granted as US11,279,930 March 22, 2022) and USSN 16 / 999,759 (filed August 21, 2020, and claiming priority to 62 / 891,185 (filed August 23, 2019), all of which are incorporated herein by reference.

[0230] Adhesion function part In other embodiments, the RNA silencer may be modified with one or more functional moieties. A functional moiety is a molecule that imparts one or more additional activities to the RNA silencer. In some embodiments, the functional moieties enhance cellular uptake by target cells (e.g., neurons). Therefore, this disclosure includes RNA silencers conjugated or unconjugated to another moiety (e.g., at its 5' and / or 3' ends), such as a non-nucleic acid moiety such as a peptide, an organic compound (e.g., a dye). Conjugation can be accomplished by methods known in the art, such as those described in Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001) (which describes nucleic acids loaded onto polyalkyl cyanoacrylate (PACA) nanoparticles); Fattal et al., J. Control Release 53(1-3):137-43 (1998) (which describes nucleic acids bound to nanoparticles); Schwab et al., Ann. Oncol. 5 Supplement 4:55-8 (1994) (which describes nucleic acids linked to intercalators, hydrophobic groups, polycations, or PACA nanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10 (1995) (which describes nucleic acids linked to nanoparticles).

[0231] In one embodiment, the functional portion is a hydrophobic portion. In one embodiment, the hydrophobic portion is selected from the group consisting of: fatty acids, steroids, open-ring steroids, lipids, gangliosides and nucleoside analogs, endocannabinoids, and vitamins. In one embodiment, the steroid is selected from the group consisting of cholesterol and lithocholic acid (LCA). In one embodiment, the fatty acid is selected from the group consisting of: eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanoic acid (DCA). In one embodiment, the vitamin is selected from the group consisting of: choline, vitamin A, vitamin E, and their derivatives or metabolites. In one embodiment, the vitamin is selected from the group consisting of retinoic acid and α-tocopherol succinate.

[0232] In one embodiment, the RNA silencing agent of this disclosure is conjugated to a lipophilic moiety. In one embodiment, the lipophilic moiety is a ligand comprising a cationic group. In another embodiment, the lipophilic moiety is attached to one or both strands of the siRNA. In one 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 some 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 one exemplary embodiment, the lipophilic moiety is cholesterol. Other lipophilic moieties include cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O-(hexadecyl)glycerol, geraniol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholic acid, dimethoxytriphenylmethyl, or phenoxazine.

[0233] In some embodiments, the functional portion may include one or more ligands linked to the RNA silencer to enhance stability, hybridization thermodynamics with the target nucleic acid, targeting a specific tissue or cell type, or cell permeability, such as through endocytosis-dependent or independent mechanisms. The ligand and associated modifications may also increase sequence specificity, thereby reducing ectopic targeting. The tethered ligand may include one or more modified bases or sugars that can be used as intercalators. These may be located in internal regions, such as in the protrusions of the RNA silencer / target duplex. The intercalator may be an aromatic compound, such as a polycyclic aromatic compound or a heterocyclic aromatic compound. Polycyclic intercalators may have stacking capabilities and may include systems with 2, 3, or 4 fused rings. The universal bases described herein may be included on the ligand. In one embodiment, the ligand may include a cleaving group that facilitates the repression of the target gene by cleaving the target nucleic acid. The cleavage group can 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. The metal ion chelating group can include, for example, 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 by free metal ions (such as Lu(III)) at the protrusion site. In some embodiments, the peptide ligand can be conjugated to an RNA silencing agent to promote the cleavage of the target RNA, for example, in the protrusion region. For example, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (cyclam) can be conjugated to a peptide (e.g., via an amino acid derivative) to promote the cleavage of the target RNA. The tethering ligand can be an aminoglycoside ligand, which can impart improved hybridization properties or improved sequence specificity to the RNA silencer. Exemplary aminoglycosides include glycosylated polylysine, galactosylated polylysine, neomycin B, tobramycin, kanamycin A, and acridine conjugates of aminoglycosides, such as neo-N-acrididine, neo-S-acrididine, neo-C-acrididine, Tobra-N-acrididine, and KanaA-N-acrididine. The use of acridine analogs can increase sequence specificity. For example, neomycin B has a high affinity for RNA but low sequence specificity compared to DNA. The acridine analog neo-5-acrididine increases affinity for HIV Rev response elements (RREs). In some embodiments, a guanidine analog of the aminoglycoside ligand (guanidinoglycoside) is tethered to the RNA silencer. In guanidinoglycosides, the amino group on the amino acid is exchanged for a guanidino group. Attachment of guanidine analogs can enhance the cellular permeability of RNA silencing agents. Tethering ligands can be polyarginine peptides, peptide-like substances, or peptide-like compounds, which can enhance the cellular uptake of oligonucleotide reagents.

[0234] Exemplary ligands are coupled directly or indirectly to a ligand-conjugated carrier via an inserted ligand. In some embodiments, coupling occurs via covalent bonds. In some embodiments, the ligand is linked to the vector via an inserted ligand. In some embodiments, the ligand alters the distribution, targeting, or lifetime of the RNA silencing agent it incorporates. In some embodiments, the ligand provides enhanced affinity for selected targets, such as molecules, cells or cell types, compartments (e.g., cell or organ compartments, tissues, organs, or body regions), compared to species without such ligands.

[0235] Exemplary ligands can improve transport, hybridization, and specificity properties, and can also improve the nuclease resistance of resulting natural or modified RNA silencing agents or polymer molecules containing any combination of monomers and / or natural or modified ribonucleotides described herein. Ligands can generally include therapeutic modifiers, such as those for enhancing uptake; diagnostic compounds or reporter groups, such as those for monitoring distribution; cross-linking agents; nuclease resistance-conferring moieties; and natural or unusual nucleobases. General examples include lipophilic substances, lipids, steroids (e.g., urobilinogen, hecoside, diosgenin), terpenes (e.g., triterpenes, such as sarsaponin, limonene, epi-limonene alcohol-derived lithocholic acid), vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins, protein-binding agents, integrin-targeting molecules, polycations, peptides, polyamines, and peptide mimics. Ligands can include naturally occurring substances (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, amylopectin, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); amino acids; or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, like 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-co-glycolic acid) 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 polyphosphazene. Examples of polyamines include: polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, pseudopeptide polyamines, dendritic polymer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.

[0236] Ligands may also include targeting groups, such as cell or tissue targets, such as lectins, glycoproteins, lipids, or proteins, like antibodies that bind to specific cell types such as kidney cells. Targeting groups can be thyroid-stimulating hormone, melanocyte-stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polylactose, polygalactose, N-acetylgalactosamine (GalNAc) or its derivatives, N-acetylglucosamine, polymannose, polyfucose, glycosylated polyamino acids, polygalactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, biotin, or RGD peptides or RGD peptide mimics. Other examples of ligands include dyes, intercalating agents (e.g., acridine and substituted acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine, phenanthroline, pyrene), lys-tyr-lys tripeptides, aminoglycosides, guanidine aminoglycosides, artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol (and its thioanalytes), cholic acids, cholanonic acids, lithocholic acids, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, glycerol (e.g., esters (e.g., mono-, di-, or tri-fatty acid esters, e.g., C), and esters (e.g., mono-, di-, or tri-fatty acid esters, e.g., C). 10 C 11 C 12 C 13 C 14 C 15 C 16 C 17 C 18 C 19 Or C 20 Fatty acids and their ethers, such as C 10 C 11 C 12 C 13 C 14 C 15 C 16 C 17 C 18 C 19 Or C 20Alkyl groups; for example, 1,3-bis-O-hexadecylglycerol, 1,3-bis-O-octadecylglycerol), geraniol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, stearic acid (e.g., glyceryl distearate), oleic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytriphenylmethyl or phenoxazine), and peptide conjugates (e.g., tentacles, Tat peptides), alkylating agents, phosphates, amino groups, thiol groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl groups, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption promoters (e.g., aspirin, naproxen, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, diimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, tetrazamacyclohexane Eu). 3+ (Complex), dinitrophenyl, HRP, or AP. In some embodiments, the ligand is GalNAc or a derivative thereof.

[0237] Ligands can be proteins, such as glycoproteins, or peptides, such as molecules with a specific affinity for a coligand, or antibodies, such as antibodies that bind to specific cell types, such as cancer cells, endothelial cells, or osteoblasts. Ligands may also include hormones and hormone receptors. They can also include non-peptide substances, such as lipids, lectins, carbohydrates, vitamins, cofactors, polylactose, polygalactose, N-acetylgalactosamine, N-acetylglucosamine, polymannose, or polyfucose. Ligands can be, for example, lipopolysaccharides, activators of p38MAP kinase, or NF-κB activators.

[0238] Ligands can be substances, such as drugs, that can increase the uptake of RNA silencers into cells, for example, by disrupting the cell's cytoskeleton, such as by disrupting microtubules, microfilaments, and / or intermediate filaments. Drugs can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin. For example, ligands can increase the uptake of RNA silencers into cells by activating an inflammatory response. Exemplary ligands with this effect include tumor necrosis factor-α (TNFα), interleukin-1 β, or gamma interferon. In another aspect, ligands are lipids or lipid-based molecules. Such lipids or lipid-based molecules can bind serum proteins, such as human serum albumin (HSA). HSA-binding ligands allow the conjugate to be distributed to target tissues, such as non-renal target tissues of the body. For example, target tissues could be the liver, including hepatic parenchymal cells. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. Lipids or lipid-based ligands can (a) increase resistance to conjugate degradation, (b) increase targeting or transport to target cells or cell membranes, and / or (c) be used to modulate binding to serum proteins such as HSA. Lipid-based ligands can be used to modulate, for example, control the binding of the conjugate to target tissues. For example, lipids or lipid-based ligands that bind more strongly to HSA are less likely to target the kidneys and therefore less likely to be cleared from the body. Lipids or lipid-based ligands that bind less strongly to HSA can be used to target the conjugate to the kidneys. In one embodiment, a lipid-based ligand binds to HSA. The lipid-based ligand can bind HSA with sufficient affinity such that the conjugate will be distributed to non-renal tissues. However, it is anticipated that HSA-ligand binding can be reversible, meaning the affinity for lipid-based ligands will not be strong enough to prevent HSA-ligand binding from reversing. In another embodiment, the lipid-based ligand binds weakly or not at all to HSA, allowing the conjugate to distribute into the kidney. Targeting other parts of renal cells can also be used to replace or supplement lipid-based ligands.

[0239] On the other hand, ligands are the portions absorbed by target cells, such as proliferating cells, and are like vitamins. These can be used to treat diseases characterized by unwanted cell proliferation, such as malignant or non-malignant cell types, such as cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients absorbed by cancer cells. Also included are HSA and low-density lipoprotein (LDL).

[0240] In another aspect, the ligand is a cell permeabilizer, such as a helical cell permeabilizer. In some embodiments, the reagent is amphiphilic. Exemplary reagents are peptides, such as tat or tentacles. If the reagent is a peptide, it can be modified, including peptide mimics, transformants, non-peptide or pseudopeptide bonds, and the use of D-amino acids. The helical agent can be an α-helical agent, which can have both lipophilic and lipophobic phases.

[0241] The ligand can be a peptide or a peptide mimic. A peptide mimic (also referred to herein as an oligopeptide mimic) is a molecule capable of folding into a defined three-dimensional structure similar to that of a natural peptide. The conjugation of peptides and peptide mimics to oligonucleotide reagents can influence the pharmacokinetic distribution of RNA silencing agents, for example, by enhancing cellular recognition and uptake. The peptide or peptide mimic moiety can be about 5–50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. The peptide or peptide mimic can be, for example, a cell-permeable peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (e.g., primarily composed of Tyr, Trp, or Phe). The peptide moiety can be a dendritic polymeric peptide, a bound peptide, or a cross-linked peptide. The peptide moiety can be an L-peptide or a D-peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). Peptides or peptide mimics may be encoded by random sequences of DNA, such as peptides identified from phage display libraries or single-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature 354:82-84, 1991). In exemplary embodiments, peptides or peptide mimics linked to RNA silencing agents via incorporated monomeric units are cell-targeting peptides, such as arginine-glycine-aspartic acid (RGD)-peptides or RGD mimics. The length of the peptide moiety can range from about 5 amino acids to about 40 amino acids. The peptide moiety may have structural modifications, such as to increase stability or direct conformational properties. Any structural modifications described below may be used.

[0242] In some embodiments, the functional portion is attached to the 5' end and / or the 3' end of the RNA silencing agent of the present invention. In some embodiments, the functional portion is attached to the 5' end and / or the 3' end of the antisense strand of the RNA silencing agent of the present invention. In some embodiments, the functional portion is attached to the 5' end and / or the 3' end of the sense strand of the RNA silencing agent of the present invention. In some embodiments, the functional portion is attached to the 3' end of the sense strand of the RNA silencing agent of the present invention.

[0243] In some embodiments, the functional portion is linked to an RNA silencing agent via a adapter. In some embodiments, the functional portion is linked to an antisense strand and / or a sense strand via a adapter. In some embodiments, the functional portion is linked to the 3' end of the sense strand via a adapter. In some embodiments, the adapter comprises a bivalent or trivalent adapter. In some embodiments, the adapter comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a thiophosphate, an aminophosphate, an amide, a carbamate, or a combination thereof. In some embodiments, the bivalent or trivalent adapter is selected from: ; ; ; ;or Where n is 1, 2, 3, 4 or 5.

[0244] In some embodiments, the connector further comprises a phosphate diester or a phosphate diester derivative. In some embodiments, the phosphate diester or phosphate diester derivative is selected from the group consisting of: ; (Zc1); ; (Zc2); ;and (Zc3) ; (Zc4) Where X is O, S or BH3.

[0245] The various functional components of this disclosure, and the methods of conjugating them with RNA silencing agents, are described in further detail in WO2017 / 030973A1 and WO2018 / 031933A2, which are incorporated herein by reference.

[0246] Branched oligonucleotides Two or more RNA silencing agents, such as oligonucleotide constructs (e.g., siRNAs), as disclosed above, can be linked together by one or more portions independently selected from linkers, spacers, and branching points to form a branched oligonucleotide RNA silencing agent. In some embodiments, the branched oligonucleotide RNA silencing agent consists of two siRNAs to form a bibranched siRNA (“dual siRNA”) scaffold for delivering two siRNAs. In a representative embodiment, each of the nucleic acids of the branched oligonucleotides contains an antisense strand (or a portion thereof), wherein the antisense strand is sufficiently complementary to the target mRNA to mediate an RNA-mediated silencing mechanism (e.g., RNAi).

[0247] In exemplary embodiments, the branched oligonucleotide may have two to eight RNA silencers linked by a linker. The linker may be hydrophobic. In one embodiment, the branched oligonucleotide of this application has two to three oligonucleotides. In one embodiment, the oligonucleotide independently possesses significant chemical stability (e.g., at least 40% of the constituent bases are chemically modified). In one exemplary embodiment, the oligonucleotide possesses 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 two to twenty nucleotides. In a non-limiting embodiment, each single-stranded tail has two to ten nucleotides.

[0248] In some embodiments, branched oligonucleotides are characterized by three properties: (1) a branched structure, (2) complete metabolic stability, and (3) the presence of a single-chain tail containing a phosphate thioester linker. In some embodiments, the branched oligonucleotide has two or three branches. It is believed that the increased overall size of the branched structure promotes increased uptake. Furthermore, without being bound by specific activity theories, it is thought that multiple adjacent branches (e.g., two or three) allow each branch to work synergistically, thereby significantly improving the rates of internalization, transport, and release.

[0249] Branched oligonucleotides are provided in various structurally different embodiments. In some embodiments, the nucleic acids linked at the branching points are single-stranded or double-stranded and consist of miRNA inhibitors, gapmers, mixmers, SSOs, PMOs, or PNAs. These single strands can be linked at their 3' or 5' ends. Combinations of siRNA and single-stranded oligonucleotides can also be used for dual functionality. In another embodiment, short nucleic acids complementary to gapmers, mixmers, miRNA inhibitors, SSOs, PMOs, and PNAs are used to carry these active single-stranded nucleic acids and enhance distribution and internalization within the cell. The short double-stranded regions have a low melting temperature (Tm ~37°C) for rapid dissociation during the internalization of the branched structure into the cell.

[0250] Di-siRNA branched oligonucleotides can contain chemically diverse conjugates, such as the functional moieties described above. Conjugated bioactive ligands can 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 linked to the di-siRNA via linker or spacer, or added via an additional linker or spacer attached to the end of another free siRNA.

[0251] Compared to unbranched compounds with the same chemical composition, the presence of branched structures increased tissue retention levels in the brain by more than 100-fold, indicating a novel mechanism for cellular retention and distribution. The branched oligonucleotides were unexpectedly uniformly distributed throughout the spinal cord and brain. Furthermore, the branched oligonucleotides exhibited surprisingly efficient systemic delivery to multiple tissues and very high levels of tissue accumulation.

[0252] Branched oligonucleotides contain a variety of therapeutic nucleic acids, including siRNA, ASO, miRNA, miRNA inhibitors, splicing conversion, PMO, and PNA. In some embodiments, branched oligonucleotides further include conjugated hydrophobic moieties and exhibit unprecedented silencing and efficacy both in vitro and in vivo.

[0253] connector In one embodiment of the branched oligonucleotide, each linker is independently selected from ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphate esters, phosphonates, aminophosphates, esters, amides, triazoles, and combinations thereof; wherein any carbon or oxygen atom of the linker is optionally replaced by a nitrogen atom, has a hydroxyl substituent, or has an oxygen 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 a phosphate ester. In another embodiment, each linker is a phosphonate. In another embodiment, each linker is an aminophosphate ester. In another embodiment, each linker is an ester. In another embodiment, each linker is an amide. In another embodiment, each linker is a triazole.

[0254] Branched oligonucleotides, including methods of synthesis and use, are described in more detail in WO2017 / 132669, which is incorporated herein by reference.

[0255] Methods of introducing RNA silencing agents The RNA silencing agent disclosed herein can be directly introduced into cells (e.g., nerve cells) (i.e., intracellularly); or introduced extracellularly into cavities, interstitial spaces, the circulation of an organism, or orally; or introduced by immersing cells or an organism in a solution containing nucleic acids. Blood vessels or extravascular circulation, the blood or lymphatic system, and cerebrospinal fluid are sites where nucleic acids can be introduced.

[0256] The RNA silencing agent of this disclosure can be introduced using nucleic acid delivery methods known in the art, including injecting a solution containing nucleic acid, bombarding with particles coated with nucleic acid, immersing cells or organisms in a nucleic acid solution, or electroporating the cell membrane in the presence of nucleic acid. Other methods known in the art for introducing nucleic acids into cells can be used, such as lipid-mediated carrier transport, chemically mediated transport, and cationic liposome transfection, such as with calcium phosphate. The nucleic acid can be introduced together with other components that perform one or more of the following activities: enhancing cellular uptake of nucleic acids or otherwise increasing repression of target genes.

[0257] Physical methods for introducing nucleic acids include injecting a solution containing RNA, bombarding with particles coated with RNA, immersing cells or organisms in an RNA solution, or electroporating the cell membrane in the presence of RNA. Other methods known in the art for introducing nucleic acids into cells can be used, such as lipid-mediated carrier transport, chemically mediated transport, such as calcium phosphate, etc. Therefore, RNA can be introduced along with other components that perform one or more of the following activities: enhancing cellular uptake of RNA, inhibiting single-strand annealing, stabilizing single strands, or otherwise increasing repression of target genes.

[0258] RNA can be introduced directly into cells (i.e., intracellularly); or extracellularly into cavities, interstitial spaces, the body's circulation, or 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 where RNA can be introduced.

[0259] Cells carrying target genes can originate from germline or somatic cells, totipotent or pluripotent cells, dividing or non-dividing cells, parenchymal or epithelial cells, immortalized or transformed cells, etc. Cells can 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 endocrine or exocrine gland cells.

[0260] Depending on the specific target gene and the dosage of the delivered double-stranded RNA material, the process may result in partial or complete loss of function of the target gene. Reduced or lost gene expression in at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more of the target cells is exemplary. Inhibition of gene expression refers to the absence (or observable reduction) of the levels of protein and / or mRNA products from the target gene. Specificity refers to the ability to inhibit a target gene without significantly affecting other genes in the cell. The consequences of inhibition can be confirmed by examining extrinsic properties of the cell or organism (as illustrated in the examples below) or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring using microarrays, antibody binding, enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence-activated cell sorting (FACS)).

[0261] For RNA-mediated repression in cell lines or the whole organism, gene expression can be conveniently measured using reporter genes or drug resistance genes whose protein products are readily measurable. Such reporter genes include acetylhydroxylase (AHAS), alkaline phosphatase (AP), β-galactosidase (LacZ), β-glucuronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), caustic solanine synthase (NOS), octopus solanine synthase (OCS), and their derivatives. A variety of optional markers are available to confer resistance to ampicillin, bleomycin, chloramphenicol, gentamicin, hygromycin, kanamycin, lincomycin, methotrexate, phosphatidylinosin, puromycin, and tetracycline. Quantification of gene expression levels, based on assays, allows for the determination of repression levels greater than 10%, 33%, 50%, 90%, 95%, or 99% compared to cells not treated according to this disclosure. Lower doses of injectable materials and longer durations of RNAi agent administration may result in inhibition of a smaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%, or 95% of target cells). Quantification of gene expression in cells may show similar levels of inhibition at the accumulation of target mRNA or the translational level of target protein. For example, inhibitory efficiency can be determined by assessing the amount of gene product in cells; mRNA can be detected with hybridization probes having nucleotide sequences outside the region for repressive double-stranded RNA, or translated peptides can be detected with antibodies generated from peptide sequences targeting that region.

[0262] RNA can be introduced in amounts that allow delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500, or 1000 copies per cell) of the material can produce more effective inhibition; lower doses may also be useful for specific applications.

[0263] In one exemplary aspect, the efficacy of the RNAi agents disclosed herein (e.g., siRNAs targeting a target sequence of interest) is tested to determine their ability to specifically degrade mutant mRNAs (e.g., the production of target mRNAs and / or target proteins) in cells, particularly in neurons (e.g., striatal or cortical neuronal clonal lines and / or primary neurons). Other readily transfectable cell types are also suitable for cell-based validation assays, such as HeLa cells or COS cells. 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-circular mRNA is co-transfected. Selective reduction in target mRNAs and / or target proteins is measured. The reduction in target mRNAs or proteins can be compared to the levels of target mRNAs or proteins in the absence of an RNAi agent or in the presence of an RNAi agent that does not target the target mRNA. For comparative purposes, exogenously introduced mRNAs or proteins (or endogenous mRNAs or proteins) can be analyzed. When using neurons that are known to be resistant to standard transfection techniques, it may be necessary to introduce RNAi agents (e.g., siRNA) through passive uptake.

[0264] Treatment As used herein, “treatment” or “treatment” is defined as the application or administration of a therapeutic agent (e.g., an RNA agent) to a patient, or the application or administration of a therapeutic agent to an isolated tissue or cell line from a patient who has a disease or condition, has symptoms of a disease or condition, or is susceptible to a disease or condition, with the aim of curing, healing, alleviating, reducing, altering, remedying, improving, enhancing, or influencing the disease or condition, its symptoms, or susceptibility to the disease.

[0265] In one aspect, this disclosure provides a method for preventing the disease or condition as described above in a subject by administering a therapeutic agent (e.g., an RNAi agent or vector or a transgene encoding them) to the subject. Subjects at risk of disease can be identified by, for example, any diagnostic or prognostic assay or a combination thereof described herein. Administration of the preventative agent can occur prior to the manifestation of characteristic symptoms of the disease or condition, thereby preventing the disease or condition, or alternatively delaying its progression.

[0266] Another aspect of this disclosure relates to a method for therapeutically treating a subject, namely, altering the onset of symptoms of a disease or condition.

[0267] Regarding preventative and therapeutic approaches to treatment, such treatments can be specifically tailored or modified based on knowledge gained 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 studying how a patient's genes determine his or her response to a drug (e.g., a patient's "drug response phenotype" or "drug response genotype"). Therefore, another aspect of this disclosure provides methods for tailoring preventative or therapeutic treatments to an individual based on their drug response genotype, using the target gene molecules or target gene modulators of this disclosure. Pharmacogenomics allows clinicians or physicians to target preventative or therapeutic treatments to patients who will benefit most from the treatment and to avoid treating patients who will experience drug-related toxic side effects.

[0268] Therapeutic agents can be tested in appropriate animal models. For example, RNAi agents (or expression vectors or transgenes encoding them) as described herein can be used in animal models to determine the efficacy, toxicity, or side effects of treatment using said agents. Alternatively, therapeutic agents can be used in animal models to determine the mechanism of action of such agents.

[0269] Pharmaceutical Compositions and Administration This disclosure relates to the use of the aforementioned reagents for prophylactic and / or therapeutic treatments as described below. Therefore, the modifiers of this disclosure (e.g., RNAi agents) can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise nucleic acid molecules, proteins, antibodies, or modulatory compounds and pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents compatible with drug administration. The use of such media and reagents for pharmaceutically active substances is well known in the art. Unless any conventional media or reagent is incompatible with the active compound, its use in the composition is contemplated. Complementary active compounds may also be incorporated into the composition.

[0270] The pharmaceutical compositions of this disclosure are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, intraperitoneal, intramuscular, oral (e.g., inhalation), transdermal (topical), and transmucosal administration. In some exemplary embodiments, the pharmaceutical compositions of this disclosure are administered intravenously and are capable of crossing the blood-brain barrier to enter the central nervous system. In some exemplary embodiments, the pharmaceutical compositions of this disclosure are delivered to cerebrospinal fluid (CSF) via routes of administration including, but not limited to, intrastriatal (IS) administration, intraventricular (ICV) administration, and intrathecal (IT) administration (e.g., via pump, infusion, etc.).

[0271] In some embodiments, compositions comprising compounds of this disclosure can be delivered to the nervous system of a subject via a variety of routes. Exemplary routes include intrathecal, parenchymal (e.g., in the brain), nasal, and ocular delivery. The compositions can also be delivered systemically, such as via intravenous, subcutaneous, or intramuscular injection. One route of delivery is direct delivery to the brain, such as the ventricles or hypothalamus, or lateral or dorsal regions of the brain. Compounds intended for delivery to nerve cells can be incorporated into pharmaceutical compositions suitable for administration.

[0272] For example, the composition may include one or more compounds of this disclosure and a pharmaceutically acceptable carrier. The pharmaceutical compositions of this disclosure can be administered in a variety of ways, depending on whether local or systemic treatment is required and depending on the area to be treated. Administration may be local (including ocular, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous infusion, subcutaneous, intraperitoneal, or intramuscular injection, intrathecal, or intraventricular (e.g., intracerebral) administration. In some exemplary embodiments, the RNA silencing agents of this disclosure are delivered across the blood-brain barrier (BBB) ​​using a variety of suitable compositions and methods described herein.

[0273] The route of delivery may depend on the patient's condition. In addition to the compounds disclosed herein, secondary therapies, such as palliative care and / or disease-specific therapies, may be administered to the patient. Secondary therapies may be, for example, symptomatic treatment (e.g., for symptom relief), neuroprotective treatment (e.g., for slowing or stopping disease progression), or rehabilitative treatment (e.g., for reversing disease progression). Other therapies may include psychotherapy, physical therapy, speech therapy, communication and memory assistance, social support services, and dietary advice.

[0274] The compounds disclosed herein can be delivered to nerve cells in the brain. In some embodiments, the compounds of this disclosure can be delivered to the brain without direct administration to the central nervous system, i.e., the compounds can be delivered intravenously and cross the blood-brain barrier to enter the brain. Delivery methods that do not require the composition to cross the blood-brain barrier can be utilized. For example, a pharmaceutical composition containing a compound of this disclosure can be delivered to a patient by direct injection into an area containing disease-affected cells. For example, the pharmaceutical composition can be delivered to the brain by direct injection. The injection can be stereotactically injected into a specific area of ​​the brain (e.g., substantia nigra, cortex, hippocampus, striatum, or globus pallidus). The compound can be delivered to multiple areas of the central nervous system (e.g., multiple areas of the brain, and / or into the spinal cord). The compound can be delivered to diffuse areas of the brain (e.g., diffuse delivery to the cerebral cortex).

[0275] In one embodiment, the compound can be delivered via a cannula or other delivery device, one end of which is implanted in tissue, such as the brain, including the substantia nigra, cortex, hippocampus, striatum, or globus pallidus. The cannula can be connected to a reservoir containing the compound. Flow or delivery can be mediated by a pump (e.g., an osmotic pump or a micropump, such as the Alzet pump (Durect, Cupertino, CA)). In one embodiment, the pump and reservoir are implanted in an area remote from the tissue, such as in the abdomen, and delivery is achieved via a catheter leading from the pump or reservoir to a release site. Devices for delivery to the brain are described, for example, in U.S. Patent Nos. 6,093,180 and 5,814,014.

[0276] It will be apparent to those skilled in the art that other suitable modifications and adaptations can be made to the methods described herein using appropriate equivalents without departing from the scope of the embodiments disclosed herein. Certain embodiments have been described in detail, and a clearer understanding of the embodiments will be provided by referring to the following examples, which are included herein for illustrative purposes only and are not intended to limit the invention.

[0277] Example 1. Test for ethylene glycol modification in siRNA Ethylene glycol modification was tested in siRNA. Specifically, Figure 3The relative HTT mRNA levels in cells incubated with various chemically modified siRNAs (including those with one or two ethylene glycol modifications at the 3' end of the antisense strand) at different doses were described. These results showed that siRNAs containing one or two ethylene glycol modifications (named "HEG-1", "HEG-2", and "HEG-3" and corresponding to oligonucleotide IDs 30912, 30913, and 30914 in Table 6) exhibited improved target silencing compared to chemically modified siRNAs without ethylene glycol modifications, such as alkyl-modified siRNAs (e.g., 5x or 3x butane-modified siRNAs).

[0278] Table 6 below provides exemplary siRNAs with various chemical modifications.

[0279] Table 6 – Chemically modified siRNAs.

[0280] In Table 6, “P” corresponds to 5’ phosphate ester, “m” corresponds to 2’-OMe modification, “f” corresponds to 2’-fluorine modification, “#” corresponds to the internucleotide bond of thiophosphate ester, “HEG” corresponds to ethylene glycol modification with 6 ethylene glycol units (hexaethylene glycol), and “but” corresponds to alkyl modification with butyl.

[0281] By incorporating via reference All references cited throughout this application (including references, patents, patent applications, and websites) are expressly incorporated herein by reference in their entirety for any purpose, and the cited references are also incorporated herein by reference. Unless otherwise stated, this disclosure will employ conventional techniques of immunology, molecular biology, and cell biology well known in the art.

[0282] This disclosure also incorporates, by reference, 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.) John Wiley & Sons, NY (1993); Edited by Ausubel, FM et al. (4th edition 1999) John Wiley & Sons, NY. (ISBN 0-471-32938-X); Smolen and Ball (eds.), Wiley, New York (1984); Giege, R. and Ducruix, A. Barrett, , a Practical Approach, 2nd ed., pp. 201-16, Oxford University Press, New York, New York, (1999); Goodson, Volume 2, pp. 115-138 (1984); Hammerling et al. 563-681 (Elsevier, NY, 1981; Harlow et al. , (Cold Spring Harbor Laboratory Press, 2nd Edition 1988); Kabat et al. (National Institutes of Health, Bethesda, Md. (1987) and (1991); Kabat, E.A., et al. (1991) , 5th edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Edited by Kontermann and Dubel (2001) Springer-Verlag. New York. Page 790 (ISBN 3-540-41354-5). Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); Edited by Lu and Weiner (2001) BioTechniques Press. Westborough, MA. Page 298 (ISBN 1-881299-21-X). Langer and Wise (eds.), CRCPres., Boca Raton, Fla. (1974); Old, RW&S.B. Primrose, (3rd edition, 1985) Blackwell Scientific Publications, Boston. Studies in Microbiology; V.2: p. 409 (ISBN 0-632-01318-4). Edited by Sambrook, J. et al. (2nd edition, 1989) Cold Spring Harbor Laboratory Press, NY. Pages 1-3. (ISBN 0-87969-309-6). , edited by JR Robinson, Marcel Dekker, Inc., New York, 1978 Winnacker, EL (1987) VCH Publishers, NY (translated by Horst Ibelgaufts), p. 634 (ISBN 0-89573-614-4). Equivalent solution This disclosure may be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered illustrative in all respects and not limiting of the invention. The scope of this disclosure is therefore indicated by the appended claims rather than by the foregoing description, and thus all variations within the meaning and equivalence of the claims are encompassed within the claims.

Claims

1. An RNA molecule comprising a 5' end and a 3' end, wherein the RNA molecule comprises at least one ethylene glycol modification of one or both of the 5' end and the 3' end, wherein the at least one ethylene glycol modification comprises 1-20 ethylene glycol units.

2. The RNA molecule of 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 of claim 1 or 2, wherein the RNA molecule comprises 2-5 ethylene glycol modifications.

4. The RNA molecule of claim 3, wherein the RNA molecule comprises two ethylene glycol modifications.

5. The RNA molecule of claim 3, wherein the RNA molecule comprises three ethylene glycol modifications.

6. The RNA molecule of claim 3, wherein the RNA molecule comprises four ethylene glycol modifications.

7. The RNA molecule of claim 3, wherein the RNA molecule comprises 5 ethylene glycol modifications.

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

9. The RNA molecule according to any one of claims 1-8, wherein, relative to an RNA molecule that does not contain the at least one ethylene glycol modification at the same position within the RNA molecule, the at least one ethylene glycol modification does not replace the nucleotide at the position within the RNA molecule.

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

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

12. The RNA molecule of claim 11, wherein the at least one alkyl modification comprises C1-C 10 alkyl.

13. The RNA molecule of claim 11 or 12, wherein the at least one alkyl modification comprises a C4 alkyl group.

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

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

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

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

18. The RNA molecule of claim 14, wherein the RNA molecule comprises 5 alkyl modifications.

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

20. The RNA molecule of any one of claims 11-19, wherein, relative to an RNA molecule that does not contain the at least one alkyl modification at the same position within the RNA molecule, the at least one alkyl modification does not replace the nucleotide at the position within the RNA molecule.

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

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

23. The RNA molecule of claim 22, wherein the RNA molecule comprises dsRNA having an antisense strand and a sense strand, the antisense strand having a 5' end and a 3' end, and the sense strand having a 5' end and a 3' end.

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

25. The RNA molecule of 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 of claim 25, wherein the antisense strand comprises 2-5 ethylene glycol modifications.

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

28. The RNA molecule of claim 26, wherein the antisense strand comprises three ethylene glycol modifications.

29. The RNA molecule of claim 26, wherein the antisense strand comprises four ethylene glycol modifications.

30. The RNA molecule of claim 26, wherein the antisense strand comprises 5 ethylene glycol modifications.

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

32. The RNA molecule according to any one of claims 24-31, wherein, relative to an RNA molecule that does not contain the at least one ethylene glycol modification at the same position within the RNA molecule, the at least one ethylene glycol modification does not replace the nucleotide at the position within the RNA molecule.

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

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

35. The RNA molecule of claim 34, wherein the at least one alkyl modification comprises C1-C 10 alkyl.

36. The RNA molecule of claim 34 or 35, wherein the at least one alkyl modification comprises a C4 alkyl group.

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

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

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

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

41. The RNA molecule of claim 37, wherein the antisense strand comprises 5 alkyl modifications.

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

43. The RNA molecule according to any one of claims 34-42, wherein, relative to an RNA molecule that does not contain the at least one alkyl modification at the same position within the RNA molecule, the at least one alkyl modification does not replace the nucleotide at the position within the RNA molecule.

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

45. The RNA molecule of any one of claims 23-44, wherein the length of the antisense strand is between 15 and 25 nucleotides.

46. ​​The RNA molecule of claim 45, wherein the antisense strand is 18, 19, 20, 21, 22, or 23 nucleotides in length.

47. The RNA molecule of any one of claims 23-46, wherein the length of the sense strand is between 15 and 25 nucleotides.

48. The RNA molecule of claim 47, wherein the sense strand is 14, 15, 16 or 17 nucleotides in length.

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

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

51. The RNA molecule according to any one of claims 22-50, wherein the RNA molecule further comprises at least one chemically modified nucleotide other than the at least one alkyl modification or the at least one ethylene glycol modification.

52. The RNA molecule of claim 51, wherein the at least one chemically modified nucleotide comprises a 2'-O-methyl modified nucleotide, a 2'-deoxy-2'-fluorine modified nucleotide, a 2'-deoxy modified nucleotide, a lock nucleotide, a debased nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholinonucleotide, an aminophosphate, a nucleotide containing a non-natural base, or a mixture thereof.

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

54. The RNA molecule of claim 53, wherein the modified nucleotide inter-bond comprises a phosphate thioester nucleotide inter-bond.

55. The RNA molecule according to any one of claims 22-54, wherein the RNA molecule comprises 4-16 phosphate thionucleotide bonds.

56. The RNA molecule according to any one of claims 22-55, wherein the RNA molecule comprises 8-13 phosphate thionucleotide bonds.

57. The RNA molecule of any one of claims 22-56, wherein the dsRNA comprises a blunt end.

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

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

60. The RNA molecule of claim 58, wherein the dsRNA comprises a single-stranded nucleotide overhang of 2 nucleotides.

61. The RNA molecule of claim 58, wherein the dsRNA comprises a single-stranded nucleotide overhang of 3 nucleotides.

62. The RNA molecule of claim 58, wherein the dsRNA comprises a single-stranded nucleotide overhang of 4 nucleotides.

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

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

65. The RNA molecule of claims 58-64, wherein the single-stranded nucleotide overhang comprises 2, 3, 4 or 5 alkyl modifications.

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

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

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 of one or both of the 5' end and the 3' end, wherein the at least one ethylene glycol modification comprises 1-20 ethylene glycol units.

69. The dsRNA of claim 68, wherein the antisense strand is between 15 and 25 nucleotides in length.

70. The dsRNA of claim 69, wherein the antisense strand is 18, 19, 20, 21, 22, or 23 nucleotides in length.

71. The dsRNA of any one of claims 68-70, wherein the length of the sense strand is between 15 and 25 nucleotides.

72. The dsRNA of claim 71, wherein the sense strand is 14, 15, 16 or 17 nucleotides in length.

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

74. The dsRNA according to any one of claims 68-73, wherein the dsRNA further comprises at least one alkyl modification.

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

76. The dsRNA according to any one of claims 68-75, wherein the dsRNA further comprises at least one chemically modified nucleotide other than the at least one alkyl modification or the at least one ethylene glycol modification.

77. The dsRNA of claim 76, wherein the at least one chemically modified nucleotide comprises a 2'-O-methyl modified nucleotide, a 2'-deoxy-2'-fluorine modified nucleotide, a 2'-deoxy modified nucleotide, a lock nucleotide, a debased nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholinonucleotide, an aminophosphate, a nucleotide containing a non-natural base, or a mixture thereof.

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

79. The dsRNA of claim 78, wherein the modified nucleotide inter-bond comprises a phosphate thioester nucleotide inter-bond.

80. The dsRNA according to any one of claims 68-79, wherein the dsRNA comprises 4-16 phosphate thionucleotide internucleotide bonds.

81. The dsRNA according to any one of claims 68-80, wherein the dsRNA comprises 8-13 phosphate thionucleotide internucleotide bonds.

82. The dsRNA of any one of claims 68-81, wherein the dsRNA comprises a blunt end.

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

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

85. The dsRNA of claim 83, wherein the dsRNA comprises a single-stranded nucleotide overhang of 2 nucleotides.

86. The dsRNA of claim 83, wherein the dsRNA comprises a single-stranded nucleotide overhang of 3 nucleotides.

87. The dsRNA of claim 83, wherein the dsRNA comprises a single-stranded nucleotide overhang of 4 nucleotides.

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

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

90. The dsRNA of any one of claims 83-89, wherein the single-stranded nucleotide overhang comprises 2, 3, 4 or 5 alkyl modifications.

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

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

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

94. The dsRNA of claim 93, wherein the single-stranded nucleotide overhang comprises 2, 3, 4 or 5 nucleotide modifications consisting of ethylene glycol modifications.

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

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

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

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

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

100. The method of 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 prior to administration of the RNA molecule or the dsRNA.

101. The method of claim 99 or 100, wherein the reduction in expression of the target mRNA lasts 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 the dsRNA.

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