Adipose-selective branched RNA compounds

Abstract

This disclosure relates to branched RNA compounds comprising an alkyl linker with enhanced target RNA silencing in adipose tissue.

Description

[0001] ADIPOSE-SELECTIVE BRANCHED RNA COMPOUNDS

[0002] Related Applications

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 639,190, filed April 26, 2024, the entire disclosure of which is hereby incorporated herein by reference.

[0004] Field of the Disclosure

[0005] This disclosure relates to branched RNA compounds comprising two or more RNA molecules linked via an alkyl linker.

[0006] Background

[0007] Adipose tissue holds significant scientific interest for numerous reasons. For the first time in history, the global burden of ovemutrition surpasses that of undernutrition, with over 1 billion adults classified as obese. In the United States alone, obesity-related ailments impose a financial burden exceeding US$190 billion on the healthcare system. Besides its association with obesity, fat tissue serves as the largest endocrine organ in humans. It plays a crucial role in the production and maintenance of hormone levels, regulating a wide array of processes such as reproduction, immunology, ageing, and nutritional homeostasis. Adipocytes are traditionally categorized into white adipose tissue (WAT) and brown adipose tissue (BAT). WAT is distributed throughout the body, with greater accumulation in specific depots, predominantly serving as sites for lipid storage. Generally, WAT can be further divided into subcutaneous and visceral depots. The localization and functionality of these depots differ among species, which complicates research efforts. As a therapeutic target, adipose tissue currently holds a central role in metabolic disorder research, including obesity and diabetes. However, due to its multifunctionality and involvement also in diseases like cancer, targeting adipose tissue is increasingly gaining interest for a broader range of disease types.

[0008] Accordingly, there exists a need for adipose tissue targeted therapeutics.

[0009] Summary

[0010] In one aspect, the disclosure provides a branched RNA compound comprising: two or more RNA molecules comprising 15 to 35 nucleotides in length, and a sequence substantially complementary to a target mRNA, wherein the two or more RNA molecules are connected to one another by an alkyl linker comprising a C1-C14 alkyl. In some embodiments, the alkyl linker is a C12 alkyl linker.

[0011] In some embodiments, the alkyl linker links the alkyl linker links the two or more RNA molecules by their 3’ ends.

[0012] In some embodiments, the branched RNA compound comprising the alkyl linker has increased target mRNA silencing in adipose tissue relative to a branched RNA compound that lacks the alkyl linker.

[0013] In some embodiments, the branched RNA compound comprising the alkyl linker has increased target mRNA silencing in adipose tissue relative to a branched RNA compound comprising two or more RNA molecules that are connected to one another by an ethylene glycol linker.

[0014] In some embodiments, the ethylene glycol linker is a triethylene glycol linker or a tetraethylene glycol linker.

[0015] In some embodiments, said RNA molecule comprises one or both of single stranded RNA (ssRNA) and double stranded RNA (dsRNA).

[0016] In some embodiments, said RNA molecule comprises an antisense oligonucleotide.

[0017] In some embodiments, each RNA molecule comprises 15 to 25 nucleotides in length.

[0018] In some embodiments, each RNA molecule comprises a dsRNA comprising a sense strand and an antisense strand, wherein each antisense strand independently comprises a sequence substantially complementary to the target mRNA.

[0019] In some embodiments, the alkyl linker links two or more dsRNAs (e.g., siRNAs) via their sense strands. In some embodiments, the alkyl linker links two or more dsRNAs (e.g., siRNAs) via the 3’ ends of their sense strands.

[0020] In some embodiments, the antisense strand comprises full complementary to the target mRNA.

[0021] In some embodiments, the antisense strand and / or sense strand comprises about 15 nucleotides to 25 nucleotides in length. In some embodiments, the antisense strand is 20 nucleotides in length. In some embodiments, the antisense strand is 21 nucleotides in length. In some embodiments, the antisense strand is 22 nucleotides in length. In some embodiments, the sense strand is 15 nucleotides in length. In some embodiments, the sense strand is 16 nucleotides in length. In some embodiments, the sense strand is 18 nucleotides in length. In some embodiments, the sense strand is 20 nucleotides in length. In some embodiments, the dsRNA comprises a double-stranded region of 15 base pairs 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.

[0022] In some embodiments, the dsRNA comprises a blunt-end. In some embodiments, the dsRNA comprises at least one single stranded nucleotide overhang. In some embodiments, the dsRNA comprises between a 2-nucleotide to 5-nucleotide single stranded nucleotide overhang.

[0023] In some embodiments, the dsRNA comprises naturally occurring nucleotides.

[0024] In some embodiments, the dsRNA comprises at least one modified nucleotide.

[0025] In some embodiments, said modified nucleotide comprises a 2'-O-methyl modified nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy -modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, or a non-natural base comprising nucleotide.

[0026] In some embodiments, the dsRNA comprises at least one modified intemucleotide linkage.

[0027] In some embodiments, said modified internucleotide linkage comprises a phosphorothioate intemucleotide linkage. In some embodiments, the dsRNA comprises 4-16 phosphorothioate intemucleotide linkages. In some embodiments, the dsRNA comprises 8-13 phosphorothioate intemucleotide linkages.

[0028] In some embodiments, said dsRNA comprises at least one modified intemucleotide linkage of Formula I:

[0029] (i); wherein:

[0030] B is a base pairing moiety;

[0031] W is selected from the group consisting of O, OCH2, OCH, CH2, and CH; X is selected from the group consisting of halo, hydroxy, and Ci-6 alkoxy;

[0032] Y is selected from the group consisting of O , OH, OR, NH , NH2, S ", and SH;

[0033] Z is selected from the group consisting of O and CH2;

[0034] R is a protecting group; and

[0035] — is an optional double bond.

[0036] In some embodiments, said dsRNA comprises at least 80% chemically modified nucleotides.

[0037] In some embodiments, said dsRNA is fully chemically modified.

[0038] In some embodiments, the antisense strand comprises a 5’ phosphate, a 5’ -alkyl phosphonate, a 5’ alkylene phosphonate, a 5’ alkenyl phosphonate, or a mixture thereof.

[0039] In some embodiments, the antisense strand comprises a 5’ vinyl phosphonate.

[0040] In some embodiments, a functional moiety is linked to the 5’ end and / or 3’ end of the antisense strand.

[0041] In some embodiments, a functional moiety is linked to the 5’ end and / or 3’ end of the sense strand.

[0042] In some embodiments, a functional moiety is linked to the 3’ end of the sense strand.

[0043] In some embodiments, the functional moiety comprises a hydrophobic moiety.

[0044] In some embodiments, the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and a mixture thereof.

[0045] In some embodiments, the steroid is selected from the group consisting of cholesterol and Lithocholic acid (LCA).

[0046] In some embodiments, the fatty acid is selected from the group consisting of Eicosapentaenoic acid (EP A), Docosahexaenoic acid (DHA) and Docosanoic acid (DCA).

[0047] In some embodiments, the vitamin is selected from the group consisting of choline, vitamin A, vitamin E, derivatives thereof, and metabolites thereof.

[0048] In some embodiments, the vitamin is selected from the group consisting of retinoic acid and alpha-tocopheryl succinate.

[0049] In some embodiments, the functional moiety is linked to the antisense strand and / or sense strand by a linker.

[0050] In some embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphodiester, a phosphorothioate, a phosphorami date, an amide, a carbamate, or a combination thereof. In some embodiments, the linker comprises a divalent or trivalent linker.

[0051] In some embodiments, the divalent or trivalent linker is selected from the group consisting of: wherein n is 1, 2, 3, 4, or 5.

[0052] In some embodiments, when the linker is a trivalent linker, the linker further links a phosphodiester or phosphodiester derivative.

[0053] In some embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of: and wherein X is O, S or BH3.

[0054] In some embodiments, the nucleotides at positions 1 and 2 from the 3’ end of sense strand, and the nucleotides at positions 1 and 2 from the 5’ end of antisense strand, are connected to adjacent ribonucleotides via phosphorothioate linkages.

[0055] In one aspect, the disclosure provides a pharmaceutical composition for inhibiting the expression of a target mRNA in an organism, comprising the branched RNA compound described herein, and a pharmaceutically acceptable carrier.

[0056] In one aspect, the disclosure provides a method for inhibiting expression of a target mRNA in adipose tissue in a subject, the method comprising administering to the subject the branched RNA compound described herein, thereby inhibiting expression of the target mRNA in the adipose tissue in the subject.

[0057] In one aspect, the disclosure provides a method of treating or preventing a disease of adipose tissue in a subject, comprising administering to the subject in need of such treatment or prevention a therapeutically effective amount of the branched RNA compound described herein, thereby treating or preventing the disease of adipose tissue in the subject.

[0058] In some embodiments, the branched RNA compound is administered to the subject by one or both of intravenous (IV) injection or subcutaneous (SQ) injection.

[0059] In some embodiments, the adipose tissue is epidydimal adipose tissue (eWAT), perirenal adipose tissue (PR), brown adipose tissue (BAT), Subcutaneous adipose tissue (SC), or a combination thereof.

[0060] In some embodiments, the branched RNA compound inhibits expression of the target mRNA in one or more of liver, kidney, lung, spleen, and heart in the subject by 20% or less relative to a subject that is not administered the branched RNA compound.

[0061] Brief Description of the Drawings

[0062] The foregoing and other features and advantages of the present disclosure will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0063] FIG. 1A depicts a synthesis strategy to build up various tethering chemistry during divalent-sense strand synthesis. FIG. IB depicts a schematic of divalent-siRNA with various tethering chemistry (i.e., tethering moieties are linkers connecting two or more RNA molecules). FIG. 2 depicts relative Htt mRNA expression in liver, kidney, lung, spleen, heart, quadricep muscle, and eWAT fat tissue (eWAT) after subcutaneous injection of either divalent Htt siRNA, divalent non-targeting control siRNA, or PBS in eight weeks old female mice on a standard chow diet. Divalent siRNA with single carbon-12 (DI0-C12), triple C12 (DIO- C12x3), single tri-ethyl-glycol (DIO-TEG), triple TEG (TEGx3), or a combination of TEG and C12 linkers (DIO-TEG-C 12x3 -TEG) was tested. Mice were injected with 500 pg divalent siRNA (n = 5). Htt mRNA was measured by Quantigene assay as a percentage of the PBS treated control two weeks post-injection. All results were compared to the non-targeting divalent siRNA control for each tissue. Mean ± SD. Statistical analysis was performed using one-way ANOVA with Dunnett’s correction for multiple comparisons, ns = not significant, *P < 0.05, or ***P < 0.001.

[0064] FIG. 3 depicts relative Mecp2 mRNA expression after subcutaneous injection of either divalent Mecp2 siRNA carrying a carbon 12 (C12) linker or monovalent Mecp2 siRNA carrying a 3’-Ci2 linker in eight-month-old female mice on a standard chow diet. Mice were injected with either 500 pg divalent siRNA (n = 5), 500 pg monovalent siRNA (n = 4), or divalent C12 siRNA non-targeting control (n = 4). Mecp2 mRNA measured by Quantigene assay as percentage of the PBS control in four different adipose tissues; Subcutaneous (SC), epidydimal (eWAT), perirenal (PR), or brown adipose tissue (BAT). All results are compared to the nontargeting divalent siRNA control. Mean ± SD. Statistical analysis was performed using oneway ANOVA with Dunnett’s correction for multiple comparisons for each adipose tissue, ns = not significant, *P < 0.05, or **P < 0.01.

[0065] FIG. 4 depicts silencing in liver, kidney, heart, and quadricep muscle after systemic delivery of either divalent Mecp2 siRNA carrying a carbon 12 (C12) linker or monovalent Mecp2 siRNA carrying a 3’-C12 linker. All data are from female mice 8 months of age at the time of injection kept on a standard chow diet. Mice were injected with either 500 pg divalent siRNA (middle data point, blue, n = 5), 500 pg monovalent siRNA (right data point, black, n = 4), or divalent C 12 siRNA non-targeting control (left data point, green, n = 4). Mecp2 mRNA measured by Quantigene assay as percentage of the PBS control in liver, kidney, heart, and muscle. All results are compared to the non-targeting divalent siRNA control. Mean ± SD. Statistical analysis was performed using one-way ANOVA with Dunnett’s correction for multiple comparisons for each adipose tissue, ns = not significant, or *P < 0.05. Detailed Description

[0066] Unless otherwise specified, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Unless otherwise specified, the methods and techniques provided herein are performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, delivery, and treatment of patients.

[0067] Unless otherwise defined herein, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and / or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.

[0068] So that the disclosure may be more readily understood, certain terms are first defined.

[0069] The term “nucleoside” refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine and thymidine. Additional exemplary nucleosides include inosine, 1-methyl inosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine and N2,N2-dimethylguanosine (also referred to as “rare” nucleosides). The term “nucleotide” refers to a nucleoside having one or more phosphate groups joined in ester linkages to the sugar moiety. Exemplary nucleotides include nucleoside monophosphates, diphosphates and triphosphates. The terms “polynucleotide” and “nucleic acid molecule” are used interchangeably herein and refer to a polymer of nucleotides joined together by a phosphodiester or phosphorothioate linkage between 5' and 3' carbon atoms. 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., by DNA replication or transcription of DNA, 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 single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis when ribosomes bind to the mRNA.

[0070] As used herein, the term "small interfering RNA" ("siRNA") (also referred to in the art as "short interfering RNAs") refers to an RNA (or RNA analog) comprising between about 10-50 nucleotides (or nucleotide analogs), which is capable of directing or mediating RNA interference. In certain embodiments, a siRNA comprises between about 15-30 nucleotides or nucleotide analogs, or between about 16-25 nucleotides (or nucleotide analogs), or between about 18-23 nucleotides (or nucleotide analogs), or between about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21 or 22 nucleotides or nucleotide analogs). The term "short" siRNA refers to a siRNA comprising about 21 nucleotides (or nucleotide analogs), for example, 19, 20, 21 or 22 nucleotides. The term "long" siRNA refers to a siRNA comprising about 24- 25 nucleotides, for example, 23, 24, 25 or 26 nucleotides. Short siRNAs may, in some instances, include fewer than 19 nucleotides, e.g., 16, 17 or 18 nucleotides, provided that the shorter siRNA retains the ability to mediate RNAi. Likewise, long siRNAs may, in some instances, include more than 26 nucleotides, provided that the longer siRNA retains the ability to mediate RNAi absent further processing, e.g., enzymatic processing, to a short siRNA.

[0071] The term "nucleotide analog" or "altered nucleotide" or "modified nucleotide" refers to a non-standard nucleotide, including non-naturally occurring ribonucleotides or deoxyribonucleotides. Exemplary nucleotide analogs are modified at any position so as to alter certain chemical properties of the nucleotide yet retain the ability of the nucleotide analog to perform its intended function. Examples of positions of the nucleotide, which may be derivatized include: the 5 position, e g., 5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine, 5-propenyl uridine, etc.; the 6 position, e.g., 6-(2-amino)propyl uridine; and the 8- position for adenosine and / or guanosines, e.g., 8-bromo guanosine, 8-chloro guanosine, 8- fluoroguanosine, etc. Nucleotide analogs also include deaza nucleotides, e.g., 7-deaza- adenosine; O- and N-modified (e.g., alkylated, e.g., N6-methyl adenosine, or as otherwise known in the art) nucleotides; and other heterocyclically modified nucleotide analogs, such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug. 10(4):297-310.

[0072] Nucleotide analogs may also comprise modifications to the sugar portion of the nucleotides. 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, wherein R is substituted or unsubstituted Ci-Ce alkyl, alkenyl, alkynyl, aryl, etc. Other possible modifications include those described in U.S. Pat. Nos. 5,858,988, and 6,291,438.

[0073] The phosphate group of the nucleotide may also be modified, e.g., by substituting one or more of the oxygens of the phosphate group with sulfur (e.g., phosphorothioates), or by making other substitutions, which allow the nucleotide to perform its intended function, such as described in, for example, Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr. 10(2): 117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000 Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct. 11(5): 317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001 Apr. 11(2):77-85, and U.S. Pat. No. 5,684,143. Certain of the above-referenced modifications (e.g., phosphate group modifications) decrease the rate of hydrolysis of, for example, polynucleotides comprising said analogs in vivo or in vitro.

[0074] The term "oligonucleotide" refers to a short polymer of nucleotides and / or nucleotide analogs.

[0075] The term "RNA analog" refers to a polynucleotide (e.g., a chemically synthesized polynucleotide) having at least one altered or modified nucleotide as compared to a corresponding unaltered or unmodified RNA, but retaining the same or similar nature or function as the corresponding unaltered or unmodified RNA. As discussed above, the oligonucleotides may be linked with linkages, which result in a lower rate of hydrolysis of the RNA analog as compared to an RNA molecule with phosphodiester linkages. For example, the nucleotides of the analog may comprise methylenediol, ethylene diol, oxymethylthio, oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phosphoroamidate, and / or phosphorothioate linkages. Some RNA analogues include sugar- and / or backbone-modified ribonucleotides and / or deoxyribonucleotides. Such alterations or modifications can further include addition of non-nucleotide material, such as to the end(s) of the RNA or internally (at one or more nucleotides of the RNA). An RNA analog need only be sufficiently similar to natural RNA that it has the ability to mediate RNA interference. As used herein, the term "RNA interference" ("RNAi") refers to a selective intracellular degradation of RNA. RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAi proceeds via fragments cleaved from free dsRNA, which direct the degradative mechanism to other similar RNA sequences. Alternatively, RNAi can be initiated by the hand of man, for example, to silence the expression of target genes.

[0076] An RNAi agent, e.g., an RNA silencing agent, having a strand, which is "sequence sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi)" means that the strand has a sequence sufficient to trigger the destruction of the target mRNA by the RNAi machinery or process.

[0077] As used herein, the term “isolated RNA” (e g., "isolated siRNA" or "isolated siRNA precursor") refers to RNA molecules, which are substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0078] As used herein, the term “RNA silencing” refers to a group of sequence-specific regulatory mechanisms (e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression) mediated by RNA molecules, which result in the inhibition or "silencing" of the expression of a corresponding protein-coding gene. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

[0079] The term "discriminatory RNA silencing" refers to the ability of an RNA molecule to substantially inhibit the expression of a "first" or "target" polynucleotide sequence while not substantially inhibiting the expression of a "second" or "non-target" polynucleotide sequence," e.g., when both polynucleotide sequences are present in the same cell. In certain embodiments, the target polynucleotide sequence corresponds to a target gene, while the non-target polynucleotide sequence corresponds to a non-target gene. In other embodiments, the target polynucleotide sequence corresponds to a target allele, while the non-target polynucleotide sequence corresponds to a non-target allele. In certain embodiments, the target polynucleotide sequence is the DNA sequence encoding the regulatory region (e.g. promoter or enhancer elements) of a target gene. In other embodiments, the target polynucleotide sequence is a target mRNA encoded by a target gene.

[0080] The term "in vitro" has its art recognized meaning, e g., involving purified reagents or extracts, e.g., cell extracts. The term "in vivo" also has its art recognized meaning, e.g., involving living cells, e.g., immortalized cells, primary cells, cell lines, and / or cells in an organism.

[0081] As used herein, the term "transgene" refers to any nucleic acid molecule, which is inserted by artifice into a cell, and becomes part of the genome of the organism that develops from the cell. Such a transgene may include a gene that is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism. The term "transgene" also means a nucleic acid molecule that includes one or more selected nucleic acid sequences, e g., DNAs, that encode one or more engineered RNA precursors, to be expressed in a transgenic organism, e.g., animal, which is partly or entirely heterologous, i.e., foreign, to the transgenic animal, or homologous to an endogenous gene of the transgenic animal, but which is designed to be inserted into the animal's genome at a location which differs from that of the natural gene. A transgene includes one or more promoters and any other DNA, such as introns, necessary for expression of the selected nucleic acid sequence, all operably linked to the selected sequence, and may include an enhancer sequence.

[0082] A gene "involved" in a disease or disorder includes a gene, the normal or aberrant expression or function of which effects or causes the disease or disorder or at least one symptom of said disease or disorder.

[0083] As used herein, the term "target gene" is a gene whose expression is to be substantially inhibited or "silenced." This silencing can be achieved by RNA silencing, e.g., by cleaving the mRNA (i.e., the target mRNA) of the target gene or translational repression of the target gene. The term "non-target gene" is a gene whose expression is not to be substantially silenced. In one embodiment, the polynucleotide sequences of the target and non-target gene (e.g. mRNA encoded by the target and non-target genes) can differ by one or more nucleotides. In another embodiment, the target and non-target genes can differ by one or more polymorphisms (e.g., Single Nucleotide Polymorphisms or SNPs). In another embodiment, the target and non-target genes can share less than 100% sequence identity. In another embodiment, the non-target gene may be a homologue (e.g. an orthologue or paralogue) of the target gene.

[0084] As used herein, the term "RNA silencing agent" refers to an RNA, which is capable of inhibiting or "silencing" the expression of a target gene. In certain embodiments, the RNA silencing agent is capable of preventing complete processing (e.g., the full translation and / or expression) of a mRNA molecule through a post-transcriptional silencing mechanism. RNA silencing agents include small (<50 b.p.), noncoding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small noncoding RNAs can be generated. Exemplary RNA silencing agents include siRNAs, miRNAs, siRNA-like duplexes, antisense oligonucleotides, GAPMER molecules, and dual-function oligonucleotides, as well as precursors thereof. In one embodiment, the RNA silencing agent is capable of inducing RNA interference. In another embodiment, the RNA silencing agent is capable of mediating translational repression.

[0085] As used herein, the term "rare nucleotide" refers to a naturally occurring nucleotide that occurs infrequently, including naturally occurring deoxyribonucleotides or ribonucleotides that occur infrequently, e.g., a naturally occurring ribonucleotide that is not guanosine, adenosine, cytosine, or uridine. Examples of rare nucleotides include, but are not limited to, inosine, 1 -methyl inosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N- methylguanosine and 2,2N,N-dimethylguanosine.

[0086] The term "engineered," as in an engineered RNA precursor, or an engineered nucleic acid molecule, indicates that the precursor or molecule is not found in nature, in that all or a portion of the nucleic acid sequence of the precursor or molecule is created or selected by a human. Once created or selected, the sequence can be replicated, translated, transcribed, or otherwise processed by mechanisms within a cell. Thus, an RNA precursor produced within a cell from a transgene that includes an engineered nucleic acid molecule is an engineered RNA precursor.

[0087] As used herein, the term "microRNA" ("miRNA"), also known in the art as "small temporal RNAs" ("stRNAs"), refers to a small (10-50 nucleotide) RNA, which are genetically encoded (e.g., by viral, mammalian, or plant genomes) and are capable of directing or mediating RNA silencing.

[0088] As used herein, the term "antisense strand" of an RNA silencing agent, e.g., an siRNA or RNA silencing agent, refers to a strand that is substantially complementary to a section of about 10-50 nucleotides, e.g., about 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 sequence sufficiently complementary to the desired target mRNA sequence to direct target-specific silencing, e.g., complementarity sufficient to trigger the destruction of the desired target mRNA by the RNAi machinery or process (RNAi interference) or complementarity sufficient to trigger translational repression of the desired target mRNA. The term "sense strand" or "second strand" of an RNA silencing agent, e.g., an siRNA or RNA silencing agent, refers to a strand that is complementary to the antisense strand or first strand. Antisense and sense strands can also be referred to as first or second strands, the first or second strand having complementarity to the target sequence and the respective second or first strand having complementarity to said first or second strand. miRNA duplex intermediates or siRNA-like duplexes include a miRNA strand having sufficient complementarity to a section of about 10-50 nucleotides of the mRNA of the gene targeted for silencing and a miRNA* strand having sufficient complementarity to form a duplex with the miRNA strand.

[0089] As used herein, the term "guide strand" refers to a strand of an RNA silencing agent, e.g., an antisense strand of an siRNA duplex or siRNA sequence, that enters into the RISC complex and directs cleavage of the target mRNA.

[0090] As used herein, the term "asymmetry," as in the asymmetry of the duplex region of an RNA silencing agent (e g., the stem of an shRNA), refers to an inequality of bond strength or base pairing strength between the termini of the RNA silencing agent (e.g., between terminal nucleotides on a first strand or stem portion and terminal nucleotides on an opposing second strand or stem portion), such that the 5' end of one strand of the duplex is more frequently in a transient unpaired, e.g., single- stranded, state than the 5' end of the complementary strand. This structural difference determines that one strand of the duplex is preferentially incorporated into a RISC complex. The strand whose 5' end is less tightly paired to the complementary strand will preferentially be incorporated into RISC and mediate RNAi.

[0091] 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 opposing strands of an oligonucleotide duplex (e.g., an siRNA duplex), due primarily to H-bonding, van der Waals interactions, and the like, between said nucleotides (or nucleotide analogs).

[0092] As used herein, the "5' end," as in the 5' end of an antisense strand, refers to the 5' terminal nucleotides, e.g., between one and about 5 nucleotides at the 5' terminus of the antisense strand. As used herein, the "3' end," as in the 3' end of a sense strand, refers to the region, e.g., a region of between one and about 5 nucleotides, that is complementary to the nucleotides of the 5' end of the complementary antisense strand.

[0093] As used herein the term "destabilizing nucleotide" refers to a first nucleotide or nucleotide analog capable of forming a base pair with second nucleotide or nucleotide analog such that the base pair is of lower bond strength than a conventional base pair (i.e., Watson- Crick base pair). In certain embodiments, the destabilizing nucleotide is capable of forming a mismatch base pair with the second nucleotide. In other embodiments, the destabilizing nucleotide is capable of forming a wobble base pair with the second nucleotide. In yet other embodiments, the destabilizing nucleotide is capable of forming an ambiguous base pair with the second nucleotide.

[0094] As used herein, the term "base pair" refers to the interaction between pairs of nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide duplex (e.g., a duplex formed by a strand of a RNA silencing agent and a target mRNA sequence), due primarily to H-bonding, van der Waals interactions, and the like between said nucleotides (or nucleotide analogs). As used herein, the term "bond strength" or "base pair strength" refers to the strength of the base pair.

[0095] As used herein, the term "mismatched base pair" refers to a base pair consisting of non-complementary or non-Watson-Crick base pairs, for example, not normal complementary G:C, A:T or A:U base pairs. As used herein the term "ambiguous base pair" (also known as a non-discriminatory base pair) refers to a base pair formed by a universal nucleotide.

[0096] As used herein, term "universal nucleotide" (also known as a "neutral nucleotide") include those nucleotides (e.g. certain destabilizing nucleotides) having a base (a "universal base" or "neutral base") that does not significantly discriminate between bases on a complementary polynucleotide when forming a base pair. Universal nucleotides are predominantly hydrophobic molecules that can pack efficiently into antiparallel duplex nucleic acids (e.g., double-stranded DNA or RNA) due to stacking interactions. The base portion of universal nucleotides typically comprise a nitrogen-containing aromatic heterocyclic moiety.

[0097] As used herein, the terms "sufficient complementarity" or "sufficient degree of complementarity" mean that the RNA silencing agent has a sequence (e g. in the antisense strand, mRNA targeting moiety or miRNA recruiting moiety), which is sufficient to bind the desired target RNA, respectively, and to trigger the RNA silencing of the target mRNA.

[0098] As used herein, the term "translational repression" refers to a selective inhibition of mRNA translation. Natural translational repression proceeds via miRNAs cleaved from shRNA precursors. Both RNAi and translational repression are mediated by RISC. Both RNAi and translational repression occur naturally or can be initiated by the hand of man, for example, to silence the expression of target genes. Various methodologies of the instant disclosure include a step that involves comparing a value, level, feature, characteristic, property, etc. to a "suitable control," referred to interchangeably herein as an "appropriate control " A "suitable control" or "appropriate control" is any control or standard familiar to one of ordinary skill in the art useful for comparison purposes. In one embodiment, a "suitable control" or "appropriate control" is a value, level, feature, characteristic, property, etc. determined prior to performing an RNAi methodology, as described herein. For example, a transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristic or property, genotype, phenotype, etc. can be determined prior to introducing an RNA silencing agent of the disclosure into a cell or organism. In another embodiment, a "suitable control" or "appropriate control" is a value, level, feature, characteristic, property, etc. determined in a cell or organism, e.g., a control or normal cell or organism, exhibiting, for example, normal traits. In yet another embodiment, a "suitable control" or "appropriate control" is a predefined value, level, feature, characteristic, property, etc.

[0099] 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 belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and example are illustrative only and not intended to be limiting.

[0100] Various aspects of the disclosure are described in further detail in the following subsections.

[0101] Target Sequences

[0102] In certain exemplary embodiments, RNA silencing agents of the disclosure (i.e., branched RNA compounds) are capable of targeting and silencing a target mRNA. In certain exemplary embodiments, RNA silencing agents of the disclosure (i.e., branched RNA compounds) are capable of targeting and silencing a target mRNA in adipose tissue. In certain exemplary embodiments, RNA silencing agents of the disclosure (i.e., branched RNA compounds) are capable of targeting and silencing a target mRNA in adipose tissue while minimally silencing (e.g., 20% or less of the silencing in adipose tissue) the target mRNA in non-adipose tissue, such as liver, kidney, lung, spleen, and heart.

[0103] Branched RNA Compound - siRNA

[0104] The branched RNA compounds of the disclosure comprise two or more RNA molecules. In some embodiments, the RNA molecules are dsRNA, such as siRNA. An siRNA molecule of the application is a duplex made of a sense strand and complementary antisense strand, the antisense strand having sufficient complementary to a target mRNA to mediate RNAi. In certain embodiments, the siRNA molecule has a length from about 10-50 or more nucleotides, i.e., each strand comprises 10-50 nucleotides (or nucleotide analogs). In other embodiments, the siRNA molecule has a length from about 15-30, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is sufficiently complementary to a target region. In certain embodiments, the strands are aligned such that there are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases at the end of the strands, which do not align (i.e., for which no complementary bases occur in the opposing strand), such that an overhang of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues occurs at one or both ends of the duplex when strands are annealed.

[0105] In some embodiments, siRNAs are designed as follows. First, a portion of the target gene is selected. Cleavage of mRNA at these sites should eliminate translation of corresponding protein. Antisense strands were designed based on the target sequence and sense strands were designed to be complementary to the antisense strand. Hybridization of the antisense and sense strands forms the siRNA duplex. The antisense strand includes about 19 to 25 nucleotides, e.g., 19, 20, 21, 22, 23, 24 or 25 nucleotides. In other embodiments, the antisense strand includes 20, 21, 22 or 23 nucleotides. The sense strand includes about 14 to 25 nucleotides, e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. In other embodiments, the sense strand is 15 nucleotides. In other embodiments, the sense strand is 18 nucleotides. In other embodiments, the sense strand is 20 nucleotides. The skilled artisan will appreciate, however, that siRNAs having a length of less than 19 nucleotides or greater than 25 nucleotides can also function to mediate RNAi. Accordingly, siRNAs of such length are also within the scope of the instant disclosure, provided that they retain the ability to mediate RNAi. Longer RNAi agents have been demonstrated to elicit an interferon or PKR response in certain mammalian cells, which may be undesirable. In certain embodiments, the RNAi agents of the disclosure do not elicit a PKR response (i.e., are of a sufficiently short length). However, longer RNAi agents may be useful, for example, in cell types incapable of generating a PKR response or in situations where the PKR response has been down-regulated or dampened by alternative means.

[0106] The sense strand sequence can be designed such that the target sequence is essentially in the middle of the strand. Moving the target sequence to an off-center position can, in some instances, reduce efficiency of cleavage by the siRNA. Such compositions, i.e., less efficient compositions, may be desirable for use if off-silencing of the wild-type mRNA is detected.

[0107] The antisense strand can be the same length as the sense strand and includes complementary nucleotides. In one embodiment, the strands are fully complementary, i.e., the strands are blunt-ended when aligned or annealed. In another embodiment, the strands align or anneal such that 1-, 2-, 3-, 4-, 5-, 6-, 7-, or 8-nucleotide overhangs are generated, i.e., the 3' end of the sense strand extends 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides further than 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 further than the 5' end of the sense strand. Overhangs can comprise (or consist of) nucleotides corresponding to the target gene sequence (or complement thereof). Alternatively, overhangs can comprise (or consist of) deoxyribonucleotides, for example dTs, or nucleotide analogs, or other suitable non-nucleotide material.

[0108] To facilitate entry of the antisense strand into RISC (and thus increase or improve the efficiency of target cleavage and silencing), the base pair strength between the 5' end of the sense strand and 3' end of the antisense strand can be altered, e.g., lessened or reduced, as described in detail in U.S. Patent Nos. 7,459,547, 7,772,203 and 7,732,593, entitled “Methods and Compositions for Controlling Efficacy of RNA Silencing” (filed Jun. 2, 2003) and U.S. Patent Nos. 8,309,704, 7,750,144, 8,304,530, 8,329,892 and 8,309,705, entitled “Methods and Compositions for Enhancing the Efficacy and Specificity of RNAi” (filed Jun. 2, 2003), the contents of which are incorporated in their entirety by this reference. In one embodiment of these aspects of the disclosure, the base-pair strength is less due to fewer G:C base pairs between the 5' end of the first or antisense strand and the 3' end of the second or sense strand than between the 3' end of the first or antisense strand and the 5' end of the second or sense strand. In another embodiment, the base pair strength is less due to at least one mismatched base pair between the 5' end of the first or antisense strand and the 3' end of the second or sense strand. In certain exemplary embodiments, the mismatched base pair is selected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C and U:U. In another embodiment, the base pair strength is less due to at least one wobble base pair, e.g., G:U, between the 5' end of the first or antisense strand and the 3' end of the second or sense strand. In another embodiment, the base pair strength is less due to at least one base pair comprising a rare nucleotide, e g., inosine (I). In certain exemplary embodiments, the base pair is selected from the group consisting of an I:A, I:U and I:C. In yet another embodiment, the base pair strength is less due to at least one base pair comprising a modified nucleotide. In certain exemplary embodiments, the modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

[0109] To validate the effectiveness by which, the siRNA can be incubated with cDNA in a Drosophila-based in vitro mRNA expression system. Radiolabeled with32P, newly synthesized mRNAs are detected autoradiographically on an agarose gel. The presence of cleaved mRNA indicates mRNA nuclease activity. Suitable controls include omission of siRNA. Alternatively, control siRNAs are selected having the same nucleotide composition as the selected siRNA, but without significant sequence complementarity to the appropriate target gene. Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected siRNA; a homology search can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome. In addition, negative control siRNAs can be designed by introducing one or more base mismatches into the sequence. Sites of siRNA-mRNA complementation are selected which result in optimal mRNA specificity and maximal mRNA cleavage.

[0110] The siRNA molecules of the disclosure have sufficient complementarity with the target sequence such that the siRNA can mediate RNAi. In general, siRNA containing nucleotide sequences sufficiently complementary to a target sequence portion of the target gene to effect RISC-mediated cleavage of the target gene are contemplated. Accordingly, in a certain embodiment, the antisense strand of the siRNA is designed to have a sequence sufficiently complementary to a portion of the target. For example, the antisense strand may have 100% complementarity to the target site. However, 100% complementarity is not required. Greater than 80% identity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% complementarity, between the antisense strand and the target RNA sequence is contemplated. The present application has the advantage of being able to tolerate certain sequence variations to enhance efficiency and specificity of RNAi. In one embodiment, the antisense strand has 4, 3, 2, 1, or 0 mismatched nucleotide(s) with a target region, such as a target region that differs by at least one base pair between a wild-type and mutant allele, e.g., a target region comprising the gain- of-function mutation, and the other strand is identical or substantially identical to the first strand. Moreover, siRNA sequences with small insertions or deletions of 1 or 2 nucleotides may also be effective for mediating RNAi. Alternatively, siRNA sequences with nucleotide analog substitutions or insertions can be effective for inhibition.

[0111] Sequence identity may be determined by sequence comparison and alignment algorithms known in the art. To determine the percent identity of two nucleic acid sequences (or of two amino acid sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The nucleotides (or amino acid residues) at corresponding nucleotide (or amino acid) positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), optionally penalizing the score for the number of gaps introduced and / or length of gaps introduced.

[0112] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the alignment generated over a certain portion of the sequence aligned having sufficient identity but not over portions having low degree of identity (i.e., a local alignment). A non-limiting example of a local alignment algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the BLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.

[0113] In another embodiment, the alignment is optimized by introducing appropriate gaps and the percent identity is determined over the length of the aligned sequences (i.e., a gapped alignment). To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul etal., (1997) Nucleic Acids Res. 25(17):3389-3402. In another embodiment, the alignment is optimized by introducing appropriate gaps and percent identity is determined over the entire length of the sequences aligned (i.e., a global alignment). A nonlimiting example of a mathematical algorithm utilized for the global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0114] The antisense or guide strand of the siRNA is routinely the same length as the sense strand and includes complementary nucleotides. In one embodiment, the guide and sense strands are fully complementary, i.e., the strands are blunt-ended when aligned or annealed. In another embodiment, the strands of the siRNA can be paired in such a way as to have a 3' overhang of 1 to 7 (e.g., 2, 3, 4, 5, 6 or 7), or 1 to 4, e.g., 2, 3 or 4 nucleotides. Overhangs can comprise (or consist of) nucleotides corresponding to the target gene sequence (or complement thereof). Alternatively, overhangs can comprise (or consist of) deoxyribonucleotides, for example dTs, or nucleotide analogs, or other suitable non-nucleotide material. Thus, in another embodiment, the nucleic acid molecules may have a 3' overhang of 2 nucleotides, such as TT. The overhanging nucleotides may be either RNA or DNA. As noted above, it is desirable to choose a target region wherein the mutant:wild type mismatch is a purine:purine mismatch.

[0115] Using any method known in the art, compare the potential targets to the appropriate genome database (human, mouse, rat, etc.) and eliminate from consideration any target sequences with significant homology to other coding sequences. One such method for such sequence homology searches is known as BLAST, which is available at National Center for Biotechnology Information website.

[0116] Further general information about the design and use of siRNA may be found in "The siRNA User Guide, " available at The Max-Pl ank-Insti tut fur Biophysikalische Chemie website.

[0117] Alternatively, the siRNA may be defined functionally as a nucleotide sequence (or oligonucleotide sequence) that is capable of hybridizing with the target sequence (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 °C or 70 °C hybridization for 12-16 hours; followed by washing). Additional hybridization conditions include hybridization at 70 °C in IxSSC or 50 °C in IxSSC, 50% formamide followed by washing at 70 °C in 0.3xSSC or hybridization at 70 °C in 4xSSC or 50 °C in 4xSSC, 50% formamide followed by washing at 67 °C in IxSSC. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10 °C less than the melting temperature (Tm) of the hybrid, where Tmis determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(°C)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs in length, Tm(°C)=81.5+16.6(log 10[Na+])+0.41(% G+C)-(600 / N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for lxSSC=0.165 M). Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference.

[0118] Negative control siRNAs should have the same nucleotide composition as the selected siRNA, but without significant sequence complementarity to the appropriate genome. Such negative controls may be designed by randomly scrambling the nucleotide sequence of the selected siRNA. A homology search can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome. In addition, negative control siRNAs can be designed by introducing one or more base mismatches into the sequence. siRNA-Like Molecules

[0119] The branched RNA compounds of the disclosure comprise two or more RNA molecules. In some embodiments, the RNA molecules are siRNA-like molecules. siRNA-like molecules of the disclosure have a sequence (i.e., have a strand having a sequence) that is "sufficiently complementary" to a target sequence of a target mRNA to direct gene silencing either by RNAi or translational repression. siRNA-like molecules are designed in the same way as siRNA molecules, but the degree of sequence identity between the sense strand and target RNA approximates that observed between a miRNA and its target. In general, as the degree of sequence identity between a miRNA sequence and the corresponding target gene sequence is decreased, the tendency to mediate post-transcriptional gene silencing by translational repression rather than RNAi is increased. Therefore, in an alternative embodiment, where post-transcriptional gene silencing by translational repression of the target gene is desired, the miRNA sequence has partial complementarity with the target gene sequence. In certain embodiments, the miRNA sequence has partial complementarity with one or more short sequences (complementarity sites) dispersed within the target mRNA (e g. within the 3'-UTR of the target mRNA) (Hutvagner and Zamore, Science, 2002; Zeng et al., Mol. Cell, 2002; Zeng et al., RNA, 2003; Doench et al., Genes & Dev., 2003). Since the mechanism of translational repression is cooperative, multiple complementarity sites (e.g., 2, 3, 4, 5, or 6) may be targeted in certain embodiments.

[0120] The capacity of a siRNA-like duplex to mediate RNAi or translational repression may be predicted by the distribution of non-identical nucleotides between the target gene sequence and the nucleotide sequence of the silencing agent at the site of complementarity. In one embodiment, where gene silencing by translational repression is desired, at least one nonidentical nucleotide is present in the central portion of the complementarity site so that duplex formed by the miRNA guide strand and the target mRNA contains a central "bulge" (Doench J G et al., Genes & Dev., 2003). In another embodiment 2, 3, 4, 5, or 6 contiguous or noncontiguous non-identical nucleotides are introduced. The non-identical nucleotide may be selected such that it forms a wobble base pair (e.g., G:U) or a mismatched base pair (G: A, C: A, C:U, G:G, A:A, C:C, U:U). In a further embodiment, the "bulge" is centered at nucleotide positions 12 and 13 from the 5' end of the miRNA molecule.

[0121] Short Hairpin RNA (shRNA) Molecules

[0122] The branched RNA compounds of the disclosure comprise two or more RNA molecules. In some embodiments, the RNA molecules are shRNAs. In certain featured embodiments, the instant disclosure provides shRNAs capable of mediating RNA silencing of the target sequence with enhanced selectivity. In contrast to siRNAs, shRNAs mimic the natural precursors of micro RNAs (miRNAs) and enter at the top of the gene silencing pathway. For this reason, shRNAs are believed to mediate gene silencing more efficiently by being fed through the entire natural gene silencing pathway. miRNAs are noncoding RNAs of approximately 22 nucleotides, which can regulate gene expression at the post transcriptional or translational level during plant and animal development. One common feature of miRNAs is that they are all excised from an approximately 70 nucleotide precursor RNA stem-loop termed pre-miRNA, probably by Dicer, an RNase Ill-type enzyme, or a homolog thereof. Naturally-occurring miRNA precursors (pre- miRNA) have a single strand that forms a duplex stem including two portions that are generally complementary, and a loop, that connects the two portions of the stem. In typical pre-miRNAs, the stem includes one or more bulges, e.g., extra nucleotides that create a single nucleotide "loop" in one portion of the stem, and / or one or more unpaired nucleotides that create a gap in the hybridization of the two portions of the stem to each other. Short hairpin RNAs, or engineered RNA precursors, of the present application are artificial constructs based on these naturally occurring pre-miRNAs, but which are engineered to deliver desired RNA silencing agents (e.g., siRNAs of the disclosure). By substituting the stem sequences of the pre-miRNA with sequence complementary to the target mRNA, a shRNA is formed. The shRNA is processed by the entire gene silencing pathway of the cell, thereby efficiently mediating RNAi.

[0123] The requisite elements of a shRNA molecule include a first portion and a second portion, having sufficient complementarity to anneal or hybridize to form a duplex or doublestranded stem portion. The two portions need not be fully or perfectly complementary. The first and second "stem" portions are connected by a portion having a sequence that has insufficient sequence complementarity to anneal or hybridize to other portions of the shRNA. This latter portion is referred to as a "loop" portion in the shRNA molecule. The shRNA molecules are processed to generate siRNAs. shRNAs can also include one or more bulges, i.e., extra nucleotides that create a small nucleotide "loop" in a portion of the stem, for example a one-, two- or three-nucleotide loop. The stem portions can be the same length, or one portion can include an overhang of, for example, 1-5 nucleotides. The overhanging nucleotides can include, for example, uracils (Us), e.g., all Us. Such Us are notably encoded by thymidines (Ts) in the shRNA-en coding DNA which signal the termination of transcription.

[0124] In shRNAs (or engineered precursor RNAs) of the instant disclosure, one portion of the duplex stem is a nucleic acid sequence that is complementary (or anti-sense) to the APP target sequence. In certain embodiments, one strand of the stem portion of the shRNA is sufficiently complementary (e.g., antisense) to a target RNA (e.g., mRNA) sequence to mediate degradation or cleavage of said target RNA via RNA interference (RNAi). Thus, engineered RNA precursors include a duplex stem with two portions and a loop connecting the two stem portions. The antisense portion can be on the 5' or 3' end of the stem. The stem portions of a shRNA are about 15 to about 50 nucleotides in length. In certain embodiments, the two stem portions are about 18 or 19 to about 21, 22, 23, 24, 25, 30, 35, 37, 38, 39, or 40 or more nucleotides in length. In certain embodiments, the length of the stem portions should be 21 nucleotides or greater. When used in mammalian cells, the length of the stem portions should be less than about 30 nucleotides to avoid provoking non-specific responses like the interferon pathway. In non-mammalian cells, the stem can be longer than 30 nucleotides. In fact, the stem can include much larger sections complementary to the target mRNA (up to, and including the entire mRNA). In fact, a stem portion can include much larger sections complementary to the target mRNA (up to, and including the entire mRNA). The two portions of the duplex stem must be sufficiently complementary to hybridize to form the duplex stem. Thus, the two portions can be, but need not be, fully or perfectly complementary. In addition, the two stem portions can be the same length, or one portion can include an overhang of 1, 2, 3, or 4 nucleotides. The overhanging nucleotides can include, for example, uracils (Us), e.g., all Us. The loop in the shRNAs or engineered RNA precursors may differ from natural pre-miRNA sequences by modifying the loop sequence to increase or decrease the number of paired nucleotides, or replacing all or part of the loop sequence with a tetraloop or other loop sequences. Thus, the loop in the shRNAs or engineered RNA precursors can be 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g., 15 or 20, or more nucleotides in length.

[0125] The loop in the shRNAs or engineered RNA precursors may differ from natural pre- miRNA sequences by modifying the loop sequence to increase or decrease the number of paired nucleotides, or replacing all or part of the loop sequence with a tetraloop or other loop sequences. Thus, the loop portion in the shRNA can be about 2 to about 20 nucleotides in length, i.e., about 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g., 15 or 20, or more nucleotides in length. In certain embodiments, a loop consists of or comprises a "tetraloop" sequence. Exemplary tetraloop sequences include, but are not limited to, the sequences GNRA, where N is any nucleotide and R is a purine nucleotide, GGGG, and UUUU.

[0126] In certain embodiments, shRNAs of the present application include the sequences of a desired siRNA molecule described supra. In other embodiments, the sequence of the antisense portion of a shRNA can be designed essentially as described above or generally by selecting an 18, 19, 20, 21 nucleotide, or longer, sequence from within the target, for example, from a region 100 to 200 or 300 nucleotides upstream or downstream of the start of translation. In general, the sequence can be selected from any portion of the target RNA (e.g., mRNA) including the 5' UTR (untranslated region), coding sequence, or 3' UTR. This sequence can optionally follow immediately after a region of the target gene containing two adjacent AA nucleotides. The last two nucleotides of the nucleotide sequence can be selected to be UU. This 21 or so nucleotide sequence is used to create one portion of a duplex stem in the shRNA. This sequence can replace a stem portion of a wild-type pre-miRNA sequence, e.g., enzymatically, or is included in a complete sequence that is synthesized. For example, one can synthesize DNA oligonucleotides that encode the entire stem-loop engineered RNA precursor, or that encode just the portion to be inserted into the duplex stem of the precursor, and using restriction enzymes to build the engineered RNA precursor construct, e.g., from a wild-type pre-miRNA. Engineered RNA precursors include, in the duplex stem, the 21-22 or so nucleotide sequences of the siRNA or siRNA-like duplex desired to be produced in vivo. Thus, the stem portion of the engineered RNA precursor includes at least 18 or 19 nucleotide pairs corresponding to the sequence of an exonic portion of the gene whose expression is to be reduced or inhibited. The two 3' nucleotides flanking this region of the stem are chosen so as to maximize the production of the siRNA from the engineered RNA precursor and to maximize the efficacy of the resulting siRNA in targeting the corresponding mRNA for translational repression or destruction by RNAi in vivo and in vitro.

[0127] In certain embodiments, shRNAs of the disclosure include miRNA sequences, optionally end-modified miRNA sequences, to enhance entry into RISC. The miRNA sequence can be similar or identical to that of any naturally occurring miRNA (see e.g. The miRNA Registry; Griffiths-Jones S, Nuc. Acids Res., 2004). Over one thousand natural miRNAs have been identified to date and together they are thought to comprise about 1% of all predicted genes in the genome. Many natural miRNAs are clustered together in the introns of pre-mRNAs and can be identified in silico using homology-based searches (Pasquinelli et al., 2000; Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001) or computer algorithms (e.g. MiRScan, MiRSeeker) that predict the capability of a candidate miRNA gene to form the stem loop structure of a pri-mRNA (Grad et al., Mol. Cell., 2003; Lim et al., Genes Dev., 2003; Lim et al., Science, 2003; Lai E C et al., Genome Bio., 2003). An online registry provides a searchable database of all published miRNA sequences (The miRNA Registry at the Sanger Institute website; Griffiths-Jones S, Nuc. Acids Res., 2004). Exemplary, natural miRNAs include lin-4, let-7, miR-10, mirR-15, miR-16, miR-168, miR-175, miR-196 and their homologs, as well as other natural miRNAs from humans and certain model organisms including Drosophila melanogaster , Caenorhabditis elegans, zebrafish, Arabidopsis thalania Mus musculus, and Rattus norvegicus as described in International PCT Publication No. WO 03 / 029459.

[0128] Naturally-occurring miRNAs are expressed by endogenous genes in vivo and are processed from a hairpin or stem-loop precursor (pre-miRNA or pri -miRNAs) by Dicer or other RNAses (Lagos-Quintana et al., Science, 2001; Lau et al., Science, 2001; Lee and Ambros, Science, 2001; Lagos-Quintana et al., Curr. Biol., 2002; Mourelatos et al., Genes Dev., 2002; Reinhart et al., Science, 2002; Ambros et al., Curr. Biol., 2003; Brennecke et al., 2003; Lagos- Quintana et al., RNA, 2003; Lim et al., Genes Dev., 2003; Lim et al., Science, 2003). miRNAs can exist transiently in vivo as a double-stranded duplex, but only one strand is taken up by the RISC complex to direct gene silencing. Certain miRNAs, e.g., plant miRNAs, have perfect or near-perfect complementarity to their target mRNAs and, hence, direct cleavage of the target mRNAs Other miRNAs have less than perfect complementarity to their target mRNAs and, hence, direct translational repression of the target mRNAs. The degree of complementarity between a miRNA and its target mRNA is believed to determine its mechanism of action. For example, perfect or near-perfect complementarity between a miRNA and its target mRNA is predictive of a cleavage mechanism (Yekta et al., Science, 2004), whereas less than perfect complementarity is predictive of a translational repression mechanism. In certain embodiments, the miRNA sequence is that of a naturally-occurring miRNA sequence, the aberrant expression or activity of which is correlated with a miRNA disorder.

[0129] Modified RNA Silencing Agents

[0130]

[0001] In certain aspects of the disclosure, an RNA silencing agent (or any portion thereof) of the present application, as described supra, may be modified, such that the activity of the agent is further improved. For example, the RNA silencing agents described in Section II supra, may be modified with any of the modifications described infra. The modifications can, in part, serve to further enhance target discrimination, to enhance stability of the agent (e.g., to prevent degradation), to promote cellular uptake, to enhance the target efficiency, to improve efficacy in binding (e.g., to the targets), to improve patient tolerance to the agent, and / or to reduce toxicity.

[0131] 1) Modifications to Enhance Target Discrimination

[0132]

[0002] In certain embodiments, the RNA silencing agents of the present application may be substituted with a destabilizing nucleotide to enhance single nucleotide target discrimination (see U.S. application Ser. No. 11 / 698,689, filed Jan. 25, 2007 and U.S. Provisional Application No. 60 / 762,225 filed Jan. 25, 2006, both of which are incorporated herein by reference). Such a modification may be sufficient to abolish the specificity of the RNA silencing agent for a non-target mRNA (e.g. wild-type mRNA), without appreciably affecting the specificity of the RNA silencing agent for a target mRNA (e.g. gain-of-function mutant mRNA).

[0133]

[0003] In certain embodiments, the RNA silencing agents of the present application are modified by the introduction of at least one universal nucleotide in the antisense strand thereof. Universal nucleotides comprise base portions that are capable of base pairing indiscriminately with any of the four conventional nucleotide bases (e.g. A, G, C, U). A universal nucleotide is contemplated because it has relatively minor effect on the stability of the RNA duplex or the duplex formed by the guide strand of the RNA silencing agent and the target mRNA. Exemplary universal nucleotides include those having an inosine base portion or an inosine analog base portion selected from the group consisting of deoxyinosine (e.g. 2'- deoxyinosine), 7-deaza-2'-deoxyinosine, 2'-aza-2'-deoxyinosine, PNA-inosine, morpholinoinosine, LNA-inosine, phosphoramidate-inosine, 2'-O-methoxyethyl-inosine, and 2'-0Me- inosine. In certain embodiments, the universal nucleotide is an inosine residue or a naturally occurring analog thereof.

[0134]

[0004] In certain embodiments, the RNA silencing agents of the disclosure are modified by the introduction of at least one destabilizing nucleotide within 5 nucleotides from a specificity-determining nucleotide (i.e., the nucleotide which recognizes the disease-related polymorphism). For example, the destabilizing nucleotide may be introduced at a position that is within 5, 4, 3, 2, or 1 nucleotide(s) from a specificity-determining nucleotide. In exemplary embodiments, the destabilizing nucleotide is introduced at a position which is 3 nucleotides from the specificity-determining nucleotide (i.e., such that there are 2 stabilizing nucleotides between the destablilizing nucleotide and the specificity-determining nucleotide). In RNA silencing agents having two strands or strand portions (e.g. siRNAs and shRNAs), the destabilizing nucleotide may be introduced in the strand or strand portion that does not contain the specificity-determining nucleotide. In certain embodiments, the destabilizing nucleotide is introduced in the same strand or strand portion that contains the specificity-determining nucleotide.

[0135] 2) Modifications to Enhance Efficacy and Specificity

[0136]

[0005] In certain embodiments, the RNA silencing agents of the disclosure may be altered to facilitate enhanced efficacy and specificity in mediating RNAi according to asymmetry design rules (see U.S. Patent Nos. 8,309,704, 7,750,144, 8,304,530, 8,329,892 and 8,309,705). Such alterations facilitate entry of the antisense strand of the siRNA (e.g., a siRNA designed using the methods of the present application or an siRNA produced from a shRNA) into RISC in favor of the sense strand, such that the antisense strand preferentially guides cleavage or translational repression of a target mRNA, and thus increasing or improving the efficiency of target cleavage and silencing. In certain embodiments, the asymmetry of an RNA silencing agent is enhanced by lessening the base pair strength between the antisense strand 5' end (AS 5') and the sense strand 3' end (S 3') of the RNA silencing agent relative to the bond strength or base pair strength between the antisense strand 3' end (AS 3') and the sense strand 5' end (S '5) of said RNA silencing agent.

[0137]

[0006] In one embodiment, the asymmetry of an RNA silencing agent of the present application may be enhanced such that there are fewer G:C base pairs between the 5' end of the first or antisense strand and the 3' end of the sense strand portion than between the 3' end of the first or antisense strand and the 5' end of the sense strand portion. In another embodiment, the asymmetry of an RNA silencing agent of the disclosure may be enhanced such that there is at least one mismatched base pair between the 5' end of the first or antisense strand and the 3' end of the sense strand portion. In certain 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 asymmetry of an RNA silencing agent of the disclosure may be enhanced such that there is at least one wobble base pair, e.g., G:U, between the 5' end of the first or antisense strand and the 3' end of the sense strand portion. In another embodiment, the asymmetry of an RNA silencing agent of the disclosure may be enhanced such that there is at least one base pair comprising a rare nucleotide, e g., inosine (I). In certain embodiments, the base pair is selected from the group consisting of an I:A, I:U and I:C. In yet another embodiment, the asymmetry of an RNA silencing agent of the disclosure may be enhanced such that there is at least one base pair comprising a modified nucleotide. In certain 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.

[0138] 3) RNA Silencing Agents with Enhanced Stability

[0139]

[0007] The RNA silencing agents of the present application can be modified to improve stability in serum or in growth medium for cell cultures. In order to enhance the stability, the 3 '-residues may be stabilized against degradation, e.g., they may be selected such that they consist of purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine by 2'-deoxythymidine is tolerated and does not affect the efficiency of RNA interference.

[0008] In a one aspect, the present application features RNA silencing agents that include first and second strands wherein the second strand and / or first strand is modified by the substitution of internal nucleotides with modified nucleotides, such that in vivo stability is enhanced as compared to a corresponding unmodified RNA silencing agent. As defined herein, an "internal" nucleotide is one occurring at any position other than the 5' end or 3' end of nucleic acid molecule, polynucleotide or oligonucleotide. An internal nucleotide can be within a single-stranded molecule or within a strand of a duplex or double-stranded molecule. In one embodiment, the sense strand and / or antisense strand is modified by the substitution of at least one internal nucleotide. In another embodiment, the sense strand and / or antisense strand is modified by the substitution of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more internal nucleotides. In another embodiment, the sense strand and / or antisense strand is modified by the substitution of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the internal nucleotides. In yet another embodiment, the sense strand and / or antisense strand is modified by the substitution of all of the internal nucleotides.

[0140]

[0009] In one aspect, the present application features RNA silencing agents that are at least 80% chemically modified. In certain embodiments, the RNA silencing agents may be fully chemically modified, i.e., 100% of the nucleotides are chemically modified. In another aspect, the present application features RNA silencing agents comprising 2’-OH ribose groups that are at least 80% chemically modified. In certain embodiments, the RNA silencing agents comprise 2’-OH ribose groups that are about 80%, 85%, 90%, 95%, or 100% chemically modified.

[0141]

[0010] In certain embodiments, the RNA silencing agents may contain at least one modified nucleotide analogue. The nucleotide analogues may be located at positions where the target-specific silencing activity, e.g., the RNAi mediating activity or translational repression activity is not substantially affected, e g., in a region at the 5'-end and / or the 3'-end of the siRNA molecule. Moreover, the ends may be stabilized by incorporating modified nucleotide analogues.

[0142]

[0011] Exemplary nucleotide analogues include sugar- and / or backbone-modified ribonucleotides (i.e., include modifications to the phosphate-sugar backbone). For example, the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. In exemplary backbone-modified ribonucleotides, the phosphoester group connecting to adjacent ribonucleotides is replaced by a modified group, e.g., of phosphothioate group. In exemplary sugar-modified ribonucleotides, the 2' OH-group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or ON, wherein R is Ci-Ce alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.

[0143]

[0012] In certain embodiments, the modifications are 2'-fluoro, 2'-amino and / or 2'- thio modifications. Modifications include 2'-fluoro-cytidine, 2'-fluoro-uridine, 2'-fluoro- adenosine, 2'-fluoro-guanosine, 2'-amino-cytidine, 2'-amino-uridine, 2'-amino-adenosine, 2'- amino-guanosine, 2,6-diaminopurine, 4-thio-uridine, and / or 5-amino-allyl-uridine. In a certain embodiment, the 2'-fluoro ribonucleotides are every uridine and cytidine. Additional exemplary modifications include 5-bromo-uridine, 5 -iodo-uridine, 5-methyl-cytidine, ribothymidine, 2-aminopurine, 2'-amino-butyryl-pyrene-uridine, 5-fluoro-cytidine, and 5-fluoro- uridine. 2'-deoxy-nucleotides and 2'-0me nucleotides can also be used within modified RNA- silencing agents moities of the instant disclosure. Additional modified residues include, deoxy- abasic, inosine, N3-methyl-uridine, N6,N6-dimethyl-adenosine, pseudouridine, purine ribonucleoside and ribavirin. In a certain embodiment, the 2' moiety is a methyl group such that the linking moiety is a 2'-O-methyl oligonucleotide.

[0144]

[0013] In a certain embodiment, the RNA silencing agent of the present application comprises Locked Nucleic Acids (LNAs). LNAs comprise sugar-modified nucleotides that resist nuclease activities (are highly stable) and possess single nucleotide discrimination for mRNA (Elmen et al., Nucleic Acids Res., (2005), 33(1): 439-447; Braasch et al. (2003) Biochemistry 42:7967-7975, Petersen et al. (2003) Trends Biotechnol 21 :74-81). These molecules have 2'-O,4'-C-ethylene-bridged nucleic acids, with possible modifications such as 2'-deoxy-2"-fluorouridine. Moreover, LNAs increase the specificity of oligonucleotides by constraining the sugar moiety into the 3'-endo conformation, thereby pre-organizing the nucleotide for base pairing and increasing the melting temperature of the oligonucleotide by as much as 10 °C per base.

[0145]

[0014] In another exemplary embodiment, the RNA silencing agent of the present application comprises Peptide Nucleic Acids (PNAs). PNAs comprise modified nucleotides in which the sugar-phosphate portion of the nucleotide is replaced with a neutral 2-amino ethylglycine moiety capable of forming a polyamide backbone , which is highly resistant to nuclease digestion and imparts improved binding specificity to the molecule (Nielsen, et al., Science, (2001), 254: 1497-1500).

[0015] Also contemplated are nucleobase-modified ribonucleotides, i.e., ribonucleotides, containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Bases may be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine; adenosine and / or guanosines modified at the 8 position, e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. It should be noted that the above modifications may be combined.

[0146]

[0016] In other embodiments, cross-linking can be employed to alter the pharmacokinetics of the RNA silencing agent, for example, to increase half-life in the body. Thus, the present application includes RNA silencing agents having two complementary strands of nucleic acid, wherein the two strands are crosslinked. The present application also includes RNA silencing agents which are conjugated or unconjugated (e.g., at its 3' terminus) to another moiety (e.g. a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye), or the like). Modifying siRNA derivatives in this way may improve cellular uptake or enhance cellular targeting activities of the resulting siRNA derivative as compared to the corresponding siRNA, are useful for tracing the siRNA derivative in the cell, or improve the stability of the siRNA derivative compared to the corresponding siRNA.

[0147]

[0017] Other exemplary modifications include: (a) 2' modification, e.g., provision of a 2' OMe moiety on a U in a sense or antisense strand, but especially on a sense strand, or provision of a 2' OMe moiety in a 3' overhang, e.g., at the 3' terminus (3' terminus means at the 3' atom of the molecule or at the most 3' moiety, e.g., the most 3' P or 2' position, as indicated by the context); (b) modification of the backbone, e.g., with the replacement of an 0 with an S, in the phosphate backbone, e.g., the provision of a phosphorothioate modification, on the U or the A or both, especially on an antisense strand; e g., with the replacement of a O with an S; (c) replacement of the U with a C5 amino linker; (d) replacement of an A with a G (sequence changes can be located on the sense strand and not the antisense strand in certain embodiments); and (d) modification at the 2', 6', 7', or 8' position. Exemplary embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications. Yet other exemplary modifications include the use of a methylated P in a 3' overhang, e.g., at the 3' terminus; combination of a 2' modification, e.g., provision of a 2' O Me moiety and modification of the backbone, e.g., with the replacement of a O with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P, in a 3' overhang, e g., at the 3' terminus; modification with a 3' alkyl; modification with an abasic pyrrolidone in a 3' overhang, e g., at the 3' terminus; modification with naproxen, ibuprofen, or other moi eties which inhibit degradation at the 3' terminus.

[0148] Heavily modified RNA silencing agents

[0149]

[0018] In certain embodiments, the RNA silencing agent comprises at least 80% chemically modified nucleotides. In certain embodiments, the RNA silencing agent is fully chemically modified, i.e., 100% of the nucleotides are chemically modified.

[0150]

[0019] In certain embodiments, the RNA silencing agent is 2’-O-methyl rich, i.e., comprises greater than 50% 2’-O-methyl content. In certain embodiments, the RNA silencing agent comprises at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% 2’- O-methyl nucleotide content. In certain embodiments, the RNA silencing agent comprises at least about 70% 2’-O-methyl nucleotide modifications. In certain embodiments, the RNA silencing agent comprises between about 70% and about 90% 2’-O-methyl nucleotide modifications. In certain embodiments, the RNA silencing agent is a dsRNA comprising an antisense strand and sense strand. In certain embodiments, the antisense strand comprises at least about 70% 2’-O-methyl nucleotide modifications. In certain embodiments, the antisense strand comprises between about 70% and about 90% 2’-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises at least about 70% 2’-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises between about 70% and about 90% 2’-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises between 100% 2’-O-methyl nucleotide modifications.

[0151]

[0020] 2’-O-methyl rich RNA silencing agents and specific chemical modification patterns are further described in U.S. 20200087663A1 and U.S. 20210115442A1, each of which is incorporated herein by reference.

[0152] Internucleotide linkage modifications

[0153]

[0021] In certain embodiments, at least one internucleotide linkage, intersubunit linkage, or nucleotide backbone is modified in the RNA silencing agent. In certain embodiments, all of the internucleotide linkages in the RNA silencing agent are modified. In certain embodiments, the modified internucleotide linkage comprises a phosphorothioate internucleotide linkage. In certain embodiments, the RNA silencing agent comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 phosphorothioate internucleotide linkages. In certain embodiments, the RNA silencing agent comprises 4-16 phosphorothioate internucleotide linkages. In certain embodiments, the RNA silencing agent comprises 8-13 phosphorothioate internucleotide linkages. In certain embodiments, the RNA silencing agent is a dsRNA comprising an antisense strand and a sense strand, each comprising a 5’ end and a 3’ end. In certain embodiments, the nucleotides at positions 1 and 2 from the 5’ end of sense strand are connected to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, the nucleotides at positions 1 and 2 from the 3’ end of sense strand are connected to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, the nucleotides at positions 1 and 2 from the 5’ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, the nucleotides at positions 1-2 to 1-8 from the 3’ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, the nucleotides at positions 1-2, 1-3, 1-4, 1- 5, 1-6, 1-7, or 1-8 from the 3’ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, the nucleotides at positions 1-2 to 1-7 from the 3’ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate internucleotide linkages.

[0154]

[0022] In one aspect, the disclosure provides a modified oligonucleotide, said oligonucleotide having a 5’ end, a 3’ end, that is complementary to a target, wherein the oligonucleotide comprises a sense and antisense strand, and at least one modified intersubunit linkage of Formula (I):

[0155] (i); wherein:

[0156] B is a base pairing moiety;

[0157] W is selected from the group consisting of O, OCH2, OCH, CH2, and CH;

[0158] X is selected from the group consisting of halo, hydroxy, and C1-6 alkoxy;

[0159] Y is selected from the group consisting of O , OH, OR, NH , NH2, S ", and SH;

[0160] Z is selected from the group consisting of O and CH2;

[0161] R is a protecting group; and

[0162] ~ is an optional double bond.

[0163]

[0023] In an embodiment of Formula (I), when W is CH, = is a double bond.

[0164]

[0024] In an embodiment of Formula (I), when W selected from the group consisting of O, OCH2, OCH, CH2, ~ is a single bond.

[0165]

[0025] In an embodiment of Formula (I), when Y is O , either Z or W is not O.

[0166]

[0026] In an embodiment of Formula (I), Z is CH2 and W is CH2. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula (II):

[0167]

[0027] In an embodiment of Formula (I), Z is CH2 and W is O. In another embodiment, wherein the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula (III):

[0168]

[0169]

[0028] In an embodiment of Formula (I), Z is O and W is CH2. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula (IV):

[0170] (IV).

[0171]

[0029] In an embodiment of Formula (I), Z is O and W is CH. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula V:

[0172] (V).

[0173]

[0030] In an embodiment of Formula (I), Z is O and W is OCH2. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula VI:

[0174] (VI).

[0175]

[0031] In an embodiment of Formula (I), Z is CH2 and W is CH. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula VII:

[0176]

[0032] In an embodiment of Formula (I), the base pairing moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0033] In an embodiment, the modified oligonucleotide is incorporated into siRNA, said modified siRNA having a 5’ end, a 3’ end, that is complementary to a target, wherein the siRNA comprises a sense and antisense strand, and at least one modified intersubunit linkage of any one or more of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), or Formula (VII).

[0034] In an embodiment, the modified oligonucleotide is incorporated into siRNA, said modified siRNA having a 5’ end, a 3 ’ end, that is complementary to a target and comprises a sense and antisense strand, wherein the siRNA comprises at least one modified intersubunit linkage is of Formula VIII:

[0177] (VIII); wherein:

[0178] D is selected from the group consisting of O, OCH2, OCH, CH2, and CH;

[0179] C is selected from the group consisting of O , OH, OR1, NH", NH2, S", and SH;

[0180] A is selected from the group consisting of O and CH2;

[0181] R1is a protecting group;

[0182] ~ is an optional double bond; and the intersubunit is bridging two optionally modified nucleosides.

[0183]

[0035] In an embodiment, when C is O , either A or D is not O.

[0184]

[0036] In an embodiment, D is CH2. In another embodiment, the modified intersubunit linkage of Formula VIII is a modified intersubunit linkage of Formula (IX):

[0185] (IX).

[0186]

[0037] In an embodiment, D is O. In another embodiment, the modified intersubunit linkage of Formula VIII is a modified intersubunit linkage of Formula (X):

[0187]

[0038] In an embodiment, D is CH2. In another embodiment, the modified intersubunit linkage of Formula (VIII) is a modified intersubunit linkage of Formula (XI):

[0188] (XI).

[0189]

[0039] In an embodiment, D is CH. In another embodiment, the modified intersubunit linkage of Formula VIII is a modified intersubunit linkage of Formula (XII):

[0190]

[0040] In another embodiment, the modified intersubunit linkage of Formula (VII) is a modified intersubunit linkage of Formula (XIV):

[0191] (XIV).

[0192]

[0041] In an embodiment, D is OCH2. In another embodiment, the modified intersubunit linkage of Formula (VII) is a modified intersubunit linkage of Formula (XIII):

[0193]

[0042] In another embodiment, the modified intersubunit linkage of Formula (VII) is a modified intersubunit linkage of Formula (XXa):

[0194] (XXa).

[0195]

[0043] In an embodiment of the modified siRNA linkage, each optionally modified nucleoside is independently, at each occurrence, selected from the group consisting of adenosine, guanosine, cytidine, and uridine.

[0196]

[0044] In certain exemplary embodiments of Formula (I), W is O. In another embodiment, W is CH2. In yet another embodiment, W is CH.

[0197]

[0045] In certain exemplary embodiments of Formula (I), X is OH. In another embodiment, X is OCH3. In yet another embodiment, X is halo.

[0198]

[0046] In a certain embodiment of Formula (I), the modified siRNA does not comprise a 2’-fluoro substituent.

[0199]

[0047] In an embodiment of Formula (I), Y is O". In another embodiment, Y is OH. In yet another embodiment, Y is OR. In still another embodiment, Y is NH". In an embodiment, Y is NH2. In another embodiment, Y is S . In yet another embodiment, Y is SH.

[0200]

[0048] In an embodiment of Formula (I), Z is O. In another embodiment, Z is CH2.

[0201]

[0049] In an embodiment, the modified intersubunit linkage is inserted on position 1-2 of the antisense strand. In another embodiment, the modified intersubunit linkage is inserted on position 6-7 of the antisense strand. In yet another embodiment, the modified intersubunit linkage is inserted on position 10-11 of the antisense strand. In still another embodiment, the modified intersubunit linkage is inserted on position 19-20 of the antisense strand. In an embodiment, the modified intersubunit linkage is inserted on positions 5-6 and 18-19 of the antisense strand.

[0202]

[0050] In an exemplary embodiment of the modified siRNA linkage of Formula (VIII), C is O . In another embodiment, C is OH. In yet another embodiment, C is OR1. In still another embodiment, C is NH". In an embodiment, C is NH2. In another embodiment, C is S". In yet another embodiment, C is SH.

[0203]

[0051] In an exemplary embodiment of the modified siRNA linkage of Formula (VIII), A is O. In another embodiment, A is CH2. In yet another embodiment, C is OR1. In still another embodiment, C is NH . In an embodiment, C is NH2. In another embodiment, C is S . In yet another embodiment, C is SH.

[0204]

[0052] In a certain embodiment of the modified siRNA linkage of Formula (VIII), the optionally modified nucleoside is adenosine. In another embodiment of the modified siRNA linkage of Formula (VIII), the optionally modified nucleoside is guanosine. In another embodiment of the modified siRNA linkage of Formula (VUI), the optionally modified nucleoside is cytidine. In another embodiment of the modified siRNA linkage of Formula (VIII), the optionally modified nucleoside is uridine.

[0205]

[0053] In an embodiment of the modified siRNA linkage, wherein the linkage is inserted on position 1-2 of the antisense strand. In another embodiment, the linkage is inserted on position 6-7 of the antisense strand. In yet another embodiment, the linkage is inserted on position 10-11 of the antisense strand. In still another embodiment, the linkage is inserted on position 19-20 of the antisense strand. In an embodiment, the linkage is inserted on positions 5-6 and 18-19 of the antisense strand.

[0206]

[0054] In certain embodiments of Formula (I), the base pairing moiety B is adenine. In certain embodiments of Formula (I), the base pairing moiety B is guanine. In certain embodiments of Formula (I), the base pairing moiety B is cytosine. In certain embodiments of Formula (I), the base pairing moiety B is uracil.

[0207]

[0055] In an embodiment of Formula (I), W is O. In an embodiment of Formula (I), W is CH2. In an embodiment of Formula (I), W is CH.

[0208]

[0056] In an embodiment of Formula (I), X is OH. In an embodiment of Formula (I), X is OCH3. In an embodiment of Formula (I), X is halo.

[0209]

[0057] In an exemplary embodiment of Formula (I), the modified oligonucleotide does not comprise a 2’-fluoro substituent.

[0210]

[0058] In an embodiment of Formula (I), Y is O . In an embodiment of Formula (I), Y is OH. In an embodiment of Formula (I), Y is OR. In an embodiment of Formula (I), Y is NH . In an embodiment of Formula (I), Y is NH2. In an embodiment of Formula (I), Y is S". In an embodiment of Formula (I), Y is SH.

[0211]

[0059] In an embodiment of Formula (I), Z is O. In an embodiment of Formula (I), Z is CH2

[0060] In an embodiment of the Formula (I), the linkage is inserted on position 1-2 of the antisense strand. In another embodiment of Formula (I), the linkage is inserted on position 6-7 of the antisense strand In yet another embodiment of Formula (I), the linkage is inserted on position 10-11 of the antisense strand. In still another embodiment of Formula (I), the linkage is inserted on position 19-20 of the antisense strand. In an embodiment of Formula (I), the linkage is inserted on positions 5-6 and 18-19 of the antisense strand.

[0212]

[0061] Modified intersubunit linkages are further described in U.S. Patent Publication No. 2020 / 0385740A1, and U.S.S.N. 17 / 213,852, each of which is incorporated herein by reference.

[0213] 4) Conjugated Functional Moieties

[0214]

[0062] In other embodiments, RNA silencing agents may be modified with one or more functional moieties. A functional moiety is a molecule that confers one or more additional activities to the RNA silencing agent. In certain embodiments, the functional moieties enhance cellular uptake by target cells (e.g., neuronal cells). Thus, the disclosure includes RNA silencing agents which are conjugated or unconjugated (e.g., at its 5’ and / or 3' terminus) to another moiety (e g. a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye), or the like. The conjugation can be accomplished by methods known in the art, e.g., using the methods of Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001) (describes nucleic acids loaded to polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al., J. Control Release 53(1-3): 137-43 (1998) (describes nucleic acids bound to nanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4:55-8 (1994) (describes nucleic acids linked to intercalating agents, hydrophobic groups, polycations or PACA nanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10 (1995) (describes nucleic acids linked to nanoparticles).

[0215]

[0063] In a certain embodiment, the functional moiety is a hydrophobic moiety. In a certain embodiment, the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides and nucleoside analogs, endocannabinoids, and vitamins. In a certain embodiment, the steroid selected from the group consisting of cholesterol and Lithocholic acid (LCA). In a certain embodiment, the fatty acid selected from the group consisting of Eicosapentaenoic acid (EP A), Docosahexaenoic acid (DHA) and Docosanoic acid (DCA). In a certain embodiment, the vitamin selected from the group consisting of choline, vitamin A, vitamin E, and derivatives or metabolites thereof. In a certain embodiment, the vitamin is selected from the group consisting of retinoic acid and alpha- tocopheryl succinate.

[0216]

[0064] In a certain embodiment, an RNA silencing agent of disclosure is conjugated to a lipophilic moiety. In one embodiment, the lipophilic moiety is a ligand that includes a cationic group. In another embodiment, the lipophilic moiety is attached to one or both strands of an siRNA. In an exemplary embodiment, the lipophilic moiety is attached to one end of the sense strand of the siRNA. In another exemplary embodiment, the lipophilic moiety is attached to the 3' end of the sense strand. In certain embodiments, the lipophilic moiety is selected from the group consisting of cholesterol, vitamin E, vitamin K, vitamin A, folic acid, a cationic dye (e.g., Cy3). In an exemplary embodiment, the lipophilic moiety is cholesterol. Other lipophilic moi eties include cholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyl oxy hexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

[0217]

[0065] In certain embodiments, the functional moieties may comprise one or more ligands tethered to an RNA silencing agent to improve stability, hybridization thermodynamics with a target nucleic acid, targeting to a particular tissue or cell-type, or cell permeability, e.g., by an endocytosis-dependent or -independent mechanism. Ligands and associated modifications can also increase sequence specificity and consequently decrease off-site targeting. A tethered ligand can include one or more modified bases or sugars that can function as intercalators. These can be located in an internal region, such as in a bulge of RNA silencing agent / target duplex. The intercalator can be an aromatic, e.g., a polycyclic aromatic or heterocyclic aromatic compound. A polycyclic intercalator can have stacking capabilities, and can include systems with 2, 3, or 4 fused rings. The universal bases described herein can be included on a ligand. In one embodiment, the ligand can include a cleaving group that contributes to target gene inhibition by cleavage of the target nucleic acid. The cleaving group can be, for example, a bleomycin (e.g., bleomycin-A5, bleomycin-A2, or bleomycin-B2), pyrene, phenanthroline (e g., O-phenanthroline), a polyamine, a tripeptide (e g., lys-tyr-lys tripeptide), or a metal ion chelating group. The metal ion chelating group can include, e.g., an Lu(III) or EU(III) macrocyclic complex, a Zn(II) 2,9-dimethylphenanthroline derivative, a Cu(II) terpyridine, or acridine, which can promote the selective cleavage of target RNA at the site of the bulge by free metal ions, such as Lu(III). In some embodiments, a peptide ligand can be tethered to a RNA silencing agent to promote cleavage of the target RNA, e.g., at the bulge region. For example, l,8-dimethyl-l,3,6,8,10,13-hexaazacyclotetradecane (cyclam) can be conjugated to a peptide (e.g., by an amino acid derivative) to promote target RNA cleavage. A tethered ligand can be an aminoglycoside ligand, which can cause an RNA silencing agent to have improved hybridization properties or improved sequence specificity. Exemplary aminoglycosides include glycosylated polylysine, galactosylated polylysine, neomycin B, tobramycin, kanamycin A, and acridine conjugates of aminoglycosides, such as Neo-N- acridine, Neo-S-acridine, Neo-C-acridine, Tobra-N-acridine, and KanaA-N-acridine. Use of an acridine analog can increase sequence specificity. For example, neomycin B has a high affinity for RNA as compared to DNA, but low sequence-specificity. An acridine analog, neo- 5-acridine, has an increased affinity for the HIV Rev-response element (RRE). In some embodiments, the guanidine analog (the guanidinoglycoside) of an aminoglycoside ligand is tethered to an RNA silencing agent. In a guanidinoglycoside, the amine group on the amino acid is exchanged for a guanidine group. Attachment of a guanidine analog can enhance cell permeability of an RNA silencing agent. A tethered ligand can be a poly-arginine peptide, peptoid or peptidomimetic, which can enhance the cellular uptake of an oligonucleotide agent.

[0218]

[0066] Exemplary ligands are coupled, either directly or indirectly, via an intervening tether, to a ligand-conjugated carrier. In certain embodiments, the coupling is through a covalent bond. In certain embodiments, the ligand is attached to the carrier via an intervening tether. In certain embodiments, a ligand alters the distribution, targeting or lifetime of an RNA silencing agent into which it is incorporated. In certain embodiments, a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand.

[0219]

[0067] Exemplary ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified RNA silencing agent, or a polymeric molecule comprising any combination of monomers described herein and / or natural or modified ribonucleotides. Ligands in general can include therapeutic modifiers, e g., for enhancing uptake; diagnostic compounds or reporter groups e g , for monitoring distribution; cross-linking agents; nuclease-resistance conferring moieties; and natural or unusual nucleobases. General examples include lipophiles, lipids, steroids (e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid), vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins, protein binding agents, integrin targeting molecules, polycationics, peptides, polyamines, and peptide mimics. Ligands can include a naturally occurring substance, (e g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); amino acid, or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L- glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

[0220]

[0068] Ligands can also include targeting groups, e g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine (GalNAc) or derivatives thereof, N-acetyl-glucosamine, multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B 12, biotin, or an RGD peptide or RGD peptide mimetic. Other examples of ligands include dyes, intercalating agents (e.g. acridines and substituted acridines), crosslinkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine, phenanthroline, pyrenes), lys-tyr- lys tripeptide, aminoglycosides, guanidium aminoglycodies, artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g, cholesterol (and thio analogs thereof), cholic acid, cholanic acid, lithocholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, glycerol (e.g., esters (e.g., mono, bis, or tris fatty acid esters, e.g., Cio, Cn, C12, C13, C14, C15, Ci6, C17, Cis, C19, or C20 fatty acids) and ethers thereof, e.g., Cio, Cn, C12, C13, C14, C15, Ci6, C17, Cis, C19, or C2o alkyl; e.g., l,3-bis-O(hexadecyl)glycerol, l,3-bis-O(octaadecyl)glycerol), geranyl oxy hexyl group, hexadecylglycerol, borneol, menthol, 1,3 -propanediol, heptadecyl group, palmitic acid, stearic acid (e.g., glyceryl distearate), oleic acid, myristic acid, 03- (oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport / absorption facilitators (e.g., aspirin, naproxen, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP or AP. In certain embodiments, the ligand is GalNAc or a derivative thereof.

[0221]

[0069] Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N- acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-kB.

[0222]

[0070] The ligand can be a substance, e.g., a drug, which can increase the uptake of the RNA silencing agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin. The ligand can increase the uptake of the RNA silencing agent into the cell by activating an inflammatory response, for example. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNFD), interleukin- 1 beta, or gamma interferon. In one aspect, the ligand is a lipid or lipid- based molecule. Such a lipid or lipid-based molecule can bind a serum protein, e g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and / or (c) can be used to adjust binding to a serum protein, e.g., HSA. A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney. In a certain embodiment, the lipid based ligand binds HSA. A lipid-based ligand can bind HSA with a sufficient affinity such that the conjugate will be distributed to a non-kidney tissue. However, it is contemplated that the affinity not be so strong that the HSA-ligand binding cannot be reversed. In another embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.

[0223]

[0071] In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These can be useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL).

[0224]

[0072] In another aspect, the ligand is a cell-permeation agent, such as a helical cellpermeation agent. In certain embodiments, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent can be an alpha-helical agent, which may have a lipophilic and a lipophobic phase.

[0225]

[0073] The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to oligonucleotide agents can affect pharmacokinetic distribution of the RNA silencing agent, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. The peptide moiety can be an L-peptide or D- peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one- compound (OBOC) combinatorial library (Lam et al., Nature 354:82-84, 1991). In exemplary embodiments, the peptide or peptidomimetic tethered to an RNA silencing agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

[0226]

[0074] In certain embodiments, the functional moiety is linked to the 5’ end and / or 3’ end of the RNA silencing agent of the disclosure. In certain embodiments, the functional moiety is linked to the 5’ end and / or 3’ end of an antisense strand of the RNA silencing agent of the disclosure. In certain embodiments, the functional moiety is linked to the 5’ end and / or 3’ end of a sense strand of the RNA silencing agent of the disclosure. In certain embodiments, the functional moiety is linked to the 3 ’ end of a sense strand of the RNA silencing agent of the disclosure.

[0227]

[0075] In certain embodiments, the functional moiety is linked to the RNA silencing agent by a linker. In certain embodiments, the functional moiety is linked to the antisense strand and / or sense strand by a linker. In certain embodiments, the functional moiety is linked to the 3’ end of a sense strand by a linker. In certain embodiments, the linker comprises a divalent or trivalent linker. In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof. In certain embodiments, the divalent or trivalent linker is selected from: is 1, 2, 3, 4, or 5.

[0076] In certain embodiments, the linker further comprises a phosphodiester or phosphodiester derivative. In certain embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of: nd wherein X is O, S or BH3.

[0228]

[0077] The various functional moieties of the disclosure and means to conjugate them to RNA silencing agents are described in further detail in W02017 / 030973A1 and

[0229] WO2018 / 031933A2, incorporated herein by reference.

[0230] Branched RNA Compounds

[0231]

[0078] Two or more RNA silencing agents as disclosed supra, may be connected to one another by a linker to form a branched RNA compound. In certain embodiments, the branched RNA compound consists of two siRNAs to form a di-branched siRNA (“di-siRNA”) scaffolding for delivering two siRNAs. In representative embodiments, the nucleic acids of the branched RNA compound each comprise an antisense strand (or portions thereof), wherein the antisense strand has sufficient complementarity to a target mRNA to mediate an RNA- mediated silencing mechanism (e.g. RNAi).

[0079] In exemplary embodiments, the branched RNA compounds may have two to eight RNA silencing agents attached through a linker. In branched RNA compounds of the present application have two to three RNA molecules. In an embodiment, the RNA molecules independently have substantial chemical stabilization (e.g., at least 40% of the constituent bases are chemically-modified, such as 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In an exemplary embodiment, the RNA molecules have full chemical stabilization (i.e., all the constituent bases are chemically-modified). In some embodiments, branched RNA compounds comprise one or more single-stranded phosphorothioated tails, each independently having two to twenty nucleotides. In a non-limiting embodiment, each single-stranded tail has two to ten nucleotides.

[0232]

[0080] In certain embodiments, branched RNA compounds are characterized by three properties: (1) a branched structure, (2) full chemical stabilization, and (3) the presence of a single-stranded tail comprising phosphorothioate linkers. In certain embodiments, branched RNA compounds have 2 or 3 branches. It is believed that the increased overall size of the branched structures promotes increased uptake. Also, without being bound by a particular theory of activity, multiple adjacent branches (e.g., 2 or 3) are believed to allow each branch to act cooperatively and thus dramatically enhance rates of internalization, trafficking and release.

[0233]

[0081] Branched RNA compounds are provided in various structurally diverse embodiments. In some embodiments nucleic acids attached 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 attached at their 3’ or 5’ end. Combinations of siRNA and single stranded oligonucleotides could also be used for dual function. In another embodiment, short nucleic acids complementary to the gapmers, mixmers, miRNA inhibitors, SSOs, PMOs, and PNAs are used to carry these active single-stranded nucleic acids and enhance distribution and cellular internalization. The short duplex region has a low melting temperature (Tm ~37 °C) for fast dissociation upon internalization of the branched structure into the cell.

[0234]

[0082] The Di-siRNA branched RNA compounds may comprise chemically diverse conjugates, such as the functional moieties described above. Conjugated bioactive ligands may be used to enhance cellular specificity and to promote membrane association, internalization, and serum protein binding. Examples of bioactive moieties to be used for conjugation include DHA, GalNAc, and cholesterol. These moieties can be attached to Di-siRNA either through the connecting linker or spacer, or added via an additional linker or spacer attached to another free siRNA end.

[0235]

[0083] The presence of a branched structure improves the level of tissue retention in target tissues compared to non-branched compounds of identical chemical composition. Branched RNA compounds exhibit unexpectedly efficient systemic delivery to a variety of tissues, and very high levels of tissue accumulation.

[0236]

[0084] Branched oligonucleotides comprise a variety of therapeutic nucleic acids, including siRNAs, ASOs, miRNAs, miRNA inhibitors, splice switching, PMOs, PNAs. In some embodiments, branched oligonucleotides further comprise conjugated hydrophobic moieties and exhibit unprecedented silencing and efficacy in vitro and in vivo.

[0237] Alkyl Linkers

[0238]

[0085] The branched RNA compounds of the disclosure comprise two or more RNA molecules, wherein the two or more RNA molecules are connected to one another by an alkyl linker comprising a C1-C14 alkyl (i.e., a single carbon (Ci) up to a chain of 14 consecutive carbons (Cu). In some embodiments, the alkyl linker is a linear alkyl linker. In other embodiments, the alkyl linker is a branched alkyl linker. The alkyl linker of the disclosure promotes adipose tissue uptake and target mRNA silencing. In some embodiments, the alkyl linker promotes adipose tissue uptake and target mRNA silencing after systemic administration to a subject. In some embodiments, the alkyl linker is a Ci alkyl linker, a C2 alkyl linker, a C3 alkyl linker, a C4 alkyl linker, a C5 alkyl linker, a Cs alkyl linker, a Q, alkyl linker, a C7 alkyl linker, a Cs alkyl linker, a C9 alkyl linker, a C10 alkyl linker, a Cn alkyl linker, a C12 alkyl linker, a C13 alkyl linker, or a C14 alkyl linker. In some embodiments, the alkyl linker is a C12 alkyl linker (e.g., dodecane). In some embodiments, the alkyl linker connects two or more RNA molecules via their 3’ ends.

[0239]

[0086] In some embodiments, the branched RNA compound comprising the alkyl linker has increased target mRNA silencing in adipose tissue relative to a branched RNA compound that lacks the alkyl linker.

[0240]

[0087] In some embodiments, the branched RNA compound comprising the alkyl linker has increased target mRNA silencing in adipose tissue relative to a branched RNA compound comprising two or more RNA molecules that are connected to one another by an ethylene glycol linker.

[0088] In some embodiments, the ethylene glycol linker is a triethylene glycol linker or a tetraethylene glycol linker.

[0241]

[0089] In some embodiments, the branched RNA compound comprising the alkyl linker is adipose-tissue selective, such that target mRNA silencing in adipose tissue is at least 50% higher (e.g., 50%, 60%, 70%, 80%, 90%, 100%, or more) than target mRNA silencing in non-adipose tissue, such as one or more or liver, kidney, lung, spleen, and heart.

[0242]

[0090] Branched oligonucleotides, including synthesis and methods of use, are described in greater detail in WO2017 / 132669, incorporated herein by reference.

[0243] Methods of Treatment and Prevention / Methods of Target Silencing

[0244] In one aspect, the present disclosure provides for both prophylactic and therapeutic methods of treating a subject with a disease of adipose tissue. As used herein, a “disease of adipose tissue” refers to a disease that involves disfunction of adipose tissue. Non-limiting examples include insulin resistance, diabetes, dysfunctional hunger and satiety signals, obesity, hypertension, fatty liver disease, lipedema, and Dercum’s disease.

[0245] In another aspect, the present disclosure provides a method for inhibiting expression of a target mRNA in adipose tissue in a subject, the method comprising administering to the subject the branched RNA compound described herein, thereby inhibiting expression of the target mRNA in the adipose tissue in the subject.

[0246] In some embodiments, the branched RNA compound is administered to the subject by one or both of intravenous (IV) injection or subcutaneous (SQ) injection.

[0247] In some embodiments, the adipose tissue is epidydimal adipose tissue (eWAT), perirenal adipose tissue (PR), brown adipose tissue (BAT), Subcutaneous adipose tissue (SC), or a combination thereof.

[0248] In some embodiments, the branched RNA compound inhibits expression of the target mRNA in one or more of liver, kidney, lung, spleen, and heart in the subject by 20% or less relative to a subject that is not administered the branched RNA compound.

[0249] "Treatment," or "treating," as used herein, is defined as the application or administration of a therapeutic agent (e.g., a RNA agent or vector or transgene encoding same) to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has the disease or disorder, a symptom of disease or disorder or a predisposition toward a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder, or the predisposition toward disease.

[0250] In one aspect, the disclosure provides a method for preventing in a subject, a disease or disorder as described above, by administering to the subject a therapeutic agent (e g., an RNAi agent or vector or transgene encoding same). Subjects at risk for the disease can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the disease or disorder, such that the disease or disorder is prevented or, alternatively, delayed in its progression.

[0251] Pharmaceutical Compositions and Methods of Administration

[0252] The disclosure pertains to uses of the above-described agents for prophylactic and / or therapeutic treatments as described infra. Accordingly, the modulators (e g., RNAi agents) of the present disclosure can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, antibody, or modulatory compound and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0253] A pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, intraperitoneal, intramuscular, transdermal (topical), and transmucosal administration. In certain exemplary embodiments, the pharmaceutical composition of the disclosure is administered intravenously. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following example, which is included for purposes of illustration only and is not intended to be limiting.

[0254] EXAMPLES

[0255] Example 1. Design and synthesis of divalent siRNA with various tethering chemistries

[0256] The chemical modification pattern employed for the tested siRNAs consisted of alternating 2'-0Me and 2'-F sugar modifications with terminal phosphorothioate internucleotide modifications for both antisense and sense strands. An asymmetric siRNA scaffold was used in the studies because it enables robust gene silencing compared to blunt scaffold in vivo. To assess the impact of chemical and physical properties of a tethering moiety (i.e., a linker) of divalent-siRNA, commercially available hydrophilic and hydrophobic spacer phosphoramidites were used (TEG and Ci2-spacer phosphoramidites, respectively). This enables building up of various tethering structures by just changing the type and numbers of spacer-phosphoramidite coupling without custom-synthesizing a spacer moiety so that the impact of length and hydrophobicity of the tethering moiety can be evaluated in semithroughput manner. The tethering chemistry was changed from the original single TEG tether to longer TEG tethers (TEGx3). Changing from single TEG to hydrophobic tethers such as a single C12 tether, more hydrophobic and longer multi-Cn tethers (Ci2x3), and further longer TEG-Chx3-TEG tether were incorporated in between two sense strands, respectively (FIG. 1A and FIG. IB)

[0257] Example 2. Tethering chemistry modulates gene silencing in mice fat tissue

[0258] To assess the impact of tethering chemistry on gene silencing in various tissues, Htt mRNA levels were quantified in heart, liver, lung, quadriceps muscle, kidney, spleen, and adipose tissue two weeks after the injection of 500 pg divalent Htt siRNA with five different linkers (FIG. 2).

[0259] The choice of tethering chemistry significantly influenced gene silencing in different tissues. Incorporating a carbon-12 (C12) tethering moiety between the two siRNA strands, remarkably exhibited specific gene silencing in adipose tissue (eWAT), without impacting gene expression in liver, kidney, lung, spleen, and heart, and with only slight silencing in quadriceps muscle.

[0260] In adipose tissue, the mean knockdown seemingly depends on the type of linker. After a single subcutaneous injection of 500 pg divalent siRNA (DIO), the DIO-C12 achieved a substantial knockdown of Htt mRNA in epidi dymal WAT (eWAT), resulting in a -45% mRNA reduction two weeks post-injection compared to the divalent siRNA control. In comparison, single TEG, triple C12, and triple TEG linkers demonstrated only approximately 25% silencing efficiency. Interestingly, the longer TEG-C12-C12-C12-TEG linker did not affect gene silencing compared to the non -targeting divalent siRNA control.

[0261] Remarkably, the DIO-C12 Htt-targeting compound exhibited specific gene silencing in adipose tissue (eWAT), with only slight silencing observed in quadriceps muscle. Significant enhancement in adipose selective gene silencing was observed (DIO-C12 siRNA gives silencing of target mRNA in adipose tissue of about 55%, while only having about 5% silencing of target mRNA in liver tissue. This is in contrast to prior work with monovalent siRNA conjugated to a hydrophobic moiety, such as a DCA-siRNA, which gives silencing in adipose tissue of about 50 %, but a similar level of silencing in liver. Thus the alkyl linkers employed herein are effective to adipose selective silencing of target mRNA.

[0262] To further assess the reproducibility of these discovery and characterize the phenomena in detail, a second gene was silenced, other fat deposits were explored, and the importance of di-valency by comparing monovalent-siRNAs was investigated (FIG. 3). Mecp2 was selected since it is a ubiquitously expressed housekeeping gene with high expression in adipocytes. Thus, we synthesized both monovalent siRNA with a C12 tether and divalent siRNA with a C12 tether (Mono-Ci2 Mecp2 and DIO-C12 Mecp2, respectively) and isolated four different fat tissue deposits from eight-month-old mice.

[0263] The silencing efficiency varied across different fat deposits (FIG. 3). For the subcutaneous (SC) fat, no significant silencing was observed, although both the monovalent and divalent siRNAs showed a trend towards reduced Mecp2 expression. In all other adipose tissues, only the divalent construct showed significant silencing. Although tendencies of silencing in various adipose type were observed with the monovalent construct, the efficacy was not as significant as DIO-C12 Mecp2. In the eWAT deposit, the DIO-C12 Mecp2 compound achieved 60% mRNA silencing, while the Mono-Ci2 Mecp2 compound only achieved 23%. The perirenal (PR) fat deposit showed comparable silencing for the DIO-C12 Mecp2 compound as observed in eWAT, whereas the Mono-Ci2 Mecp2 yielded an average silencing of 7%. For brown adipose tissue (BAT), located near the injection site, the divalent compound resulted in 60% average silencing. The monovalent siRNA showed comparable average silencing of 55% in BAT, although the silencing was not significantly different compared to the control group. Moreover, the divalent siRNA only showed a knockdown in muscle, whereas the monovalent siRNA resulted in knockdown in both muscle and kidney (FIG. 4).

[0264] Incorporation by Reference

[0265] The contents of all cited references (including literature references, patents, patent applications, and websites) that maybe cited throughout this application are hereby expressly incorporated by reference in their entirety for any purpose, as are the references cited therein. The disclosure will employ, unless otherwise indicated, conventional techniques of immunology, molecular biology and cell biology, which are well known in the art.

[0266] The present disclosure also incorporates by reference in their entirety techniques well known in the field of molecular biology and drug delivery. These techniques include, but are not limited to, techniques described in the following publications:

[0267] Atwell et al. J. Mol. Biol. 1997, 270: 26-35;

[0268] Ausubel et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &Sons, NY (1993);

[0269] Ausubel, F.M. et al. eds., SHORT PROTOCOLS IN MOLECULAR BIOLOGY (4th Ed. 1999) John Wiley & Sons, NY. (ISBN 0-471 -32938-X);

[0270] CONTROLLED DRUG BIOAVAILABILITY, DRUG PRODUCT DESIGN AND PERFORMANCE, Smolen and Ball (eds.), Wiley, New York (1984);

[0271] Giege, R. and Ducruix, A. Barrett, CRYSTALLIZATION OF NUCLEIC ACIDS AND PROTEINS, a Practical Approach, 2nd ea., pp. 20 1-16, Oxford University Press, New York, New York, (1999);

[0272] Goodson, in MEDICAL APPLICATIONS OF CONTROLLED RELEASE, vol. 2, pp. 115-138 (1984);

[0273] Hammerling, et al., in: MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS 563-681 (Elsevier, N.Y., 1981; Harlow et al., ANTIBODIES: ALABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);

[0274] Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST (National Institutes of Health, Bethesda, Md. (1987) and (1991);

[0275] Kabat, E. A., et al. (1991 ) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242;

[0276] Kontermann and Dubel eds., ANTIBODY ENGINEERING (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).

[0277] Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990);

[0278] Lu and Weiner eds., CLONING AND EXPRESSION VECTORS FOR GENE FUNCTION ANALYSIS (2001) BioTechniques Press. Westborough, MA. 298 pp. (ISBN 1-881299-21-X).

[0279] MEDICAL APPLICATIONS OF CONTROLLED RELEASE, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974);

[0280] Old, R.W. & S.B. Primrose, PRINCIPLES OF GENE MANIPULATION: AN INTRODUCTION To GENETIC ENGINEERING (3d Ed. 1985) Blackwell Scientific Publications, Boston. Studies in Microbiology; V.2:409 pp. (ISBN 0-632-01318-4).

[0281] Sambrook, J. et al. eds., MOLECULAR CLONING: A LABORATORY MANUAL (2d Ed. 1989) Cold Spring Harbor Laboratory Press, NY. Vols. 1-3. (ISBN 0-87969-309-6).

[0282] SUSTAINED AND CONTROLLED RELEASE DRUG DELIVERY SYSTEMS, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978

[0283] Winnacker, E.L. FROM GENES TO CLONES: INTRODUCTION TO GENE TECHNOLOGY (1987) VCH Publishers, NY (translated by Horst Ibelgaufts). 634 pp. (ISBN 0-89573-614-4).

[0284] Equivalents

[0285] The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting of the disclosure. Scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced herein.

Claims

ClaimsWhat is claimed:

1. A branched RNA compound comprising: two or more RNA molecules comprising 15 to 35 nucleotides in length, and a sequence substantially complementary to a target mRNA, wherein the two or more RNA molecules are connected to one another by an alkyl linker comprising a C1-C14 alkyl.

2. The branched RNA compound of claim 1, wherein the alkyl linker is a C12 alkyl linker.

3. The branched RNA compound of claim 1 or 2, wherein the branched RNA compound comprising the alkyl linker has increased target mRNA silencing in adipose tissue relative to a branched RNA compound that lacks the alkyl linker.

4. The branched RNA compound of any one of claims 1-3, wherein the branched RNA compound comprising the alkyl linker has increased target mRNA silencing in adipose tissue relative to a branched RNA compound comprising two or more RNA molecules that are connected to one another by an ethylene glycol linker.

5. The branched RNA compound of claim 4, wherein the ethylene glycol linker is a triethylene glycol linker or a tetraethylene glycol linker.

6. The branched RNA compound of any one of claims 1-5, wherein said RNA molecule comprises one or both of single stranded RNA (ssRNA) and double stranded RNA (dsRNA).

7. The branched RNA compound of any one of claims 1-6, wherein said RNA molecule comprises an antisense oligonucleotide.

8. The branched RNA compound of any one of claims 1-7, wherein each RNA molecule comprises 15 to 25 nucleotides in length.

9. The branched RNA compound of any one of claims 1-8, wherein each RNA molecule comprises a dsRNA comprising a sense strand and an antisense strand, wherein each antisense strand independently comprises a sequence substantially complementary to the target mRNA.

10. The branched RNA compound of claim 9, wherein the antisense strand comprises full complementary to the target mRNA.

11. The branched RNA compound of claim 9 or 10, wherein the antisense strand and / or sense strand comprises about 15 nucleotides to 25 nucleotides in length.

12. The branched RNA compound of any one of claims 9-11, wherein the antisense strand is 20 nucleotides in length.

13. The branched RNA compound of any one of claims 9-11, wherein the antisense strand is 21 nucleotides in length.

14. The branched RNA compound of any one of claims 9-11, wherein the antisense strand is 22 nucleotides in length.

15. The branched RNA compound of any one of claims 9-14, wherein the sense strand is15 nucleotides in length.

16. The branched RNA compound of any one of claims 9-14, wherein the sense strand is16 nucleotides in length.

17. The branched RNA compound of any one of claims 9-14, wherein the sense strand is 18 nucleotides in length.

18. The branched RNA compound of any one of claims 9-14, wherein the sense strand is 20 nucleotides in length.

19. The branched RNA compound of any one of claims 9-18, wherein the dsRNA comprises a double-stranded region of 15 base pairs to 20 base pairs.

20. The branched RNA compound of any one of claims 9-19, wherein the dsRNA comprises a double-stranded region of 15 base pairs.

21. The branched RNA compound of any one of claims 9-19, wherein the dsRNA comprises a double-stranded region of 16 base pairs.

22. The branched RNA compound of any one of claims 9-19, wherein the dsRNA comprises a double-stranded region of 18 base pairs.

23. The branched RNA compound of any one of claims 9-19, wherein the dsRNA comprises a double-stranded region of 20 base pairs.

24. The branched RNA compound of any one of claims 9-23, wherein the dsRNA comprises a blunt-end.

25. The branched RNA compound of any one of claims 9-24, wherein the dsRNA comprises at least one single stranded nucleotide overhang.

26. The branched RNA compound of any one of claims 9-25, wherein the dsRNA comprises between a 2-nucleotide to 5-nucleotide single stranded nucleotide overhang.

27. The branched RNA compound of any one of claims 9-26, wherein the dsRNA comprises naturally occurring nucleotides.

28. The branched RNA compound of any one of claims 9-27, wherein the dsRNA comprises at least one modified nucleotide.

29. The branched RNA compound of claim 28, wherein said modified nucleotide comprises a 2'-O-methyl modified nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'- deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, or a non-natural base comprising nucleotide.

30. The branched RNA compound of any one of claims 9-29, wherein the dsRNA comprises at least one modified intemucleotide linkage.

31. The branched RNA compound of claim 30, wherein said modified intemucleotide linkage comprises a phosphorothioate intemucleotide linkage.

32. The branched RNA compound of any one of claims 9-31, wherein the dsRNA comprises 4-16 phosphorothioate intemucleotide linkages.

33. The branched RNA compound of any one of claims 9-32, wherein the dsRNA comprises 8-13 phosphorothioate intemucleotide linkages.

34. The branched RNA compound of any one of claims 9-33, wherein said dsRNA comprises at least one modified intemucleotide linkage of Formula I:(i); wherein:B is a base pairing moiety;W is selected from the group consisting of O, OCH2, OCH, CH2, and CH;X is selected from the group consisting of halo, hydroxy, and Ci-6 alkoxy;Y is selected from the group consisting of O , OH, OR, NH , NH2, S ", and SH;Z is selected from the group consisting of O and CH2;R is a protecting group; and— is an optional double bond.

35. The branched RNA compound of any one of claims 9-34, wherein said dsRNA comprises at least 80% chemically modified nucleotides.

36. The branched RNA compound of any one of claims 9-35, wherein said dsRNA is fully chemically modified.

37. The branched RNA compound of any one of claims 9-35, wherein the antisense strand comprises a 5’ phosphate, a 5 ’-alkyl phosphonate, a 5’ alkylene phosphonate, a 5’ alkenyl phosphonate, or a mixture thereof.

38. The branched RNA compound of claim 37, wherein the antisense strand comprises a 5’ vinyl phosphonate.

39. The branched RNA compound of any one of claims 9-38, wherein a functional moiety is linked to the 5’ end and / or 3’ end of the antisense strand.

40. The branched RNA compound of any one of claims 9-39, wherein a functional moiety is linked to the 5’ end and / or 3’ end of the sense strand.

41. The branched RNA compound of any one of claims 9-40, wherein a functional moiety is linked to the 3’ end of the sense strand.

42. The branched RNA compound of any one of claims 39-41, wherein the functional moiety comprises a hydrophobic moiety.

43. The branched RNA compound of claim 42, wherein the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and a mixture thereof.

44. The branched RNA compound of claim 43, wherein the steroid is selected from the group consisting of cholesterol and Lithocholic acid (LCA).

45. The branched RNA compound of claim 43, wherein the fatty acid is selected from the group consisting of Eicosapentaenoic acid (EPA), Docosahexaenoic acid (DHA) and Docosanoic acid (DCA).

46. The branched RNA compound of claim 43, wherein the vitamin is selected from thegroup consisting of choline, vitamin A, vitamin E, derivatives thereof, and metabolites thereof.

47. The branched RNA compound of claim 46, wherein the vitamin is selected from the group consisting of retinoic acid and alpha-tocopheryl succinate.

48. The branched RNA compound of any one of claims 39-47, wherein the functional moiety is linked to the antisense strand and / or sense strand by a linker.

49. The branched RNA compound of claim 49, wherein the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphodiester, a phosphor othioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.

50. The branched RNA compound of claim 48 or 49, wherein the linker comprises a divalent or trivalent linker.

51. The branched RNA compound of claim 50, wherein the divalent or trivalent linker is selected from the group consisting of:wherein n is 1, 2, 3, 4, or 5.

52. The branched RNA compound of claim 51 , wherein when the linker is a trivalent linker, the linker further links a phosphodiester or phosphodiester derivative.

53. The branched RNA compound of claim 52, wherein the phosphodiester or phosphodiester derivative is selected from the group consisting of:andwherein X is O, S or BH3.

54. The branched RNA compound of any one of claims 9-53, wherein the nucleotides at positions 1 and 2 from the 3’ end of sense strand, and the nucleotides at positions 1 and 2 from the 5’ end of antisense strand, are connected to adjacent ribonucleotides via phosphorothioate linkages.

55. A pharmaceutical composition for inhibiting the expression of a target mRNA in an organism, comprising the branched RNA compound of any one of claims 1-54, and a pharmaceutically acceptable carrier.

56. A method for inhibiting expression of a target mRNA in adipose tissue in a subject, the method comprising administering to the subject the branched RNA compound of any one of claims 1-54, thereby inhibiting expression of the target mRNA in the adipose tissue in the subject.

57. A method of treating or preventing a disease of adipose tissue in a subject, comprising administering to the subject in need of such treatment or prevention a therapeutically effectiveamount of the branched RNA compound of any one of claims 1-54, thereby treating or preventing the disease of adipose tissue in the subject.

58. The method of claim 56 or 57, wherein the branched RNA compound is administered to the subject by one or both of intravenous (IV) injection or subcutaneous (SQ) injection.

59. The method of any one of claims 56-58, wherein the adipose tissue is epidydimal adipose tissue (eWAT), perirenal adipose tissue (PR), brown adipose tissue (BAT), Subcutaneous adipose tissue (SC), or a combination thereof.

60. The method of any one of claims 56-59, wherein the branched RNA compound inhibits expression of the target mRNA in one or more of liver, kidney, lung, spleen, and heart in the subject by 20% or less relative to a subject that is not administered the branched RNA compound.

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