Methods and compositions for treating obesity and other disorders

Abstract

Provided herein are CB1R targeting inhibitory RNA oligonucleotides and compositions thereof. Also provided herein are methods for treating CB1R associated disorders with the CB1R targeting inhibitory RNA oligonucleotides and compositions described herein. Also provided herein are inhibitory RNA oligonucleotides specific for ZNF423 mRNA, or TLE3 mRNA, and methods of use thereof.

Description

Attorney Docket: 090147-0584673 METHODS AND COMPOSITIONS FOR TREATING OBESITY AND OTHER DISORDERS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Nos. 63 / 676,596, filed on July 29, 2024, and 63 / 692,899, filed on September 10, 2024, the entire contents of which are incorporated herein by reference. BACKGROUND

[0002] Severe obesity is a global epidemic with serious health consequences. Current treatment options are limited and often associated with adverse side effects. The endocannabinoid system (ECS) plays a crucial role in regulating energy balance, metabolism, and food intake. Cannabinoid receptor 1 (CB1R) is a key component of the ECS, and its overactivation has been linked to obesity and metabolic dysregulation. However, current approaches (e.g., small molecules) targeting CB1R result in neurological side effects due to expression of CB1R in the central nervous system. Accordingly, alternative therapeutics are needed. SUMMARY

[0003] Provided herein are inhibitory RNA oligonucleotides specific for cannabinoid receptor 1 (CB1R) mRNA, as well as compositions comprising such inhibitory RNA oligonucleotides and therapeutic methods of using such inhibitory oligonucleotides to treat various diseases and disorders, including obesity and other metabolic disorders. In certain embodiments, the inhibitory RNA oligonucleotides provided herein are used as part of a combination therapy with additional therapeutic agents provided herein.

[0004] In some aspects, provided herein is an inhibitory RNA oligonucleotide comprising a 10-30 nucleotide long antisense strand comprising a nucleotide sequence complementary to at least 10 contiguous nucleotides of a cannabinoid receptor 1 (CB1R) mRNA targeting sequence selected from Tables 1-13.

[0005] In some embodiments, the nucleotide sequence is fully complementary to the CB1R mRNA targeting sequence.

[0006] In some embodiments, the antisense strand is 19-23 nucleotides in length.

[0007] In some embodiments, the nucleotide sequence is at least 90% identical to an antisense strand sequence selected from Tables 1-13. In some embodiments, the nucleotide sequence is an antisense strand sequence selected from Tables 1-13.

[0008] In some embodiments, the oligonucleotide is a single-stranded oligonucleotide. In some embodiments, the oligonucleotide is a double-stranded oligonucleotide comprising a 1Attorney Docket: 090147-0584673 sense strand that forms a duplex with the antisense strand. In some embodiments, the sense strand comprises a sense strand sequence that is at least 50% complementary to at least 10 contiguous nucleotides of the antisense strand. In some embodiments, the sense strand is 10- 50 nucleotides in length. In some embodiments, the double-stranded oligonucleotide comprises a blunt end at the 5’ and / or 3’ of the antisense strand. In some embodiments, the double-stranded oligonucleotide comprises an overhang at the 5’ and / or 3’ end of the oligonucleotide, and wherein the overhang comprises 1-4 nucleotides. In some embodiments, the double-stranded oligonucleotide is a substrate for Dicer.

[0009] In some embodiments, at least one nucleotide of the oligonucleotide comprises a modification. In some embodiments, the modification is a ribose modification, a base modification, or a phosphate modification. In some embodiments, the ribose modification is a 2’-modification. In some embodiments, the 2’-modification is selected from 2’-fluoro, 2’-O- methyl, 2’-aminoethyl, 2’-O-methoxyethyl, and 2’-deoxy-2’ -fluoro- β -d-arabinonucleic acid.

[0010] In some embodiments, the nucleotide sequence is complementary to one or more CB1R isoforms of CB1R mRNA. In some embodiments, the one or more CB1R isoforms is selected from CB1R, CB1aR, CB1bR, and combinations thereof. In some embodiments, the nucleotide sequence is complementary to a CB1a isoform of CB1R mRNA. In some embodiments, the nucleotide sequence is complementary to a CB1b isoform of CB1R mRNA.

[0011] In some aspects, provided herein is a composition comprising the inhibitory RNA oligonucleotide. In some embodiments, the inhibitory RNA oligonucleotide is formulated in a lipid nanoparticle, a polymeric nanoparticle, or an exosome.

[0012] In some aspects, provided herein is a composition comprising a means for reducing CB1R mRNA levels and / or activity and a pharmaceutically acceptable carrier. In some embodiments, the means is formulated in a lipid nanoparticle.

[0013] In some embodiments, a composition provided herein comprises a second agent. In some embodiments, the second agent is a thermogenic agent. In some embodiments, the thermogenic agent is an mRNA encoding creatine kinase B (CKB). In some embodiments, the second agent is an inhibitory RNA oligonucleotide targeting zinc finger protein 423 (ZNF423, or also ZFP423), or an mRNA encoding creatine kinase B (CKB), leptin, or leptin receptor. In some embodiments, the second agent is an inhibitory RNA oligonucleotide targeting diacylglycerol lipase (DAGL). In some embodiments, the second agent is an inhibitory RNA oligonucleotide targeting transducing-like enhancer of split 3 (TLE3). 2Attorney Docket: 090147-0584673

[0014] In some aspects, provided herein is a method for reducing a CB1R mRNA in a cell, comprises contacting the cell with the inhibitory RNA oligonucleotide or the composition described herein. In some embodiments, the cell is in a subject. In some embodiments the CB1R mRNA is a CB1a isoform mRNA. In some embodiments, the CB1R mRNA is a CB1b isoform mRNA. In some embodiments, the cell is in adipose tissue, liver, pancreas or kidney. In some embodiments, mitochondrial activity is increased in the cell.

[0015] In some aspects, provided herein is a method for treating a disorder in a subject, comprises administering to the subject the inhibitory RNA oligonucleotide or the composition described herein . In some embodiments, the disorder is obesity, leptin resistance, insulin resistance, glucose tolerance, diabetes, renal fibrosis, glucose responsiveness in pancreas through modulation of β-cell function, Liver cirrhosis, fatty liver disease (FLD), cardiovascular, and inflammatory diseases

[0016] In some aspects, provided herein is a method for treating a disorder in a subject, comprising administering a means for reducing CB1R mRNA levels and / or activity. In some embodiments, the disorder is obesity, leptin resistance, insulin resistance, glucose tolerance, diabetes, renal fibrosis, glucose responsiveness in pancreas through modulation of β-cell function, Liver cirrhosis, fatty liver disease (FLD), cardiovascular, and inflammatory diseases.

[0017] In some embodiments, the method comprises administering a second agent sequentially or simultaneously. In some embodiments, the second agent is a thermogenic agent, an inhibitory RNA oligonucleotide targeting ZNF423, or an mRNA encoding CKB, leptin, or leptin receptor. In some embodiments, the second agent is an inhibitory RNA oligonucleotide targeting diacylglycerol lipase (DAGL). In some embodiments, the second agent is an inhibitory RNA oligonucleotide targeting transducing-like enhancer of split 3 (TLE3).

[0018] In some embodiments, white adipose tissue is converted to brown adipose tissue or beige adipose tissue in the subject. In some embodiments, non-shivering thermogenesis is increased in the subject. In some embodiments, lean body mass is preserved in the subject.

[0019] In some embodiments, CB1R mRNA expression is reduced in adipose tissue, optionally CB1R mRNA expression is not reduced in tissue of the central nervous system.

[0020] In some embodiments, the disorder is obesity. In some embodiments, the obesity is caused by a genetic disorder and / or diet. In some embodiments, the genetic disorder is a leptin deficiency or a leptin receptor deficiency. 3Attorney Docket: 090147-0584673

[0021] In some aspects, provided herein, is a method for treating a metabolic disorder in a subject, comprising administering to the subject the inhibitory RNA oligonucleotide or the composition described herein.

[0022] In some aspects, provided herein is a method for treating a metabolic disorder in a subject, comprising administering to the subject a means for reducing CB1R mRNA levels and / or activity.

[0023] In some embodiments, the method comprises administering a second agent sequentially or simultaneously. In some embodiments, the second agent is a thermogenic agent, an inhibitory RNA oligonucleotide targeting ZNF423, or an mRNA encoding creatine kinase B (CKB), leptin, or leptin receptor. In some embodiments, the second agent is an inhibitory RNA oligonucleotide targeting diacylglycerol lipase (DAGL). In some embodiments, the second agent is an inhibitory RNA oligonucleotide targeting transducing- like enhancer of split 3 (TLE3).

[0024] In some embodiments, the metabolic disorder is obesity, leptin resistance, insulin resistance, glucose tolerance, diabetes, renal fibrosis, glucose responsiveness in pancreas through modulation of β-cell function, Liver cirrhosis, fatty liver disease (FLD), cardiovascular, and inflammatory diseases.

[0025] Also provided herein are inhibitory RNA oligonucleotides specific for ZNF423 mRNA, as well as compositions comprising such inhibitory RNA oligonucleotides and therapeutic methods of using such inhibitory oligonucleotides to treat various diseases and disorders, including obesity and other metabolic disorders. In certain embodiments, the inhibitory RNA oligonucleotides provided herein are used as part of a combination therapy with additional therapeutic agents provided herein.

[0026] Also provided herein are inhibitory RNA oligonucleotides specific for DAGL mRNA, as well as compositions comprising such inhibitory RNA oligonucleotides and therapeutic methods of using such inhibitory oligonucleotides to treat various diseases and disorders, including obesity and other metabolic disorders. In certain embodiments, the inhibitory RNA oligonucleotides provided herein are used as part of a combination therapy with additional therapeutic agents provided herein.

[0027] Also provided herein are inhibitory RNA oligonucleotides specific for TLE3 mRNA, as well as compositions comprising such inhibitory RNA oligonucleotides and therapeutic methods of using such inhibitory oligonucleotides to treat various diseases and disorders, including obesity and other metabolic disorders. In certain embodiments, the inhibitory RNA 4Attorney Docket: 090147-0584673 oligonucleotides provided herein are used as part of a combination therapy with additional therapeutic agents provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIGS.1A-H graphs illlustrateexamplary data showing that CCT-217 treatment induced significant weight loss and fat mass reduction in mice. In these graphs, the effects of CCT-217 treatment in diet-induced obese (DIO) C57BL / 6J mice across metabolic, histopathological, biochemical, and inflammatory parameters were compared to control non- diabetic DIO mice. FIG.1A is a line graph showing body weight measurements in treated (“CCT-217 Treated DIO”) and in control mice (“Non Diabetic DIO Control”). FIG.1B is a line graph showing cumulative feed intake in treated mice (“CCT-217 Treated DIO”) and in control mice (“Non Diabetic DIO Control”). FIG.1C is a bar graph showing fat mass composition percentage in treated mice (“CCT-217 Treated DIO”) and in control mice (“Non Diabetic DIO Control”). FIG.1D is a bar graph showing lean mass composition percentage as measured by Echo MRI in treated mice and in control mice. FIG.1E is a bar graph showing inguinal fat weight in treated mice (“CCT-217 Treated DIO”) and in control mice (“Non Diabetic DIO Control”). FIG.1F is a bar graph showing retroperitoneal, gonadal, and mesenteric fat pad weights in treated mice (“CCT-217 Treated DIO”) and in control mice (“Non Diabetic DIO Control”). FIG.1G is a bar graph showing COX1-1 expression levels in inguinal white adipose tissue (iWAT) of treated mice (“CCT-217 Treated DIO”) and in control mice (“Non Diabetic DIO Control”). FIG.1H is a bar graph showing UCP1 expression levels in iWAT of treated mice (“CCT-217 Treated Mice (Increased COX1 expression)”) and in control mice (“Control DIO mice”).

[0029] FIGS.2A–2J graphs illlustrate exemplary data showing various molecular and physiological parameters measured in CCT-217 treated DIO C57BL / 6J mice compared to control non-diabetic DIO mice. FIG.2A is a bar graph showing CB1R expression levels in treated mice (“CCT-217 Treatment Effect On CB1R Expression”) and in control mice (“Vehicle Control”). FIG.2B is a bar graph showing ZFP423 expression levels in treated mice (“CCT-217 Treatment Effects On ZFP423 Expression”) and in control mice (“Baseline DIO Control Values”). FIG.2C is a bar graph showing leptin levels in treated mice (“CCT- 217 Treatment Effects On Leptin Levels”) and in control mice (“Baseline DIO Control Values”). FIG.2D is a bar graph showing C-reactive protein (CRP) levels in treated mice (“CCT-217 Treatment Effects On CRP Levels”) and in control mice (“Baseline DIO Control Values”). FIG.2E is a bar graph showing insulin sensitivity markers in treated mice (“CCT- 217 Treatment Effects On Insulin Levels”) and in control mice (“Baseline DIO Control 5Attorney Docket: 090147-0584673 Values”). FIG.2F is a bar graph showing alkaline phosphatasse (ALP) levels in treated mice (“CCT-217 Treatment Effects On ALP Levels”) and in control mice (“Baseline DIO Control Values”). FIG.2G is a bar graph showing total cholesterol levels in treated mice (“CCT-217 Treatment Effects On CHOL Levels”) and in control mice (“Baseline DIO Control Values”). FIG.2H is a bar graph showing thyroid-stimulating hormone (TSH) levels in treated mice (“CCT-217 Treated DIO”) and in control mice (“Non Diabetic DIO Control”). FIG.2I is a bar graph showing triiodothyronine (T3) levels in treated mice (“Elevated T3 in CCT-217 Treated Mice”) and in control mice (“Control T3 Levels”). FIG.2J is a bar graph showing thyroxine (T4) levels in treated mice (“CCT-217 Treated DIO”) and in control mice (Non Diabetic DIO Control”).

[0030] FIGS.3A-3D illlustratesexemplary data showing that CCT-217 treatment induced adipose remodeling, protects organs, and enhanced bone marrow composition. FIG.3A is a set of microscopy pictures of Hematoxylin and Eosin (H&E)-stained sections of inguinal white adipose tissue (iWAT), Liver, Bone Marrow, and Kidney from CCT-217 treated DIO C57BL / 6J mice (“CCT-217”) compared to control non-diabetic DIO mice (“Control”). FIG. 3B is a bar graph showing average adipocyte cell count per field in the H&E-stained sections of inguinal white adipose tissue (iWAT). In this bar graph, the left and right bar respectively show the average cell count per field in treated mice prior to CCT-217 treatment (“Pre- treatment or control measurements”, left bar) and in mice post CCT-217 treament (“Post- treatment measurements”, right bar). FIG.3C is a bar graph showing average adipocyte cell size in the H&E-stained sections of inguinal white adipose tissue (iWAT). In this bar graph, the left and right bar respectively show the average cell size in treated mice prior to CCT-217 treatment (“Pre-treatment or control measurements”, left bar) and in mice post CCT-217 treament (“Post-treatment measurements”, right bar). FIG.3D is a line graph showing average body temperature over time in treated mice prior to CCT-217 treatment (“Pre- treatment or control measurements”) and in mice post CCT-217 treament (“Post-treatment measurements”), by rectal measurement. DETAILED DESCRIPTION

[0031] In some aspects, provided herein are inhibitory RNA oligonucleotides targeting cannabinoid receptor 1 (CB1R), as well as compositions (e.g., pharmaceutical compositions) comprising such inhibitory RNA oligonucleotides. In some aspects, provided herein are methods for treating obesity and / or metabolic disorders, e.g., type 2 diabetes, leptin resistance, insulin resistance, inflammation, lipid metabolism, and hepatic steatosis, by administering the inhibitory RNA oligonucleotides and / or pharmaceutical compositions 6Attorney Docket: 090147-0584673 provided herein. In some embodiments, the inhibitory RNA oligonucleotides and compositions described herein are used in combination with other agents, such as mRNA encoding creatine kinase B, mRNA encoding leptin, mRNA encoding leptin receptor, and / or an inhibitory oligonucleotide targeting ZPF423.

[0032] CB1R is a component of the endocannabinoid system, influencing various physiological processes, including thermogenesis and energy balance. CB1R gene expression promotes lipogenesis and the maintenance of white adipose tissue, thereby decreasing mitochondrial genesis and energy expenditure. Multiple isoforms of CB1R exist, each with a unique open reading frame (ORF). Without wishing to be bound by theory, targeting these ORFs using inhibitory RNA oligonucleotides described herein offers a therapeutic approach to modulating CB1R activity, promoting thermogenesis, increasing energy expenditure, and enhancing mitochondrial genesis, which can effectively address obesity and other metabolic diseases.

[0033] The present disclosure describes inhibitory RNA oligonucleotides, compositions and methods for modulating the expression of a CB1R gene. In some embodiments, expression and / or activity of CB1R is reduced or inhibited using a CB1R-specific inhibitory RNA oligonucleotide, thereby leading to reduced lipogenesis, increased mitochondrial genesis, and increased energy expenditure. Thus, inhibition of CB1R gene expression or activity using the inhibitory RNA oligonucleotides featured in the disclosure can be a useful approach to therapies aimed at weight loss and metabolic health. Such inhibition can be useful for treating severe obesity and metabolic disorders. Definitions

[0034] In order that the present disclosure may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this disclosure.

[0035] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element, e.g., a plurality of elements.

[0036] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to".

[0037] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise. 7Attorney Docket: 090147-0584673

[0038] As used herein, the term "about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, about means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to + / - 10% of the specified value. In embodiments, about includes the specified value.

[0039] "Nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof in either single-, double- or multiple-stranded form, or complements thereof; or nucleosides (e.g., deoxyribonucleosides or ribonucleosides). In embodiments, “nucleic acid” does not include nucleosides. The terms “polynucleotide,” “oligonucleotide,” “oligo” or the like refer, in the usual and customary sense, to a linear sequence of nucleotides. The term “nucleoside” refers, in the usual and customary sense, to a glycosylamine including a nucleobase and a five-carbon sugar (ribose or deoxyribose). Non limiting examples, of nucleosides include, cytidine, uridine, adenosine, guanosine, thymidine and inosine. The term “nucleotide” refers, in the usual and customary sense, to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA. Examples of nucleic acid, e.g. polynucleotides contemplated herein include any types of RNA, e.g. mRNA, siRNA, miRNA, and guide RNA and any types of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragments thereof. The term “duplex” in the context of polynucleotides refers, in the usual and customary sense, to double strandedness. Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides or the nucleic acids can be branched, e.g., such that the nucleic acids comprise one or more arms or branches of nucleotides. Optionally, the branched nucleic acids are repetitively branched to form higher ordered structures such as dendrimers and the like.

[0040] Nucleic acids, including e.g., nucleic acids with a phosphothioate backbone, can include one or more reactive moieties. As used herein, the term reactive moiety includes any group capable of reacting with another molecule, e.g., a nucleic acid or polypeptide through covalent, non-covalent or other interactions. By way of example, the nucleic acid can include an amino acid reactive moiety that reacts with an amino acid on a protein or polypeptide through a covalent, non-covalent or other interaction. 8Attorney Docket: 090147-0584673

[0041] The terms also encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non- naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphodiester derivatives including, e.g., phosphoramidate, phosphorodiamidate, phosphorothioate (also known as phosphothioate having double bonded sulfur replacing oxygen in the phosphate), phosphorodithioate, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boron phosphonate, or O-methylphosphoroamidite linkages (see Eckstein, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, Oxford University Press) as well as modifications to the nucleotide bases such as in 5-methyl cytidine or pseudouridine.; and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, modified sugars, and non-ribose backbones (e.g. phosphorodiamidate morpholino oligos or locked nucleic acids (LNA) as known in the art), including those described in U.S. Patent Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CARBOHYDRATE MODIFICATIONS IN ANTISENSE RESEARCH, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. In embodiments, the internucleotide linkages in DNA are phosphodiester, phosphodiester derivatives, or a combination of both.

[0042] Nucleic acids can include nonspecific sequences. As used herein, the term "nonspecific sequence" refers to a nucleic acid sequence that contains a series of residues that are not designed to be complementary to or are only partially complementary to any other nucleic acid sequence. By way of example, a nonspecific nucleic acid sequence is a sequence of nucleic acid residues that does not function as an inhibitory nucleic acid when contacted with a cell or organism.

[0043] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) 9Attorney Docket: 090147-0584673 for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0044] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site http: / / www.ncbi.nlm.nih.gov / BLAST / or the like). Such sequences are then said to be "substantially identical." This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0045] As used herein, "CB1R" refers to the gene or protein of cannabinoid receptor 1. CB1R is expressed in a variety of tissues, including the central nervous system and peripheral tissues such as adipose. The gene encoding CB1R produces multiple isoforms having different tissue expression profiles. Exemplary CB1R isoforms are provided in NM_001160226.1, NM_001160258.1, NM_001160259.1, NM_016083.4, and NM_033181.3. The term "CB1R" encompasses all isoforms unless specifically indicated otherwise.

[0046] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a CB1R gene.

[0047] "G," "C," "A" "T" and "U" each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymine and uracil as a base, respectively. However, it will be understood that the term "ribonucleotide" or "nucleotide" or "deoxyribonucleotide" can also refer to a modified nucleotide, as further detailed below. In some embodiments, guanine, cytosine, adenine, and / or uracil are replaced by other moieties without substantially altering 10Attorney Docket: 090147-0584673 the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of the disclosure by a nucleotide containing, for example, inosine.

[0048] The term "inhibitory RNA oligonucleotide” refers to an oligonucleotide comprising ribonucleotides, and optionally deoxyribonucleotides, that inhibits translation of a target RNA sequence. An inhibitory RNA oligonucleotide can be double-stranded or single- stranded.

[0049] In some embodiments, an inhibitory RNA oligonucleotide of the disclosure includes a single stranded oligonucleotide that interacts with a target RNA sequence, e.g., a CB1R target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev.15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev.15: 188). Thus, in one aspect the disclosure relates to a single stranded RNA (siRNA) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target gene, i.e., a CB1R gene. Accordingly, the term "siRNA" is also used herein to refer to an inhibitory RNA oligonucleotide as described above.

[0050] In some embodiments, the inhibitory RNA oligonucleotide may be a single-stranded oligonucleotide that is introduced into a cell or organism to inhibit a target mRNA. Single- stranded inhibitory RNA oligonucleotides bind to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. The design and testing of single-stranded oligonucleotides are described in U.S. Patent No.8,101,348 and in Lima et al., (2012) Cell 150: 883-894, the entire contents of each of which are hereby incorporated herein by reference. Any of the antisense nucleotide sequences described herein may be used as a single-stranded oligonucleotide as described herein or as chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894. 11Attorney Docket: 090147-0584673

[0051] An "antisense nucleic acid" or “antisense oligonucleotide” as referred to herein is a nucleic acid (e.g., DNA or RNA molecule) that is complementary to at least a portion of a specific target nucleic acid and is capable of reducing transcription of the target nucleic acid (e.g. mRNA from DNA), reducing the translation of the target nucleic acid (e.g. mRNA), altering transcript splicing (e.g. single stranded morpholino oligo), or interfering with the endogenous activity of the target nucleic acid. See, e.g., Weintraub, Scientific American, 262:40 (1990). Typically, synthetic antisense nucleic acids (e.g. oligonucleotides) are generally between 15 and 25 bases in length. Thus, antisense nucleic acids are capable of hybridizing to (e.g. selectively hybridizing to) a target nucleic acid. In embodiments, the antisense nucleic acid hybridizes to the target nucleic acid in vitro. In embodiments, the antisense nucleic acid hybridizes to the target nucleic acid in a cell. In embodiments, the antisense nucleic acid hybridizes to the target nucleic acid in an organism. In embodiments, the antisense nucleic acid hybridizes to the target nucleic acid under physiological conditions. Antisense nucleic acids may comprise naturally occurring nucleotides or modified nucleotides such as, e.g., phosphorothioate, methylphosphonate, and -anomeric sugar- phosphate, backbonemodified nucleotides.

[0052] In some embodiments, the present disclosure provides single-stranded antisense oligonucleotide molecules targeting CB1R. A "single-stranded antisense oligonucleotide molecule" is complementary to a sequence within the target mRNA (i.e., CB1R). Single- stranded antisense oligonucleotide molecules can inhibit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. Alternatively, the single-stranded antisense oligonucleotide molecules inhibit a target mRNA by hybridizing to the target and cleaving the target through an RNaseH cleavage event. The single-stranded antisense oligonucleotide molecule may be about 10 to about 30 nucleotides in length and have a sequence that is complementary to a target sequence. For example, the single-stranded antisense oligonucleotide molecule may comprise a sequence that is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense nucleotide sequences described herein, e.g., the sequences provided in any one of Tables 1- 13, or bind any of the target sites described herein. The single-stranded antisense oligonucleotide molecules may comprise modified RNA, DNA, or a combination thereof.

[0053] In some embodiments, an "inhibitory RNA oligonucleotide" is a double-stranded RNA and is referred to herein as a "double stranded inhibitory RNA oligonucleotide," "double-stranded RNA (dsRNA) molecule," "dsRNA agent," or "dsRNA". The term 12Attorney Docket: 090147-0584673 "dsRNA", refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having "sense" and "antisense" orientations with respect to a target RNA, i.e., a CB1R gene. In some embodiments of the disclosure, a double-stranded RNA (dsRNA) triggers the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.

[0054] The term "antisense strand" refers to the strand of a single or double stranded inhibitory RNA oligonucleotide which includes a region that is substantially complementary to a target sequence (e.g., a human CB1R mRNA). Where the region of complementarity is not fully complementary to the target sequence, the mismatches are most tolerated in the terminal regions and, if present, are generally in a terminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5' and / or 3' terminus. In some embodiments, the antisense strand is at least 80%, 85%, 90%, 95%, or 100% complementary to a target mRNA sequence.

[0055] The term "sense strand," as used herein, refers to the strand of a double stranded inhibitory RNA oligonucleotide that includes a region that is sufficiently complementary to a region of the antisense strand such that it forms a duplex region with the antisense strand. In some embodiments, the sense strand is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to a sense strand sequence.

[0056] As described herein the complementarity of sequences may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing. Thus, two sequences that are complementary to each other, may have a specified percentage of nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region).

[0057] As used herein, and unless otherwise indicated, the term "complementary," when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. For example, a complementary sequence is sufficient to allow the relevant function of the nucleic acid to 13Attorney Docket: 090147-0584673 proceed, e.g., RNAi. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

[0058] Sequences can be "fully complementary" with respect to each when there is base- pairing of the nucleotides of the first nucleotide sequence with the nucleotides of the second nucleotide sequence over the entire length of the first and second nucleotide sequences. However, where a first sequence is referred to as "substantially complementary" with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as "fully complementary" for the purposes described herein.

[0059] "Complementary" sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but not limited to, G:U Wobble or Hoogsteen base pairing.

[0060] The terms "complementary," "fully complementary" and "substantially complementary" herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of an inhibitory RNA oligonucleotide and a target sequence, as will be understood from the context of their use.

[0061] The term "inhibiting," as used herein, is used interchangeably with "reducing," "silencing," "downregulating," "suppressing" and other similar terms, and includes any level of inhibition.

[0062] The phrase "inhibiting expression of a CB1R," as used herein, includes inhibition of expression (i.e., inhibition of the formation of a CB1R gene product, such as a protein product) of a CB1R gene (such as, e.g., a human CB1R gene) as well as variants, (e.g., naturally occurring variants), isoforms, or mutants of a CB1R gene. Thus, the CB1R gene 14Attorney Docket: 090147-0584673 may be a wild-type CB1R gene, a mutant CB1R gene, or a transgenic CB1R gene in the context of a genetically manipulated cell, group of cells, or organism.

[0063] "Inhibiting expression of a CB1R gene" includes any level of inhibition of a CB1R gene, e.g., at least partial suppression of the expression of a CB1R gene, such as an inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0064] The expression of a CB1R gene may be assessed based on the level of any variable associated with CB1R gene expression, e.g., CB1R mRNA level or CB1R protein level. Inhibition may be assessed by a decrease in an absolute or relative level of one or more of these variables compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).

[0065] The phrase "contacting a cell with an inhibitory RNA oligonucleotide," as used herein, includes contacting a cell by any possible means. Contacting a cell with an inhibitory RNA oligonucleotide includes contacting a cell in vitro with the oligonucleotide or contacting a cell in vivo with the oligonucleotide. The contacting may be done directly or indirectly. Thus, for example, an inhibitory RNA oligonucleotide may be put into physical contact with the cell by the individual performing the method, or alternatively, the oligonucleotide may be put into a situation that will permit or cause it to subsequently come into contact with the cell.

[0066] Contacting a cell in vitro may be done, for example, by incubating the cell with the inhibitory RNA oligonucleotide. Contacting a cell in vivo may be done, for example, by injecting the inhibitory RNA oligonucleotide into or near the tissue where the cell is located, or by injecting the oligonucleotide into another area, the bloodstream or the subcutaneous space, such that the oligonucleotide will subsequently reach the tissue where the cell to be contacted is located. For example, the inhibitory RNA oligonucleotide may contain and / or be coupled to a ligand that directs the inhibitory RNA oligonucleotide to a site of interest. Combinations of in vitro and in vivo methods of contacting are also possible. In connection with the methods of the disclosure, a cell might also be contacted in vitro with an inhibitory RNA oligonucleotide and subsequently transplanted into a subject. 15Attorney Docket: 090147-0584673

[0067] A "patient" or "subject," as used herein, is intended to include either a human or non- human animal, preferably a mammal, e.g., human or a monkey. Most preferably, the subject or patient is a human.

[0068] A " CB1R associated disorder", as used herein, is intended to include any disorder that can be treated or prevented, or the symptoms of which can be alleviated, by inhibiting the expression of CB1R. CB1R associated disorders include, but are not limited to, obesity, Type 2 Diabetes Mellitus, insulin resistance, Beta Cell dysfunction, elevated HbA1c, fatty liver disease, prediabetes, elevated lipid profiles, gastrointestinal motility, hyperphagia, renal fibrosis, reduction in glucose responsiveness of the pancreas, liver cirrhosis, fatty liver disease, cardiovascular disease, inflammatory disease, leptin resistance, leptin deficiency, and leptin receptor deficiency.

[0069] "Therapeutically effective amount," as used herein, is intended to include the amount of an inhibitory RNA oligonucleotide that, when administered to a patient for treating a CB1R associated disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease). The "therapeutically effective amount" may vary depending on the inhibitory RNA oligonucleotide, how the oligonucleotide is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, stage of pathological processes mediated by CB1R expression, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

[0070] A "therapeutically-effective amount" also includes an amount of an inhibitory RNA oligonucleotide that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. Inhibitory RNA oligonucleotides employed in the methods of the present disclosure may be administered in a sufficient amount to produce a reasonable benefit / risk ratio applicable to such treatment.

[0071] As used herein, the term "administering" means oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. In embodiments, the 16Attorney Docket: 090147-0584673 administering does not include administration of any active agent other than the recited active agent.

[0072] "Co-administer" it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies. The compounds provided herein can be administered alone or can be coadministered to the patient. Coadministration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound). Thus, the preparations can also be combined, when desired, with other active substances (e.g. to reduce metabolic degradation). The compositions of the present disclosure can be delivered transdermally, by a topical route, or formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0073] A “cell” as used herein, refers to a cell carrying out metabolic or other function sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaroytic cells. Prokaryotic cells include but are not limited to bacteria. Eukaryotic cells include but are not limited to yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g., spodoptera) and human cells. Cells may be useful when they are naturally nonadherent or have been treated not to adhere to surfaces, for example by trypsinization.

[0074] The term "sample," as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samples may include samples from tissues, organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g., whole liver or certain segments of liver or certain types of cells in the liver, such as, e.g., hepatocytes). In preferred embodiments, a "sample derived from a subject" refers to blood or plasma drawn from the subject. In further embodiments, a "sample derived from a subject" refers to liver tissue (or subcomponents thereof) derived from the subject. 17Attorney Docket: 090147-0584673 Targeting Inhibitory RNA Oligonucleotides

[0075] The disclosure provides, inter alia, oligonucleotides that inhibit expression or a target gene (e.g., CB1R) (i.e., inhibitory RNA oligonucleotides). In some embodiments, an oligonucleotide that inhibits expression of a target gene is targeted to a target mRNA. CB1R Target Sequences

[0076] In some embodiments, the inhibitory RNA oligonucleotide is targeted to a target sequence comprising a CB1R mRNA. In some embodiments, the CB1R mRNA is selected from any one of the sequences set forth in transcript accession numbers NM_001370546.1, NM_001424094.1, NM_001370547.1, NM_00142096.1, NM_001424097.1, NM_001424095.1, NM_001370545.1, NM_0168083.6, NM_001160226.3, NM_001365870.2, NM_001365869.2, NM_001160258.3, NM_001424098.1, XM_047418173.1, NM_001365872.2, XM_047418171.1, NM_001160259.3, NM_001365874.3, XM_047418172.1, NM_033181.4, XM_054354204.1, XM_054354206.1, and XM_054354205.1, each sequence is incorporated by reference herein. In some embodiments, the oligonucleotide, or a portion, fragment or strand thereof (e.g., an antisense strand of a double-stranded oligonucleotide) binds or anneals to a target sequence comprising a CB1R mRNA, thereby inhibiting CB1R expression. In some embodiments, the oligonucleotide, or a portion, fragment or strand thereof (e.g., an antisense strand of a double- stranded oligonucleotide) binds or anneals to a target sequence comprising an isoform of CB1R mRNA. In some embodiments, the oligonucleotide, or a portion, fragment or strand thereof (e.g., an antisense strand of a double-stranded oligonucleotide) binds or anneals to a target sequence comprising an isoform of CB1R mRNA selected from the sequences set forth in NM_001160226.1, NM_001160258.1, NM_001160259.1, NM_016083.4, and NM_033181.3.

[0077] In some embodiments, the inhibitory RNA oligonucleotide selectively targets an isoform of CB1R. In some embodiments, the CB1R isoform is full-length CB1R. In some embodiments, the CB1R isoform is CB1a. In some embodiments the CB1R isoform is CB1b. In some embodiments, the inhibitory RNA oligonucleotide selectively targets the full-length CB1R and no other isoforms. In some embodiments, the inhibitory RNA oligonucleotide selectively targets the CB1a isoform and no other isoforms. In some embodiments, the inhibitory RNA oligonucleotide selectively targets the CB1b isoform and no other isoforms. In some embodiments, the inhibitory RNA oligonucleotide selectively targets the CB1R isoform provided in NM_001160226.1. In some embodiments, the inhibitory RNA 18Attorney Docket: 090147-0584673 oligonucleotide selectively targets the CB1R isoform provided in NM_001160258.1. In some embodiments, the inhibitory RNA oligonucleotide selectively targets the CB1R isoform provided in NM_001160259.1. In some embodiments, the inhibitory RNA oligonucleotide selectively targets the CB1R isoform provided in NM_016083.4. In some embodiments, the inhibitory RNA oligonucleotide selectively targets the CB1R isoform provided in NM_033181.3. The expression profiles of CB1R isoforms are summarized in the following table. Isoform Predominant Expression Full-length CB1R Central Nervous System (CNS), , , sequence selected from the target sequences set forth in Tables 1-13. In some embodiments, a CB1R target sequence comprises, or consists of, a target sequence having at least 90% (e.g., at least 95%, at least 100%) identity to a target sequence selected from Tables 1-13.

[0079] The inhibitory RNA oligonucleotides described herein comprise an antisense strand comprising a nucleotide sequence complementary to a CB1R target sequence described herein (e.g., any one of the CB1R target sequences in Tables 1-13). In some embodiments, the nucleotide sequence of the antisense strand is complementary to at least 10 contiguous nucleotides of a CB1R target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 10-30 contiguous nucleotides of a CB1R target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 15-30 contiguous nucleotides of a CB1R target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 15-25 contiguous nucleotides of a CB1R target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 15 contiguous nucleotides of a CB1R target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 16 contiguous nucleotides of a CB1R target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 17 contiguous nucleotides of a CB1R target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is 19Attorney Docket: 090147-0584673 complementary to 18 contiguous nucleotides of a CB1R target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 19 contiguous nucleotides of a CB1R target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 20 contiguous nucleotides of a CB1R target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 21 contiguous nucleotides of a CB1R target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 22 contiguous nucleotides of a CB1R target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 23 contiguous nucleotides of a CB1R target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 24 contiguous nucleotides of a CB1R target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 25 contiguous nucleotides of a CB1R target sequence described herein.

[0080] In some embodiments, the antisense strand comprises one, two or three mismatches as compared to the corresponding region of the target sequence, depending on the length of the region of the mRNA that is targeted, and as such may be not fully complementary. Methods to analyze and identify inhibitory RNA oligonucleotides with sufficient sequence identity to effectively inhibit expression of a specific target sequence are known in the art. Sequence identity may be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith- Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). In some embodiments, provided herein are inhibitory RNA oligonucleotides that target any one of the CB1R target sequences set forth in Tables 1-13. ZNF423 Target Sequences

[0081] In some aspects, provided herein are inhibitory RNA oligonucleotides targeting ZNF423. The ZNF423 targeting inhibitory RNA oligonucleotides can be formulated or administered alone or in combination in any of the compositions or methods described herein. Methods for identifying inhibitory RNA oligonucleotide are known in the art and described herein. 20Attorney Docket: 090147-0584673

[0082] In some embodiments, the inhibitory RNA oligonucleotides and compositions provided herein, are co-formulated and / or administered with an inhibitory RNA oligonucleotide targeting ZNF423.

[0083] In some embodiments, the inhibitory RNA oligonucleotide is targeted to a target sequence comprising a ZNF423 mRNA. In some embodiments, the ZNF423 mRNA is selected from any one of the sequences set forth in transcript accession numbers NM_001379286.1, XM_047433808.1, XM_047433805.1, XM_047433804.1, NM_015069.5, XM_006721171.5, XM_047433806.1, XM_047433803.1, XM_047433807.1, XM_005255856.5, XM_017023078.2, NM_001330533.2, XM_047433810.1, NM_001271620.2, XM_054379860.1, XM_054379859.1, XM_054379855.1, XM_054379854.1, XM_054379857.1, XM_054379856.1, XM_054379853.1, XM_054379858.1, XM_054379861.1, XM_054379862.1, and XM_054379863.1, each sequence is incorporated by reference herein. In some embodiments, the oligonucleotide, or a portion, fragment or strand thereof (e.g., an antisense strand of a double-stranded oligonucleotide) binds or anneals to a target sequence comprising a ZNF423 mRNA, thereby inhibiting ZNF423 expression. In some embodiments, the oligonucleotide, or a portion, fragment or strand thereof (e.g., an antisense strand of a double-stranded oligonucleotide) binds or anneals to a target sequence comprising an isoform of ZFP423 mRNA selected from the sequences set forth in NM_015069.5, NM_001271620.2, NM_001330533.2, and NM_001379286.1.

[0084] In some embodiments, the inhibitory RNA oligonucleotide selectively targets an isoform of ZFP423. In some embodiments, the ZFP423 isoform is full-length ZFP423. In some embodiments, the ZFP423 isoform is zinc finger protein isoform 1. In some embodiments the ZFP423 isoform is zinc finger protein isoform 2. In some embodiments the ZFP423 isoform is zinc finger protein isoform 3. In some embodiments the ZFP423 isoform is zinc finger protein isoform 4. In some embodiments, the inhibitory RNA oligonucleotide selectively targets the full-length ZFP423 and no other isoforms. In some embodiments, the ZFP423 isoform is zinc finger protein isoform 1 and no other isoform. In some embodiments the ZFP423 isoform is zinc finger protein isoform 2 and no other isoform. In some embodiments the ZFP423 isoform is zinc finger protein isoform 3 and no other isoform. In some embodiments the ZFP423 isoform is zinc finger protein isoform 4 and no other isoform.

[0085] In some embodiments, the inhibitory RNA oligonucleotide selectively targets the ZFP423 isoform provided in NM_015069.5. In some embodiments, the inhibitory RNA oligonucleotide selectively targets the ZFP423 isoform provided in NM_001271620.2. In 21Attorney Docket: 090147-0584673 some embodiments, the inhibitory RNA oligonucleotide selectively targets the ZFP423 isoform provided in NM_001330533.2. In some embodiments, the inhibitory RNA oligonucleotide selectively targets the ZFP423isoform provided in NM_001379286.1.

[0086] In some embodiments, a ZNF423 target sequence comprises, or consists of, a target sequence selected from the target sequences set forth in Tables 14-26. In some embodiments, a ZNF423 target sequence comprises, or consists of, a target sequence having at least 90% (e.g., at least 95%, at least 100%) identity to a target sequence selected from Tables 14-26.

[0087] The inhibitory RNA oligonucleotides described herein comprise an antisense strand comprising a nucleotide sequence complementary to a ZNF423 target sequence described herein (e.g., any one of the ZNF423 target sequences in Tables 14-26). In some embodiments, the nucleotide sequence of the antisense strand is complementary to at least 10 contiguous nucleotides of a ZNF423 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 10-30 contiguous nucleotides of a ZNF423 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 15-30 contiguous nucleotides of a ZNF423 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 15-25 contiguous nucleotides of a ZNF423 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 15 contiguous nucleotides of a ZNF423 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 16 contiguous nucleotides of a ZNF423 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 17 contiguous nucleotides of a ZNF423 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 18 contiguous nucleotides of a ZNF423 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 19 contiguous nucleotides of a ZNF423 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 20 contiguous nucleotides of a ZNF423 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 21 contiguous nucleotides of a ZNF423 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 22 contiguous nucleotides of a ZNF423 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 23 contiguous nucleotides of a ZNF423 target 22Attorney Docket: 090147-0584673 sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 24 contiguous nucleotides of a ZNF423 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 25 contiguous nucleotides of a ZNF423 target sequence described herein. In some embodiments, provided herein are inhibitory RNA oligonucleotides that target any one of the ZNF423 target sequences set forth in Tables 14-26.

[0088] In some embodiments, the ZNF423 targeting inhibitory RNA oligonucleotide comprises an antisense strand having a sequence at least 90% identical to an antisense strand set forth in any one of Tables 14-26. In some embodiments, the ZNF423 targeting inhibitory RNA oligonucleotide comprises an antisense strand having a sequence of an antisense strand set forth in any one of Tables 14-26. In some embodiments, the ZNF423 targeting inhibitory RNA oligonucleotide is double-stranded. In some embodiments, the ZNF423 targeting inhibitory RNA oligonucleotide is double-stranded and comprises an antisense sense strand having a sequence at least 90% identical to an antisense strand set forth in any one of Tables 14-26, and a sense strand that forms a duplex region with the antisense strand. In some embodiments, the ZNF423 targeting inhibitory RNA oligonucleotide is double-stranded and comprises an antisense sense strand having a sequence of an antisense strand set forth in any one of Tables 14-26, and a sense strand that forms a duplex region with the antisense strand. DAGL Target Sequences

[0089] In some aspects, provided herein are inhibitory RNA oligonucleotides targeting DAGL. The DAGL targeting inhibitory RNA oligonucleotides can be formulated or administered alone or in combination in any of the compositions or methods described herein.

[0090] In some embodiments, the inhibitory RNA oligonucleotide is targeted to a target sequence comprising a DAGL mRNA. In some embodiments, the DAGLA mRNA is selected from any one of the sequences set forth in transcript accession numbers NM_006133.3, XM_047427544.1, XM_047427543.1, XM_047427541.1, XM_047427542.1, XM_047427540.1, XM_047427545.1, XM_054369855.1, XM_054369854.1, XM_054369853.1, XM_054369852.1, XM_054369856.1, each sequence is incorporated by reference herein. In some embodiments, the oligonucleotide, or a portion, fragment or strand thereof (e.g., an antisense strand of a double-stranded oligonucleotide) binds or anneals to a target sequence comprising a DAGLA mRNA, thereby inhibiting DAGLA expression.

[0091] In some embodiments, the inhibitory RNA oligonucleotide is targeted to a target sequence comprising a DAGLB mRNA. In some embodiments, the DAGLB mRNA is 23Attorney Docket: 090147-0584673 selected from any one of the sequences set forth in transcript accession numbers NM_001142936.2 and NM_139179.4, each sequence is incorporated by reference herein. In some embodiments, the oligonucleotide, or a portion, fragment or strand thereof (e.g., an antisense strand of a double-stranded oligonucleotide) binds or anneals to a target sequence comprising a DAGLB mRNA, thereby inhibiting DAGLB expression.

[0092] In some embodiments, the nucleotide sequence of the antisense strand is complementary to at least 10 contiguous nucleotides of a DAGL target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 10-30 contiguous nucleotides of a DAGL target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 15-30 contiguous nucleotides of a DAGL target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 15-25 contiguous nucleotides of a DAGL target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 15 contiguous nucleotides of a DAGL target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 16 contiguous nucleotides of a DAGL target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 17 contiguous nucleotides of a DAGL target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 18 contiguous nucleotides of a DAGL target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 19 contiguous nucleotides of a DAGL target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 20 contiguous nucleotides of a DAGL target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 21 contiguous nucleotides of a DAGL target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 22 contiguous nucleotides of a DAGL target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 23 contiguous nucleotides of a DAGL target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 24 contiguous nucleotides of a DAGL target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 25 contiguous nucleotides of a DAGL target sequence described herein. 24Attorney Docket: 090147-0584673 TLE3 Target Sequences

[0093] In some aspects, provided herein are inhibitory RNA oligonucleotides targeting TLE3. The TLE3 targeting inhibitory RNA oligonucleotides can be formulated or administered alone or in combination in any of the compositions or methods described herein.

[0094] In some embodiments, the inhibitory RNA oligonucleotide is targeted to a target sequence comprising a TLE3 mRNA. In some embodiments, the TLE3 mRNA is selected from any one of the sequences set forth in transcript accession numbers NM_001105192.3, NM_001282979.2, NM_001282980.2, NM_001282981.2, NM_001282982.2, NM_001438147.1, NM_001438148.1, NM_001438836.1, NM_001438837.1, NM_001438838.1, NM_005078.4, NM_020908.3, XM_005254622.6, XM_005254623.5, XM_005254625.5, XM_005254628.5, XM_005254633.4, XM_006720665.5, XM_011521976.4, XM_011521977.4, XM_011521978.4, XM_011521979.4, XM_011521980.4, XM_011521981.3, XM_011521982.4, XM_011521983.4, XM_017022532.3, XM_047432992.1, XM_047432993.1, XM_054378718.1, XM_054378719.1, XM_054378720.1, XM_054378721.1, XM_054378722.1, XM_054378723.1, XM_054378724.1, XM_054378725.1, XM_054378726.1, XM_054378727.1, XM_054378728.1, XM_054378729.1, XM_054378730.1, XM_054378731.1, XM_054378732.1, XM_054378733.1, XM_054378734.1, each sequence is incorporated by reference herein. In some embodiments, the oligonucleotide, or a portion, fragment or strand thereof (e.g., an antisense strand of a double-stranded oligonucleotide) binds or anneals to a target sequence comprising a TLE3 mRNA, thereby inhibiting TLE3 expression.

[0095] In some embodiments, a TLE3 target sequence comprises, or consists of, a target sequence selected from the target sequences set forth in Tables 27-34. In some embodiments, a TLE3 target sequence comprises, or consists of, a target sequence having at least 90% (e.g., at least 95%, at least 100%) identity to a target sequence selected from Tables 27-34.

[0096] The inhibitory RNA oligonucleotides described herein comprise an antisense strand comprising a nucleotide sequence complementary to a TLE3 target sequence described herein (e.g., any one of the TLE3 target sequences in Tables 27-34). In some embodiments, the nucleotide sequence of the antisense strand is complementary to at least 10 contiguous nucleotides of a TLE3 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 10-30 contiguous nucleotides of a TLE3 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 15-30 contiguous 25Attorney Docket: 090147-0584673 nucleotides of a TLE3 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 15-25 contiguous nucleotides of a TLE3 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 15 contiguous nucleotides of a TLE3 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 16 contiguous nucleotides of a TLE3 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 17 contiguous nucleotides of a TLE3 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 18 contiguous nucleotides of a TLE3 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 19 contiguous nucleotides of a TLE3 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 20 contiguous nucleotides of a TLE3 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 21 contiguous nucleotides of a TLE3 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 22 contiguous nucleotides of a TLE3 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 23 contiguous nucleotides of a TLE3 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 24 contiguous nucleotides of a TLE3 target sequence described herein. In some embodiments, the nucleotide sequence of the antisense strand is complementary to 25 contiguous nucleotides of a TLE3 target sequence described herein. In some embodiments, provided herein are inhibitory RNA oligonucleotides that target any one of the TLE3 target sequences set forth in Tables 27-34.

[0097] In some embodiments, the TLE3 targeting inhibitory RNA oligonucleotide comprises an antisense strand having a sequence at least 90% identical to an antisense strand set forth in any one of Tables 27-34. In some embodiments, the TLE3 targeting inhibitory RNA oligonucleotide comprises an antisense strand having a sequence of an antisense strand set forth in any one of Tables 27-34. In some embodiments, the TLE3 targeting inhibitory RNA oligonucleotide is double-stranded. In some embodiments, the TLE3 targeting inhibitory RNA oligonucleotide is double-stranded and comprises an antisense sense strand having a sequence at least 90% identical to an antisense strand set forth in any one of Tables 27-34, and a sense strand that forms a duplex region with the antisense strand. In some 26Attorney Docket: 090147-0584673 embodiments, the TLE3 targeting inhibitory RNA oligonucleotide is double-stranded and comprises an antisense sense strand having a sequence of an antisense strand set forth in any one of Tables 27-34, and a sense strand that forms a duplex region with the antisense strand. Inhibitory RNA Oligonucleotides

[0098] In some embodiments, the inhibitory RNA oligonucleotides are short interfering RNA (siRNA). In some embodiments, the inhibitory RNA oligonucleotides are single-stranded. In some embodiments, the inhibitory RNA oligonucleotides are double-stranded. In some embodiments, the inhibitory RNA oligonucleotides are microRNA (miRNA). In some embodiments, the inhibitory RNA oligonucleotides are short hairpin RNA (shRNA).

[0099] In some embodiments, the inhibitory RNA oligonucleotide is single stranded. In some embodiments, the single stranded inhibitory RNA oligonucleotide is 10-30 nucleotides in length. In some embodiments, the single stranded inhibitory RNA oligonucleotide is 15-30 nucleotides in length, 17-30 nucleotides in length, 19-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.

[0100] In some embodiments, the inhibitory RNA oligonucleotide is double stranded. In some embodiments, the double stranded inhibitory RNA oligonucleotide comprises a sense strand and an antisense strand. Each strand of the inhibitory RNA oligonucleotide may range from 10-30 nucleotides in length. For example, in some embodiments each strand is 15-30 nucleotides in length, 17-30 nucleotides in length, 19-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.

[0101] The sense strand and antisense strand typically form a duplex double stranded RNA ("dsRNA") The duplex region of an inhibitory RNA oligonucleotide may be 10-30 nucleotide pairs in length. For example, in some embodiments, the duplex region is 15-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17 - 23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19- 21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another 27Attorney Docket: 090147-0584673 example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

[0102] In some embodiments, the two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3'-end of one strand and the 5'-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a "hairpin loop." Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3'-end of one strand and the 5'-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a "linker." The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex.

[0103] Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev.15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev.15: 188). As used herein, a "nucleotide overhang" refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of an inhibitory RNA oligonucleotide when a 3'-end of one strand of the oligonucleotide extends beyond the 5'-end of the other strand, or vice versa. "Blunt" or "blunt end" means that there are no unpaired nucleotides at that end of the double stranded RNAi agent, i.e., no nucleotide overhang. A "blunt ended" inhibitory RNA oligonucleotide is a dsRNA that is double-stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule. The inhibitory RNA oligonucleotides of the disclosure include oligonucleotides with nucleotide overhangs at one end (i.e., agents with one overhang and one blunt end) or with nucleotide overhangs at both ends.

[0104] Similar to siRNA, shRNA is processed through the RNAi pathway. Specifically, shRNAs are comprised of a set of ‘sense’ and ‘anti-sense’ complementary nucleotide sequences, separated by a short unpaired loop that combine to form a hairpin structure. While 28Attorney Docket: 090147-0584673 the siRNA duplex is delivered directly into the cytosol, shRNA can be introduced into cells through viral vectors where it is subsequently integrated into the host genome and transcribed by polymerase II or III. The shRNA is then processed into siRNA duplexes by Dicer which binds the target mRNA and mediates degradation through interaction with the RISC complex. Further, while siRNA can lead to transient downregulation of the target gene, shRNA ensures long-term silencing and can be used in transfection-resistant cell types.

[0105] In some embodiments, the inhibitory RNA oligonucleotide comprises one or more overhang regions and / or capping groups at the 3'-end, 5'-end, or both ends of one or both strands. In some embodiments, the overhang is 1-6 nucleotides in length, for instance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target mRNA, or it can be complementary to the gene sequences being targeted or can be another sequence. The first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

[0106] In some embodiments, the nucleotides in the overhang region of the inhibitory RNA oligonucleotide are each independently be a modified or unmodified nucleotide including, but no limited to 2'-sugar modified, such as, 2-F, 2'-O-methyl, thymidine (T), 2‵-O-methoxyethyl- 5-methyluridine (Teo), 2‵-O-methoxyethyladenosine (Aeo), 2‵-O-methoxyethyl-5- methylcytidine (m5Ceo), and any combinations thereof. For example, in some embodiments, TT is an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA, or it can be complementary to the gene sequences being targeted or can be another sequence.

[0107] The 5'- or 3'- overhangs at the sense strand, antisense strand or both strands of the inhibitory RNA oligonucleotide may be phosphorylated. In some embodiments, the overhang region(s) contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In some embodiments, the overhang is present at the 3'-end of the sense strand, antisense strand, or both strands. In some embodiments, this 3'-overhang is present in the antisense strand. In one embodiment, this 3'- overhang is present in the sense strand.

[0108] The inhibitory RNA oligonucleotide may contain only a single overhang, which can strengthen the interference activity of the inhibitory RNA oligonucleotide, without affecting its overall stability. For example, the single-stranded overhang may be located at the 3'- 29Attorney Docket: 090147-0584673 terminal end of the sense strand or, alternatively, at the 3'-terminal end of the antisense strand. The inhibitory RNA oligonucleotide may also have a blunt end, located at the 5'-end of the antisense strand (or the 3'-end of the sense strand) or vice versa. Generally, the antisense strand of the inhibitory RNA oligonucleotide has a nucleotide overhang at the 3'- end, and the 5'-end is blunt. While not wishing to be bound by theory, the asymmetric blunt end at the 5'-end of the antisense strand and 3'-end overhang of the antisense strand favor the guide strand loading into RISC process. Oligonucleotide Modifications

[0109] In some embodiments, all the nucleotides of the oligonucleotide are ribonucleotides. In some embodiments, the oligonucleotide comprises ribonucleotides and non- ribonucleotides, e.g., a deoxyribonucleotide and / or a modified nucleotide.

[0110] In some aspects, the disclosure provides inhibitory RNA oligonucleotides comprising chemical modifications. In some embodiments, substantially all of the nucleotides of an inhibitory RNA oligonucleotide of the disclosure are modified. In other embodiments, all of the nucleotides of an inhibitory RNA of the disclosure are modified. Inhibitory RNA oligonucleotides of the disclosure in which "substantially all of the nucleotides are modified" are largely but not wholly modified and can include not more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0111] Any of the nucleic acids featured in the disclosure can be synthesized and / or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5'-end modifications (phosphorylation, conjugation, inverted linkages) or 3'-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2'-position or 4'-position) or replacement of the sugar; and / or backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of inhibitory RNA oligonucleotides useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the 30Attorney Docket: 090147-0584673 backbone. In some embodiments, a modified inhibitory RNA oligonucleotide will have a phosphorus atom in its internucleoside backbone.

[0112] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5'-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included.

[0113] Representative U.S. patents that teach the preparation of the above phosphorus- containing linkages include, but are not limited to, U.S. Patent Nos.3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6, 239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and US Pat RE39464, the entire contents of each of which are hereby incorporated herein by reference.

[0114] Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.

[0115] Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 31Attorney Docket: 090147-0584673 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.

[0116] In other embodiments, suitable RNA mimetics are contemplated for use in inhibitory RNA oligonucleotides, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos.5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the inhibitory RNA oligonucleotides of the disclosure are described in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0117] Some embodiments featured in the disclosure include inhibitory RNA oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular╌CH2╌NH╌ CH2-,╌CH2╌N(CH3)╌O╌CH2╌[known as a methylene (methylimino) or MMI backbone],╌ CH2╌O╌N(CH3)╌CH2╌,╌CH2╌N(CH3)╌ N(CH3)╌CH2╌ and╌N(CH3)╌CH2╌CH2╌ [wherein the native phosphodiester backbone is represented as╌ O╌ P╌ O╌ CH2╌] of the above-referenced U.S. Patent No.5,489,677, and the amide backbones of the above-referenced U.S. Patent No.5,602,240. In some embodiments, the inhibitory RNA oligonucleotides featured herein have morpholino backbone structures of the above-referenced U.S. Patent No.5,034,506.

[0118] Modified inhibitory RNA oligonucleotides can also contain one or more substituted sugar moieties. In some embodiments, the inhibitory RNA oligonucleotides comprise one of the following at the 2'-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N- alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2)nO] mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2) nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In other embodiments, inhibitory RNA oligonucleotides include one of the following at the 2' 32Attorney Docket: 090147-0584673 position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O- aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an inhibitory RNA oligonucleotide, or a group for improving the pharmacodynamic properties of an inhibitory RNA oligonucleotides, and other substituents having similar properties. In some embodiments, the modification includes a 2'- methoxyethoxy (2'-O╌ CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in examples herein below, and 2'- dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'- DMAEOE), i.e., 2'-O╌CH2╌O╌CH2╌N(CH2)2.

[0119] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'- OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an inhibitory RNA oligonucleotides, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked inhibitory RNA oligonucleotides and the 5' position of 5' terminal nucleotide. Inhibitory RNA oligonucleotides can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos.4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, the entire contents of each of the foregoing are hereby incorporated herein by reference.

[0120] An inhibitory RNA oligonucleotide can also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as deoxy-thymine (dT), 5-methylcytosine (5- me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil 33Attorney Docket: 090147-0584673 (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8- substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No.3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp.276-278) and are exemplary base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

[0121] Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Patent Nos.3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.

[0122] The inhibitory RNA oligonucleotide can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in the 3'-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 34Attorney Docket: 090147-0584673 33(l):439-447; Mook, OR. et al., (2007) Mol Cane Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0123] Representative U.S. Patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos.6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, the entire contents of each of which are hereby incorporated herein by reference.

[0124] Potentially stabilizing modifications to the ends of RNA molecules can include N- (acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"- phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in PCT Publication No. WO 2011 / 005861. Exemplary CB1R Oligonucleotides

[0125] Provided herein, are inhibitory RNA oligonucleotides targeting the CB1R gene. In some embodiments, the inhibitory RNA oligonucleotides of the disclosure selectively target an isoform of CB1R. In some embodiments, inhibitory RNA oligonucleotides of the disclosure selectively target full length CB1R. In some embodiments, inhibitory RNA oligonucleotides of the disclosure selectively target CB1aR. In some embodiments, inhibitory RNA oligonucleotides of the disclosure selectively target CB1bR.

[0126] In some embodiments, the inhibitory RNA oligonucleotide comprises an antisense strand having a sequence at least 90% identical to an antisense strand set forth in any one of Tables 1-13. In some embodiments, the inhibitory RNA oligonucleotide comprises an antisense strand having a sequence of an antisense strand set forth in any one of Tables 1-13. In some embodiments, the inhibitory RNA oligonucleotide is double-stranded. In some embodiments, the inhibitory RNA oligonucleotide is double-stranded and comprises an antisense sense strand having a sequence at least 90% identical to an antisense strand set forth in any one of Tables 1-13, and a sense strand that forms a duplex region with the antisense strand. In some embodiments, the inhibitory RNA oligonucleotide is double-stranded and comprises an antisense sense strand having a sequence of an antisense strand set forth in any one of Tables 1-13, and a sense strand that forms a duplex region with the antisense strand. 35Attorney Docket: 090147-0584673 Formulations and Delivery

[0127] Provided herein, are pharmaceutical compositions and formulations comprising the inhibitory RNA oligonucleotides of the present disclosure. In some embodiments, provided herein are pharmaceutical compositions comprising an inhibitory RNA oligonucleotide, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical compositions containing the inhibitory RNA oligonucleotide are useful for treating a CB1R associated disease or disorder, e.g. a metabolic disorder. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) delivery.

[0128] The pharmaceutical compositions comprising inhibitory RNA oligonucleotides of the disclosure may be, for example, solutions with or without a buffer, or compositions containing pharmaceutically acceptable carriers. Such compositions include, for example, aqueous or crystalline compositions, liposomal formulations, micellar formulations, emulsions, and gene therapy vectors.

[0129] In some embodiments, the inhibitory RNA oligonucleotide may be administered in a solution. A free inhibitory RNA oligonucleotide may be administered in an unbuffered solution, e.g., in saline or in water. Alternatively, a free siRNA may also be administered in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In a preferred embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolality of the buffer solution containing the inhibitory RNA oligonucleotide can be adjusted such that it is suitable for administering to a subject.

[0130] In some embodiments, the buffer solution further comprises an agent for controlling the osmolality of the solution, such that the osmolarity is kept at a desired value, e.g., at the physiologic values of the human plasma. Solutes which can be added to the buffer solution to control the osmolarity include, but are not limited to, proteins, peptides, amino acids, non- metabolized polymers, vitamins, ions, sugars, metabolites, organic acids, lipids, or salts. In some embodiments, the agent for controlling the osmolarity of the solution is a salt. In certain embodiments, the agent for controlling the osmolarity of the solution is sodium chloride or potassium chloride.

[0131] The pharmaceutical compositions of the present disclosure can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., by a transdermal patch), pulmonary, 36Attorney Docket: 090147-0584673 e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular administration.

[0132] The inhibitory RNA oligonucleotide can be delivered in a manner to target a particular tissue, such as adipose tissue.

[0133] Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable. Coated condoms, gloves and the like can also be useful. Suitable topical formulations include those in which the inhibitory RNA oligonucleotides provided herein are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). Inhibitory RNA oligonucleotides featured in the disclosure can be encapsulated within liposomes or can form complexes thereto, in particular to cationic liposomes. Alternatively, inhibitory RNA oligonucleotides can be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1- dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C1-20 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof). Topical formulations are described in detail in U.S. Patent No.6,747,014, which is incorporated herein by reference.

[0134] Further provided herein are inhibitory RNA oligonucleotides and compositions thereof that can be delivered to a cell. In some embodiments, the cell is an isolated cell. In some embodiments, the cell is within a subject. In some embodiments, the cell is in adipose tissue.

[0135] The delivery of an inhibitory RNA oligonucleotide of the disclosure to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a 37Attorney Docket: 090147-0584673 subject having a CB1R associated disorder, such as a metabolic disorder) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an inhibitory RNA oligonucleotide of the disclosure either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an inhibitory RNA oligonucleotide, e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the inhibitory RNA oligonucleotide. These alternatives are discussed further below.

[0136] In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with an inhibitory RNA oligonucleotide of the disclosure (see e.g., Akhtar S. and Julian RL. (1992) Trends Cell. Biol.2(5): 139-144 and WO94 / 02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider in order to deliver an inhibitory RNA oligonucleotide molecule include, for example, biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. The non-specific effects of an inhibitory RNA oligonucleotide can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the inhibitory RNA oligonucleotide to be administered. For administering an inhibitory RNA oligonucleotide systemically for the treatment of a disease, the oligonucleotide can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the oligonucleotide by endo- and exo-nucleases in vivo. Modification of the oligonucleotide or the pharmaceutical carrier can also permit targeting of the inhibitory RNA oligonucleotide composition to the target tissue and avoid undesirable off-target effects. In some embodiments, inhibitory RNA oligonucleotides are modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation.

[0137] In some embodiments, the inhibitory RNA oligonucleotide can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an inhibitory RNA oligonucleotide (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an inhibitory RNA oligonucleotide by the cell. Cationic lipids, dendrimers, or polymers can either be bound to 38Attorney Docket: 090147-0584673 an inhibitory RNA oligonucleotide, or induced to form a vesicle or micelle (see e.g., Kim SH., et al (2008) Journal oƒ Controlled Release 129(2): 107-116) that encases an inhibitory RNA oligonucleotide. The formation of vesicles or micelles further prevents degradation of the inhibitory RNA oligonucleotide when administered systemically. Methods for making and administering cationic- inhibitory RNA oligonucleotide complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, DR., et al (2003) J. Mol. Biol 327:761- 766; Verma, UN., et al (2003) Clin. Cancer Res.9: 1291-1300; Arnold, AS et al (2007) J. Hypertens.25: 197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of inhibitory RNA oligonucleotides include DOTAP (Sorensen, DR., et al (2003), supra; Verma, UN., et al (2003), supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, TS., et al (2006) Nature 441: 111-114), cardiolipin (Chien, PY., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol.26: 1087-1091), polyethyleneimine (Bonnet ME., et al (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol.71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm.3:472-487), and polyamidoamines (Tomalia, DA., et al (2007) Biochem. Soc. Trans.35:61-67; Yoo, H., et al (1999) Pharm. Res.16: 1799-1804). In some embodiments, an inhibitory RNA oligonucleotide forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of inhibitory RNA oligonucleotides and cyclodextrins can be found in U.S. Patent No.7,427,605, which is herein incorporated by reference in its entirety. Lipid Nanoparticles

[0138] In some embodiments, inhibitory RNA oligonucleotides of the disclosure are formulated in a lipid formulation, e.g., a lipid nanoparticle (LNP), or other nucleic acid-lipid particle.

[0139] As used herein, the term "LNP" refers to a stable nucleic acid-lipid particle. LNPs contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). LNPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). LNPs include "pSPLP," which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00 / 03683. The particles of the present disclosure typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm 39Attorney Docket: 090147-0584673 to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid- lipid particles of the present disclosure are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Patent Nos.5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Publication No.2010 / 0324120 and PCT Publication No. WO 96 / 40964.

[0140] In some embodiments, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to oligonucleotide ratio) will be in the range of from about 1: 1 to about 50: 1, from about 1: 1 to about 25: 1, from about 3: 1 to about 15: 1, from about 4: 1 to about 10: 1, from about 5: 1 to about 9: 1, or about 6: 1 to about 9: 1. Ranges intermediate to the above recited ranges are also contemplated to be part of the disclosure.

[0141] In some embodiments, a lipid nanoparticle comprises a cationic lipid. The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N- distearyl-N,N-dimethylammonium bromide (DDAB), N-(I -(2,3-dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTAP), N-(I -(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2- Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3- (dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3- dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3- (N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N- dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N- dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca- 9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2- (bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1- yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid can 40Attorney Docket: 090147-0584673 comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.

[0142] In some embodiments, a lipid nanoparticle comprises an ioniziable / non-cationic lipid. The ionizable / non-cationic lipid can be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, 1 -stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid can be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.

[0143] In some embodiments, a lipid nanoparticle comprises a conjugated lipid that inhibits aggregation of particles. The conjugated lipid that inhibits aggregation of particles can be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG- diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG- ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, a PEG- dilauryloxypropyl (Ci2), a PEG-dimyristyloxypropyl (Ci4), a PEG-dipalmityloxypropyl (Ci6), or a PEG-distearyloxypropyl (C]8). The conjugated lipid that prevents aggregation of particles can be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.

[0144] In some embodiments, the LNP further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

[0145] In some embodiments, agents that enhance uptake of inhibitory RNA oligonucleotides at the cellular level are added to the pharmaceutical and other compositions of the present disclosure. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97 / 30731), are also known to enhance the cellular uptake of dsRNAs. Examples of commercially available transfection reagents include, for example Lipofectamine™ (Invitrogen; Carlsbad, CA), Lipofectamine 2000™ (Invitrogen; Carlsbad, CA), 293fectin™ (Invitrogen; Carlsbad, CA), Cellfectin™ (Invitrogen; Carlsbad, CA), 41Attorney Docket: 090147-0584673 DMRIE-C™ (Invitrogen; Carlsbad, CA), FreeStyle™ MAX (Invitrogen; Carlsbad, CA), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, CA), Lipofectamine™ (Invitrogen; Carlsbad, CA), RNAiMAX (Invitrogen; Carlsbad, CA), Oligofectamine™ (Invitrogen; Carlsbad, CA), Optifect™ (Invitrogen; Carlsbad, CA), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega; Madison, WI), TransFast™ Transfection Reagent (Promega; Madison, WI), Tfx™-20 Reagent (Promega; Madison, WI), Tfx™-50 Reagent (Promega; Madison, WI), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPassa D1 Transfection Reagent (New England Biolabs; Ipswich, MA, USA), LyoVec™ / LipoGen™ (Invitrogen; San Diego, CA, USA), PerFectin Transfection Reagent (Genlantis; San Diego, CA, USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), GenePORTER Transfection reagent (Genlantis; San Diego, CA, USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, CA, USA), Cytofectin Transfection Reagent (Genlantis; San Diego, CA, USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), TroganPORTER™ transfection Reagent (Genlantis; San Diego, CA, USA ), RiboFect (Bioline; Taunton, MA, USA), PlasFect (Bioline; Taunton, MA, USA), UniFECTOR (B-Bridge International; Mountain View, CA, USA), SureFECTOR (B-Bridge International; Mountain View, CA, USA), or HiFect™ (B-Bridge International, Mountain View, CA, USA), among others.

[0146] Other agents can be utilized to enhance the penetration of the administered oligonucleotides, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone. Vectors

[0147] Inhibitory RNA oligonucleotides targeting the CB1R gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00 / 22113, Conrad, International PCT Publication No. WO 00 / 22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be 42Attorney Docket: 090147-0584673 constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92: 1292).

[0148] The individual strand or strands of an inhibitory RNA oligonucleotide can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co- introduced (e.g., by transfection or infection) into a target cell. Alternatively, each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as inverted repeat polynucleotides joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

[0149] Inhibitory RNA oligonucleotide expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of an inhibitory RNA oligonucleotide as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of inhibitory RNA oligonucleotide expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex -planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.

[0150] Inhibitory RNA oligonucleotide expression plasmids can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid- based carriers (e.g., Transit-TKO™). Multiple lipid transfections for inhibitory RNA oligonucleotide-mediated knockdowns targeting different regions of a target RNA over a period of a week or more are also contemplated by the disclosure. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

[0151] Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno- associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) 43Attorney Docket: 090147-0584673 polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired.

[0152] Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of an inhibitory RNA oligonucleotide will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the inhibitory RNA oligonucleotide in target cells. Other aspects to consider for vectors and constructs are further described below.

[0153] Vectors useful for the delivery of an inhibitory RNA oligonucleotide will include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the inhibitory RNA oligonucleotide in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated / inducible expression.

[0154] Expression of the inhibitory RNA oligonucleotide can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of dsRNA expression in cells or in mammals include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1 - thiogalactopyranoside (IPTG). A person skilled in the art would be able to choose the appropriate regulatory / promoter sequence based on the intended use of the inhibitory RNA oligonucleotide transgene.

[0155] Viral vectors that contain nucleic acid sequences encoding an inhibitory RNA oligonucleotide can be used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol.217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding an inhibitory RNA oligonucleotide are cloned into one or more vectors, which facilitate delivery of the nucleic acid into a patient. More detail about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291- 302 (1994), which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest.93:644-651 (1994); Kiem et al., Blood 83: 1467-1473 (1994); Salmons and 44Attorney Docket: 090147-0584673 Gunzberg, Human Gene Therapy 4: 129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel.3: 110-114 (1993). Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in U.S. Patent Nos.6,143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference.

[0156] Adenoviruses are also contemplated for use in delivery of inhibitory RNA oligonucleotide of the disclosure. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68: 143-155 (1992); Mastrangeli et al., J. Clin. Invest.91:225-234 (1993); PCT Publication WO94 / 12649; and Wang, et al., Gene Therapy 2:775-783 (1995).

[0157] Adeno-associated virus (AAV) vectors may also be used to delivery an inhibitory RNA oligonucleotide of the disclosure (Walsh et al., Proc. Soc. Exp. Biol. Med.204:289-300 (1993); U.S. Pat. No.5,436,146). In one embodiment, the inhibitory RNA oligonucleotide can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in the disclosure, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol.61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No.5,252,479; U.S. Pat. No.5,139,941; International Patent Application No. WO 94 / 13788; and International Patent Application No. WO 93 / 24641, the entire disclosures of which are herein incorporated by reference.

[0158] Another viral vector suitable for delivery of an inhibitory RNA oligonucleotide of the disclosure is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.

[0159] The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors can be made to target different cells by engineering the vectors to express 45Attorney Docket: 090147-0584673 different capsid protein serotypes; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.

[0160] The pharmaceutical preparation of a vector can include the vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system. Molecular Assemblies

[0161] An inhibitory RNA oligonucleotide described herein disclosure can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the inhibitory RNA oligonucleotide composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the inhibitory RNA oligonucleotide composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the inhibitory RNA oligonucleotide are delivered into the cell where the inhibitory RNA oligonucleotide can specifically bind to a target RNA and can mediate RNAi. In some cases the liposomes are also specifically targeted, e.g., to direct the inhibitory RNA oligonucleotide to particular cell types.

[0162] A liposome containing an inhibitory RNA oligonucleotide can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The inhibitory RNA oligonucleotide preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the inhibitory RNA oligonucleotide t and condense around the inhibitory RNA oligonucleotide to form a liposome. After condensation, 46Attorney Docket: 090147-0584673 the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of inhibitory RNA oligonucleotide.

[0163] If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation.

[0164] Methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide / cationic lipid complex as structural components of the delivery vehicle, are further described in, e.g., WO 96 / 37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include one or more aspects of exemplary methods described in Feigner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No.4,897,355; U.S. Pat. No.5,171,678; Bangham, et al. M. Mol. Biol.23:238, 1965; Olson, et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775: 169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al. Endocrinol.115:757, 1984. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858: 161, 1986). Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775: 169, 1984). These methods are readily adapted to packaging RNAi agent preparations into liposomes.

[0165] Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged nucleic acid molecules to form a stable complex. The positively charged nucleic acid / liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0166] Liposomes which are pH-sensitive or negatively-charged, entrap nucleic acids rather than complex with it. Since both the nucleic acid and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture.

[0167] Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274). 47Attorney Docket: 090147-0584673

[0168] One major type of liposomal composition includes phospholipids other than naturally- derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and / or phosphatidylcholine and / or cholesterol.

[0169] Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. No.5,283,185; U.S. Pat. No.5,171,678; WO 94 / 00569; WO 93 / 24640; WO 91 / 16024; Feigner, J. Biol. Chem.269:2550, 1994; Nabel, Proc. Natl. Acad. Sci.90: 11307, 1993; Nabel, Human Gene Ther.3:649, 1992; Gershon, Biochem.32:7143, 1993; and Strauss EMBO J.11:417, 1992.

[0170] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4(6) 466).

[0171] Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside GM1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0172] Various liposomes comprising one or more glycolipids are known in the art. 48Attorney Docket: 090147-0584673

[0173] Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside GM1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No.4,837,028 and WO 88 / 04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside GM1 or a galactocerebroside sulfate ester. U.S. Pat. No.5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn- dimyristoylphosphatidylcholine are disclosed in WO 97 / 13499 (Lim et al).

[0174] In some embodiments, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver RNAi agents to macrophages.

[0175] Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated RNAi agents in their internal compartments from metabolism and degradation (Rosoff, in "Pharmaceutical Dosage Forms," Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p.245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

[0176] A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of inhibitory RNA oligonucleotide (see, e.g., Feigner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No.4,897,355 for a description of DOTMA and its use with DNA).

[0177] A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles.

[0178] Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell 49Attorney Docket: 090147-0584673 surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2- bis(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Indiana) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.

[0179] Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (Transfectam™, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide ("DPPES") (see, e.g., U.S. Pat. No.5,171,678).

[0180] Another cationic lipid conjugate includes derivatization of the lipid with cholesterol ("DC-Choi") which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun.179:280, 1991). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98 / 39359 and WO 96 / 37194.

[0181] Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer RNAi agent into the skin. In some implementations, liposomes are used for delivering RNAi agent to epidermal cells and also to enhance the penetration of RNAi agent into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., Journal oƒ Drug Targeting, 1992, vol.2,405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259- 265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276.1987; Nicolau, C. et al. Meth. Enz.149: 157-176, 1987; Straubinger, R. M. 50Attorney Docket: 090147-0584673 and Papahadjopoulos, D. Meth. Enz.101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

[0182] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with an inhibitory RNA oligonucleotide are useful for treating a dermatological disorder.

[0183] Liposomes that include inhibitory RNA oligonucleotide can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes.

[0184] Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include an inhibitory RNA oligonucleotide can be delivered, for example, subcutaneously by infection in order to deliver RNAi agent to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self- repairing, and can frequently reach their targets without fragmenting, and often self-loading.

[0185] Other formulations amenable to the present disclosure are described in United States provisional application serial Nos.61 / 018,616, filed January 2, 2008; 61 / 018,611, filed January 2, 2008; 61 / 039,748, filed March 26, 2008; 61 / 047,087, filed April 22, 2008 and 61 / 051,528, filed May 8, 2008. PCT application no PCT / US2007 / 080331, filed October 3, 2007 also describes formulations that are amenable to the present disclosure.

[0186] Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to 51Attorney Docket: 090147-0584673 deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0187] The inhibitory RNA oligonucleotide described herein can also be provided as micellar formulations. "Micelles" are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

[0188] A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the inhibitory RNA oligonucleotide, an alkali metal C8 to C22 alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles.

[0189] In one method a first micellar composition is prepared which contains the inhibitory RNA oligonucleotide and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the inhibitory RNA oligonucleotide, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.

[0190] Phenol and / or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and / or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition. 52Attorney Docket: 090147-0584673

[0191] For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.

[0192] Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen- containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

[0193] The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract. Methods of Use

[0194] The present disclosure provides methods of inhibiting expression of CB1R in a cell. The methods include contacting a cell with an inhibitory RNA oligonucleotide in an amount effective to inhibit expression of the CB1R in the cell, thereby inhibiting expression of the CB1R in the cell.

[0195] In some embodiments, contacting of a cell with an inhibitory RNA oligonucleotide is done in vitro or in vivo. In some embodiments, contacting a cell in vivo with the inhibitory RNA oligonucleotide comprises contacting a cell or group of cells within a subject, e.g., a human subject, with the inhibitory RNA oligonucleotide. Combinations of in vitro and in vivo methods of contacting are also possible. In some embodiments, contacting a cell is accomplished via a targeting ligand, including any ligand described herein or known in the art. In some embodiments, the targeting ligand is a carbohydrate moiety, e.g., a GalNAc ligand, or any other ligand that directs the inhibitory RNA oligonucleotide to a site of interest, e.g., the liver of a subject.

[0196] In some embodiments, contacting a cell with an inhibitory RNA oligonucleotide comprising administering the inhibitory RNA oligonucleotide through a suitable route of administration as known in the art or described herein. For example, in some embodiments, the inhibitory RNA oligonucleotide is administered via topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; 53Attorney Docket: 090147-0584673 intratracheal, intranasal, epidermal and transdermal, oral or parenteral administration. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular administration.

[0197] In some embodiments, inhibition is assessed by a decrease in an absolute or relative level of one or more variables that are associated with CB1R expression compared with a control level. In some embodiments, the control level is any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).

[0198] In some embodiments, expression of a CB1R gene is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0199] In some embodiments, inhibition of the expression of a CB1R gene is manifested by a reduction of the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which a CB1R gene is transcribed and which has or have been treated (e.g., by contacting the cell or cells with an inhibitory RNA oligonucleotide of the disclosure, or by administering an inhibitory RNA oligonucleotide of the disclosure to a subject in which the cells are or were present) such that the expression of a CB1R gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or have not been so treated (control cell(s)).

[0200] In some embodiments, inhibition of the expression of a CB1R gene is assessed in terms of a reduction of a parameter that is functionally linked to CB1R gene expression. CB1R gene silencing may be determined in any cell expressing CB1R, either constitutively or by genomic engineering, and by any assay known in the art.

[0201] In some embodiments, inhibition of the expression of a CB1R protein is manifested by a reduction in the level of the CB1R protein that is expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject). As explained above for the assessment of mRNA suppression, the inhibition of protein expression levels in a 54Attorney Docket: 090147-0584673 treated cell or group of cells may similarly be expressed as a percentage of the level of protein in a control cell or group of cells.

[0202] In some embodiments, a control cell or group of cells that are used to assess the inhibition of the expression of a CB1R gene comprises a cell or group of cells that has not yet been contacted with an inhibitory RNA oligonucleotide of the disclosure. In some embodiments, the control cell or group of cells is derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an inhibitory RNA oligonucleotide.

[0203] The level of CB1R mRNA that is expressed by a cell or group of cells may be determined using any method known in the art for assessing mRNA expression. In one embodiment, the level of expression of CB1R in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the CB1R gene. In some embodiments, RNA is extracted from cells using RNA extraction techniques including, for example, using acid phenol / guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kits (Qiagen) or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT- PCR, RNase protection assays (Melton et al., Nuc. Acids Res.12:7035), Northern blotting, in situ hybridization, and microarray analysis.

[0204] In some embodiments, the level of expression of CB1R is determined using a nucleic acid probe. The term "probe", as used herein, refers to any molecule that is capable of selectively binding to a specific CB1R. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0205] Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction (PCR) analyses and probe arrays. One method for the determination of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to CB1R mRNA. In some embodiments, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In some embodiments, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in determining the level of CB1R mRNA. 55Attorney Docket: 090147-0584673

[0206] In some embodiments, a method for determining the level of expression of CB1R in a sample involves the process of nucleic acid amplification and / or reverse transcriptase (to prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No.4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88: 189-193), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio / Technology 6: 1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In some embodiments, the level of expression of CB1R is determined by quantitative fluorogenic RT-PCR (i.e., the TaqMan™ System).

[0207] In some embodiments, expression levels of CB1R mRNA may be monitored using a membrane blot (such as used in hybridization analysis such as Northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U.S. Pat. Nos.5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference. The determination of CB1R expression level may also comprise using nucleic acid probes in solution.

[0208] In some embodiments, the level of mRNA expression is assessed using branched DNA (bDNA) assays or real time PCR (qPCR).

[0209] In some embodiments, the level of CB1R protein expression is determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), Immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, electrochemiluminescence assays, and the like.

[0210] Provided herein are methods of treating a disease or disorder in a subject comprising administering inhibitory RNA oligonucleotides to reduce expression of CB1R. In some embodiments, administration of an inhibitory RNA oligonucleotide provided herein reduces expression of CB1R in adipose tissue. In some embodiments, administration of an inhibitory RNA oligonucleotide provided herein reduces expression of CB1R in the liver. In some 56Attorney Docket: 090147-0584673 embodiments, administration of an inhibitory RNA oligonucleotide provided herein reduces expression of CB1R in the pancreas. In some embodiments, administration of an inhibitory RNA oligonucleotide provided herein reduces expression of CB1R in the kidney. In some embodiments, administration of an inhibitory RNA oligonucleotide provided herein reduces activity of CB1R in adipose tissue. In some embodiments, administration of an inhibitory RNA oligonucleotide provided herein reduces activity of CB1R in the liver. In some embodiments, administration of an inhibitory RNA oligonucleotide provided herein reduces activity of CB1R in the pancreas. In some embodiments, administration of an inhibitory RNA oligonucleotide provided herein reduces activity of CB1R in the kidney. In some embodiments, administration of an inhibitory RNA oligonucleotide provided herein does not reduce expression of CB1R in the tissue of the central nervous system. In some embodiments, administration of an inhibitory RNA oligonucleotide provided herein does not reduce activity of CB1R in the tissue of the central nervous system.

[0211] In some embodiments, administration of an inhibitory RNA oligonucleotide provided herein induces the conversion of white adipose tissue to brown adipose tissue or beige adipose tissue. In some embodiments, administration of an inhibitory RNA oligonucleotide provided herein increases non-shivering thermogenesis. Non-shivering thermogenesis can be evaluated by determining energy expenditure, heat production, oxygen consumption, and / or carbon dioxide production in a subject. In some embodiments, lean body mass is preserved in a subject following administration of an inhibitory RNA oligonucleotide provided herein. In some embodiments, mitogenesis is increased in cells following administration of an inhibitory RNA oligonucleotide provided herein.

[0212] In some embodiments, the inhibitory RNA oligonucleotide induces in localized thermogenesis at the site of injection. Accordingly, in some aspects, provided herein are methods for targeted adipose tissue reduction comprising administering the inhibitory RNA oligonucleotide.

[0213] The present disclosure also provides methods for treating or preventing diseases and disorders that can be modulated by CB1R gene expression. In some embodiments, the disease or disorder is selected from, obesity, a metabolic dysfunction-associated steatotic liver disease (MASLD, e.g., non-alcoholic fatty liver disease (NAFLD), a metabolic-associated steatohepatitis (MASH; e.g., non-alcoholic steatohepatitis), Type 2 Diabetes Mellitus, insulin resistance, Beta Cell dysfunction, elevated HbA1c, fatty liver disease, prediabetes, elevated lipid profiles, gastrointestinal motility, hyperphagia, renal fibrosis, reduction in glucose responsiveness of the pancreas, liver cirrhosis, cardiovascular disease, inflammatory disease, 57Attorney Docket: 090147-0584673 leptin resistance, leptin deficiency, and leptin receptor deficiency. In some embodiments, administration of an inhibitory RNA oligonucleotide is used as a therapy in weight management. In some embodiments, administration of an inhibitory RNA oligonucleotide is used as a therapy to prevent cardiovascular disease.

[0214] In some aspects, the disclosure provides methods for reducing drug seeking behavior in a subject, comprising administering to the subject an inhibitory RNA oligonucleotide provided herein. In some aspects, the disclosure provides methods for treating drug addiction in a subject, comprising administering to the subject an inhibitory RNA oligonucleotide provided herein. CB1R is believed to be involved in the rewarding effects of drugs of abuse, including cocaine and heroin. Accordingly, without wishing to be bound by theory, the inhibitory RNA oligonucleotides targeting CB1R provided herein are expected to reduce the rewarding effects of such drugs, thereby decreasing the likelihood of relapse and drug- seeking behavior.

[0215] In some aspects, the disclosure provides methods for treating chronic neuropathic pain in a subject, comprising administering to the subject an inhibitory RNA oligonucleotide provided herein. As CB1R is expressed in pain pathways in the peripheral and central nervous systems, the inhibitory RNA oligonucleotides targeting CB1R provided herein are expected to provide relief from chronic neuropathic pain by silencing the CB1R gene.

[0216] The methods of the disclosure further relate to the use of an inhibitory RNA oligonucleotide or a pharmaceutical composition thereof, e.g., for treating obesity or a metabolic disorder, in combination with other pharmaceuticals and / or other therapeutic methods, e.g., with known pharmaceuticals and / or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders.

[0217] In some embodiments, the inhibitory RNA oligonucleotide and an additional therapeutic agent are administered in the same composition, e.g., parenterally, or the additional therapeutic agent is administered as part of a separate composition or by another method described herein. Administration of the inhibitory RNA oligonucleotide and the additional therapeutic agent can be at the same time, or at different times and, in any order.

[0218] In some embodiments, efficacy of treatment or prevention of disease is assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to 58Attorney Docket: 090147-0584673 monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters.

[0219] In some embodiments, administration of the inhibitory RNA oligonucleotides the disclosure enhances mitogenesis in cells. In some embodiments, mitogenesis is enhanced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, mitogenesis is enhanced for 7 days, 10 days, 20 days, 30 days, or more following administration.

[0220] In some embodiments, mitogenesis is evaluated by quantifying mitochondrial biogenesis markers. In some embodiments, a mitochondrial biogenesis marker is PGC-1α. In some embodiments, a mitochondrial biogenesis marker is NRF-1. Methods to analyze biomarkers are known to those with ordinary skill in the art. In some embodiments, mitochondrial biogenesis markers are quantified via RT-PCR, 2D-PAGE, mass spectrometry, western blot, and ELISA among others.

[0221] In some embodiments, a treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. In some embodiments, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more is indicative of effective treatment. Efficacy for a given inhibitory RNA oligonucleotide drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.

[0222] In some embodiments, the efficacy is measured by a reduction in the severity of disease as determined by one skilled in the art of diagnosis based on a clinically accepted disease severity grading scale. Combinatorial Therapies

[0223] In some aspects of the disclosure, the inhibitory RNA oligonucleotides and compositions provided herein, are formulated or administered with a second agent. In some embodiments, an inhibitory RNA oligonucleotide provided herein is administered to a subject with a second agent to treat a disease or disorder. In some embodiments, an inhibitory RNA oligonucleotide provided herein is administered to a subject with a second agent to prevent a disease or disorder. 59Attorney Docket: 090147-0584673

[0224] In some embodiments, a subject is administered an inhibitory RNA oligonucleotide of the disclosure prior to administration of a second agent. In some embodiments, an inhibitory RNA oligonucleotide is co-formulated with a second agent and administered to a subject. In some embodiments, a subject is administered an inhibitory RNA oligonucleotide and second agent simultaneously. In some embodiments, a subject is administered an inhibitory RNA oligonucleotide and second agent sequentially. In some embodiments, a subject is administered a second agent prior to administration of an inhibitory RNA oligonucleotide of the disclosure.

[0225] In some embodiments, the disclosure provides a method for treating a disorder described herein in a subject that has received or is receiving treatment with an inhibitory RNA oligonucleotide described herein, comprising administering a second agent described herein to the subject. In some embodiments, the disclosure provides a method for treating a disorder described herein in a subject that has received or is receiving treatment with a second agent described herein, comprising administering an inhibitory RNA oligonucleotide described herein to the subject.

[0226] In some embodiments, the second agent is a therapeutic for treating diabetes, hypertension, or inflammation.

[0227] In some embodiments, the second agent is a thermogenic agent. In some embodiments, the thermogenic agent is an mRNA encoding a protein associated with futile cycles or metabolic effects. Methods for generating mRNA compositions are known to those of skill in the art.

[0228] As established know to those of skill in the art, mRNA generally contains a 5′ untranslated region (5′-UTR), a peptide coding region and a 3′ untranslated region (3′-UTR). In some embodiments, the RNA is produced by in vitro transcription or chemical synthesis. In some embodiments, the mRNA is produced by in vitro transcription using a DNA template where DNA refers to a nucleic acid that contains deoxyribonucleotides.

[0229] In some embodiments, the mRNA comprises a microRNA (miR) binding site to control expression of the mRNA. In some embodiments the microRNA binding sites are of the 3p and 5p arms of the same microRNA. In some embodiments the microRNA binding sites are located in the 5′ UTR, 3′ UTR, or both the 5′ UTR and 3′ UTR of the mRNA.

[0230] In some embodiments the mRNA has a 5′ terminal cap that comprises a Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guano sine, 8-oxo- guanosine, 2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5′ methylG cap, or an analog thereof. 60Attorney Docket: 090147-0584673

[0231] In some embodiments the mRNA comprises a poly-A region. The poly-A region may have about 10 to about 200, about 20 to about 180, about 50 to about 160, about 70 to about 140, or about 80 to about 120 nucleotides in length.

[0232] In some embodiments the mRNA comprises at least one chemically modified nucleobase, sugar, backbone, or any combination thereof as known in the art and / or described herein. In embodiments the at least one chemically modified nucleobase is selected from the group consisting of pseudouracil (ψ), N1-methylpseudouracil (m1ψ), 1-ethylpseudouracil, 2- thiouracil (s2U), 4′-thiouracil, 5-methylcytosine, 5-methyluracil, 5-methoxyuracil, and any combination thereof.

[0233] In some embodiments at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% of the guanines, adenines, uracils or thymines are chemically modified.

[0234] In some embodiments, methods for preparing mRNA are described in US Publication Nos. US20200254086, US20180085474, US20240009238, US20180237786, US20220340641, US20150258174; the contents of each of which are herein incorporated by reference in their entirety.

[0235] In some embodiments, the mRNA is formulated in a delivery vehicle suitable for mRNA delivery and / or described herein. In some embodiments, the mRNA is formulated in a lipid nanoparticle as known in the art and / or described herein. Creatine Kinase B

[0236] Creatine kinase B (CKB) is an enzyme involved in energy metabolism within brown adipose tissue (BAT), also known as brown fat. BAT is specialized for generating heat (thermogenesis) through the burning of calories. Without being bound by theory, increasing CKB expression in BAT, enhances BAT activity and thus increases thermogenesis. Administering an inhibitory RNA oligonucleotide targeting CB1R and mRNA encoding CKB is believed to provide a synergistic effect that drives thermogenesis thus contributing to weight loss.

[0237] Accordingly, in some embodiments, the inhibitory RNA oligonucleotides and compositions provided herein, are co-formulated and / or administered with a CKB polypeptide or an mRNA encoding a CKB polypeptide. In some embodiments, the mRNA encoding a CKB polypeptide comprises a nucleotide sequence having at least 80% identity to 61Attorney Docket: 090147-0584673 the sequence set forth in SEQ ID NO: 1. In some embodiments, the mRNA encoding a CKB polypeptide comprises a nucleotide sequence having at least 85% identity to the sequence set forth in SEQ ID NO: 1. In some embodiments, the mRNA encoding a CKB polypeptide comprises a nucleotide sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 1. In some embodiments, the mRNA encoding a CKB polypeptide comprises a nucleotide sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 1. In some embodiments, the mRNA encoding a CKB polypeptide comprises a nucleotide sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 1.In some embodiments, the mRNA encoding CKB comprises a sequence set forth in SEQ ID NO: 1.

[0238] In some embodiments, the disclosure provides a method for treating a disorder described herein in a subject that has received or is receiving treatment with an inhibitory RNA oligonucleotide described herein, comprising administering an mRNA encoding a CKB polypeptide described herein to the subject. In some embodiments, the disclosure provides a method for treating a disorder described herein in a subject that has received or is receiving treatment with an mRNA encoding a CKB polypeptide described herein, comprising administering an inhibitory RNA oligonucleotide described herein to the subject. ZNF423

[0239] Zinc Finger Protein 423, ZNF423, alternatively referred to as ZFP423, is a transcription factor that plays a key role in the development and function of white adipose tissue (WAT). Accordingly, silencing the expression of the ZNF423 gene is expected to decrease WAT and increase in brown adipose tissue (BAT). Without being bound by theory, an increase in BAT activity leads to an increase in energy expenditure and a decrease in body weight. Administering an inhibitory RNA oligonucleotide targeting CB1R and siRNA targeting ZNF423 is believed to provide synergistic effect that drives thermogenesis.

[0240] Four isoforms of ZFP423 are found on the NCBI database: NM_015069.5)

[0241] In some embodiments, the inhibitory RNA oligonucleotides and compositions provided herein, are co-formulated and / or administered with an inhibitory RNA oligonucleotide targeting ZNF423. Methods for identifying inhibitory RNA oligonucleotide are known in the art and described herein.

[0242] In some embodiments, the disclosure provides a method for treating a disorder described herein in a subject that has received or is receiving treatment with a CB1R targeting inhibitory RNA oligonucleotide described herein, comprising administering a ZNF423 targeting inhibitory RNA oligonucleotide described herein to the subject. In some 62Attorney Docket: 090147-0584673 embodiments, the disclosure provides a method for treating a disorder described herein in a subject that has received or is receiving treatment with a ZNF423 targeting inhibitory RNA oligonucleotide described herein, comprising administering an inhibitory RNA oligonucleotide described herein to the subject.

[0243] In some aspects, provided herein are inhibitory RNA oligonucleotides targeting ZNF423. The ZNF423 targeting inhibitory RNA oligonucleotides can be formulated or administered alone or in combination in any of the compositions or methods described herein. Leptin

[0244] Leptin Deficiency Disorder (LDD) is a rare genetic disorder characterized by a deficiency in the hormone leptin. Leptin is produced by adipose tissue and plays a crucial role in regulating appetite, energy expenditure, and metabolism. Leptin Receptor (LEPR) Deficiency Disorder is a rare genetic disorder characterized by impaired leptin signaling due to mutations in the LEPR gene. Individuals with LDD or LEPR Deficiency Disorder experience severe obesity, hyperphagia (excessive hunger), and a variety of metabolic complications. Current treatment for LDD and LEPR Deficiency Disorder involves daily injections of recombinant human leptin (Metreleptin ®). While Metreleptin® can effectively reduce appetite and weight, it has several limitations, including the need for daily injections, high cost, and potential for developing leptin resistance. Inhibition of CB1R in the peripheral tissue by a small molecule has been shown to reduce appetite, increase energy expenditure, and improve insulin sensitivity (Tam, Joseph et al. Cell Metabolism, vol.16(2): 167-179, July 26, 2012). Without being bound by theory, administering an inhibitory RNA oligonucleotide of the disclosure with a leptin (LEP) polypeptide or an mRNA encoding a LEP polypeptide offers a synergistic approach to treating LDD or LEPR Deficiency Disorder.

[0245] Accordingly, in some embodiments, the inhibitory RNA oligonucleotides and compositions provided herein, are co-formulated and / or administered with a an LEP polypeptide or an mRNA encoding a LEP polypeptide. In some embodiments, the mRNA encoding LEP comprises 80% identity to the sequence set forth in SEQ ID NO: 2. In some embodiments, the mRNA encoding LEP comprises 85% identity to the sequence set forth in SEQ ID NO: 2. In some embodiments, the mRNA encoding LEP comprises 90% identity to the sequence set forth in SEQ ID NO: 2. In some embodiments, the mRNA encoding LEP comprises 95% identity to the sequence set forth in SEQ ID NO: 2. In some embodiments, the mRNA encoding LEP comprises 99% identity to the sequence set forth in SEQ ID NO: 2. 63Attorney Docket: 090147-0584673 In some embodiments, the mRNA encoding LEP comprises a sequence set forth in SEQ ID NO: 2.

[0246] In some embodiments, the inhibitory RNA oligonucleotides and compositions provided herein, are co-formulated and / or administered with a LEPR polypeptide or an mRNA encoding a LEPR polypeptide.

[0247] Further, leptin resistance has been implicated in Bardet-Biedl syndrome (BSS) and proopiomelanocortin (POMC) deficiency. Without being bound by theory, administering an inhibitory RNA oligonucleotide of the disclosure with a LEPR polypeptide or an mRNA encoding a LEPR polypeptide can be used to treat leptin-related disorders including, BBS and POMC.

[0248] In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 3. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 85% identity to the sequence set forth in SEQ ID NO: 3. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 3. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 3. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 3. In some embodiments, the mRNA encoding LEPR comprises a sequence set forth in SEQ ID NO: 3. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 4. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 85% identity to the sequence set forth in SEQ ID NO: 4. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 4. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 4. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 4. In some embodiments, the mRNA encoding LEPR comprises a sequence set forth in SEQ ID NO: 4. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 5. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 85% identity to the sequence set forth in SEQ ID NO: 5. In some embodiments, the mRNA 64Attorney Docket: 090147-0584673 encoding a LEPR polypeptide comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 5. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 5. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 5. In some embodiments, the mRNA encoding LEPR comprises a sequence set forth in SEQ ID NO: 5. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 6. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 85% identity to the sequence set forth in SEQ ID NO: 6. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 6. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 6. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 6. In some embodiments, the mRNA encoding LEPR comprises a sequence set forth in SEQ ID NO: 6. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 7. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 85% identity to the sequence set forth in SEQ ID NO: 7. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 7. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 7. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 7. In some embodiments, the mRNA encoding LEPR comprises a sequence set forth in SEQ ID NO: 7. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 8. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 85% identity to the sequence set forth in SEQ ID NO: 8. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 8. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 8. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a 65Attorney Docket: 090147-0584673 sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 8. In some embodiments, the mRNA encoding LEPR comprises a sequence set forth in SEQ ID NO: 8. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 9. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 85% identity to the sequence set forth in SEQ ID NO: 9. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 9. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 9. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 9. In some embodiments, the mRNA encoding LEPR comprises a sequence set forth in SEQ ID NO: 9. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 10. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 85% identity to the sequence set forth in SEQ ID NO: 10. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 10. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 10. In some embodiments, the mRNA encoding a LEPR polypeptide comprises a sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 10. In some embodiments, the mRNA encoding LEPR comprises a sequence set forth in SEQ ID NO: 10.

[0249] In some embodiments, the disclosure provides a method for treating a disorder described herein in a subject that has received or is receiving treatment with an inhibitory RNA oligonucleotide described herein, comprising administering an mRNA encoding a LEP polypeptide described herein to the subject. In some embodiments, the disclosure provides a method for treating a disorder described herein in a subject that has received or is receiving treatment with an mRNA encoding a LEP polypeptide described herein, comprising administering an inhibitory RNA oligonucleotide described herein to the subject.

[0250] In some embodiments, the disclosure provides a method for treating a disorder described herein in a subject that has received or is receiving treatment with an inhibitory RNA oligonucleotide described herein, comprising administering an mRNA encoding a LEPR polypeptide described herein to the subject. In some embodiments, the disclosure provides a method for treating a disorder described herein in a subject that has received or is 66Attorney Docket: 090147-0584673 receiving treatment with an mRNA encoding a LEPR polypeptide described herein, comprising administering an inhibitory RNA oligonucleotide described herein to the subject. DAGL

[0251] Diaclyglycerol lipase (DAGL) is the primary enzyme responsible for synthesizing 2- arachidonoylglycerol (2-AG), the most abundant cannabinoid receptor ligand. Thus, silencing DAGL is expected to reduce endocannabinoid synthesis, which decreases signaling through CB1R. Inhibiting CB1R expression alone can lead to enhanced dependence on alternative cannabinoid signaling pathways. However, without wishing to be bound by theory, the combined administration of an inhibitory RNA oligonucleotide targeting CB1R and an inhibitory RNA oligonucleotide targeting DAGL is believed to provide a complementary approach by both inhibiting CB1R expression and decreasing overall endocannabinoid levels. In some embodiments, the inhibitory RNA oligonucleotides and compositions provided herein, are co-formulated and / or administered with an inhibitory RNA oligonucleotide targeting DAGL isoform alpha (DAGLA) or beta (DAGLB). Methods for identifying inhibitory RNA oligonucleotide are known in the art and described herein.

[0252] In some embodiments, the disclosure provides a method for treating a disorder described herein in a subject that has received or is receiving treatment with a CB1R targeting inhibitory RNA oligonucleotide described herein, comprising administering a DAGL targeting inhibitory RNA oligonucleotide described herein to the subject. In some embodiments, the disclosure provides a method for treating a disorder described herein in a subject that has received or is receiving treatment with a DAGL targeting inhibitory RNA oligonucleotide described herein, comprising administering an inhibitory RNA oligonucleotide described herein to the subject.

[0253] In some aspects, provided herein are inhibitory RNA oligonucleotides targeting DAGL. The DAGL targeting inhibitory RNA oligonucleotides can be formulated or administered alone or in combination in any of the compositions or methods described herein. TLE3

[0254] Transducing-like enhancer of split 3 (TLE3) is a highly conserved transcriptional regulator that promotes differentiation of white adipose tissue (WAT) in part by suppressing genes associated with brown adipose tissue (BAT). Accordingly, inhibiting expression of the TLE3 gene is expected to decrease WAT and increase BAT. Without wishing to be bound by theory, administering an inhibitory RNA oligonucleotide targeting CB1R and an inhibitory 67Attorney Docket: 090147-0584673 RNA oligonucleotide targeting TLE3 is believed to provide a synergistic effect that promotes browning of adipose tissue. In some embodiments, the inhibitory RNA oligonucleotides and compositions provided herein, are co-formulated and / or administered with an inhibitory RNA oligonucleotide targeting TLE3. Methods for identifying inhibitory RNA oligonucleotide are known in the art and described herein.

[0255] In some embodiments, the disclosure provides a method for treating a disorder described herein in a subject that has received or is receiving treatment with a CB1R targeting inhibitory RNA oligonucleotide described herein, comprising administering a TLE3 targeting inhibitory RNA oligonucleotide described herein to the subject. In some embodiments, the disclosure provides a method for treating a disorder described herein in a subject that has received or is receiving treatment with a TLE3 targeting inhibitory RNA oligonucleotide described herein, comprising administering an inhibitory RNA oligonucleotide described herein to the subject.

[0256] In some aspects, provided herein are inhibitory RNA oligonucleotides targeting TLE3. The TLE3 targeting inhibitory RNA oligonucleotides can be formulated or administered alone or in combination in any of the compositions or methods described herein. Kits

[0257] The present disclosure also provides kits for using any of the inhibitory RNA oligonucleotides and / or performing any of the methods of the disclosure. Such kits include one or more inhibitory RNA oligonucleotide(s) and instructions for use, e.g., instructions for inhibiting expression of a CB1R in a cell by contacting the cell with the inhibitory RNA oligonucleotide (s) in an amount effective to inhibit expression of the CB1R. The kits may optionally further comprise means for contacting the cell with the inhibitory RNA oligonucleotide (e.g., an injection device), or means for measuring the inhibition of CB1R (e.g., means for measuring the inhibition of CB1R mRNA or CB1R protein). Such means for measuring the inhibition of CB1R may comprise a means for obtaining a sample from a subject, such as, e.g., a plasma sample. The kits of the disclosure may optionally further comprise means for administering the inhibitory RNA oligonucleotide (s) to a subject or means for determining the therapeutically effective or prophylactically effective amount.

[0258] In some embodiments, the present disclosure provides a kit comprising an inhibitory RNA oligonucleotide as described herein, and instructions for administering the inhibitory RNA oligonucleotide to a subject that has received or is receiving treatment with a second agent as described herein. In some embodiments, the present disclosure provides a kit 68Attorney Docket: 090147-0584673 comprising a second agent described herein, and instructions for administering the second agent to a subject that has received or is receiving treatment with an inhibitory RNA oligonucleotide described herein.

[0259] 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 inhibitory RNA oligonucleotides and methods featured in the 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 examples are illustrative only and not intended to be limiting. TABLES Table 1. CB1R Target and Antisense Strand Sequences with 18 Bases Target Sequence on mRNA of Human Antisense strand GC Content CB1R69Attorney Docket: 090147-0584673 AAGACCCUGGUCCUGAUC GAUCAGGACCAGGGUCUU 55.56% AAGACGGUGUUUGCAUUC GAAUGCAAACACCGUCUU 44.44%70Attorney Docket: 090147-0584673 GAAGAGCAUCAUCAUCCA UGGAUGAUGAUGCUCUUC 44.44% GAAGAUGAACAAGCUCAU AUGAGCUUGUUCAUCUUC 38.89%Nucleotide Sequence on Antisense strand GRCh38 / hg38 Target Uniqueness Human CB1R ORF Genome sequence for CB1R EAttorney Docket: 090147-0584673 88144149 AAGCTCATTAAGACGGTGT ACACCGUCUUAAUGAGCUU Chr6 88144140- 88144158 E E EAttorney Docket: 090147-0584673 AAGACATCAAAGGTGACAT AUGUCACCUUUGAUGUCUU Chr6 88145165- NON 88145183 UNIQUE AATGACATTCAGTACGAAG CUUCGUACUGAAUGUCAUU Chr6 88145181- E E E E E E E E EAttorney Docket: 090147-0584673 88147758 AAGTGATCAAAAAGGAACA UGUUCCUUUUUGAUCACUU Chr6 88147715- 88147733 E ETarget Sequence on mRNA of Human Antisense strand GC CB1R ContentAttorney Docket: 090147-0584673 AACUUCCUUUAGGGGAAGU ACUUCCCCUAAAGGAAGUU 42.11% AAGACAUCAAAGGUGACAU AUGUCACCUUUGAUGUCUU 36.84%75Attorney Docket: 090147-0584673 CAGCGUACUGCUUCUGUUC GAACAGAAGCAGUACGCUG 52.63% CAGCUUCAUUGACUUCCAC GUGGAAGUCAAUGAAGCUG 47.37%Nucleotide Sequence on Antisense strand GRCh38 / hg38 Target Uniqueness Human CB1R ORF Genome sequence for CB1R76Attorney Docket: 090147-0584673 AAAGATAGCCGCAAC AACACGUUGCGGCUAUChr688144709- GTGTTCUUU 88144728AAAGCTGCATCAAGA CGUGCUCUUGAUGCAG 88143916-Attorney Docket: 090147-0584673 AAGATAGCCGCAACG AAACACGUUGCGGCUAChr688144708- TGTTTUCUU88144727 AAGATGAACAAGCTC UUAAUGAGCUUGUUCA 88144148-Attorney Docket: 090147-0584673 AAACCTCCATCAGTA CCUCCUACUGAUGGAGchr688150590- GGAGGGUUU88150609 AACCTCCATCAGTAG GCCUCCUACUGAUGGA 88150589-Attorney Docket: 090147-0584673 AAGTGGAGGTGGCAG ACAUUCUGCCACCUCCch88147622- NON AATGTACUUr688147641 UNIQUE AAGGATTGCCCCCTG ACCCACAGGGGGCAAU 88145321-Target Sequence on mRNA of Human Antisense strand GC CB1R Content80Attorney Docket: 090147-0584673 AAGAUGAACAAGCUCAUUAA UUAAUGAGCUUGUUCAUCUU 30.00% AAGAUGACUGCGGGAGACAA UUGUCUCCCGCAGUCAUCUU 50.00% AAGA GCCAAGG AACCA A GG ACC GGCAA C 4000%81Attorney Docket: 090147-0584673 CAGGCCUUCCUACCACUUCA UGAAGUGGUAGGAAGGCCUG 55.00% CAGGUGAACAUUACAGAAUU AAUUCUGUAAUGUUCACCUG 35.00% CAG A GC C GCC GC GA CAGCAGGCAGAGCA AC G 00%Nucleotide Sequence on Antisense strand GRCh38 / hg38 Target Uniqueness Human CB1R ORF Genome sequence for CB1R82Attorney Docket: 090147-0584673 AACAAGCTCATTAAG CACCGUCUUAAUGA Chr6 88144141- ACGGTG GCUUGUU 88144161 AACAAGTCTCTCTCG GAAGGACGAGAGAG Chr6 88145008-83Attorney Docket: 090147-0584673 AAGGAGAATGAGGA GAUGUUCUCCUCAU Chr6 88144987- GAACATC UCUCCUU 88145007 AAGGTAACCATGTCT GGACACAGACAUGG Chr6 88143883-84Attorney Docket: 090147-0584673 AAGATTACCCCCACA CACAGCUGUGGGGG chr6 88147738- GCTGTG UAAUCUU 88147758 AAGTGATCAAAAAGG CCUGUUCCUUUUUG chr6 88147713-Target Sequence on mRNA of Antisense strand GC Human CB1R Content85Attorney Docket: 090147-0584673 AACCUACCUGAUGUUCUGGAU AUCCAGAACAUCAGGUAGGUU 42.86% AACGUGUUUCUGUUCAAACUG CAGUUUGAACAGAAACACGUU 38.10% AACUCCACCGUGAACCCCAUC GAUGGGGUUCACGGUGGAGUU 5714%86Attorney Docket: 090147-0584673 CACCAGCGUACUGCUUCUGUU AACAGAAGCAGUACGCUGGUG 52.38% CACGCUUUCCGGAGCAUGUUU AAACAUGCUCCGGAAAGCGUG 52.38% CAGAAAUUCCCUUUAACUUCC GGAAGUUAAAGGGAAUUUCUG 3810%87Attorney Docket: 090147-0584673 CACAGAAAUUCCCUUUGAUUU AAAUCAAAGGGAAUUUCUGUG 33.33% AGGUGAACAUUACGGAGUUUU AAAACUCCGUAAUGUUCACCU 38.10%Nucleotide Sequence on Antisense strand GRCh38 / hg38 Target sequence Uniqueness Human CB1R ORF Genome coordinates for CB1R Assembl ORF88Attorney Docket: 090147-0584673 AACGTGTTTCTGTTC CCAGUUUGAACAGAC88144695- AAACTGGAACACGUUhr688144716AACTCCACCGTGAAC UGAUGGGGUUCACGh88144089-89Attorney Docket: 090147-0584673 AATGACATTCAGTAC UGUCUUCGUACUGAC88145178- GAAGACAAUGUCAUUhr688145199AATGAGGAGAACATC CACACUGGAUGUUCh88144980-90Attorney Docket: 090147-0584673 AAAAAGGAACAGGA UUCUCUGGUCCUGU 88147704- N CAGAGAAUCCUUUUUcON Chr688147725 UNIQUE AAAAGGAACAGGAC CUUCUCUGGUCCUGh88147703-Target Sequence on mRNA of Human Antisense strand GC CB1R Content % % % % % % % % % % % % % % % % % %Attorney Docket: 090147-0584673 AACCUCCUGGUGCUGUGCGUCA UGACGCACAGCACCAGGAGGUU 59.09% AACGUGUUUCUGUUCAAACUGG CCAGUUUGAACAGAAACACGUU 40.91% AACUCCACCGUGAACCCCAUCA UGAUGGGGUUCACGGUGGAGUU 5455% % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % %92Attorney Docket: 090147-0584673 CAGAAUUUUACAACAAGUCUCU AGAGACUUGUUGUAAAAUUCUG 31.82% CAGACAUUUUCCCACACAUUGA UCAAUGUGUGGGAAAAUGUCUG 40.91% CAGACCAAGCCCGCAUGGACAU AUGUCCAUGCGGGCUUGGUCUG 5909% % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % %. Nucleotide Sequence on Antisense strand GRCh38 / hg38 Target Uniqueness Human CB1R ORF Genome se uence for CB1RAttorney Docket: 090147-0584673 AAACTGCAATCTGTT UCUGAGCAAACAGAUUCh88144478- NON TGCTCAGAGCAGUUUr688144500 UNIQUE AAAGATAGCCGCAA AGAAACACGUUGCGGCh88144706-94Attorney Docket: 090147-0584673 AAGATAGCCGCAAC CAGAAACACGUUGCGGC88144705- GTGTTTCTGCUAUCUUhr688144727AAGATGAACAAGCT GUCUUAAUGAGCUUGUh88144145-95Attorney Docket: 090147-0584673 AAGAGAGAGGTTTT UUCGUAUAUAAAACCUchr88150526- NON ATATACGAACUCUCUU688150548 UNIQUE AATATGGAACTGTT CACAUUGUGAACAGUUh88150505-96Attorney Docket: 090147-0584673 Table 11. CB1R Target and Antisense Strand Sequences with 23 Bases Target Sequence on mRNA of Human Antisense strand GC CB1R Content AAACAAUGCAGCCAGUGUUCACA UGUGAACACUGGCUGCAUUGUUU 43.48%97Attorney Docket: 090147-0584673 AAUCAUGGUGUAUGAUGUCUUUG CAAAGACAUCAUACACCAUGAUU 34.78% AAUCUGUUUGCUCAGACAUUUUC GAAAAUGUCUGAGCAAACAGAUU 34.78% AA GACA CAG ACGAAGACA A G C CG AC GAA G CA 34 8%98Attorney Docket: 090147-0584673 GAGUAAGGACCUGCGACACGCUU AAGCGUGUCGCAGGUCCUUACUC 56.52% GAGUGUUUCAUGGUCCUGAACCC GGGUUCAGGACCAUGAAACACUC 52.17% GA AGCCGCAACG G C G AACAGAAACACG GCGGC A C 4 83%Target Sequence on mRNA of Human Antisense strand GC CB1R ContentAttorney Docket: 090147-0584673 AACACGCAAACAAUGCAGCCAGUG CACUGGCUGCAUUGUUUGCGUG 50.00% UU AACAUCCAGUGUGGGGAGAACUUC GAAGUUCUCCCCACACUGGAUG 5000%Attorney Docket: 090147-0584673 AAGGCACUGCGCAGCCUCUGGAUA UAUCCAGAGGCUGCGCAGUGCC 58.33% UU AAGGUAACCAUGUCUGUGUCCACA UGUGGACACAGACAUGGUUACC 4583%Attorney Docket: 090147-0584673 CAGAAAGCUGCAUCAAGAGCACGG CCGUGCUCUUGAUGCAGCUUUC 54.17% UG CAGAAAUUCCCUUUAACUUCCUUU AAAGGAAGUUAAAGGGAAUUU 3333%Attorney Docket: 090147-0584673 GAAUGAGGAGAACAUCCAGUGUGG CCACACUGGAUGUUCUCCUCAU 50.00% UC GAAUUUUACAACAAGUCUCUCUCG CGAGAGAGACUUGUUGUAAAAU 3750%. Target Sequence on mRNA of Human Antisense strand GC CB1R C103Attorney Docket: 090147-0584673 AAACCUACCUGAUGUUCUGGAUCG CCGAUCCAGAACAUCAGGUAGG 48.00% G UUU AAACUGCAAUCUGUUUGCUCAGAC UGUCUGAGCAAACAGAUUGCAG 4000%Attorney Docket: 090147-0584673 AAGAGCACGGUCAAGAUUGCCAAG CCUUGGCAAUCUUGACCGUGCUC 52.00% G UU AAGAGCAUCAUCAUCCACACGUCU CAGACGUGUGGAUGAUGAUGCU 4800%Attorney Docket: 090147-0584673 CAAACAAUGCAGCCAGUGUUCACA CUGUGAACACUGGCUGCAUUGU 48.00% G UUG CAAAGAUAGCCGCAACGUGUUUCU CAGAAACACGUUGCGGCUAUCU 4800%Attorney Docket: 090147-0584673 GAAACCUACCUGAUGUUCUGGAUC CGAUCCAGAACAUCAGGUAGGU 48.00% G UUC GAAAGCUGCAUCAAGAGCACGGUC UGACCGUGCUCUUGAUGCAGCU 5200%Attorney Docket: 090147-0584673 UAGGGUACUUCCCACAGAAAUUCC GGGAAUUUCUGUGGGAAGUACC 48.00% C CUA UAGGUUAGCCAAGACCCUGGUCCU CAGGACCAGGGUCUUGGCUAAC 5600%Target Sequence on mRNA of Human Antisense strand GC ZNF423 Content AAAAAGAACAAGGAGCAU AUGCUCCUUGUUCUUUUU 3333%Attorney Docket: 090147-0584673 AACCACGAGUGUAAGCUG CAGCUUACACUCGUGGUU 50.00% AACCAGAAACACAAGUGC GCACUUGUGUUUCUGGUU 44.44% AACCAGA G CGAC CC GGAG CGAACA C GG 000%109Attorney Docket: 090147-0584673 AAGAGGUGGCUCAGUGUG CACACUGAGCCACCUCUU 55.56% AAGAGUCCAUUACCCAAG CUUGGGUAAUGGACUCUU 44.44% AAGA CCACC GAAGACC GG C CAGG GGA C 000%110Attorney Docket: 090147-0584673 AAGUCGGAGAAGGAAGCC GGCUUCCUUCUCCGACUU 55.56% AAGUGCAACGUUUGUUCA UGAACAAACGUUGCACUU 38.89% AAG GCACC AC GCAGC GC GCAG AGG GCAC 6%111Attorney Docket: 090147-0584673 CAAGAUCCAUGGCACCUU AAGGUGCCAUGGAUCUUG 50.00% CAAGAUCUACGACUGCUC GAGCAGUCGUAGAUCUUG 50.00% CAAGCAACAGG GGAG AAC CCACC G GC G 000%112Attorney Docket: 090147-0584673 CAGUGCAUCAAGUGCCAG CUGGCACUUGAUGCACUG 55.56% CAGUGCAUUACAUGACCA UGGUCAUGUAAUGCACUG 44.44% CAG GCA CAC GCCC AGGGCAG GAA GCAC G 6%113Attorney Docket: 090147-0584673 GAGAUCCAAAUCCACGUU AACGUGGAUUUGGAUCUC 44.44% GAGAUGGUUGUGACCUCG CGAGGUCACAACCAUCUC 55.56% GAGCGAGGAGGAG A A AAAC CC CC CGC C 000%Nucleotide Sequence on Human Antisense strand GRCh38 / hg38 ZNF423 ORF GenomeAttorney Docket: 090147-0584673 AAAAATTAGTTTAGCCCCT AGGGGCUAAACUAAUUUUU Chr16 AAAACAATCCCAGAGAAGT ACUUCUCUGGGAUUGUUUU Chr16 AAAACCTCGCAGAAAGTAG CUACUUUCUGCGAGGUUUU Chr16Attorney Docket: 090147-0584673 AAAGACATGGATGCATAAG CUUAUGCAUCCAUGUCUUU Chr16 AAAGACTCGAAGAGTCCAT AUGGACUCUUCGAGUCUUU Chr16 AAAGAGAGATGGATGGGTG CACCCAUCCAUCUCUCUUU Chr16Attorney Docket: 090147-0584673 AAATTGTACTGTACAGGAA UUCCUGUACAGUACAAUUU Chr16 AAATTGTCAGTTTCTAGCG CGCUAGAAACUGACAAUUU Chr16 AACAAGGAGCATCTGGCCA UGGCCAGAUGCUCCUUGUU Chr16Attorney Docket: 090147-0584673 AACGAGGAGATTAACCTCA UGAGGUUAAUCUCCUCGUU Chr16 AACGCTCCTTTTAAATTCA UGAAUUUAAAAGGAGCGUU Chr16 AACGTGCAGCTTTCCCAAG CUUGGGAAAGCUGCACGUU Chr16Attorney Docket: 090147-0584673 AAGAAGGCTGAGTTTATCA UGAUAAACUCAGCCUUCUU Chr16 AAGAAGTATCACTGCCACG CGUGGCAGUGAUACUUCUU Chr16 AAGAAGTCCAAGGCCGAGC GCUCGGCCUUGGACUUCUU Chr16Attorney Docket: 090147-0584673 AAGCAATTTTCCTCGGTGG CCACCGAGGAAAAUUGCUU Chr16 AAGCACAAGAGGAGCCGTG CACGGCUCCUCUUGUGCUU Chr16 AAGCACACACACACACGTC GACGUGUGUGUGUGUGCUU Chr16Attorney Docket: 090147-0584673 AAGGATGTTGCGTCACCCA UGGGUGACGCAACAUCCUU Chr16 AAGGCATCAACCACGAGTG CACUCGUGGUUGAUGCCUU Chr16 AAGGCATCTTACATATGTG CACAUAUGUAAGAUGCCUU Chr16Attorney Docket: 090147-0584673 AAGTGCACTGTGTGCAAGC GCUUGCACACAGUGCACUU Chr16 AAGTGCATCTTCTGTGGGG CCCCACAGAAGAUGCACUU Chr16 AAGTGCCAGATGACCTTCG CGAAGGUCAUCUGGCACUU Chr16Attorney Docket: 090147-0584673 AATCTGACTGCAGTAACTA UAGUUACUGCAGUCAGAUU Chr16 AATCTGGGAAGTTTGGCCA UGGCCAAACUUCCCAGAUU Chr16 AATCTGGGCTCCATCTCCC GGGAGAUGGAGCCCAGAUU Chr16Attorney Docket: 090147-0584673 AATTTTGCCCAGGCTGGGA UCCCAGCCUGGGCAAAAUU Chr16 AAAAAAGTTTCCAAGAGAC GUCUCUUGGAAACUUUUUU chr16 AAAAAATTAGTTTAGCCCC GGGGCUAAACUAAUUUUUU chr16Attorney Docket: 090147-0584673 AACCTCAGGGAGAGATGGG CCCAUCUCUCCCUGAGGUU chr16 AACGAGACCATGCAGAGAT AUCUCUGCAUGGUCUCGUU chr16 AACGAGGAGATTAACCTCA UGAGGUUAAUCUCCUCGUU chr16Attorney Docket: 090147-0584673 AAGTCAAGAGGAGAGAAAT AUUUCUCUCCUCUUGACUU chr16 AAGTGGAAAGAAGGAAAGA UCUUUCCUUCUUUCCACUU chr16 AAGTTTAGGTGTGTTAGGA UCCUAACACACCUAAACUU chr16Target Sequence on mRNA of Antisense strand GC Human ZNF423 Content126Attorney Docket: 090147-0584673 AAAGGAGUCAUUUCAACCC GGGUUGAAAUGACUCCUUU 42.11% AAAGGCACCCAGACAUCGC GCGAUGUCUGGGUGCCUUU 57.89%127Attorney Docket: 090147-0584673 AAGAACAAGGAGCAUCUGG CCAGAUGCUCCUUGUUCUU 47.37% AAGAACAUUCCACUGGCCC GGGCCAGUGGAAUGUUCUU 52.63%128Attorney Docket: 090147-0584673 AAGCUGCACAAGAACCAUG CAUGGUUCUUGUGCAGCUU 47.37% AAGCUGCAUACGGGCGACA UGUCGCCCGUAUGCAGCUU 57.89%129Attorney Docket: 090147-0584673 AAUCCACGUUGCCAACCAC GUGGUUGGCAACGUGGAUU 52.63% AAUCCCCAUUUUAUAGAUG CAUCUAUAAAAUGGGGAUU 31.58%130Attorney Docket: 090147-0584673 CACAGUGCCCUCAGAAGUU AACUUCUGAGGGCACUGUG 52.63% CACCAACUCCCCCAUCUUU AAAGAUGGGGGAGUUGGUG 52.63%131Attorney Docket: 090147-0584673 GAACGAGAGAGAGAUCCAA UUGGAUCUCUCUCUCGUUC 47.37% GAAGAAGAUGUACCGCUGC GCAGCGGUACAUCUUCUUC 52.63%132Attorney Docket: 090147-0584673 Table 17. ZNF423 Target and Antisense Strand Sequences with 20 Bases Nucleotide Sequence on Human Antisense strand GRCh38 / hg38 ZNF423 ORF Genome Assembly33Attorney Docket: 090147-0584673 AAGGCGGACCTGCAGTGCAT AUGCACUGCAGGUCCGCCUU chr16 AAGGCCTTCCACGCCATCAT AUGAUGGCGUGGAAGGCCUU chr16 AAGGCATCAACCACGAGTGT ACACUCGUGGUUGAUGCCUU chr16Attorney Docket: 090147-0584673 AAGAGGAGAGAAATGAGGAT AUCCUCAUUUCUCUCCUCUU chr16 AAGAGGACTTTGACTCCCAG CUGGGAGUCAAAGUCCUCUU chr16 AAGACTCGAAGAGTCCATTA UAAUGGACUCUUCGAGUCUU chr16Attorney Docket: 090147-0584673 AACCACACGATGAGCCAGCA UGCUGGCUCAUCGUGUGGUU chr16 AACCACAAGAACATTCCACT AGUGGAAUGUUCUUGUGGUU chr16 AACATTCCACTGGCCCACAG CUGUGGGCCAGUGGAAUGUU chr16Attorney Docket: 090147-0584673 AAAAAGGAGTCATTTCAACC GGUUGAAAUGACUCCUUUUU chr16 AAAAAGTATGAGGAAGAAGA UCUUCUUCCUCAUACUUUUU chr16 AAAAAGTTTCCAAGAGACAG CUGUCUCUUGGAAACUUUUU chr16Attorney Docket: 090147-0584673 AACCAGGAACTGACATTTAT AUAAAUGUCAGUUCCUGGUU chr16 AACCCTGGATGGACTGGCTG CAGCCAGUCCAUCCAGGGUU chr16 AACCCTTACCTGCCTGGCGA UCGCCAGGCAGGUAAGGGUU chr16Attorney Docket: 090147-0584673 AAGGTACAGGCAATCTGGGA UCCCAGAUUGCCUGUACCUU chr16 AAGGTCACTCATGGTTGGGG CCCCAACCAUGAGUGACCUU chr16 AAGGTTACACAGCAAGCAAC GUUGCUUGCUGUGUAACCUU chr16Target Sequence on mRNA of Antisense strand GC Human ZNF423 Content139Attorney Docket: 090147-0584673 AAACUAGAGUCUCCGGUGGU ACCACCGGAGACUCUAGUUU 50.00% AAACUCACCAAGCACAUCAA UUGAUGUGCUUGGUGAGUUU 40.00%140Attorney Docket: 090147-0584673 AACGUGCGAAGAGGUGGCUC GAGCCACCUCUUCGCACGUU 60.00% AACGUGGUAACGUGCGAAGA UCUUCGCACGUUACCACGUU 50.00%141Attorney Docket: 090147-0584673 AAGAUGCCCCUGCAGAGCGA UCGCUCUGCAGGGGCAUCUU 60.00% AAGAUGCGGGAUGACGGGCA UGCCCGUCAUCCCGCAUCUU 60.00%142Attorney Docket: 090147-0584673 AAGGCUCACAAGUGCAUCUU AAGAUGCACUUGUGAGCCUU 45.00% AAGGCUGAGUUUAUCAAGGG CCCUUGAUAAACUCAGCCUU 45.00%143Attorney Docket: 090147-0584673 AAUUUACACCUGCGAUCACU AGUGAUCGCAGGUGUAAAUU 40.00% AAUUUUCCUCGGUGGAUGAC GUCAUCCACCGAGGAAAAUU 45.00%144Attorney Docket: 090147-0584673 CAGAAAAAAGGAGUCAUUUC GAAAUGACUCCUUUUUUCUG 35.00% CAGAAACACAAGUGCCCCAU AUGGGGCACUUGUGUUUCUG 50.00%145Attorney Docket: 090147-0584673 GAAAGGCACCCAGACAUCGC GCGAUGUCUGGGUGCCUUUC 60.00% GAACAAGGAGCAUCUGGCCA UGGCCAGAUGCUCCUUGUUC 55.00%146Attorney Docket: 090147-0584673 GAGCUCAAGAUCCAUGGCAC GUGCCAUGGAUCUUGAGCUC 55.00% GAGCUGCAGUGCCACAUCAC GUGAUGUGGCACUGCAGCUC 60.00%Nucleotide Sequence on Human ZNF423 Antisense strand GRCh38 / hg38 ORF Genome147Attorney Docket: 090147-0584673 AAAACCAGCCGTGCGCTGGAA UUCCAGCGCACGGCUGGUU chr16 UU AAAACTTCCTGGAGAAGGATG CAUCCUUCUCCAGGAAGUU chr16Attorney Docket: 090147-0584673 AACAAGTTGCAGCAGCACATC GAUGUGCUGCUGCAACUU chr16 GUU AACACAAGGTGACCCACAGCA UGCUGUGGGUCACCUUGU chr16Attorney Docket: 090147-0584673 AACTACTGCCCCGAGATGTTC GAACAUCUCGGGGCAGUA chr16 GUU AACTAGAGTCTCCGGTGGTGC GCACCACCGGAGACUCUAG chr16Attorney Docket: 090147-0584673 AAGACCATCCACGCGGACAAG CUUGUCCGCGUGGAUGGUC chr16 UU AAGACCCACAGCTCCAGCAAG CUUGCUGGAGCUGUGGGU chr16Attorney Docket: 090147-0584673 AAGCGCCAACTCCATCTCCAA UUGGAGAUGGAGUUGGCG chr16 CUU AAGCGGGACTTTAACAGCCTG CAGGCUGUUAAAGUCCCGC chr16Attorney Docket: 090147-0584673 AAGGTGGTCTATAGCTGCCCC GGGGCAGCUAUAGACCACC chr16 UU AAGGTGTCCATCCAGGTGCAC GUGCACCUGGAUGGACACC chr16Attorney Docket: 090147-0584673 AATGAGAAGAAGATGTACCGC GCGGUACAUCUUCUUCUCA chr16 UU AATGAGGATGATGAAGACATG CAUGUCUUCAUCAUCCUCA chr16Attorney Docket: 090147-0584673 AAACAGACCTGGGCAGATGTG CACAUCUGCCCAGGUCUGU chr16 UU AAACAGCAAACAGACCTGGGC GCCCAGGUCUGUUUGCUGU chr16Attorney Docket: 090147-0584673 AACAGACCTGGGCAGATGTGG CCACAUCUGCCCAGGUCUG chr16 UU AACAGCAAACAGACCTGGGCA UGCCCAGGUCUGUUUGCUG chr16Attorney Docket: 090147-0584673 AAGAGAGATGGATGGGTGATC GAUCACCCAUCCAUCUCUC chr16 UU AAGAGAGTGGGGTGCCGATCT AGAUCGGCACCCCACUCUC chr16Attorney Docket: 090147-0584673 AAGGGAATTTTCCGCACACAT AUGUGUGCGGAAAAUUCC chr16 CUU AAGGGCCATTGGGAGTATCAG CUGAUACUCCCAAUGGCCC chr16Attorney Docket: 090147-0584673 AATGGGTGAGTGCGTGTGATT AAUCACACGCACUCACCCA chr16 UU AATGTGCATGTGTTATGCCTG CAGGCAUAACACAUGCACA chr16Target Sequence on mRNA of Human Antisense strand GC ZNF423 Content159Attorney Docket: 090147-0584673 AAAGACAUGGAUGCAUAAGAA UUCUUAUGCAUCCAUGUCUU 33.33% U AAAGACUCGAAGAGUCCAUUA UAAUGGACUCUUCGAGUCUU 38.10%Attorney Docket: 090147-0584673 AACCAGAUGUUCGACUCCCCG CGGGGAGUCGAACAUCUGGU 57.14% U AACCAGUGCUCCAUGGGUUUC GAAACCCAUGGAGCACUGGU 52.38%Attorney Docket: 090147-0584673 AAGAACCCUGAGGCACCUAAC GUUAGGUGCCUCAGGGUUCU 52.38% U AAGAAGAAAGACAUGGAUGCA UGCAUCCAUGUCUUUCUUCU 38.10%Attorney Docket: 090147-0584673 AAGCAAUUUUCCUCGGUGGAU AUCCACCGAGGAAAAUUGCU 42.86% U AAGCACAAGAGGAGCCGUGAC GUCACGGCUCCUCUUGUGCU 57.14%Attorney Docket: 090147-0584673 AAGGAGCAUCUGGCCAAGUCG CGACUUGGCCAGAUGCUCCU 57.14% U AAGGAGUCAUUUCAACCCUUA UAAGGGUUGAAAUGACUCC 38.10%Attorney Docket: 090147-0584673 AAGUUCUGCAGCAAGGCCUUC GAAGGCCUUGCUGCAGAACU 52.38% U AAGUUCUUCUUCCAGACCGAG CUCGGUCUGGAAGAAGAACU 47.62%Attorney Docket: 090147-0584673 CAAAUGCCCCGUGUGUUUCAC GUGAAACACACGGGGCAUUU 52.38% G CAACAAGUUGCAGCAGCACAU AUGUGCUGCUGCAACUUGUU 47.62%Attorney Docket: 090147-0584673 CAAGCUGCACAAGAACCAUGC GCAUGGUUCUUGUGCAGCUU 52.38% G CAAGCUGCAUACGGGCGACAA UUGUCGCCCGUAUGCAGCUU 57.14%Attorney Docket: 090147-0584673 CAGAUGAUCGGAGAUGGUUGU ACAACCAUCUCCGAUCAUCU 47.62% G CAGCAACCACAGUGUCAGUCC GGACUGACACUGUGGUUGCU 57.14%Attorney Docket: 090147-0584673 CAGUUCUGCGACAAGUCCUUC GAAGGACUUGUCGCAGAACU 52.38% G CAUCAAGUGCCAGAUGACCUU AAGGUCAUCUGGCACUUGAU 47.62%Attorney Docket: 090147-0584673 GAAGUAUAACUGUAAGUUCUG CAGAACUUACAGUUAUACUU 33.33% C GAAGUUCUUCUUCCAGACCGA UCGGUCUGGAAGAAGAACU 47.62%Attorney Docket: 090147-0584673 GAGUCCAUUACCCAAGGUUAC GUAACCUUGGGUAAUGGAC 47.62% UC GAGUGUAAGCUGUGCAACCAG CUGGUUGCACAGCUUACACU 52.38%Attorney Docket: 090147-0584673 ACGCAAGAAGGCUGGGUUUGUACAAACCCAGCCUUCUUGCGU52.38%ACGUUUGUUCACGGGCUUUUUAAAAAGCCCGUGAACAAACGNucleotide Sequence on Human Antisense strand GRCh38 / ZNF423 ORF hg38 y172Attorney Docket: 090147-0584673 AACACACTGCTCGCCCATATCC GGAUAUGGGCGAGCAGUGUGUU chr16 AACAGCGTGACAAGTCAAGAGG CCUCUUGACUUGUCACGCUGUU chr16 AACAGGTGGAGTTTATGATCAA UUGAUCAUAAACUCCACCUGUU chr16Attorney Docket: 090147-0584673 AAGACATGGAGGATGAATCAAT AUUGAUUCAUCCUCCAUGUCUU chr16 AAGACATGGATGCATAAGAAGA UCUUCUUAUGCAUCCAUGUCUU chr16 AAGACCATCCACGCGGACAAGC GCUUGUCCGCGUGGAUGGUCUU chr16Attorney Docket: 090147-0584673 AAGGATGGATTTTACCCTGTAA UUACAGGGUAAAAUCCAUCCUU chr16 AAGGATGTTGCGTCACCCACGC GCGUGGGUGACGCAACAUCCUU chr16 AAGGCACCCAGACATCGCCAGT ACUGGCGAUGUCUGGGUGCCUU chr16Attorney Docket: 090147-0584673 AAAAAAGTTTCCAAGAGACAGG CCUGUCUCUUGGAAACUUUUUU chr16 AAAAAATTAGTTTAGCCCCTGG CCAGGGGCUAAACUAAUUUUUU chr16 AAAAAGAGAGATGGATGGGTGA UCACCCAUCCAUCUCUCUUUUU chr16Attorney Docket: 090147-0584673 AACCACAAAAAAAGTATGAGGA UCCUCAUACUUUUUUUGUGGUU chr16 AACCACATGTATAACCAGGAAC GUUCCUGGUUAUACAUGUGGUU chr16 AACCAGATGGAAGCACACACAC GUGUGUGUGCUUCCAUCUGGUU chr16Attorney Docket: 090147-0584673 AAGGTACAGGCAATCTGGGAAG CUUCCCAGAUUGCCUGUACCUU chr16 AAGGTCACTCATGGTTGGGGGC GCCCCCAACCAUGAGUGACCUU chr16 AAGGTTACACAGCAAGCAACAG CUGUUGCUUGCUGUGUAACCUU chr16Target Sequence on mRNA of Human Antisense strand GC ZNF423 Content178Attorney Docket: 090147-0584673 AAAAGAAGAAAGACAUGGAUGC GCAUCCAUGUCUUUCUUCUU 36.36% UU AAAAGACAUACCAGUGCAUCAA UUGAUGCACUGGUAUGUCUU 3636%Attorney Docket: 090147-0584673 AACACAAGGUGACCCACAGCAA UUGCUGUGGGUCACCUUGUG 50.00% UU AACACAAGUGCCCCAUGUGCCC GGGCACAUGGGGCACUUGUG 5909%Attorney Docket: 090147-0584673 AACGUGUGAAGAGGUGGCUCAG CUGAGCCACCUCUUCACACG 54.55% UU AACGUUUGUUCACGGACUUUCU AGAAAGUCCGUGAACAAACG 4091%Attorney Docket: 090147-0584673 AAGACAUACCAGUGCAUCAAGU ACUUGAUGCACUGGUAUGUC 40.91% UU AAGACAUGGAGGAUGAAUCAAU AUUGAUUCAUCCUCCAUGUC 3636%Attorney Docket: 090147-0584673 AAGCAGGACCUGGUGAAGCUUG CAAGCUUCACCAGGUCCUGC 54.55% UU AAGCCAAGAAGGACGACUUCAU AUGAAGUCGUCCUUCUUGGC 4545%Attorney Docket: 090147-0584673 AAGGAUGGAUUUUACCCUGUAA UUACAGGGUAAAAUCCAUCC 36.36% UU AAGGAUGUUGCGUCACCCACGC GCGUGGGUGACGCAACAUCC 5909%Attorney Docket: 090147-0584673 AAGUUGAAGAGGGGGAGGCCUC GAGGCCUCCCCCUCUUCAACU 59.09% U AAGUUGCAGCAGCACAUCUUUG CAAAGAUGUGCUGCUGCAAC 4545%Attorney Docket: 090147-0584673 CAACCAGAAACACAAGUGCCCC GGGGCACUUGUGUUUCUGGU 54.55% UG CAACCAGAUGUUCGACUCCCCG CGGGGAGUCGAACAUCUGGU 5909%Attorney Docket: 090147-0584673 CAAGUGCAACGUUUGUUCACGG CCGUGAACAAACGUUGCACU 50.00% UG CAAGUGCACUGUGUGCAAGCGC GCGCUUGCACACAGUGCACU 5909%Attorney Docket: 090147-0584673 CAGCAGCACAUCUUUGCCGUGC GCACGGCAAAGAUGUGCUGC 59.09% UG CAGCAGGACUUCGAGUCUCUGG CCAGAGACUCGAAGUCCUGC 5909%Attorney Docket: 090147-0584673 GAAGACAGGAACAGCGUGACAA UUGUCACGCUGUUCCUGUCU 50.00% UC GAAGACAUGGAGGAUGAAUCAA UUGAUUCAUCCUCCAUGUCU 4091%Attorney Docket: 090147-0584673 GAGCCACACAUGUCAGAUCUGC GCAGAUCUGACAUGUGUGGC 54.55% UC GAGCUCAAGAUCCAUGGCACCU AGGUGCCAUGGAUCUUGAGC 5455%Nucleotide Sequence on Human Antisense strand GRCh38 / hg38 ZNF423 ORF Genome90Attorney Docket: 090147-0584673 AAAACTTCCTGGAGAAGGAT UCCAUCCUUCUCCAGGAAG chr16 GGA UUUU AAAAGAACAAGGAGCATCTG GGCCAGAUGCUCCUUGUUC chr16Attorney Docket: 090147-0584673 AACACAAGGTGACCCACAGC CUUGCUGUGGGUCACCUUG chr16 AAG UGUU AACACAAGTGCCCCATGTGCC AGGGCACAUGGGGCACUUG chr16Attorney Docket: 090147-0584673 AACTCACCAAGCACATCAAG CUCCUUGAUGUGCUUGGUG chr16 GAG AGUU AACTCCATCTCCAATGGGGAG UACUCCCCAUUGGAGAUGG chr16Attorney Docket: 090147-0584673 AAGAGGACTTTGACTCCCAGG CUCCUGGGAGUCAAAGUCC chr16 AG UCUU AAGAGGAGAGAAATGAGGAT AUCAUCCUCAUUUCUCUCC chr16Attorney Docket: 090147-0584673 AAGCTGCATACGGGCGACAA UUCUUGUCGCCCGUAUGCA chr16 GAA GCUU AAGCTGCCGTTCAAGTGCACC UAGGUGCACUUGAACGGCA chr16Attorney Docket: 090147-0584673 AAGTATCACTGCCACGAGTGC UCGCACUCGUGGCAGUGAU chr16 GA ACUU AAGTCAAGAGGAGAGAAATG CCUCAUUUCUCUCCUCUUG chr16Attorney Docket: 090147-0584673 AATGGGGAGTATCCTTGCAAT UGAUUGCAAGGAUACUCCC chr16 CA CAUU AATTTACACCTGCGATCACTG GACAGUGAUCGCAGGUGUA chr16Attorney Docket: 090147-0584673 AAACAGGATGCAGGAGCCAG GGUCUGGCUCCUGCAUCCU chr16 ACC GUUU AAACCACATGTATAACCAGG GUUCCUGGUUAUACAUGUG chr16Attorney Docket: 090147-0584673 AAATTAGTTTAGCCCCTGGTT AAAACCAGGGGCUAAACUA chr16 TT AUUU AAATTGCAAGGCTGTGGGGA UUUUCCCCACAGCCUUGCA chr16Attorney Docket: 090147-0584673 AAGACAGGAACAGCGTGACA ACUUGUCACGCUGUUCCUG chr16 AGT UCUU AAGACATGGAGGATGAATCA AAUUGAUUCAUCCUCCAUG chr16Attorney Docket: 090147-0584673 AAGGAGTCATTTCAACCCTTA GGUAAGGGUUGAAAUGACU chr16 CC CCUU AAGGCGAACTTGGCTAAGAG GAUCUCUUAGCCAAGUUCG chr16Attorney Docket: 090147-0584673 AATCTGGGAAGTTTGGCCAGT UAACUGGCCAAACUUCCCA chr16 TA GAUU AATCTGGGCTCCATCTCCCTC CAGAGGGAGAUGGAGCCCA chr16Target Sequence on mRNA of Human Antisense strand GC ZNF423 Content202Attorney Docket: 090147-0584673 AAACACAAGUGCCCCAUGUGCCC GGGCACAUGGGGCACUUGUGU 56.52% UU AAACUAGAGUCUCCGGUGGUGCA UGCACCACCGGAGACUCUAGU 52.17%Attorney Docket: 090147-0584673 AACAUCUCUGCCUUCCACUGCAA UUGCAGUGGAAGGCAGAGAUG 47.83% UU AACAUUCCACUGGCCCACAGCAA UUGCUGUGGGCCAGUGGAAUG 52.17%Attorney Docket: 090147-0584673 AACUUUGAGAGCUUCCAGACCCA UGGGUCUGGAAGCUCUCAAAG 47.83% UU AAGAAAGACAUGGAUGCAUAAGA UCUUAUGCAUCCAUGUCUUUC 34.78%Attorney Docket: 090147-0584673 AAGAUCCACCUGAAGACCCACAG CUGUGGGUCUUCAGGUGGAUC 52.17% UU AAGAUCCAUGGCACCUUCCACAU AUGUGGAAGGUGCCAUGGAUC 47.83%Attorney Docket: 090147-0584673 AAGGAAGCCAAGAAGGACGACUU AAGUCGUCCUUCUUGGCUUCC 47.83% UU AAGGACGACUUCAUGUGCGACUA UAGUCGCACAUGAAGUCGUCC 47.83%Attorney Docket: 090147-0584673 AAGUGCAUCUUCUGUGGGGAGAC GUCUCCCCACAGAAGAUGCAC 52.17% UU AAGUGCCAGAUGACCUUCGAGAA UUCUCGAAGGUCAUCUGGCAC 47.83%Attorney Docket: 090147-0584673 CAAAUCCACGUUGCCAACCACAU AUGUGGUUGGCAACGUGGAUU 47.83% UG CAAAUGCCCCGUGUGUUUCACAG CUGUGAAACACACGGGGCAUU 52.17%Attorney Docket: 090147-0584673 CAAUAGCCUGCAGGAGCACAUCC GGAUGUGCUCCUGCAGGCUAU 56.52% UG CAAUCAAUGCGACCUCAAGUUCU AGAACUUGAGGUCGCAUUGAU 43.48%Attorney Docket: 090147-0584673 CAGUCACAAGUGCAACGUUUGUU AACAAACGUUGCACUUGUGAC 43.48% UG CAGUCACUUCCACGCUUGAGCUC GAGCUCAAGCGUGGAAGUGAC 56.52%Attorney Docket: 090147-0584673 GAACACACUGCUCGCCCAUAUCC GGAUAUGGGCGAGCAGUGUGU 56.52% UC GAACCACAAGAACAUUCCACUGG CCAGUGGAAUGUUCUUGUGGU 47.83%Attorney Docket: 090147-0584673 GAGAUGUUCGCCGACAUCAAUAG CUAUUGAUGUCGGCGAACAUC 47.83% UC GAGCACGUUCGCAAGCUGCACAA UUGUGCAGCUUGCGAACGUGC 56.52%Attorney Docket: 090147-0584673 UAAGUUCUGCAGCAAGGCCUUCC GGAAGGCCUUGCUGCAGAACU 52.17% UA UACUGCACCAACUCCCCCAUCUU AAGAUGGGGGAGUUGGUGCA 52.17%Target Sequence on mRNA of Human Antisense strand GC ZNF423 Content214Attorney Docket: 090147-0584673 AAAAGGAGUCAUUUCAACCCUUAC GUAAGGGUUGAAAUGACUCCU 37.50% UUU AAACACAAGUGCCCCAUGUGCCCU AGGGCACAUGGGGCACUUGUG 5417%Attorney Docket: 090147-0584673 AACAGCCAUGAGGCCAGCGAGGAU AUCCUCGCUGGCCUCAUGGCUG 58.33% UU AACAGCGUGACAAGUCAAGAGGAG CUCCUCUUGACUUGUCACGCUG 5000%Attorney Docket: 090147-0584673 AACUAGAGUCUCCGGUGGUGCAGC GCUGCACCACCGGAGACUCUAG 58.33% UU AACUCACCAAGCACAUCAAGGAGA UCUCCUUGAUGUGCUUGGUGA 4583%Attorney Docket: 090147-0584673 AAGACAUGGAUGCAUAAGAAGAGG CCUCUUCUUAUGCAUCCAUGUC 41.67% UU AAGACCUGGAGAGCCACAUGCAGG CCUGCAUGUGGCUCUCCAGGUC 5833%Attorney Docket: 090147-0584673 AAGCCGCGCUCGGUGAAAGUUGAA UUCAACUUUCACCGAGCGCGGC 54.17% UU AAGCCGGAUCCCAGUUGAAGAGGG CCCUCUUCAACUGGGAUCCGGC 5833%Attorney Docket: 090147-0584673 AAGGGCCAUUGGGAGUAUCAGCAU AUGCUGAUACUCCCAAUGGCCC 50.00% UU AAGGUGACCCACAGCAAGAGCCUG CAGGCUCUUGCUGUGGGUCAC 5833%Attorney Docket: 090147-0584673 AAUCACCGGCUGCGGGACCACAAU AUUGUGGUCCCGCAGCCGGUG 58.33% AUU AAUCCACGUUGCCAACCACAUGAU AUCAUGUGGUUGGCAACGUGG 4583%Attorney Docket: 090147-0584673 CAAGAAGGACGACUUCAUGUGCGA UCGCACAUGAAGUCGUCCUUC 50.00% UUG CAAGAAGUAUAACUGUAAGUUCUG CAGAACUUACAGUUAUACUUC 3333%Attorney Docket: 090147-0584673 CAAUUUUCCUCGGUGGAUGACCUG CAGGUCAUCCACCGAGGAAAA 50.00% UUG CACAGCAAGCAACAGGUGGAGUUU AAACUCCACCUGUUGCUUGCU 5000%Attorney Docket: 090147-0584673 CAGGUCUCCUCAUAAGCCGGAUCC GGAUCCGGCUUAUGAGGAGAC 58.33% CUG CAGGUGCACGUCAAACACAGCCAC GUGGCUGUGUUUGACGUGCAC 5833%Attorney Docket: 090147-0584673 GAAGCACCUGCUGGACAUGCACAC GUGUGCAUGUCCAGCAGGUGC 58.33% UUC GAAGCACUGUGUGUUUGAUGCUGC GCAGCAUCAAACACACAGUGC 5000%Attorney Docket: 090147-0584673 GAGGAGGAGUUUAUUGAGCACUGC GCAGUGCUCAAUAAACUCCUCC 50.00% UC GAGGAGUUUAUUGAGCACUGCCAG CUGGCAGUGCUCAAUAAACUC 5000%Target Sequence on mRNA of Human Antisense strand GC ZNF423 Content226Attorney Docket: 090147-0584673 AAAAGAACAAGGAGCAUCUGGCCA UUGGCCAGAUGCUCCUUGUUCU 44.00% A UUU AAAAGAAGAAAGACAUGGAUGCAU UAUGCAUCCAUGUCUUUCUUCU 3200%Attorney Docket: 090147-0584673 AACACAAGGUGACCCACAGCAAGA CUCUUGCUGUGGGUCACCUUGU 52.00% G GUU AACACAAGUGCCCCAUGUGCCCUGA UCAGGGCACAUGGGGCACUUGU 5600%Attorney Docket: 090147-0584673 AACGUGGUAACGUGCGAAGAGGUG CCACCUCUUCGCACGUUACCAC 56.00% G GUU AACGUGGUAACGUGUGAAGAGGUG CCACCUCUUCACACGUUACCAC 5200%Attorney Docket: 090147-0584673 AAGAAGUAUCACUGCCACGAGUGC CGCACUCGUGGCAGUGAUACUU 52.00% G CUU AAGAAGUCCAAGGCCGAGCAGAGC GGCUCUGCUCGGCCUUGGACUU 6000%Attorney Docket: 090147-0584673 AAGCACCUGCUGGACAUGCACACCU AGGUGUGCAUGUCCAGCAGGUG 56.00% CUU AAGCACUACAUGUGUCCCAUCUGU CACAGAUGGGACACAUGUAGUG 4800%Attorney Docket: 090147-0584673 AAGGAGUCAUUUCAACCCUUACCU CAGGUAAGGGUUGAAAUGACUC 44.00% G CUU AAGGAGUUCCGCAGCAAGCAGGAC GGUCCUGCUUGCUGCGGAACUC 6000%Attorney Docket: 090147-0584673 AAGUGCGCCCUGUGCCUCAAGGAG ACUCCUUGAGGCACAGGGCGCA 60.00% U CUU AAGUUCUCCAACUUUGAGAGCUUC GGAAGCUCUCAAAGUUGGAGAA 4400%Attorney Docket: 090147-0584673 AAUUUUCCUCGGUGGAUGACCUGC UGCAGGUCAUCCACCGAGGAAA 48.00% A AUU CAAACUAGAGUCUCCGGUGGUGCA CUGCACCACCGGAGACUCUAGU 5600%Attorney Docket: 090147-0584673 CAAGGGCAGUCACAAGUGCAACGU AACGUUGCACUUGUGACUGCCC 52.00% U UUG CAAGGUGACCCACAGCAAGAGCCUG CAGGCUCUUGCUGUGGGUCACC 6000%Attorney Docket: 090147-0584673 CAGCAGAGCCACACAUGUCAGAUCU AGAUCUGACAUGUGUGGCUCUG 52.00% CUG CAGCAGGACUUCGAGUCUCUGGCA CUGCCAGAGACUCGAAGUCCUG 6000%Attorney Docket: 090147-0584673 GAAAAAAGGAGUCAUUUCAACCCU AAGGGUUGAAAUGACUCCUUUU 36.00% U UUC GAAACACAAGUGCCCCAUGUGCCCU AGGGCACAUGGGGCACUUGUGU 5600%Attorney Docket: 090147-0584673 GAGAAGAAGAUGUACCGCUGCACG CCGUGCAGCGGUACAUCUUCUU 56.00% G CUC GAGAAGCACUGUGUGUUUGAUGCU CAGCAUCAAACACACAGUGCUU 4800%Attorney Docket: 090147-0584673 GAGUACCUGCUUUUGUAGGGCCAU AAUGGCCCUACAAAAGCAGGUA 48.00% U CUC GAGUCAUUUCAACCCUUACCUGCCU AGGCAGGUAAGGGUUGAAAUG 4800%Target Sequence on mRNA of Human TLE3 Antisense Strand GC Content AAAAAAGAUGCCCCCACC GGUGGGGGCAUCUUUUUU 50.00%Attorney Docket: 090147-0584673 AACAGGCUCUGGUGACAA UUGUCACCAGAGCCUGUU 50.00% AACAGGGACAAUUACAUC GAUGUAAUUGUCCCUGUU 38.89% AACA CCCACCCCAGA G CA C GGGG GGGA G 6%240Attorney Docket: 090147-0584673 AAUACAUUGUAACAGGCU AGCCUGUUACAAUGUAUU 33.33% AAUCCAGUGCGAAUAACU AGUUAUUCGCACUGGAUU 38.89% AA CC CG C G C GA CAAGACAGACGAGGA 4444%241Attorney Docket: 090147-0584673 GAACAGGGACAAUUACAU AUGUAAUUGUCCCUGUUC 38.89% GAACAUUGAAAUGCACAA UUGUGCAUUUCAAUGUUC 33.33% GAACCACCA GAAC CGA CGAG CA GG GG C 000%. Target Sequence on mRNA of Human TLE3 Antisense Strand GC ContentAttorney Docket: 090147-0584673 AAACCAGCGUACUCAUUCC GGAAUGAGUACGCUGGUUU 47.37% AAACCGCUCUUACUAUGAG CUCAUAGUAAGAGCGGUUU 42.11%243Attorney Docket: 090147-0584673 AAGAUCUGGGACAUCAGCC GGCUGAUGUCCCAGAUCUU 52.63% AAGCAGACAGAGAUUGCGA UCGCAAUCUCUGUCUGCUU 47.37%244Attorney Docket: 090147-0584673 CAAUUACAUCCGCUCCUGC GCAGGAGCGGAUGUAAUUG 52.63% CAAUUUUAGCACAGAUCAU AUGAUCUGUGCUAAAAUUG 31.58%245Attorney Docket: 090147-0584673 GAGAUGUCCUAUGGCUUGA UCAAGCCAUAGGACAUCUC 47.37% GAGAUUGCGAAGAGACUGA UCAGUCUCUUCGCAAUCUC 47.37%Target Sequence on mRNA of Human Antisense Strand GC TLE3 Content246Attorney Docket: 090147-0584673 AAAUACAUUGUAACAGGCUC GAGCCUGUUACAAUGUAUUU 35.00% AAAUCCUCCACCCCUGGGCU AGCCCAGGGGUGGAGGAUUU 60.00%247Attorney Docket: 090147-0584673 AAGAGCCCCAUCUCCCAGCU AGCUGGGAGAUGGGGCUCUU 60.00% AAGAGUGAUGAUCUGGUGGU ACCACCAGAUCAUCACUCUU 45.00%248Attorney Docket: 090147-0584673 CAAGAGUGAUGAUCUGGUGG CCACCAGAUCAUCACUCUUG 50.00% CAAGCAGACAGAGAUUGCGA UCGCAAUCUCUGUCUGCUUG 50.00%249Attorney Docket: 090147-0584673 GACGGAGAUGCAGCGCCAUU AAUGGCGCUGCAUCUCCGUC 60.00% GACUCCCCAAUCUGCCUCUU AAGAGGCAGAUUGGGGAGUC 55.00%. Target Sequence on mRNA of Human Antisense Strand GC TLE3 C250Attorney Docket: 090147-0584673 AAACCAGCGUACUCAUUCCAU AUGGAAUGAGUACGCUGGUUU 42.86% AAACCGCUCUUACUAUGAGAU AUCUCAUAGUAAGAGCGGUUU 38.10%251Attorney Docket: 090147-0584673 AAGCAUCUAUCAUGGCAGGGU ACCCUGCCAUGAUAGAUGCUU 47.62% AAGCCUGACAAGUACCAGCUG CAGCUGGUACUUGUCAGGCUU 52.38%252Attorney Docket: 090147-0584673 CAGAGAGAGAGAAUCCAGUGC GCACUGGAUUCUCUCUCUCUG 52.38% CAGAGAUUGCGAAGAGACUGA UCAGUCUCUUCGCAAUCUCUG 47.62%253Attorney Docket: 090147-0584673 GAGACAAGAGUGAUGAUCUGG CCAGAUCAUCACUCUUGUCUC 47.62% GAGAGAACCGAGAUGGUGUUG CAACACCAUCUCGGUUCUCUC 52.38%Target Sequence on mRNA of Human Antisense Strand GC TLE3 Content254Attorney Docket: 090147-0584673 AAAUACAUUGUAACAGGCUCUG CAGAGCCUGUUACAAUGUAUUU 36.36% AAAUCCUCCACCCCUGGGCUCA UGAGCCCAGGGGUGGAGGAUUU 59.09%255Attorney Docket: 090147-0584673 AAGCAUCUAUCAUGGCAGGGUA UACCCUGCCAUGAUAGAUGCUU 45.45% AAGCCAAGAAGCGGAAGGCGGA UCCGCCUUCCGCUUCUUGGCUU 59.09%Attorney Docket: 090147-0584673 CAAGUACCAGCUGCACCUGCAC GUGCAGGUGCAGCUGGUACUUG 59.09% CAAGUCCAACACACCAACCCCA UGGGGUUGGUGUGUUGGACUU 54.55% GAttorney Docket: 090147-0584673 GACUUAGGAUGCCAGGGCGUUU AAACGCCCUGGCAUCCUAAGUC 54.55% GAGAACCCUUUGCGGAGGCUUC GAAGCCUCCGCAAAGGGUUCUC 59.09%Target Sequence on mRNA of Human Antisense Strand GC TLE3 ContentAttorney Docket: 090147-0584673 AAAGAUAACCUUCUCAACGCCUG CAGGCGUUGAGAAGGUUAUCUUU 43.48% AAAGUCUGCUUCUCCUGCUGCAG CUGCAGCAGGAGAAGCAGACUUU 52.17% %259Attorney Docket: 090147-0584673 AAGCUGGCAAACGAGAAGACGGA UCCGUCUUCUCGUUUGCCAGCUU 52.17% AAGGAAGGAAGCCGUAGAGCUCC GGAGCUCUACGGCUUCCUUCCUU 56.52% %260Attorney Docket: 090147-0584673 CAGCAUAUUCCAGGUCGGAGCAU AUGCUCCGACCUGGAAUAUGCUG 52.17% CAGCGAUGGGAACAUUGCUGUCU AGACAGCAAUGUUCCCAUCGCUG 52.17% %261Attorney Docket: 090147-0584673 UAGGUAUAAUGGCCUCGGCUCUG CAGAGCCGAGGCCAUUAUACCUA 52.17% UAGGUCCGGGGCACUUUUUUUUU AAAAAAAAAGUGCCCCGGACCUA 43.48% %Target Sequence on mRNA of Human Antisense Strand GC TLE3 Content AAAAAAGAUGCCCCCACCAGCCCU AGGGCUGGUGGGGGCAUCUUUU 5417%262Attorney Docket: 090147-0584673 AACAGGCUCUGGUGACAAGAAGGC GCCUUCUUGUCACCAGAGCCUG 54.17% UU AACAGGGACAAUUACAUCCGCUCC GGAGCGGAUGUAAUUGUCCCUG 5000%Attorney Docket: 090147-0584673 AAGACCUUGGUCAUAACGACAAAU AUUUGUCGUUAUGACCAAGGUC 37.50% UU AAGACGAAUUCCAGUUCCUGCAAG CUUGCAGGAACUGGAAUUCGUC 4583%Attorney Docket: 090147-0584673 AAUAACUCUGUGUCACCCUCGGAA UUCCGAGGGUGACACAGAGUUA 45.83% UU AAUACAUUGUAACAGGCUCUGGUG CACCAGAGCCUGUUACAAUGUA 4167%Attorney Docket: 090147-0584673 CAGACAGAGAUUGCGAAGAGACUG CAGUCUCUUCGCAAUCUCUGUC 50.00% UG CAGAUCAUGCCUUUCCUGUCACAA UUGUGACAGGAAAGGCAUGAUC 4583%Attorney Docket: 090147-0584673 GAACGCCAUCAUCGGGAAUAACUC GAGUUAUUCCCGAUGAUGGCGU 50.00% UC GAACGCUCGCCCUUGUCAGCUCUU AAGAGCUGACAAGGGCGAGCGU 5833%Attorney Docket: 090147-0584673 GAGGCAGUUUAUCUGGCACCUCCA UGGAGGUGCCAGAUAAACUGCC 54.17% UC GAGGGGCAGAGGAGGGCAAUUGUU AACAAUUGCCCUCCUCUGCCCC 5833%Target Sequence on mRNA of Human Antisense Strand GC TLE3 Content268Attorney Docket: 090147-0584673 AAACCAGCGUACUCAUUCCAUGUG UCACAUGGAAUGAGUACGCUGG 44.00% A UUU AAACCGCUCUUACUAUGAGAUGUC GGACAUCUCAUAGUAAGAGCGG 4400%Attorney Docket: 090147-0584673 AACCCCAAGGAACGACGCCCCAAC AGUUGGGGCGUCGUUCCUUGGG 60.00% U GUU AACCCCACGAGGCACGUCUACACA CUGUGUAGACGUGCCUCGUGGG 6000%Attorney Docket: 090147-0584673 AAGAGCCCCAUCUCCCAGCUGGAC AGUCCAGCUGGGAGAUGGGGCU 60.00% U CUU AAGAGUGAUGAUCUGGUGGUGGA CAUCCACCACCAGAUCAUCACUC 4800%Attorney Docket: 090147-0584673 AAUAACUCUGUGUCACCCUCGGAA UUUCCGAGGGUGACACAGAGUU 44.00% A AUU AAUACAUUGUAACAGGCUCUGGUG UCACCAGAGCCUGUUACAAUGU 4000%Attorney Docket: 090147-0584673 CAAUUACAUCCGCUCCUGCAAGCU CAGCUUGCAGGAGCGGAUGUAA 52.00% G UUG CAAUUUUAGCACAGAUCAUGCCUU AAAGGCAUGAUCUGUGCUAAAA 3600%Attorney Docket: 090147-0584673 GAAAACCAGCGUACUCAUUCCAUG ACAUGGAAUGAGUACGCUGGUU 44.00% U UUC GAAAGAUAACCUUCUCAACGCCUG CCAGGCGUUGAGAAGGUUAUCU 4800%Attorney Docket: 090147-0584673 GAGCUGCGUGCUCUCCCUCAAGUU GAACUUGAGGGAGAGCACGCAG 60.00% C CUC GAGCUGUUGGUUUUGACCCUCACC GGGUGAGGGUCAAAACCAACAG 5600%orientation) Sense Strand Modified Sequence Antisense Modified Sequence Strand f m f m U275Attorney Docket: 090147-0584673 AAAUGACA mA*mA*mAmUmGmAfCm CGCCUCCG mC*fG*mCmCmUfCmCfGfU UUCAGUAC AfUfUfCmAmGmUmAmCm UACUGAAU mAmCmUmGfAmAfUmGm GGAGGCG GmGmAmGmGmCmG GUCAUUU UmCmAmU*mU*mU f C C U f m f f f f m276Attorney Docket: 090147-0584673 UGCGAGAA mU*mG*mCmGmAmGfAm AACAAACU mA*fA*mCmAmAfAmCfUf ACUGCAGU AfAfCfUmGmCmAmGmUm GCAGUUUC GmCmAmGmUfUmUfCmU UUGUU UmUmGmUmU UCGCA mCmG*mC*mA f f f C C f f m

[0260] Table 35 provides exemplary chemically modified siRNA sequences targeting human CB1R mRNA. These sequences incorporate site-specific nucleotide modifications to enhance stability, reduce immunogenicity, and improve gene-silencing efficiency. The modifications used in these sequences are selected from the following, wherein “N” represents a nucleotide or ribonucleotide: • mN: 2′-O-methyl modified ribonucleotide; • fN: 2′-fluoro modified ribonucleotide; • *: phosphorothioate linkage between nucleotides; • 5′-(E)-Vinylphosphonate-2′-OMe-U at the 5′ end where applicable; and • 3′-terminal dTdT DNA overhangs where applicable. Table 36. Modified siRNA sequences targeting human ZFP423 mRNA (5’ to 3’ orientation) 277Attorney Docket: 090147-0584673 Sense Strand Modified Sequence Antisense Modified Sequence Strand C C A f C f f f f f f mAttorney Docket: 090147-0584673 UUCAGAAA mU*mU*mCmAmGmAfAm AAACGACC mA*fA*mAmCmGfAmCfCf AAAGGGGU AfAfAfAmGmGmGmGmUm CCUUUUUU CmCmUmUmUfUmUfUmC CGUUU CmGmUmUmU CUGAA mUmG*mA*mA f f m A f f f f f f f279Attorney Docket: 090147-0584673 AAGAAAGA mA*mA*mGmAmAmAfGm CUAUACAC mC*fU*mAmUmAfCmAfCf CAUGGGUG AfCfAfUmGmGmGmUmGm CCAUGUCU CmCmAmUmGfUmCfUmU UAUAG UmAmUmAmG UUCUU mUmC*mU*mUan ZFP423 mRNA. These sequences incorporate site-specific nucleotide modifications to enhance stability, reduce immunogenicity, and improve gene-silencing efficiency. The modifications used in these sequences are selected from the following, wherein “N” represents a nucleotide or ribonucleotide: • mN: 2′-O-methyl modified ribonucleotide; • fN: 2′-fluoro modified ribonucleotide; • 2FN: 2′-deoxy-2′-fluoro modified ribonucleotide • *: phosphorothioate linkage between nucleotides; • 5′-(E)-Vinylphosphonate-2′-OMe-U at the 5′ end where applicable; and • 3′-terminal dTdT DNA overhangs where applicable. EXAMPLES Example 1. Designing Inhibitory RNA Oligonucleotides Targeting Human CB1R

[0262] To design inhibitory RNA oligonucleotides targeting human CB1R, the mRNA sequence encoding CB1R was retrieved from the NCBI database. Five isoforms were retrieved, NM_001160226.1, NM_001160258.1, NM_001160259.1, NM_016083.4, and NM_033181.3. The 5’ untranslated region (UTR) and open reading frame (OFR) were extracted and saved as FASTA sequences. The inhibitory RNA oligonucleotide sequences were designed with Python scripts and adhered to the following selection criteria: a) GC content between 30% and 60%, b) lack of off-target effects, c) 5’ end begins with AA, and d) sequence length between 19 and 23 bases. The inhibitory RNA oligonucleotide sequences were aligned against the human genome assembly GRCh38 / hg38 via BLAST and the target uniqueness was evaluated. The CB1R target sequences, antisense strand sequences, and target uniqueness are summarized in Tables 1-13. Example 2. Evaluation of CB1R Targeting Inhibitory RNA Oligonucleotides In Vitro

[0263] Single-stranded and / or double-stranded oligonucleotides targeting the target sequences identified in Example 1 are evaluated for CB1R expression inhibition. Cells expressing CB1R (e.g., any one of cell lines hMADs, LiSa-2, primary human preadiopcytes, 280Attorney Docket: 090147-0584673 human adipose-derived stem cells, HEK293, SH-SY5Y, and HEPG2 cells) are contacted with an inhibitory RNA oligonucleotide. The level of mRNA remaining in the cells is determined. A reduction of CB1R mRNA levels indicates the inhibitory RNA oligonucleotide is effective at targeting and knocking down CB1R mRNA.

[0264] To further evaluate the efficacy of inhibitory RNA oligonucleotides targeting CB1R in vitro, Cells with relatively lower mitochondrial genesis are utilized. The cells are contacted with an inhibitory RNA oligonucleotide identified in Example 1. Mitogenesis is evaluated by quantifying the levels of mitochondrial biogenesis markers, including but not limited to, PGC-1α, NRF-1 or COX1. The levels of the mitochondrial biogenesis markers are quantified. Example 3. Increasing Leptin Expression in an ob / ob Mouse Model

[0265] To evaluate the therapeutic efficacy of inhibitory RNA oligonucleotide targeting CB1R in combination with an mRNA encoding leptin, a mouse model of severe obesity and leptin deficiency is utilized (ob / ob mice). The Ob / ob mice are deficient in leptin and exhibit severe obesity and a diabetic phenotype (see e.g., Muzzin P, et al. Correction of obesity and diabetes in genetically obese mice by leptin gene therapy. Proc Natl Acad Sci U S A. 1996;93(25):14804-14808. doi:10.1073 / pnas.93.25.14804). A total of 16 ob / ob mice, aged 8-9 weeks, are divided into two groups: 8 mice are used as controls, and 8 mice receive a combination of siRNA and mRNA. Treatments are administered via subcutaneous injections into the abdominal fat pad every 3 days for a duration of 21 days. During the study, body weight, food intake, and body temperature are monitored bi-weekly. Injection area temperature is checked at 0, 2, 6, and 24 hours after each injection during the first week. One week before the study ends, Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT) are conducted. Lean mass is measured using Echo MRI one day before sacrifice. At study termination, blood samples are collected to measure various biomarkers including leptin, insulin, lipid profile, liver enzymes, thyroid function tests, and inflammatory markers. Additionally, the adipose tissue at the injection site is collected for western blotting to analyze the expression of the UCP1 (the thermogenesis marker protein) and a mitochondrial biogenesis marker protein along with H&E staining to observe morphological changes in the white adipose tissue (WAT). RT-PCR analysis is performed to record the silencing of the CB1R gene, including its isoforms. 281Attorney Docket: 090147-0584673 Example 4. Inhibitory RNA Oligonucleotide Targeting CB1R in a Diet-Induced Obesity Mouse Model

[0266] To further evaluate therapeutic efficacy of inhibitory RNA oligonucleotide targeting CB1R, a diet-induced obesity (DIO) C57BL / 6 mouse model is used (Jackson Labs). A total of 16 DIO mice, aged 16-18 weeks, are divided into two groups: 8 mice are used as controls, and 8 mice receive CB1R siRNA. Treatments are administered via subcutaneous injections into the abdominal fat pad every 3 days for a duration of 21 days. During the study, body weight, food intake, and body temperature are monitored bi-weekly. Injection area temperature is checked at 0, 2, 6, and 24 hours after each injection during the first week. One week before the study ends, Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT) are conducted. Lean mass is measured using Echo MRI one day before sacrifice. At study termination, blood samples are collected to measure various biomarkers including leptin, insulin, lipid profile, liver enzymes, thyroid function tests, and inflammatory markers. Additionally, the adipose tissue at the injection site is collected for western blotting to analyze the expression of the UCP1 (the thermogenesis marker protein) and the mitochondrial biogenesis marker protein and H&E staining to observe morphological changes in the white adipose tissue (WAT). RT-PCR analysis is performed to record the silencing of the CB1R gene, including its isoforms. Throughout the study, DIO mice receive a commercially available 60% high-fat diet to maintain obesity. Example 5. Novel Discovery in Obesity Treatment: CCT-217, First-in-Class CB1R- ZFP423 Cross-Talk Gene Silencing Therapy Achieves Remarkable Weight Loss Through Selective Fat Loss While Building Healthy Lean Mass Introduction

[0267] Obesity is a multifactorial disease characterized by excessive adiposity, metabolic dysregulation, and increased susceptibility to comorbidities such as type 2 diabetes, cardiovascular disease, and non-alcoholic fatty liver disease (NAFLD)(1-3). CDC projects half of the U.S. adults will be classified as obese by 2030. Despite significant advancements in therapeutic interventions, current pharmacological treatments primarily target appetite suppression, often leading to unintended lean mass loss and adverse effects (4-6). The limitations of existing therapies necessitate the development of novel therapeutics that selectively reduce fat mass while preserving metabolic homeostasis. The endocannabinoid system (ECS) has emerged as a critical regulator of energy balance, lipid metabolism, and 282Attorney Docket: 090147-0584673 adipose tissue plasticity (7, 8). CB1R, a key component of the ECS, is highly expressed in the central nervous system (CNS) and peripheral metabolic tissues, including adipose tissue and the liver (7). While central CB1R activation promotes hyperphagia and weight gain, its peripheral activity is implicated in lipogenesis, insulin resistance, and adipose tissue dysfunction (9). Pharmacological CB1R antagonists such as rimonabant demonstrated efficacy in weight reduction but were withdrawn due to severe psychiatric side effects (10- 12). Consequently, selective peripheral CB1R inhibition has been proposed as a safer alternative to mitigate obesity-associated metabolic disorders while avoiding CNS-related adverse effects (13-17).

[0268] In addition to CB1R, studies have identified ZFP423 as a pivotal transcription factor governing adipocyte lineage commitment (18). ZFP423 is essential for the maintenance of white adipocyte identity and represses the transcriptional programs necessary for thermogenic activation such as Ebf2 and Prdm16 (19-22). Elevated ZFP423 expression has been linked to obesity-induced adipose dysfunction, characterized by hypertrophic, inflammation-prone white adipocytes with reduced metabolic plasticity. The inhibition of the ZFP423 has been shown to enhance adipocyte browning, promote mitochondrial biogenesis, and increase energy expenditure, making it an attractive target for anti-obesity therapeutics (21). Given the individual roles of CB1R and ZFP423 in adipose metabolism, their dual inhibition presents a promising strategy to achieve sustained weight loss through metabolic reprogramming rather than caloric restriction alone.

[0269] In this study, we developed CCT-217, a peripherally targeted siRNA-based therapeutic designed to simultaneously silence CNR1 (CB1R) and ZNF423 (ZFP423) in adipose tissue. By using lipid nanoparticle (LNP-SM102) delivery technology, we aimed to achieve efficient gene silencing while ensuring selective adipose targeting to minimize off- target effects.

[0270] Here, we present a comprehensive evaluation of CCT-217 in diet-induced obese (DIO) C57BL / 6J mice. We assessed its efficacy in reducing body weight, enhancing adipose browning, and improving systemic metabolic parameters. We further investigated the molecular interplay between CB1R and ZFP423 in regulating adipose plasticity and metabolic homeostasis. Our findings provide compelling evidence that CCT-217 represents a novel, targeted therapeutic approach capable of achieving superior fat mass reduction while preserving lean mass, positioning it as a potential next-generation treatment for obesity and metabolic disorders. 283Attorney Docket: 090147-0584673 Materials and Methods Animal Model and Study Design

[0271] Male diet-induced obese (DIO) C57BL / 6J mice, aged 19–21 weeks, were obtained from Jackson Laboratory (JAX) and housed under standard environmental conditions. A total of 12 animals were included in this study. Mice were housed in sterilized individually ventilated cages (IVCs) made of polysulfone, with stainless steel grid tops for securing water bottles and food. The animal facility maintained a room temperature of 22 ± 3°C, relative humidity of 50 ± 20%, and a 12-hour light-dark cycle controlled via an automated timer. Ventilation provided 10–15 air changes per hour. Mice were fed a high-fat diet (60 kcal%, D12492) and received ad libitum access to purified water in polypropylene bottles. Animals were acclimatized for six days, during which baseline body weight measurements were recorded, and handling stress was minimized through routine handling procedures. After acclimatization, animals were randomized into treatment groups based on body weight and received either vehicle control or CCT-217, administered subcutaneously every three days. Preparation of the Test Compounds and Vehicle

[0272] The investigational compound CCT-217 was composed of two components: Component A (CB1R siRNA SM-102 LNP formulation) and Component B (ZFP-423 siRNA SM-102 LNP formulation). The vehicle solution was prepared at 5 mL / kg. A total of 6 siRNA molecules were used, i.e., 3 each for CB1R and ZFP423. For Component A, individual siRNA vials, i.e., CCT-217-1, CCT-217-2, and CCT-217-3, were thawed, pooled in equal concentrations, and diluted in sterile saline. Component B was prepared similarly, using CCT-217-4, CCT-217-5, and CCT-217-6. Formulations were stored at 2–8°C until administration. The composition of each vial is shown in Table 38.

[0273] Table 37 below lists the siRNA sequences included in CCT-217, and Table 38 shows the chemical modifications made specifically to those same sequences as formulated in CCT- 217. Table 37. Combination of Sequences used for CCT-217 Target Sense Strand (5’ to 3’ orientation) Antisense Strand (5’ to 3’ orientation)Attorney Docket: 090147-0584673 ZFP423 CAUUAACCAUGAGUGUAAACU UUUACACUCAUGGUUAAUGCCVial Name Target Sense Strand (5’ to 3’ Antisense Strand (5’ to 3’ orientation) with modifications orientation) with modifications UCAGGG[ ] s s own n a e , t e s s were synt es ze w t a vance c emca modifications to enhance stability, reduce immunogenicity, and optimize strand selection for efficient RISC loading. The modifications include: - 2'-O-Methyl (mA, mU, mC, mG): Added at specific positions on both sense and antisense strands to improve nuclease resistance and reduce off-target effects. - Phosphorothioate Linkages (*): Incorporated between select nucleotides, particularly at the ends of the antisense strand, to further increase stability. - 2'-Fluoro Modifications (2FC, 2FU, 2FA, 2FG): Strategically placed at positions shown to enhance silencing potency and specificity.

[0275] Each gene (CB1R and ZFP423) was targeted by three distinct siRNA sequences, each binding to a different region of the mRNA. The purpose of this multi-sequence approach was to maximize knockdown efficacy and minimize the risk of escape due to sequence-specific inefficiency or target site accessibility.

[0276] The chemical modifications followed established guidelines for siRNA therapeutics, ensuring optimal pharmacokinetics and minimizing immune activation. 285Attorney Docket: 090147-0584673 Dosing Regimen and Physiological Assessments

[0277] Mice received subcutaneous injections in the inguinal (iWAT) fat pads every three days, divided equally between left and right iWAT. Body weight was recorded once every three days, and food intake was monitored daily from Day 9 onwards. The body temperature was measured using a rectal probe at 0, 2, 6, and 24 hours post-dose. Animals were observed for 1-hour post-dosing for any clinical signs. Echo MRI and Sample Collection

[0278] On Day 21, mice underwent Echo MRI analysis, after which they were transported back to the animal housing facility. On Day 22, mice were euthanized, and blood and tissue samples were collected. Blood was collected via intracardiac puncture into lithium heparinized tubes (Sigma, A32955), centrifuged at 3500 rpm for 5 minutes at 2–8°C, and plasma was stored at -60 to -80°C for biochemical analysis. Tissue samples, including inguinal white adipose tissue (iWAT), mesenteric fat, retroperitoneal fat, gonadal fat, liver, kidney, thymus, brain and pancreas, were collected for biochemical, histological, and molecular analysis. ELISA Assays for Biochemical Profiling

[0279] Plasma samples were analyzed using ELISA-based assays for TSH (Invitrogen, EEL110), T3 (Invitrogen, EIAT3C), T4 (Invitrogen, EIAT4C), leptin (R&D Systems, MOBO0B), insulin (Marcodia, 10-1247-01), IL-6 (R&D Systems, M6000B-1), TNF-α (R&D Systems, MTA00B-1), CRP (R&D Systems, MCRP00), ALT (Randox, AL3801), AST (Randox, AS3804), ALP (Randox, AP3820), cholesterol (Randox, CH3810), triglycerides (Randox, TR3823), and blood urea nitrogen (BUN). Each ELISA was conducted according to the manufacturer’s instructions using appropriate dilution factors, biotinylated detection antibodies, and HRP-conjugated secondary antibodies, with optical density measurements at 450 nm using a microplate reader. Gene Expression Analysis by RT-qPCR

[0280] Total RNA was extracted from iWAT, liver and brain tissues using the RNeasy Mini Kit (Qiagen, 74106) following homogenization in TRI reagent (Sigma, T9424). RNA integrity was assessed via NanoDrop spectrophotometry, and cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Thermofisher, 4368814). Gene expression of CB1R (Thermofisher, 4331182) and ZFP423 (Thermofisher, 4351372) was quantified via qPCR using TaqMan Fast Advanced Master Mix (Thermofisher, 4444557) and 286Attorney Docket: 090147-0584673 18S rRNA (Applied Biosystems, PN4448489) as the housekeeping gene. The ΔΔCT method was applied to determine relative gene expression levels. Western Blot Analysis for Protein Expression

[0281] Protein lysates were prepared from iWAT homogenates and quantified using the BCA Protein Assay Kit (Thermofisher, A55864). Equal amounts of protein were resolved via SDS- PAGE, transferred onto polyvinylidene difluoride membranes (PVDF) membranes, and probed with primary antibodies against COX1 (Thermofisher, MA5-32259), and Vinculin (Thermofisher, 700062). HRP-conjugated secondary antibodies were used for detection, and signals were visualized using chemiluminescence (Luminol-Peroxide mix, Thermofisher, FNN0071). Densitometric analysis was performed using ImageJ software, with Vinculin as the loading control. Histological and Chemical Reagents

[0282] Tissue samples were fixed in neutral-buffered formalin and processed for histological evaluation using hematoxylin (Sigma Aldrich, 517-28-2) and eosin Y (L.C.L.C. & Fine Chemicals, 17372-87-1) staining. Additional reagents used in biochemical and molecular procedures included DPBS (Gibco, 14190-144), chloroform (TGI, CO175), molecular biology-grade water (HiMedia, ML024), protease inhibitor cocktail (Sigma, A32955), 2- propanol (Merck Millipore, 67-63-0), and xylene (Merck Millipore, 1330-20-7). Statistical Analysis

[0283] All statistical analyses were performed using GraphPad Prism (version 10.5.0). Data are expressed as mean ± SD. Differences between groups were assessed using Student’s t-test, one-way ANOVA, or two-way ANOVA, followed by post hoc analysis where appropriate. Statistical significance was set at p < 0.05. Results CCT-217 Treatment Induces Significant and Sustained Weight Loss Through Metabolic Activation

[0284] The administration of CCT-217 in diet-induced obese (DIO) C57BL / 6J mice resulted in a profound and sustained reduction in body weight. The onset of steep weight loss was observed as early as day 9, with a progressive decline continuing throughout the study, culminating in a 26% reduction in body weight compared to vehicle-treated controls by study termination (p<0.01) (FIG.1A). Unlike conventional weight-loss therapies, which primarily rely on appetite suppression, CCT-217 administration induced weight loss through a 287Attorney Docket: 090147-0584673 mechanism largely independent of severe caloric restriction. Although a modest 18% decrease in food intake was noted, feeding experiments confirmed that the extent of weight loss in CCT-217-treated mice exceeded what could be attributed solely to reduced caloric intake, suggesting that enhanced energy expenditure was a major driving factor (FIG.1B). GLP-1 receptor agonists typically induce rapid weight loss through appetite suppression, in contrast, CCT-217 facilitated progressive and sustained weight loss while maintaining normal feeding behavior, reinforcing its unique mechanism of action that integrates adipose remodeling with enhanced systemic metabolism rather than central appetite modulation. CCT-217 Selectively Reduces Fat Mass While Enhancing Lean Mass

[0285] Body composition analysis using Echo-MRI revealed a 43.5% reduction in total fat mass while concurrently improving lean mass composition by 25% (p<0.01) (FIGs.1C and 1D). The reduction in adiposity was consistent across multiple fat depots, with inguinal WAT decreasing by 61.7%, retroperitoneal WAT by 59.4%, mesenteric WAT by 54.3%, and gonadal WAT by 42%, reinforcing that CCT-217 broadly remodels adipose tissue rather than targeting a single depot (FIGs.1E and 1F). This is particularly relevant given that visceral adiposity is strongly correlated with insulin resistance and cardiovascular disease. Histological analysis through H&E staining of inguinal white adipose tissue (iWAT) provided additional evidence of adipose remodeling. The absence of the large unilocular adipocytes in CCT-217-treated mice indicated extensive browning, a phenotype associated with enhanced mitochondrial function and lipid oxidation. Molecular analyses confirmed these changes, with a 3-fold increase in COX1 expression (p<0.01) via western blotting, suggesting elevated oxidative metabolism and mitochondrial activity (FIG.1G). Notably, UCP1 expression also showed an upregulated trend in the treated DIO group as observed through the RTqPCR (FIG.1H). Although the UCP-1 is not the only thermogenesis mechanism and alternate mechanisms have been reported for peripheral CB1R inverse agonists as well, indicating that CCT-217-induced thermogenesis is also mediated through alternative pathways independent of classical UCP1 activation (23). This aligns with emerging research on futile creatine cycling and lipid turnover as alternative thermogenic mechanisms in WAT(24), reinforcing the broad energy-expending effects of CCT-217. eR CCT-217 Treatment Reduces CB1R and ZFP423 Expression in iWAT

[0286] RT-qPCR analysis revealed a significant reduction in the expression of CB1R and ZFP423 in inguinal white adipose tissue (iWAT) of CCT-217-treated mice compared to diet- induced obese (DIO) control mice. Expression levels were normalized to 18S ribosomal 288Attorney Docket: 090147-0584673 RNA, used as the control housekeeping gene, to ensure accurate quantification. CB1R expression was elevated in the DIO control group but showed a 42-fold reduction following CCT-217 treatment (p < 0.0001), indicating strong suppression of this receptor (FIG.2A). Similarly, CCT-217 induced a 14.5-fold reduction (p < 0.0001) of the ZFP423 mRNA in the treated group compared to untreated control group (FIG.2B). This pronounced downregulation of CB1R and ZFP423 reflects CCT-217's robust inhibitory effects on key molecular pathways driving adipose tissue expansion and lipid storage. These findings align with the observed reduction in fat mass and improved adipose tissue remodeling, emphasizing the therapeutic potential of CCT-217 in targeting obesity-associated adipogenic signaling. In a pilot study conducted on healthy lean mice, we interestingly observed that treatment with ZFP423 siRNA (total 2 mg / kg body weight per week, administered subcutaneously near iWAT as equal halves in two doses over 7 days) resulted in a 26.20-fold decrease in CB1R gene expression, corresponding to 96.18% silencing, thus demonstrating the silencing effect of ZFP-423 on CB1R expression.

[0287] Similarly, in a separate study conducted by us, we observed that CB1R silencing also led to silencing of the ZFP423 gene in the mice by 3.5-fold or 71.43%. To the best of our knowledge this is first reported evidence for such a feedback loop, where silencing ZFP423 gene potentiates the silencing of CB1R and vice-versa for the browning, thermogenic activation and adipose tissue remodeling. In the pilot study we also observed that subcutaneous delivery of the CCT-217 had absolutely no effect on brain expression of the CB1R thus setting this therapy as very likely safe to avoid any psychiatric side effects. Metabolic and Endocrine Benefits of CCT-217

[0288] Plasma biochemical analysis demonstrated substantial improvements in metabolic markers following CCT-217 administration. Leptin levels decreased by 70% (p<0.001), consistent with the observed reduction in adiposity and indicative of restored leptin sensitivity. Additionally, C-reactive protein (CRP) levels declined by 21% (p<0.05), suggesting a reduction in chronic systemic inflammation, which is a hallmark of obesity- associated metabolic dysfunction. Markers of hepatic function showed significant improvement, with ALP levels decreasing by 29% (p<0.05) and total cholesterol levels dropping by 20% (p<0.01), suggesting that CCT-217 not only reduced fat mass but also improved lipid metabolism and liver health (FIGS.2C-G). CCT-217 Enhances Thyroid Hormone Regulation and Energy Expenditure 289Attorney Docket: 090147-0584673

[0289] Further investigation into endocrine modulation revealed that CCT-217 significantly reduced thyroid-stimulating hormone (TSH) levels by 37% (p<0.001) while increasing circulating T3 levels by 82% (p<0.001), with no significant alterations in T4 levels (FIGS. 2H-J). The increase in T3 suggests enhanced deiodinase activity, which facilitates greater mitochondrial biogenesis and lipid oxidation, further reinforcing the thermogenic and metabolic effects of CCT-217. Given the established role of T3 in increasing basal metabolic rate, these findings provide mechanistic insights into the metabolic benefits conferred by CCT-217. The maintenance of stable T4 levels further indicates that CCT-217 did not induce systemic thyroid dysfunction but rather optimized endogenous thyroid hormone metabolism to favor energy expenditure. CCT-217 Demonstrates a Favorable Safety Profile Across Multiple Organs

[0290] A comprehensive histopathological analysis confirmed that CCT-217 was well- tolerated across multiple organ systems, with no evidence of toxicity or structural damage in the brain, pancreas, thymus, kidney, or liver. Inguinal white adipose tissue (iWAT) stained with Hematoxylin and Eosin (H&E) revealed distinct differences between control and CCT- 217-treated groups. In the control group, adipocytes appeared large, unilocular, and relatively uniform in size, with well-defined cell membranes and flattened nuclei pushed to the periphery. The extracellular matrix was sparse, and minimal stromal or inflammatory cell presence was observed, reflecting a metabolically inactive state typical of untreated adipose tissue with high lipid content (FIG.3A). In contrast, iWAT from CCT-217-treated mice showed significant changes indicative of adipose tissue remodeling and browning, adipocytes were notably smaller and more heterogeneous in size, suggesting lipid mobilization and reduction in fat storage. Increased stromal cellularity, evidenced by a greater presence of purple-stained nuclei in the interstitial space, indicates ongoing tissue remodeling (FIGs.3B and I-J). Additionally, while a slight increase in extracellular matrix was observed, there were no signs of fibrosis or pathological tissue damage. Overall, these findings highlight the metabolic activation and favorable remodeling of iWAT following CCT-217 treatment, aligning with the observed reductions in fat mass through browning and energy expenditure improved adipose tissue health. Hepatic histology demonstrated a marked reduction in hepatocellular lipid accumulation, consistent with the biochemical findings indicating improved liver function (FIGS.3C and 3D). Histological analysis of bone marrow sections stained with Hematoxylin and Eosin (H&E) revealed clear differences between the vehicle control group and the CCT-217-treated group. In the vehicle control group, bone marrow 290Attorney Docket: 090147-0584673 displayed lower cellular density with prominent fat vacuoles occupying significant space within the marrow cavity (FIG.3E). This observation reflects marrow adiposity, a well- known consequence of prolonged high-fat diet (HFD) feeding. In the CCT217 treated group the marrow space appeared densely populated with hematopoietic cells, while fat vacuoles were significantly reduced compared to the control group indicating impressive reversal of adiposity and highlights the treatment's ability to restore bone marrow homeostasis and promote a shift from adipogenesis back to hematopoiesis (FIG.3F).

[0291] Renal histology revealed no significant pathological alterations, confirming that CCT- 217 does not negatively impact renal function (FIGS.3G and 3H). No evidence of neuroinflammation, gliosis, or CB1R-related neurotoxicity was observed in brain H&E staining, reinforcing the peripherally selective nature of CCT-217. This is a key advantage over first-generation CB1R inverse agonists, which were associated with severe psychiatric side effects due to central CB1R blockade. Unlike metabolic stress conditions that induce thymic atrophy, CCT-217-treated mice exhibited preserved thymic architecture, suggesting that the treatment does not impair immune function, a critical consideration for long-term therapeutic use. Pancreatic histology confirmed intact islet morphology, with no evidence of fibrosis or β-cell dysfunction, further supporting the metabolic safety profile of CCT-217. Body Temperature Analysis in CCT-217-Treated Mice

[0292] Rectal body temperature measurements were conducted to assess any potential hyperthermic effects of CCT-217 treatment in diet-induced obese (DIO) mice. Over the 20- day study period, body temperature remained within the normal physiological range of 36°C- 38°C for both the CCT-217-treated and control groups (FIG.3K). On Day 10 and Day 13, a modest yet statistically significant increase in body temperature (p < 0.05) was observed in the CCT-217-treated group compared to the DIO control group. However, these values were well within the normal range, indicating that the observed elevations were neither excessive nor clinically relevant. By Day 19, body temperature levels in the treated group returned to baseline values comparable to the control group. These results conclusively demonstrate that CCT-217 treatment does not induce clinical hyperthermia at all. The transient elevations in temperature on Days 10 and 13 are likely reflective of increased metabolic activity, potentially due to enhanced energy expenditure and adipose tissue remodeling, which are consistent with the physiological effects of CCT-217. Importantly, the absence of hyperthermia further supports the safety and tolerability of CCT-217 as a therapeutic intervention in obesity. 291Attorney Docket: 090147-0584673 Discussion

[0293] CCT-217’s pronounced tropism for subcutaneous adipose tissue confers a significant therapeutic advantage, as evidenced by a substantial 26% reduction in body weight within just 20 days. This is an exceptional qualitative weight loss because the treated mice had a tremendous improvement in their lean mass composition, comparable to that of healthy lean mice as observed in literature (25). Notably, this was achieved with comparatively very low and less frequent dosing (i.e., once every 3 days at a total of 3.75 mg / kg body weight dose) compared to other CB1R targeted therapies or other energy expenditure enhancing therapies. CCT-217's weight loss represents a significant improvement over existing and investigational GLP / GIPr agonists, as the latter induce primarily quantitative weight loss including substantial lean body mass reduction as reported in some studies to be as high as 20% accompanied by excessive reductions in feed intake, which expectedly may lead to nutritional depletion. On the contrary, CCT-217 promoted adipose tissue remodeling through browning and improved systemic metabolic health without lean mass loss. The CCT-217 treatment induced weight loss resulted in mild reduction in cumulative feed intake of about 18% compared to about 45% in case of Tirzepatide (26). Our study concludes that CCT-217 improved the feed intake behavior of the mice along with energy expenditure and browning, rather than over dependence on anorexic mechanisms as seen for GIP / GLPr agonists (26). Interestingly, after the 4th dose of CCT-217 in the treated mice (i.e., after day 10 onwards) the difference in the feed intake was only 12% compared to the control group’s cumulative feeding which emphasizes the browning of the WAT and energy expenditure in the adipose tissue as the major driver of this weight loss instead of hypo-phagy. The increased expression of COX-1 and UCP-1 along with the elevated T3 levels further substantiates this claim. This dual CB1R and ZFP423 targeted approach provides a compelling mechanistic rationale for superior therapeutic outcomes in obesity, particularly compared to existing mono-therapies like GLP-1 / GIP agonists, which primarily reduce fat mass but fail to preserve lean mass or enhance energy expenditure (6).

[0294] Mechanistically, CB1R is a key regulator within the endocannabinoid system (ECS) and plays an important role in energy homeostasis by controlling appetite, lipogenesis, and energy expenditure (7, 8). Its overactivation in obesity leads to hyperphagia, increased fat accumulation, and systemic inflammation. First generation CB1R targeted inhibitors induced weight loss but were also associated with psychiatric issues, while the next generation peripherally restricted CB1R inverse agonists have shown promising results in reducing 292Attorney Docket: 090147-0584673 visceral adiposity and improving insulin sensitivity, their clinical application remains limited by partial efficacy (9, 27). ZFP423, a zinc-finger transcription factor, is a potent repressor of thermogenic gene expression by inhibiting EBF2 and suppressing UCP1 (21, 22, 28, 29). By maintaining the white adipocyte phenotype, ZFP423 restricts adipose plasticity and browning potential. Knockout studies demonstrate that silencing ZFP423 can allow for the epigenetic de-repression of thermogenic programs, facilitating the activation of beige adipocytes within white adipose tissue (WAT) depots and enhancing systemic energy expenditure (21). Its suppression allows for the reprogramming of white adipocytes into thermogenically active beige cells, even in depots resistant to browning (21). In Humans, mesenchymal stem cells (MSCs) from infants of obese mothers (Ob-MSCs) exhibit increased ZFP423 expression and correlates with adipocyte hypertrophy, reduced hyperplasia, and metabolic alterations in the infants (30). Conversely, CCT-217 treatment improved hyperplasia and reduced hypertrophy in iWAT of the DIO mice may be seen as a mechanism to prevent exhaustion of the preadipocytes to excessive differentiation by reversing pathological expression of ZFP-423. A similar mechanism of quiescence in hematopoietic stem cells of Liver through ZFP-521 expression is known (31). It is interesting to indicate that ZFP-521 downregulates the expression of ZFP-423 in mesenchymal stem cells and could be a conserved mechanism to preserve the different stem cells from excessive differentiation during normal conditions. Additionally, ZNF423 upregulation in endothelial and adipose-derived stromal / stem cells (ASC) of lipedema patients correlates with excessive fat accumulation and altered distribution, reinforcing its role in pathological adiposity and human translatability (32). This dual silencing mechanism also complemented the action of CB1R silencing, which reduced adiposity and systemic inflammation. By targeting both CB1R and ZFP423, CCT-217 leveraged a synergistic effect to promote adipose tissue browning, enhanced systemic energy expenditure, to achieve superior weight loss outcomes compared to monotherapies, underscoring its transformative therapeutic potential in obesity. Our findings also revealed an unexpected crosstalk between CB1R and ZFP423, where silencing one gene reciprocally downregulated the other. Specifically, CB1R inhibition reduced ZFP423 expression, while ZFP423 silencing further suppressed CB1R. This bidirectional relationship likely reflects shared regulatory pathways governing lipid metabolism, inflammatory signaling, and adipose tissue plasticity. The combined silencing of CB1R and ZFP423 resulted in profound adipose remodeling because co-silencing of cell surface receptor and transcription factor effectively excludes any escape mechanism of the WAT from escaping browning. CCT-217 also induced significant fat loss in visceral depots (retroperitoneal (59.4%), mesenteric (54.3%), and 293Attorney Docket: 090147-0584673 gonadal (42.0%)) as well as robust browning of inguinal WAT (iWAT), evidenced by smaller, multilocular adipocytes with thermogenic characteristics. These findings align with the known role of ZFP423 suppression in facilitating adipocyte browning and systemic energy dissipation (21).

[0295] Notably, the observed weight reduction with CCT-217 was achieved with only a modest 12% reduction in feed intake from dose four onward, highlighting increased energy expenditure rather than caloric restriction as the primary driver of weight loss. This outcome, along with elevated H&E staining scores and increased expression of COX-1, UCP1, and T3, underscores the thermogenic activation of beige adipocytes as a key mechanism underlying CCT-217’s efficacy. In addition to adipose-specific effects, systemic improvements were evident across multiple metabolic organs. Hepatic pathology improved significantly, with reduced hepatocellular vacuolation and the absence of fibrosis or inflammation, suggesting hepatoprotective effects mediated by decreased lipo-toxicity and systemic inflammation. Bone marrow analyses showed adaptive myeloid activation without adverse remodeling, affirming the safety of CCT-217 therapy. Notably, in humans, ZFP423 is a genetic risk locus for reduced bone mineral density, linking its dysregulation to osteoporosis and osteopenia (33). Conclusion

[0296] CCT-217 represents a first-in-class siRNA-based dual-action therapeutic targeting CB1R and ZFP423, offering a revolutionary approach to obesity management. This study demonstrates that CCT-217 induces substantial weight loss, enhances metabolic remodeling, and preserves lean mass, presenting a compelling superior alternative to existing obesity pharmacotherapies. The observed increase in energy expenditure, activation of non-UCP1 thermogenesis, and modulation of thyroid hormone activity highlight its potential as a next- generation metabolic therapeutic. Importantly, CCT-217 exhibited a robust safety profile across multiple organ systems, with no adverse effects detected in the brain, pancreas, kidney, thymus, or liver. Given its superior efficacy and safety profile, CCT-217 holds strong potential for clinical translation as a metabolic intervention targeting obesity and its associated comorbidities. 294Attorney Docket: 090147-0584673 References 1. 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Han JH, Shin H, Park JY, Rho JG, Son DH, Kim KW, et al. A novel peripheral cannabinoid 1 receptor antagonist, AJ5012, improves metabolic outcomes and suppresses adipose tissue inflammation in obese mice. Faseb j.2019;33(3):4314-26. 12. Després J-P, Golay A, Sjöström L. Effects of Rimonabant on Metabolic Risk Factors in Overweight Patients with Dyslipidemia. New England Journal of Medicine. 2005;353(20):2121-34. 13. Engeli S, Böhnke J, Feldpausch M, Gorzelniak K, Janke Jr, Bátkai Sn, et al. Activation of the Peripheral Endocannabinoid System in Human Obesity. Diabetes. 2005;54(10):2838-43. 295Attorney Docket: 090147-0584673 14. Högberg T, Receveur J-M, Murray A, Linget J-M, Nørregaard PK, Little PB, et al. Optimizing and characterizing 4-methyl substituted pyrazol-3-carboxamides leading to the peripheral cannabinoid 1 receptor inverse agonist TM38837. Bioorganic & Medicinal Chemistry Letters.2024;98:129572. 15. Morningstar M, Kolodziej A, Ferreira S, Blumen T, Brake R, Cohen Y. 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Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. 2019;1864(1):20-8. 21. Shao M, Ishibashi J, Kusminski CM, Wang QA, Hepler C, Vishvanath L, et al. Zfp423 Maintains White Adipocyte Identity through Suppression of the Beige Cell Thermogenic Gene Program. Cell Metabolism.2016;23(6):1167-84. 22. Shao M, Zhang Q, Truong A, Shan B, Vishvanath L, Li L, et al. ZFP423 controls EBF2 coactivator recruitment and PPARγ occupancy to determine the thermogenic plasticity of adipocytes. Genes Dev.2021;35(21-22):1461-74. 23. Iyer MS, Paszkiewicz RL, Bergman RN, Richey JM, Woolcott OO, Asare-Bediako I, et al. Activation of NPRs and UCP1-independent pathway following CB1R antagonist treatment is associated with adipose tissue beiging in fat-fed male dogs. Am J Physiol Endocrinol Metab.2019;317(3):E535–E547. doi:10.1152 / ajpendo.00539.2018 24. Chouchani ET, Kazak L, Spiegelman BM. New Advances in Adaptive Thermogenesis: UCP1 and Beyond. Cell Metabolism.2019;29(1):27-37. 25. Nilsson J, Ericsson M, Joibari MM, Anderson F, Carlsson L, Nilsson SK, et al. A low-carbohydrate high-fat diet decreases lean mass and impairs cardiac function in pair-fed female C57BL / 6J mice. Nutrition & Metabolism.2016;13(1):79. 26. Coskun T, Sloop KW, Loghin C, Alsina-Fernandez J, Urva S, Bokvist KB, et al. LY3298176, a novel dual GIP and GLP-1 receptor agonist for the treatment of type 2 diabetes mellitus: From discovery to clinical proof of concept. Mol Metab.2018;18:3- 14. 296Attorney Docket: 090147-0584673 27. Silvestri C, Di Marzo V. The Endocannabinoid System in Energy Homeostasis and the Etiopathology of Metabolic Disorders. Cell Metabolism.2013;17(4):475-90. 28. Rajakumari S, Wu J, Ishibashi J, Lim H-W, Giang A-H, Won K-J, et al. EBF2 Determines and Maintains Brown Adipocyte Identity. Cell Metabolism. 2013;17(4):562-74. 29. Wang W, Kissig M, Rajakumari S, Huang L, Lim H-w, Won K-J, et al. Ebf2 is a selective marker of brown and beige adipogenic precursor cells. Proceedings of the National Academy of Sciences.2014;111(40):14466-71. 30. Keleher MR, Shubhangi S, Brown A, Duensing AM, Lixandrão ME, Gavin KM, et al. Adipocyte hypertrophy in mesenchymal stem cells from infants of mothers with obesity. Obesity (Silver Spring).2023;31(8):2090-102. 31. Li Z, Fu X, Wu W, Liu Z, Chen Z, Zhou C, et al. Zfp521 is essential for the quiescence and maintenance of adult hematopoietic stem cells under stress. iScience. 2021;24(2):102039. 32. Strohmeier K, Hofmann M, Jacak J, Narzt MS, Wahlmueller M, Mairhofer M, et al. Multi-Level Analysis of Adipose Tissue Reveals the Relevance of Perivascular Subpopulations and an Increased Endothelial Permeability in Early-Stage Lipedema. Biomedicines.2022;10(5). 33. Greenbaum J, Su KJ, Zhang X, Liu Y, Liu A, Zhao LJ, et al. A multiethnic whole genome sequencing study to identify novel loci for bone mineral density. Hum Mol Genet.2022;31(7):1067-81. 34. Fisher FM, Maratos-Flier E. Understanding the Physiology of FGF21. Annual Review of Physiology.2016;78(Volume 78, 2016):223-41. 35. Lee P, Linderman JD, Smith S, Brychta RJ, Wang J, Idelson C, et al. Irisin and FGF21 are cold-induced endocrine activators of brown fat function in humans. Cell Metab.2014;19(2):302-9. 36. Wang G-X, Zhao X-Y, Meng Z-X, Kern M, Dietrich A, Chen Z, et al. The brown fat– enriched secreted factor Nrg4 preserves metabolic homeostasis through attenuation of hepatic lipogenesis. Nature Medicine.2014;20(12):1436-43. 37. Stanford KI, Middelbeek RJ, Townsend KL, An D, Nygaard EB, Hitchcox KM, et al. Brown adipose tissue regulates glucose homeostasis and insulin sensitivity. J Clin Invest.2013;123(1):215-23. 38. Tsai VW, Macia L, Johnen H, Kuffner T, Manadhar R, Jørgensen SB, et al. TGF-b superfamily cytokine MIC-1 / GDF15 is a physiological appetite and body weight regulator. PLoS One.2013;8(2):e55174. 39. Hui X, Gu P, Zhang J, Nie T, Pan Y, Wu D, et al. Adiponectin Enhances Cold- Induced Browning of Subcutaneous Adipose Tissue via Promoting M2 Macrophage Proliferation. Cell Metabolism.2015;22(2):279-90. 297Attorney Docket: 090147-0584673 40. Svensson Katrin J, Long Jonathan Z, Jedrychowski Mark P, Cohen P, Lo James C, Serag S, et al. A Secreted Slit2 Fragment Regulates Adipose Tissue Thermogenesis and Metabolic Function. Cell Metabolism.2016;23(3):454-66. 41. Rao Rajesh R, Long Jonathan Z, White James P, Svensson Katrin J, Lou J, Lokurkar I, et al. Meteorin-like Is a Hormone that Regulates Immune-Adipose Interactions to Increase Beige Fat Thermogenesis. Cell.2014;157(6):1279-91. 42. Tian L, Li W, Yang L, Chang N, Fan X, Ji X, et al. Cannabinoid Receptor 1 Participates in Liver Inflammation by Promoting M1 Macrophage Polarization via RhoA / NF-κB p65 and ERK1 / 2 Pathways, Respectively, in Mouse Liver Fibrogenesis. Front Immunol.2017;8:1214. 43. Becher T, Palanisamy S, Kramer DJ, Eljalby M, Marx SJ, Wibmer AG, et al. 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Claims

Attorney Docket: 090147-0584673 CLAIMS 1. An inhibitory RNA oligonucleotide comprising a 10-30 nucleotide long antisense strand comprising a nucleotide sequence complementary to at least 10 contiguous nucleotides of a cannabinoid receptor 1 (CB1R) mRNA targeting sequence selected from Tables 1-13.

2. The inhibitory RNA oligonucleotide of claim 1, wherein the nucleotide sequence is fully complementary to the CB1R mRNA targeting sequence.

3. The inhibitory RNA oligonucleotide of any one of claims 1-2, wherein the antisense strand is 19-23 nucleotides in length.

4. The inhibitory RNA oligonucleotide of any one of claims 1-3, wherein the nucleotide sequence is at least 90% identical to an antisense strand sequence selected from Tables 1-13.

5. The inhibitory RNA oligonucleotide of any one of claims 1-4, wherein the nucleotide sequence is an antisense strand sequence selected from Tables 1-13.

6. The inhibitory RNA oligonucleotide of any one of claims 1-5, wherein the nucleotide sequence is an antisense strand sequence selected from Table 35.

7. The inhibitory RNA oligonucleotide of any one of claims 1-6, wherein the oligonucleotide is a single-stranded oligonucleotide.

8. The inhibitory RNA oligonucleotide of any one of claims 1-6, wherein the oligonucleotide is a double-stranded oligonucleotide comprising a sense strand that forms a duplex with the antisense strand.

9. The inhibitory RNA oligonucleotide of claim 8, wherein the sense strand comprises a sense strand sequence that is at least 50% complementary to at least 10 contiguous nucleotides of the antisense strand. 299Attorney Docket: 090147-0584673 10. The inhibitory RNA oligonucleotide of claim 9, wherein the sense strand is 10-50 nucleotides in length.

11. The inhibitory RNA oligonucleotide of any one of claims 8-10, wherein the double-stranded oligonucleotide comprises a blunt end at the 5’ and / or 3’ of the antisense strand.

12. The inhibitory RNA oligonucleotide of any one of claims 8-11, wherein the double-stranded oligonucleotide comprises an overhang at the 5’ and / or 3’ end of the oligonucleotide, and wherein the overhang comprises 1-4 nucleotides.

13. The inhibitory RNA oligonucleotide of any one of claims 8-12, wherein the double-stranded oligonucleotide is a substrate for Dicer.

14. The inhibitory RNA oligonucleotide of any one of claims 1-13, wherein at least one nucleotide of the oligonucleotide comprises a modification.

15. The inhibitory RNA oligonucleotide of claim 14, wherein the modification is a ribose modification, a base modification, or a phosphate modification.

16. The inhibitory RNA oligonucleotide of claim 15, wherein the ribose modification is a 2’-modification.

17. The inhibitory RNA oligonucleotide of claim 16, wherein the 2’-modification is selected from 2’-fluoro, 2’-O-methyl, 2’-aminoethyl, 2’-O-methoxyethyl, and 2’-deoxy-2’- fluoro-β-d-arabinonucleic acid.

18. The inhibitory RNA oligonucleotide of any one of claims 1-17, wherein the double-stranded oligonucleotide comprises a modified sequence selected from Table 35.

19. The inhibitory RNA oligonucleotide of any one of claims 1-18, wherein the double-stranded oligonucleotide comprises a modified sense sequence and a modified antisense sequence selected from Table 35. 300Attorney Docket: 090147-0584673 20. The inhibitory RNA oligonucleotide of any one of claims 1-19, wherein the nucleotide sequence is complementary to one or more CB1R isoforms of CB1R mRNA.

21. The inhibitory RNA oligonucleotide of claim 20, wherein the one or more CB1R isoforms is selected from CB1R, CB1aR, CB1bR, and combinations thereof.

22. The inhibitory RNA oligonucleotide of any one of claim 1-19, wherein the nucleotide sequence is complementary to a CB1aR isoform or a CB1bR isoform of CB1R mRNA.

23. An inhibitory RNA oligonucleotide comprising a 10-30 nucleotide long antisense strand comprising a nucleotide sequence complementary to at least 10 contiguous nucleotides of a zinc finger protein 423 (ZFP423) mRNA targeting sequence selected from Tables 14-26.

24. The inhibitory RNA oligonucleotide of claim 23, wherein the nucleotide sequence is fully complementary to the ZFP423 mRNA targeting sequence.

25. The inhibitory RNA oligonucleotide of any one of claims 23-24, wherein the antisense strand is 19-23 nucleotides in length.

26. The inhibitory RNA oligonucleotide of any one of claims 23-25, wherein the nucleotide sequence is at least 90% identical to an antisense strand sequence selected from Tables 14-26.

27. The inhibitory RNA oligonucleotide of any one of claims 23-26, wherein the nucleotide sequence is an antisense strand sequence selected from Tables 14-26.

28. The inhibitory RNA oligonucleotide of any one of claims 23-27, wherein the nucleotide sequence is an antisense strand sequence selected from Table 36.

29. The inhibitory RNA oligonucleotide of any one of claims 23-27, wherein the oligonucleotide is a single-stranded oligonucleotide. 301Attorney Docket: 090147-0584673 30. The inhibitory RNA oligonucleotide of any one of claims 23-27, wherein the oligonucleotide is a double-stranded oligonucleotide comprising a sense strand that forms a duplex with the antisense strand.

31. The inhibitory RNA oligonucleotide of claim 30, wherein the sense strand comprises a sense strand sequence that is at least 50% complementary to at least 10 contiguous nucleotides of the antisense strand.

32. The inhibitory RNA oligonucleotide of claim 31, wherein the sense strand is 10- 50 nucleotides in length.

33. The inhibitory RNA oligonucleotide of any one of claims 30-32, wherein the double-stranded oligonucleotide comprises a blunt end at the 5’ and / or 3’ of the antisense strand.

34. The inhibitory RNA oligonucleotide of any one of claims 30-33, wherein the double-stranded oligonucleotide comprises an overhang at the 5’ and / or 3’ end of the oligonucleotide, and wherein the overhang comprises 1-4 nucleotides.

35. The inhibitory RNA oligonucleotide of any one of claims 30-34, wherein the double-stranded oligonucleotide is a substrate for Dicer.

36. The inhibitory RNA oligonucleotide of any one of claims 23-35, wherein at least one nucleotide of the oligonucleotide comprises a modification.

37. The inhibitory RNA oligonucleotide of claim 36, wherein the modification is a ribose modification, a base modification, or a phosphate modification.

38. The inhibitory RNA oligonucleotide of claim 36, wherein the ribose modification is a 2’-modification.

39. The inhibitory RNA oligonucleotide of claim 38, wherein the 2’-modification is selected from 2’-fluoro, 2’-O-methyl, 2’-aminoethyl, 2’-O-methoxyethyl, and 2’-deoxy-2’- fluoro-β-d-arabinonucleic acid. 302Attorney Docket: 090147-0584673 40. The inhibitory RNA oligonucleotide of any one of claims 23-39, wherein the double-stranded oligonucleotide comprises a modified sequence selected from Table 36.

41. The inhibitory RNA oligonucleotide of any one of claims 23-40, wherein the double-stranded oligonucleotide comprises a modified sense sequence and a modified antisense sequence selected from Table 36.

42. The inhibitory RNA oligonucleotide of any one of claims 23-41, wherein the nucleotide sequence is complementary to one or more ZFP423 isoforms of ZFP423 mRNA.

43. An inhibitory RNA oligonucleotide comprising a 10-30 nucleotide long antisense strand comprising a nucleotide sequence complementary to at least 10 contiguous nucleotides of a transducing-like enhancer of split 3 (TLE3) mRNA targeting sequence selected from Tables 27-34.

44. The inhibitory RNA oligonucleotide of claim 43, wherein the nucleotide sequence is fully complementary to the TLE3 mRNA targeting sequence.

45. The inhibitory RNA oligonucleotide of any one of claims 43-44, wherein the antisense strand is 19-23 nucleotides in length.

46. The inhibitory RNA oligonucleotide of any one of claims 43-45, wherein the nucleotide sequence is at least 90% identical to an antisense strand sequence selected from Tables 27-34.

47. The inhibitory RNA oligonucleotide of any one of claims 43-46, wherein the nucleotide sequence is an antisense strand sequence selected from Tables 27-34.

48. The inhibitory RNA oligonucleotide of any one of claims 43-47, wherein the oligonucleotide is a single-stranded oligonucleotide. 303Attorney Docket: 090147-0584673 49. The inhibitory RNA oligonucleotide of any one of claims 43-47, wherein the oligonucleotide is a double-stranded oligonucleotide comprising a sense strand that forms a duplex with the antisense strand.

50. The inhibitory RNA oligonucleotide of claim 49, wherein the sense strand comprises a sense strand sequence that is at least 50% complementary to at least 10 contiguous nucleotides of the antisense strand.

51. The inhibitory RNA oligonucleotide of claim 50, wherein the sense strand is 10- 50 nucleotides in length.

52. The inhibitory RNA oligonucleotide of any one of claims 49-51, wherein the double-stranded oligonucleotide comprises a blunt end at the 5’ and / or 3’ of the antisense strand.

53. The inhibitory RNA oligonucleotide of any one of claims 49-52, wherein the double-stranded oligonucleotide comprises an overhang at the 5’ and / or 3’ end of the oligonucleotide, and wherein the overhang comprises 1-4 nucleotides.

54. The inhibitory RNA oligonucleotide of any one of claims 49-53, wherein the double-stranded oligonucleotide is a substrate for Dicer.

55. The inhibitory RNA oligonucleotide of any one of claims 43-54, wherein at least one nucleotide of the oligonucleotide comprises a modification.

56. The inhibitory RNA oligonucleotide of claim 55, wherein the modification is a ribose modification, a base modification, or a phosphate modification.

57. The inhibitory RNA oligonucleotide of claim 55, wherein the ribose modification is a 2’-modification.

58. The inhibitory RNA oligonucleotide of claim 57, wherein the 2’-modification is selected from 2’-fluoro, 2’-O-methyl, 2’-aminoethyl, 2’-O-methoxyethyl, and 2’-deoxy-2’- fluoro-β-d-arabinonucleic acid. 304Attorney Docket: 090147-0584673 59. The inhibitory RNA oligonucleotide of any one of claims 43-58, wherein the nucleotide sequence is complementary to one or more isoforms of TLE3 mRNA.

60. A combination of at least two inhibitory RNA oligonucleotides, wherein the first oligonucleotide comprises a 10-30 nucleotide long antisense strand comprising a nucleotide sequence complementary to at least 10 contiguous nucleotides of a cannabinoid receptor 1 (CB1R) mRNA targeting sequence selected from Tables 1-13, and the second oligonucleotide comprises a 10-30 nucleotide long antisense strand comprising i) a nucleotide sequence complementary to at least 10 contiguous nucleotides of a zinc finger protein 423 (ZFP423) mRNA targeting sequence selected from Tables 14-26, or ii) a nucleotide sequence complementary to at least 10 contiguous nucleotides of a transducing-like enhancer of split 3 (TLE3) mRNA targeting sequence selected from Tables 27-34.

61. The combination of at least two inhibitory RNA oligonucleotides of claim 60, wherein the nucleotide sequence of the first oligonucleotide is an antisense strand sequence selected from Table 35. 62.The combination of at least two inhibitory RNA oligonucleotides of claim 60, wherein the first oligonucleotide is a double-stranded oligonucleotide comprising a modified sequence selected from Table 35.

63. The combination of at least two inhibitory RNA oligonucleotides of claim 60 wherein the first oligonucleotide is a double-stranded oligonucleotide comprising a modified sense sequence and a modified antisense sequence selected from Table 35.

64. The combination of at least two inhibitory RNA oligonucleotides of any one of claims 60-63, wherein the nucleotide sequence of the second oligonucleotide is an antisense strand sequence selected from Table 36. 65.The combination of at least two inhibitory RNA oligonucleotides of any one of claims 60-63, wherein the second oligonucleotide is a double-stranded oligonucleotide comprising a modified sequence selected from Table 36. 305Attorney Docket: 090147-0584673 66. The combination of at least two inhibitory RNA oligonucleotides of any one of claims 60-63 wherein the second oligonucleotide is a double-stranded oligonucleotide comprising a modified sense sequence and a modified antisense sequence selected from Table 36.

67. A composition comprising the inhibitory RNA oligonucleotide of any one of claims 1-22.

68. The composition of claim 67, wherein the inhibitory RNA oligonucleotide is formulated in a lipid nanoparticle, a polymeric nanoparticle, or an exosome.

69. A composition comprising the combination of at least two inhibitory RNA oligonucleotides of any one of claims 60-66.

70. The composition of claim 69, wherein the inhibitory RNA oligonucleotides are formulated in a lipid nanoparticle, a polymeric nanoparticle, or an exosome.

71. A method for reducing a CB1R mRNA in a cell, comprising contacting the cell with the inhibitory RNA oligonucleotide of any one of claims 1-22 or the composition of any claim 67 or 68.

72. The method of claim 71, wherein the cell is in a subject.

73. The method of claim 71 or 72, wherein the CB1R mRNA is a CB1a isoform mRNA.

74. The method of claim 71 or 72, wherein the CB1R mRNA is a CB1b isoform mRNA.

75. The method of any one of claims 71-74, wherein the cell is in adipose tissue, liver, pancreas or kidney.

76. The method of any one of claims 71-75, wherein mitochondrial activity is increased in the cell. 306Attorney Docket: 090147-0584673 77. A method for treating a disorder in a subject, comprising administering to the subject the inhibitory RNA oligonucleotide of any one of claims 1-22 or the composition of claim 67 or 68.

78. The method of claim 77, wherein the disorder is obesity, leptin resistance, insulin resistance, glucose tolerance, diabetes, renal fibrosis, glucose responsiveness in pancreas through modulation of β-cell function, Liver cirrhosis, fatty liver disease (FLD), cardiovascular, and inflammatory diseases 79. A method for treating a disorder in a subject, comprising administering to the subject the combination of at least two inhibitory RNA oligonucleotides of any one of claims 60-66 or the composition of claim 69 or 70.

80. The method of claim 79, wherein the subject is administered a dose of about 0.1 to about 10.0 mg / kg of inhibitory RNA oligonucleotide complementary to CB1R mRNA, and a dose of about 0.1 to about 10.0 mg / kg of inhibitory RNA oligonucleotide complementary to ZFP423 mRNA.

81. The method of claim 79, wherein the subject is administered a dose of 2.0 mg / kg of inhibitory RNA oligonucleotide complementary to CB1R mRNA, and a dose of 1.8 mg / kg of inhibitory RNA oligonucleotide complementary to ZFP423 mRNA.

82. The method of claim 79, wherein administration to the subject is done every week to every year.

83. The method of claim 79, wherein administration to the subject is done every 6 months.

84. The method of any one of claims 79-83, wherein the disorder is obesity, leptin resistance, insulin resistance, glucose tolerance, diabetes, renal fibrosis, glucose responsiveness in pancreas through modulation of β-cell function, Liver cirrhosis, fatty liver disease (FLD), cardiovascular, and inflammatory diseases 307Attorney Docket: 090147-0584673 85. The method of any one of claims 77-84, wherein white adipose tissue is converted to brown adipose tissue or beige adipose tissue in the subject 86. The method of any one of claims 77-85, wherein non-shivering thermogenesis is increased in the subject.

87. The method of any one of claims 77-86, wherein lean body mass is preserved in the subject.

88. The method of any one of claims 77-87, wherein CB1R mRNA expression is reduced in adipose tissue, optionally wherein CB1R mRNA expression is not reduced in tissue of the central nervous system.

89. The method of any one of claims 77-88, wherein the disorder is obesity.

90. The method of claim 89, wherein the obesity is caused by a genetic disorder and / or diet.

91. The method of claim 90, wherein the genetic disorder is a leptin deficiency or a leptin receptor deficiency.

92. A method for treating a metabolic disorder in a subject, comprising administering to the subject the inhibitory RNA oligonucleotide of any one of claims 1-22 or the composition of claim 67 or 68. 308

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