Modulation of gene transcription using antisense oligonucleotides targeted to regulatory RNAs

Abstract

Described herein are methods for regulating OTC gene transcription using antisense oligonucleotides (ASOs) that target regulatory RNAs, such as promoter-associated RNAs and enhancer RNAs. These methods are useful for increasing the expression of ornithine transcarbamylase (OTC), thereby treating diseases associated with abnormal gene expression.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 240,838, filed September 3, 2021, and U.S. Provisional Application No. 63 / 292,792, filed December 22, 2021, the contents of each of which are incorporated by reference in their entirety herein.

[0002] Sequence Listing This application contains a Sequence Listing that was submitted via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy, created in XX month, 20XX, is named XXXXXUS_sequencelisting.txt and is X,XXX,XXX bytes in size.

[0003] FIELD OF THEINVENTION The present invention relates to methods of upregulating or downregulating OTC gene transcription using antisense oligonucleotides (ASOs) targeted to OTC regulatory RNAs, such as promoter-associated RNA and enhancer RNA. [Background technology]

[0004] Transcription factors bind to specific sequences in promoter and enhancer DNA elements to regulate gene transcription. It has been recently reported that active promoter and enhancer elements are themselves transcribed to generate non-coding regulatory RNAs (regRNAs), such as promoter-associated RNAs (paRNAs) and enhancer RNAs (eRNAs) (see Sartorelli and Lauberth, Nat. Struct. Mol. Biol. (2020) 27, 521-28). Unlike coding RNAs, regRNAs are transcribed bidirectionally. Various models for the function of regRNAs have been proposed, including nucleosome remodeling (see Mousavi et al., Mol. Cell (2013) 51(5):606-17 (Non-Patent Document 2)), regulation of enhancer-promoter loops (see Lai et al., Nature (2013) 494(7438):497-501 (Non-Patent Document 3)), and direct interaction with transcriptional regulators (see Sigova et al., Science (2015) 350,978-81 (Non-Patent Document 4)).

[0005] Gene expression is generally known as an undrugable biological process. Despite efforts in understanding the biology of gene transcription and regRNA, clinically relevant methods for regulating gene expression are limited. There remains a need for novel and useful methods for treating diseases associated with abnormal gene expression. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Sartorelli and Lauberth, Nat. Structure. Mol. Biol. (2020) 27, 521-28 [Non-Patent Document 2] Mousavi et al.,Mol.Cell(2013)51(5):606-17 [Non-Patent Document 3] Lai et al.,Nature(2013)494(7438):497-501 [Non-Patent Document 4] Sigova et al.,Science(2015)350,978-81 Summary of the Invention

[0007] The present invention provides antisense oligonucleotides (ASOs) that target regulatory RNAs, such as promoter-associated RNAs and enhancer RNAs, and methods of using these ASOs to regulate gene expression. These methods are useful for regulating the level of gene products, for example, regulating the expression level of disease-causing genes (e.g., ornithine transcarbamylase (OTC)), thereby treating diseases associated with abnormal gene expression (e.g., urea cycle disorders).

[0008] In one embodiment, provided herein is an antisense oligonucleotide (ASO) complementary to at least 8 consecutive nucleotides of a regulatory RNA of human ornithine transcarbamylase (OTC), the regulatory RNA having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-4 or 1077.

[0009] In some embodiments, the ASO is complementary to a sequence in the regRNA that is no more than 200 nucleotides from the 3' end of the regRNA.

[0010] In some embodiments, the ASO is complementary to a sequence in the regRNA that is no more than 200 nucleotides from the 5' end of the regRNA.

[0011] In some embodiments, the regRNA is not polyadenylated RNA.

[0012] In some embodiments, the ASO does not induce RNAse H-mediated degradation of regRNA.

[0013] In some embodiments, the regulatory RNA has the nucleotide sequence of SEQ ID NO:1 and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:6-14, 18-35, 39, 41, 75, 76, 77, 78, 87-124, or 143-892.

[0014] In some embodiments, the regulatory RNA has a nucleotide sequence of SEQ ID NO: 2 and the ASO comprises a nucleotide sequence of SEQ ID NO: 15-17, 36-38, 64-74, 125-142, or 893-1029.

[0015] In some embodiments, the regulatory RNA has a nucleotide sequence of SEQ ID NO:2 and the ASO comprises a nucleotide sequence of SEQ ID NO:17.

[0016] In some embodiments, the ASO is no more than 50, 40, 30, or 25 nucleotides in length.

[0017] In some embodiments, the ASO comprises an RNA polynucleotide that contains one or more chemical modifications.

[0018] In some embodiments, at least 3, 4, or 5 nucleotides at the 5' end and at least 3, 4, or 5 nucleotides at the 3' end of the ASO comprise ribonucleotides having one or more chemical modifications.

[0019] In some embodiments, the one or more chemical modifications are 2'-O-Ci-4 alkyl, e.g., 2'-O-methyl (2'-OMe), 2'-deoxy (2'-H), 2'-O-Ci-3 alkyl-O-Ci-3 alkyl, e.g., 2'-methoxyethyl ("2'-MOE"), 2'-fluoro ("2'-F"), 2'-amino ("2'-NH2"), 2'-arabinosyl ("2'-arabino") nucleotides, 2'- The nucleotide sugar modifications include one or more of F-arabinosyl ("2'-F-arabino") nucleotides, 2'-locked nucleic acid ("LNA") nucleotides, 2'-amide bridged nucleic acid (AmNA), 2'-unlocked nucleic acid ("ULNA") nucleotides, L-form sugars ("L-sugars"), 4'-thioribosyl nucleotides, constrained ethyl (cET), 2'-fluoro-arabino (FANA), or thiomorpholino.

[0020] In some embodiments, the one or more chemical modifications comprise an internucleotide modification comprising one or more of phosphorothioate ("PS" or (P(S))), phosphoramidate (P(NR1R2), e.g., dimethylamino phosphoramidate (P(N(CH3)2)), phosphonocarboxylate (P(CH2)nCOOR), e.g., phosphonoacetate "PACE" (P(CH2COO-)), thiophosphonocarboxylate ((S)P(CH2)nCOOR), e.g., thiophosphonoacetate "thioPACE" ((S)P(CH2COO-)), alkylphosphonate (P(C1-3 alkyl), e.g., methylphosphonate-P(CH3), boranophosphonate (P(BH3)), or phosphorodithioate (P(S)2).

[0021] In some embodiments, the one or more chemical modifications are 2-thiouracil ("2-thioU"), 2-thiocytosine ("2-thioC"), 4-thiouracil ("4-thioU"), 6-thioguanine ("6-thioG"), 2-aminoadenine ("2-aminoA"), 2-aminopurine, pseudouracil, hypoxanthine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5-methylcytosine ("5-methylC"), 5-methyluracil ("5-methylU"), 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5,6-dehydrouracil, , 5-propynylcytosine, 5-propynyluracil, 5-ethynylcytosine, 5-ethynyluracil, 5-allyluracil ("5-allylU"), 5-allylcytosine ("5-allylC"), 5-aminoallyluracil ("5-aminoallylU"), 5-aminoallyl-cytosine ("5-aminoallylC"), abasic nucleotides, Z bases, P bases, unstructured nucleic acids ("UNA"), isoguanine ("isoG"), isocytosine ("isoC"), glycerol nucleic acid (GNA), or thiophosphoramidate morpholino (TMO).

[0022] In some embodiments, the one or more chemical modifications include 2'-O-methoxyethyl, 5-methyl on cytidine, locked nucleic acid (LNA), phosphodiester (PO) internucleotide linkage, or phosphorothioate (PS) internucleotide linkage.

[0023] In some embodiments, the one or more chemical modifications include 2'-O-methoxyethyl, 5-methyl on cytidine, locked nucleic acid (LNA), phosphodiester (PO) internucleotide linkage, or phosphorothioate (PS) internucleotide linkage.

[0024] In some embodiments, the ASO comprises a nucleotide sequence of SEQ ID NO: 18-39 or 67-74.

[0025] In some embodiments, the ASO does not contain 10 or more contiguous nucleotides of unmodified DNA.

[0026] In some embodiments, the ASO does not contain deoxyribonucleotides.

[0027] In some embodiments, the ASO does not include unmodified ribonucleotides.

[0028] In some embodiments, the length of the ASO is 5×n+5 nucleotides (where n is an integer greater than or equal to 3), the nucleotide at position 5×m is an LNA-modified ribonucleotide (where m is an integer from 1 to n), and the nucleotides at the remaining positions are 2'-O-methoxyethyl-modified ribonucleotides.

[0029] In some embodiments, the ASO further comprises a GalNAc moiety.

[0030] In some embodiments, the ASO comprises the nucleotide sequence of SEQ ID NO:142.

[0031] In some embodiments, the length of the ASO is 3×n+2 nucleotides (where n is an integer greater than or equal to 6), the nucleotide at position 3×m is an LNA-modified ribonucleotide (where m is an integer from 1 to n), and the nucleotides at the remaining positions are 2'-O-methoxyethyl-modified ribonucleotides.

[0032] In some embodiments, the ASO comprises the nucleotide sequence of SEQ ID NO:21.

[0033] In some embodiments, the ASO further comprises a GalNAc moiety.

[0034] The ASO of claim 22, wherein the ASO comprises the nucleotide sequence of SEQ ID NO: 122.

[0035] In some embodiments, each ribonucleotide of the ASO is modified with 2'-O-methoxyethyl.

[0036] In some embodiments, the ASO comprises the nucleotide sequence of SEQ ID NO:25.

[0037] In some embodiments, each nucleotide of the ASO is a ribonucleotide that is modified with 2'-O-methoxyethyl.

[0038] In some embodiments, the ASO comprises the nucleotide sequence of SEQ ID NO:36.

[0039] In some embodiments, the ASO comprises 10 or more contiguous nucleotides of unmodified DNA flanked by at least three nucleotides of modified ribonucleotides on each of the 5' and 3' ends.

[0040] In some embodiments, the ASO comprises the nucleotide sequence of SEQ ID NO:18.

[0041] In some embodiments, each cytidine in the ASO is modified with 5-methyl.

[0042] In some embodiments, the regRNA is eRNA.

[0043] In one aspect, provided herein is a pharmaceutical composition comprising an ASo described herein and a pharma- ceutically acceptable carrier or excipient carrier.

[0044] In one aspect, provided herein is a method of increasing transcription of OTC in a human cell, the method comprising contacting the cell with an ASO described herein or a pharmaceutical composition described herein.

[0045] In some embodiments, the cell is a hepatocyte.

[0046] In some embodiments, the ASO increases the amount of a regulatory RNA in a cell.

[0047] In some embodiments, the ASO increases the stability of a regulatory RNA in a cell.

[0048] In one aspect, provided herein is a method for treating a urea cycle disorder, comprising administering to a subject in need thereof an effective amount of an ASO described herein or a pharmaceutical composition described herein.

[0049] In some embodiments, the ASO increases the amount of a regulatory RNA in a subject's cells.

[0050] In some embodiments, the ASO increases the stability of a regulatory RNA in a subject's cells.

[0051] In some embodiments, the cell is a hepatocyte. [Brief description of the drawings]

[0052] [Figure 1] Illustrates an exemplary schematic diagram of eRNA, paRNA, mRNA, and natural antisense transcript (NAT) of a gene on a chromosome. eRNA, paRNA, and NAT are all non-coding RNAs. eRNA is transcribed bidirectionally from the enhancer of a gene. paRNA is transcribed from the promoter of a gene in the same direction as mRNA, but in the antisense direction. NAT is transcribed from its own downstream promoter in the antisense direction, so that the transcript at least partially overlaps with the mRNA. In general, eRNA and paRNA upregulate gene expression, while NAT downregulates gene expression.

[0053] [Diagram 2]A-D show that treatment with the indicated ASOs results in a dose-dependent upregulation of OTC mRNA. A shows OTC mRNA after treatment with hOTC-ASOe1-11. B shows OTC mRNA after treatment with hOTC-ASOe1-8. C shows OTC mRNA after treatment with hOTC-ASOe2-1. D shows OTC mRNA after treatment with hOTC-ASOe1-1.

[0054] [Figure 3A] 1 shows that OTC mRNA was increased in cells from OTC-deficient donors following treatment with ASOs hOTC-ASOe1-10 and hOTC-ASOe1-2c. [Figure 3B] 1 shows that urea production was increased in cells from OTC-deficient donors following treatment with ASOs hOTC-ASOe1-10 and hOTC-ASOe1-2c. [Figure 3C] 1 shows that OTC mRNA was increased in WT cells after treatment with ASO hOTC-ASOe1-2a. [Figure 3D] 1 shows that urea production was increased in WT cells after treatment with ASO hOTC-ASOe1-2a.

[0055] [Figure 4] Figure 1 shows that the indicated mouse ASOs increased OTC mRNA levels in primary mouse liver cells from wild-type mice. One-way ANOVA *: p0.05-0.005; **: p<0.005.

[0056] [Diagram 5] Figure 1 shows that the indicated mouse ASOs increased OTC mRNA levels in spfash primary mouse liver cells. One-way ANOVA**: p<0.005.

[0057] [Figure 6]A shows upregulation of Serping1 after treatment with IFNy, and B shows downregulation of Serping1 after treatment with the JAK inhibitor tofacitinib.

[0058] [Figure 7] Correlation between Serping1 mRNA and protein secretion after IFNy induction.

[0059] [Figure 8] Shows induction of Serping1 mRNA and regRNA in mouse liver treated with IFNy.

[0060] [Figure 9] A shows Serping1 mRNA and regRNA levels in mouse hepatocytes in a time course study after treatment with IFNy. B shows Serping1 mRNA and regRNA levels in mouse hepatocytes in a time course study after treatment with IFNy.

[0061] [Figure 10] Serping1 enhancer 2 RNA and promoter 2 RNA levels after treatment with IFNg or PBS (control) are shown.

[0062] [Figure 11] Figure 11A shows a schematic of the Serping1 chromosomal vicinity, and Figure 11B shows mRNA levels of Serping1, Irf1, Ube216, and NTC-3_S following treatment with the indicated ASOs.

[0063] [Figure 12] Serping1 mRNA levels are shown following treatment with the indicated ASOs.

[0064] [Figure 13] Serping1 mRNA levels are shown 24, 48 and 72 hours after treatment with the indicated ASOs.

[0065] [Figure 14] (A) Diagram of the timeline of the in vivo mouse study. (B) Increased Serping1 mRNA expression in vivo following treatment with ASO-2.

[0066] [Figure 15] A shows the additive effect of IFNy + the indicated ASO on Serping1 mRNA expression, normalized to untreated cells.B shows the additive effect of IFNy + the indicated ASO on Serping1 mRNA expression, normalized to untreated cells.

[0067] [Figure 16] Serping1 mRNA expression following treatment with the JAK1 inhibitor tofacitinib or the JAK1 inhibitor tofacitinib + ASO-2 is shown normalized to untreated cells.

[0068] [Figure 17] We show that the indicated ASO treatment in a Serping1 knockdown system using the JAK1 inhibitor tofacitinib increased Serping1 mRNA expression.

[0069] [Figure 18A] Schematic diagrams of various human OTC ASOs with chemical modifications are shown. Light grey indicates 2'-O-(2-Methoxyethyl) (2'-MOE) modifications. Dark grey indicates Locked Nucleic Acid (LNA) modifications. Line brackets indicate phosphodiester (PO) linkages. *C indicates 5-methyl on cytidine. ^ indicates FANA nucleoside. Nucleotide sequences with specific chemical modifications shown in this figure are assigned unique sequence identifiers. [Figure 18B]Schematic diagrams of various human OTC ASOs with chemical modifications are shown. Light grey indicates 2'-O-(2-Methoxyethyl) (2'-MOE) modifications. Dark grey indicates Locked Nucleic Acid (LNA) modifications. Line brackets indicate phosphodiester (PO) linkages. *C indicates 5-methyl on cytidine. ^ indicates FANA nucleoside. Nucleotide sequences with specific chemical modifications shown in this figure are assigned unique sequence identifiers. [Figure 18C] Schematic diagrams of various mouse OTC ASOs with chemical modifications are shown. Light grey indicates 2'-O-(2-methoxyethyl) (2'-MOE) modifications. *C indicates 5-methyl on cytidine. Nucleotide sequences with specific chemical modifications shown in this diagram are assigned unique sequence identifiers.

[0070] [Figure 19] Schematic diagram of various Serping1 ASOs with chemical modifications. Light grey indicates 2'-O-(2-methoxyethyl) (2'-MOE) modifications. *C indicates 5-methyl on cytidine. Unique sequence identifiers are assigned to nucleotide sequences with specific chemical modifications shown in this figure.

[0071] [Figure 20] (A) shows that treatment with the indicated ASOs results in mRNA upregulation of human OTC in a dose-dependent manner. (B) shows that treatment with the indicated ASOs results in mRNA upregulation of OTC in a dose-dependent manner.

[0072] [Figure 21] 1 shows that treatment with the indicated ASOs results in mRNA upregulation of human OTC in a dose-dependent manner.

[0073] [Figure 22] 1 shows that treatment with the indicated ASOs results in mRNA upregulation of human OTC in a dose-dependent manner.

[0074] [Figure 23] 1 shows that hOTC-ASOe1-1a did not induce IL6, TNFa, IFNa, or IFNb cytokine release by PBMCs.

[0075] [Figure 24A] 1 shows that treatment with the indicated ASOs results in mRNA upregulation of mouse OTC in a dose-dependent manner. [Figure 24B] Figure 2 shows that Otc regRNA-targeting ASO CO-4474 did not increase mouse OTC mRNA in Otcdef mice. [Figure 24C] 1 shows that CO-4474 reduced ammonia to WT levels.

[0076] [Diagram 25] A shows upregulation of OTC gene expression after treatment with hOTC-ASOe1-10. B shows alignment and peaks of paired-end sequenced ChIP-seq library to human hg38 genome. C shows differential peaks identified in OTC enhancer, OTC promoter, and control regions (GAPDH, RPGR, TSPAN7) after treatment with hOTC-ASOe1-10.

[0077] [Figure 26] Accessible chromatin regions in the OTC promoter and the enhancer and adjacent RPGR promoter are shown (indicated by boxed regions).

[0078] [Figure 27]A shows the relative expression levels of negative-stranded regRNA (RR1) transcribed from the OTC enhancer over time after ASO treatment. B shows the relative expression levels of positive-stranded regRNA (RR2) transcribed from the OTC enhancer over time after ASO treatment. C shows the mRNA effect of OTC over time after hOTC-ASOe1-10 treatment. D shows the results of H3K27ac ChIP-qPCR after hOTC-ASOe1-10 treatment. E provides a temporal model of the transcriptional and chromatin response to OTC ASO.

[0079] [Figure 28] (A) Relative loss of binding to the indicated negative regulators following treatment with hOTC-ASOe1-10 compared to the NTC ASO (B) HDAC5 and NCOR1 binding is not reduced at the OTC enhancer in hepatocytes following RNase treatment.

[0080] [Figure 29] A shows that treatment with siHDAC5 or siNCOR1 results in at least a 50% reduction in target mRNA levels. B shows that siRNA knockdown of HDAC5 or NCOR1 knockdown leads to increased OTC mRNA expression in hepatocytes. C shows the fold change in OTC mRNA after treatment with hOTC-ASOe1-10 in untreated hepatocytes and in hepatocytes treated with siHDAC5 or siNCOR1.

[0081] [Diagram 30] Provides a model of OTC gene expression following treatment with OTC regRNA-targeting ASO.

[0082] [Diagram 31] Ammonia and urea levels in NHPs after treatment with the indicated ASOs are shown.

[0083] [Diagram 32]Relative OTC, NAGS, CPS1, ASS1, ASL, or ARG1 mRNA expression is shown after treatment with the indicated ASOs in humanized mouse models.

[0084] [Diagram 33] 1 shows that CO-5318 and CO-5319 treatment in humanized mice showed a decrease in ammonia and a corresponding increase in urea over time.

[0085] [Diagram 34] A shows that ASOs CO-3265, CO-3279, CO-2043, and CO-2051 increased Serping1 mRNA expression in a dose-dependent manner. B shows that ASOs CO-2043, CO-2051, CO-3265, CO-3419, CO-4069, and CO-3279 increased Serping1 gene expression in C1NH+ / - hepatocytes in a dose-dependent manner.

[0086] [Diagram 35] The indicated ASOs increased Serping1 mRNA in mice.

[0087] [Diagram 36] 1 shows that CO-2051 reduced the amount of dye extravasation in both the ears and colon of CINH+ / - mice.

[0088] [Figure 37] A shows that CO-2051 increased Serping1 protein expression in WT mice. B shows that CO-2051 increased Serping1 protein expression in C1NH+ / - mice. C shows that CO-2051-GalNAc increased Serping1 protein expression in C1NH+ / - mice. D shows quantification of pigment extravasation after treatment with CO-2051-GalNAc. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0089] Detailed Description The present invention provides antisense oligonucleotides (ASOs) that target regulatory RNAs, such as promoter-associated RNAs and enhancer RNAs, and methods that use these ASOs to regulate gene expression.These methods are useful for regulating the level of gene products, for example, regulating the expression level of disease-causing genes (e.g., ornithine transcarbamylase (OTC)), thereby treating diseases associated with abnormal gene expression (e.g., urea cycle disorders).

[0090] Various aspects of the polyspecific binding proteins described in this application are described in the following sections.

[0091] definition To facilitate the understanding of this application, a number of terms and phrases are defined below.

[0092] As used herein, the terms "a" and "an" mean "one or more" and include plurals unless the context is inappropriate.

[0093] As used herein, the term "ornithine transcarbamylase" or "OTC" refers to the protein of UniProt Accession No. P00480 and related isoforms and orthologs.

[0094] As used herein, the terms "regulatory RNA" and "regRNA" are used interchangeably to refer to non-coding RNA transcribed from a regulatory element of a gene (e.g., a protein-coding gene), which is not itself a non-coding RNA. Exemplary regulatory elements include, but are not limited to, promoters, enhancers, and super-enhancers. Non-coding RNA transcribed from a promoter in the antisense direction is also referred to as "promoter RNA" or "paRNA". Non-coding RNA transcribed from an enhancer or super-enhancer in either the sense or antisense direction is also referred to as "enhancer RNA" or "eRNA". It is understood that a natural antisense transcript (NAT) that is complementary to at least a portion of a transcript of a gene is not a regulatory RNA as used herein.

[0095] As used herein, the term "nascent RNA" refers to RNA that is still being transcribed or has just been transcribed by RNA polymerase and remains tethered to the transcribed DNA. RNA that has dissociated from the DNA being transcribed is also called "untethered RNA."

[0096] As used herein, the term "antisense oligonucleotide" or "ASO" refers to a single-stranded oligonucleotide having a nucleotide sequence that hybridizes with a target nucleic acid under appropriate conditions, or a conjugate containing such a single-stranded oligonucleotide.

[0097] As used herein, the stability of regRNA is inversely correlated with the degradation rate of regRNA.When ASO increases the stability of regRNA, it decreases the degradation rate of regRNA.When ASO reduces the stability of regRNA, it increases the degradation rate of regRNA.The degradation rate of regRNA can be measured by blocking the synthesis of new regRNA and evaluating the half-life of existing regRNA.

[0098] As used herein, the terms "subject" and "patient" refer to an organism that is treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., rodents, primates, monkeys, horses, cows, pigs, dogs, cats, etc.), and more preferably, humans.

[0099] As used herein, the term "effective amount" refers to an amount of a compound (e.g., a compound of the present application) sufficient to produce a beneficial or desired result. An effective amount may be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or route of administration. As used herein, the term "treat" includes any effect that results in the improvement of a condition, disease, disorder, etc., such as, for example, lowering, reducing, modulating, improving, or eliminating, or ameliorating a symptom thereof.

[0100] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent with an inert or active carrier that inherently renders the composition suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0101] As used herein, the term "pharmaceutical acceptable carrier" refers to any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, emulsions (e.g., oil-in-water or water-in-oil emulsions), and various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA (1975).

[0102] Throughout this description, where compositions are described as having, including, or comprising certain components, or processes and methods are described as having, including, or comprising certain steps, it is additionally contemplated that there are compositions described herein that consist essentially of or consist of the recited components, and that there are processes and methods according to the application that consist essentially of or consist of the recited process steps.

[0103] As a general matter, compositions specifying percentages are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, the previous definition of that variable takes precedence.

[0104] Antisense oligonucleotides The antisense oligonucleotides (ASOs) disclosed herein hybridize with regRNA transcribed from a regulatory element of a target gene. It is understood that both eRNA and paRNA are regRNAs that promote or upregulate gene expression (Figure 1). In certain embodiments, the target regRNA is an eRNA. In certain embodiments, the target regRNA is a paRNA. In certain embodiments, the target regRNA is not a polyadenylated RNA. eRNAs can be identified using methods known in the art, such as assays for transposase-accessible chromatin using sequencing (ATAC-seq), global run-on sequencing, precision run-on sequencing, cap analysis gene expression, and histone modification analysis (see, e.g., Sartorelli & Lauberth, Nat. Struct Mol. Biol. (2020) 27:521-28; PCT Application Publication No. WO2013 / 177248). paRNA is RNA transcribed from the promoter of the target gene in antisense direction (sense direction transcript is the mRNA of the target gene). They can be identified by similar methods, taking into account their specific location and orientation. In human OTC, eRNA has been identified to transcribe from the same enhancer region. In mouse SERPING1, paRNA has been identified to be transcribed from the SERPING1 promoter, but in the opposite direction to the mRNA of Serping1. The nucleotide sequence of an exemplary regRNA is shown in Table 1 below. Any of these regRNAs are contemplated as the target regRNA of the ASO disclosed herein.

[0105] Table 1. Exemplary regRNAs TIFF2024534214000002.tif119161TIFF2024534214000003.tif213158TIFF2024534214000004.tif218158TIFF2024534214000005.tif229158 TIFF2024534214000006.tif164158TIFF2024534214000007.tif220158TIFF2024534214000008.tif180161TIFF2024534214000009.tif179160

[0106] The present invention describes ASO that increases the amount or stability of target regRNA, thereby increasing the expression of target genes. This is different from the previously described ASO that is designed to inhibit eRNA (see, for example, PCT Application No. WO2013 / 177248 and PCT Application Publication No. WO2017 / 075406). Without wishing to be bound by theory, it is hypothesized that the ability of ASO to upregulate regRNA is due to the selection of target sequence in regRNA and / or chemical modification of ASO.

[0107] In some embodiments, the regulatory RNA has a nucleotide sequence of SEQ ID NO:1. In some embodiments, the regulatory RNA has a nucleotide sequence of SEQ ID NO:2. In some embodiments, the regulatory RNA has a nucleotide sequence of SEQ ID NO:3. In some embodiments, the regulatory RNA has a nucleotide sequence of SEQ ID NO:4. In some embodiments, the regulatory RNA has a nucleotide sequence of SEQ ID NO:5. In some embodiments, the regulatory RNA has a nucleotide sequence of SEQ ID NO:1073. In some embodiments, the regulatory RNA has a nucleotide sequence of SEQ ID NO:1074. In some embodiments, the regulatory RNA has a nucleotide sequence of SEQ ID NO:1075. In some embodiments, the regulatory RNA has a nucleotide sequence of SEQ ID NO:1076. In some embodiments, the regulatory RNA has a nucleotide sequence of SEQ ID NO:1077. In some embodiments, the regulatory RNA has a nucleotide sequence of SEQ ID NO:1078.

[0108] ASO sequence As disclosed herein, ASOs that bind to sequences closer to the 5' or 3' end of OTC target regRNA are more likely to upregulate regRNA.Without wishing to be bound by theory, it is hypothesized that such ASOs hybridize to the terminal portion of OTC regRNA and prevent or delay 5'→3' and / or 3'→5' RNA degradation without blocking functional regions of regRNA.In certain embodiments, the ASOs disclosed herein are complementary to sequences in target regRNA that are 300, 250, 200, 150, 100, 50, 40, 30, 20, or 10 nucleotides or less from the 5' or 3' end of target regRNA. In certain embodiments, the ASOs disclosed herein are complementary to a sequence in the target regRNA that is 300, 250, 200, 150, 100, 50, 40, 30, 20, or 10 nucleotides or less from the 5' end of the target regRNA (i.e., the 5'-most nucleotide of the regRNA sequence that forms a duplex with the ASO is 300, 250, 200, 150, 100, 50, 40, 30, 20, or 10 nucleotides or less from the 5' end of the target regRNA). In certain embodiments, the ASOs disclosed herein are complementary to a sequence in the target regRNA that is 300, 250, 200, 150, 100, 50, 40, 30, 20, or 10 nucleotides or less from the 3' end of the target regRNA (i.e., the 3'-most nucleotide of the regRNA sequence that forms a duplex with the ASO is 300, 250, 200, 150, 100, 50, 40, 30, 20, or 10 nucleotides or less from the 3' end of the target regRNA).

[0109] In certain embodiments, the ASO is no longer than 8, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides in length. In certain embodiments, the ASO is at least 8, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides in length. In certain embodiments, the ASO is at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length.

[0110] In certain embodiments, ASOs are designed to lack stable secondary structures that form within themselves or between each other, thereby increasing the amount of ASOs in single-stranded form ready to hybridize with the target regRNA. Methods for predicting secondary structure are known in the art (e.g., Seetin and Mathews, Methods Mol. Biol. (2012) 905: 99-122; Zhao et al., PLoS Comput. Biol. (2021) 17 (8): e1009291) and web-based programs (e.g., RNAfold) are publicly available to users.

[0111] For example, ASOs have been designed to target the human OTC eRNA or mouse SERPING1 paRNA, and the nucleotide sequences of these ASOs are shown in Table 2 below.

[0112] Table 2: Exemplary ASO sequences targeting regRNA TIFF2024534214000010.tif223161TIFF2024534214000011.tif75161

[0113] Tables 3 and 4 provide additional chemical modifications of hOTC-ASOe1-1 and hOTC-ASOe2-2.

[0114] (Table 3) TIFF2024534214000012.tif203161TIFF2024534214000013.tif206161TIFF2024534214000014.tif196161

[0115] (Table 4) TIFF2024534214000015.tif219161TIFF2024534214000016.tif87161

[0116] hOTC-ASOe1-1 (SEQ ID NO: 6) is complementary to a sequence 1 nucleotide away from the 3' end of human OTC eRNA-1A. SEQ ID NOs: 7-14, which overlap at least partially with SEQ ID NO: 6, are also complementary to sequences close to the 3' end of human OTC eRNA-1A. hOTC-ASOe2-1 (SEQ ID NO: 15) is complementary to a sequence 9 nucleotides away from the 3' end of human OTC eRNA-2A and 87 nucleotides away from the 3' end of human OTC eRNA-2B. SEQ ID NO: 17, which overlaps partially with SEQ ID NO: 16, is also complementary to sequences close to the 3' end of human OTC eRNA-2A and human OTC eRNA-2B. hOTC-ASOe2-2 (SEQ ID NO: 16) is complementary to a sequence 57 nucleotides away from the 5' end of human OTC eRNA-2A.

[0117] Hybridization and ΔG As used herein, the term "hybridize" or "hybridize" should be understood as two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) forming hydrogen bonds between base pairs on opposing strands, thereby forming a duplex. The affinity of the binding between two nucleic acid strands is the strength of hybridization. This is measured by the melting temperature (T), which is defined as the temperature at which half of the oligonucleotide forms a duplex with the target nucleic acid. m ) are often described in terms of physiological conditions T mis not strictly proportional to affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537). The standard state Gibbs free energy ΔG° is a more accurate representation of binding affinity, ΔG°=-RTIn(K d ) dissociation constant (K d) where R is the gas constant and T is the absolute temperature. Thus, a very low ΔG° of the reaction between an oligonucleotide and a target nucleic acid reflects strong hybridization between the oligonucleotide and the target nucleic acid. ΔG° is the free energy associated with the reaction when the aqueous solution concentration is 1M, pH is 7, and temperature is 37°C. The hybridization of an oligonucleotide to a target nucleic acid is a spontaneous reaction, and in the case of a spontaneous reaction, ΔG° is less than zero. ΔG° can be measured experimentally, for example, using the isothermal titration calorimetry (ITC) method described in Hansen et al., 1965, Chem, Comm. 36-38 and Holdgate et al., 2005, Drug Discov Today. Those skilled in the art know that commercially available equipment is available for measuring ΔG°. ΔG° can be numerically estimated using the nearest neighbor model described in Sugimoto et al., 1995, Biochemistry 34:11211-11216 and McTigue et al., 2004, Biochemistry 43:5388-5405, with appropriately derived thermodynamic parameters described in SantaLucia, 1998, Proc Natl Aced Sci USA 95:1460-1465. To have the potential to modulate its intended nucleic acid target by hybridization, the oligonucleotides of the present invention hybridize to the target nucleic acid with an estimated ΔG° value of less than -10 kcal / mol for oligonucleotides that are 10-30 nucleotides in length. In some embodiments, the degree or strength of hybridization is measured by the standard state Gibbs free energy ΔG°. The oligonucleotide may hybridize to the target nucleic acid with an estimated ΔG° value below the range of −10 kcal / mol, such as less than −15 kcal / mol, such as less than −20 kcal / mol, such as less than −25 kcal / mol, for an oligonucleotide of 8 to 30 nucleotides in length.In some embodiments, the oligonucleotide hybridizes to the target nucleic acid with an estimated ΔG° value of -10 to -60 kcal / mol, e.g., -12 to -40 kcal / mol, -15 to -30 kcal / mol, -16 to -27 kcal / mol, or -18 to -25 kcal / mol.

[0118] double stranded region The phrase "duplex region" refers to a region in two complementary or substantially complementary polynucleotides that base-pair with each other, either by Watson-Crick base pairing or any other manner that allows for a stabilized duplex between complementary or substantially complementary polynucleotide strands. For example, a polynucleotide strand having 21 nucleotide units can base-pair with another polynucleotide of 21 nucleotide units that is still complementary or fully complementary, such that the "duplex region" is 19 base pairs. The remaining bases can be present, for example, as 5' and / or 3' overhangs. Furthermore, 100% complementarity within a duplex is not required, and substantial complementarity is acceptable within a duplex. Substantial complementarity refers to 70% or greater complementarity. For example, a mismatch in a duplex of 19 base pairs results in 94.7% complementarity, giving a fully complementary duplex region. The double-stranded region can be formed by two separate oligonucleotide strands, as well as by a single oligonucleotide strand that can form a hairpin structure that includes the double-stranded region.

[0119] dsRNA comprises two RNA strands that are complementary and hybridize to form a duplex structure under the conditions in which dsRNA is used. One strand of dsRNA (antisense strand) comprises a region of complementarity that is substantially complementary to the target sequence, and generally completely complementary. The target sequence can be derived from the sequence of OTC or Serping1 regRNA, such as eRNA or paRNA. The other strand (sense strand) comprises a region that is complementary to the antisense strand, such that the two strands hybridize to form a duplex structure when combined under appropriate conditions. As described elsewhere herein and known in the art, the complementary sequence of dsRNA can be included as a self-complementary region of a single nucleic acid molecule, as opposed to being on a separate oligonucleotide. Generally, the double-stranded structure has a length of 15-50 base pairs, e.g., 15-50, 15-49, 15-48, 15-47, 15-46, 15-45, 15-44, 15-43, 15-42, 15-41, 15-40, 15-39, 15-38, 15-37, 15-36, 15-35, 15-34, 15-33, 15-32, 15-31, 15-30, 15-29, 15 ~28, 15~27, 15~26, 15~25, 15~24, 15~23, 15~22, 15~21, 15~20, 15~19, 15~18, 15~17, 18~50, 18~49, 18~48, 18~47, 18~46, 18~45, 18~44, 18~43, 18~42, 18~41, 18~40, 18~39, 18~38, 18~37, 18~36, 1 8~35, 18~34, 18~33, 18~32, 18~31, 18~30, 18~30, 18~29, 18~28, 18~27, 18~26, 18~25, 18~24, 18~23, 18~22, 18~21, 18~20, 19~50, 19~49, 19~48, 19~47, 19~46, 19~45, 19~44, 19~43, 19~42, 19~41, 19~40, 19~39, 19~38, 19~37, 19~36, 19~35, 19~34, 19~33, 19~32, 19~31, 19~30, 19~30, 19~29, 19~28, 19~27, 19~26, 19~25, 19~24, 19~23, 19~22, 19~21, 19~20, 20~50, 20~49, 20~48, 20~47, 20~46,20~45, 20~44, 20~43, 20~42, 20~41, 20~40, 20~39, 20~38, 20~37, 20~36, 20~35, 20~34, 20~33, 20~32, 20~31, 20~30, 20~30, 20~29, 20~28, 20~27, 20~26, 20~25, 20~24, 20~23, 20~22, 20~21, 21~50, 21~49, 21~48, 21~47, 21~46, 21~45, 21~44, 21~43, 21~42, 21~41, 21~40, 21~39, 21~38, 21~37, 21~36, 21~35, 21~34, 21~33, 21~32, 21~31, 21~30, 21~29, 21~28, 21~27, 21~26, 21~25, 21~24, 21~23, 21~22, 22~50, 2 2~49, 22~48, 22~47, 22~46, 22~45, 22~44, 22~43, 22~42, 22~41, 22~40, 22~39, 22~38, 22~37, 22~36, 22~35, 22~34, 22~33, 22~32, 22~31, 22~30, 22~29, 22~28, 22~27, 22~26, 22~25, 22~24, 22~23, 23~50, 23 23-49, 23-48, 23-47, 23-46, 23-45, 23-44, 23-43, 23-42, 23-41, 23-40, 23-39, 23-38, 23-37, 23-36, 23-35, 23-34, 23-33, 23-32, 23-31, 23-30, 23-29, 23-28, 23-27, 23-26, 23-25, or 23-24 base pairs in length. Ranges and lengths intermediate to the above ranges and lengths are also contemplated as part of the present invention.

[0120] Similarly, the region of complementarity to the target sequence may be 15-50 nucleotides in length, e.g., 15-50, 15-49, 15-48, 15-47, 15-46, 15-45, 15-44, 15-43, 15-42, 15-41, 15-40, 15-39, 15-38, 15-37, 15-36, 15-35, 15-34, 15-33, 15-32, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-50, 18-51, 18-52, 18-53, 18-54, 18-55, 18-56, 18-57, 18-59, 18-60, 18-61, 18-62, 18-63, 18-64, 18-65, 18-66, 18-67, 18-68, 18-69, 18-70, 18-71, 18-72, 18-73, 18-74, 18-75, 18-76, 18-77, 18-78, 18-79, 18-80, 18-81, 18-82, 18-83, 18-84, 18-8 49, 18~48, 18~47, 18~46, 18~45, 18~44, 18~43, 18~42, 18~41, 18~40, 18~39, 18~38, 18~37, 18~36, 18~35, 18~34, 18~33, 18~32, 18~31, 18~30, 18~30, 1 8~29, 18~28, 18~27, 18~26, 18~25, 18~24, 18~23, 18~22, 18~21, 18~20, 19~50, 19~49, 19~48, 19~47, 19~46, 19~45, 19~44, 19~43, 19~42, 19~41, 19~40, 19~39, 19~38, 19~37, 19~36, 19~35, 19~34, 19~33, 19~32, 19~31, 19~30, 19~30, 19~29, 19~28, 19~27, 19~26, 19~25, 19~24, 19~23, 19~22, 19~21, 19~2 0, 20~50, 20~49, 20~48, 20~47, 20~46, 20~45, 20~44, 20~43, 20~42, 20~41, 20~40, 20~39, 20~38, 20~37, 20~36, 20~35, 20~34, 20~33, 20~32, 20~31, 20~ 30, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-50, 21-49, 21-48, 21-47, 21-46, 21-45, 21-44, 21-43, 21-42, 21-41, 2 1-40, 21-39, 21-38, 21-37, 21-36, 21-35, 21-34, 21-33, 21-32, 21-31, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, 21-22, 22-50, 22-49,22~48, 22~47, 22~46, 22~45, 22~44, 22~43, 22~42, 22~41, 22~40, 22~39, 22~38, 22~37, 22~36, 22~35, 22~34, 22~33, 22~32, 22~31, 22~30, 22~29, 22~28, 22~27, 22~26, 22~25, 22~24, 22~23, 23~50, 23~49, 2 The length may be between 3-48, 23-47, 23-46, 23-45, 23-44, 23-43, 23-42, 23-41, 23-40, 23-39, 23-38, 23-37, 23-36, 23-35, 23-34, 23-33, 23-32, 23-31, 23-30, 23-29, 23-28, 23-27, 23-26, 23-25, or 23-24 nucleotides. Ranges and lengths intermediate to the above ranges and lengths are also contemplated as part of the present invention.

[0121] Chemical modification of ASOs In certain embodiments, the ASO does not consist of DNA alone. In certain embodiments, the ASO comprises at least one chemical modification to a natural nucleotide (e.g., ribonucleotide). Various chemical modifications can be included in the ASO of the present disclosure. The modifications can include one or more modifications in the ribose group, one or more modifications in the phosphate group, one or more modifications in the nucleobase, one or more terminal modifications, or a combination thereof. In some embodiments, the exemplary ASO sequences targeting regRNA shown in Table 2 are chemically modified. For example, hOTC-ASOe1-1 can be chemically modified to include any one of the modifications hOTC-ASOe1-1a to hOTC-ASOe1-1h as shown in FIG. 18A. Such modifications can be, but are not limited to, 2'-O-(2-methoxyethyl) (2'-MOE), locked nucleic acid (LNA), 5-methyl on cytidine, constrained ethyl (cET), phosphorothioate (PS) linkage, and / or phosphodiester (PO) linkage, or any combination thereof. Chemical modifications of RNA are known in the art and are described, for example, in PCT Publication No. WO2013 / 177248. In certain embodiments, each cytidine in the ASO is modified with a 5-methyl.

[0122] Various chemical modifications for use with the ASOs of the present disclosure include, but are not limited to, 3'-terminal deoxythymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformation-restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxyl modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural base containing nucleotides, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimetics.

[0123] In certain embodiments, the ASO comprises an RNA polynucleotide that is chemically modified to be resistant to one or more nuclear RNases (e.g., exosome complexes or RNase H). In some embodiments, all nucleotide bases are modified in the ASO. In certain embodiments, the chemical modifications include β-D-ribonucleosides, 2'-modified nucleosides (e.g., 2'-O-(2-methoxyethyl) (2'-MOE), 2'-O-CH 3, or 2'-fluoro-arabino (FANA)), bicyclic sugar modified nucleosides (e.g., with constrained ethyl or locked nucleic acid (LNA)), and / or one or more modified internucleotide linkages (e.g., phosphorothioate internucleotide linkages). In certain embodiments, the chemical modifications include 2'-MOE and phosphorothioate internucleotide linkages. In certain embodiments, at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of the ASO are modified with 2'-MOE. In certain embodiments, each nucleotide of the ASO is modified with 2'-MOE. In certain embodiments, at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more consecutive internucleotide linkages of the ASO are phosphorothioate internucleotide linkages. In certain embodiments, each internucleotide linkage of the ASO is a phosphorothioate internucleotide linkage.

[0124] Internucleotide linkage modifications that can be used with the ASOs of the disclosure include, but are not limited to, phosphorothioates "PS" (P(S)), phosphoramidates (P(NR1R2), e.g., dimethylamino phosphoramidate (P(N(CH3)2)), phosphonocarboxylates (P(CH2)nCOOR), e.g., phosphonoacetates "PACE" (P(CH2COO-)), thiophosphonocarboxylates ((S)P(CH2)nCOOR), e.g., thiophosphonoacetates, "thioPACE" ((S)P(CH2COO-)), alkyl phosphonates (P(C1-3 alkyl), e.g., methylphosphonic acid-P(CH3), borane phosphonic acid (P(BH3)), and phosphorodithioates (P(S)2).

[0125] In certain embodiments, the ASO comprises one or more chemical modifications at the 5' end, the 3' end, or both. Without wishing to be bound by theory, chemical modifications at one or both ends of a polynucleotide (e.g., polyribonucleotide) can stabilize the polynucleotide. In certain embodiments, the ASO comprises one or more chemical modifications at at least 1, 2, 3, 4, or 5 nucleotides at the 5' end of the ASO. In certain embodiments, the ASO comprises one or more chemical modifications at at least 1, 2, 3, 4, or 5 nucleotides at the 3' end of the ASO. In certain embodiments, the ASO comprises one or more chemical modifications at at least 1, 2, 3, 4, or 5 nucleotides at the 5' end of the ASO and one or more chemical modifications at at least 1, 2, 3, 4, or 5 nucleotides at the 3' end of the ASO.

[0126] Chemical structures may also be written out. In such cases, "M" indicates MOE; "d" indicates DNA; "L" indicates LNA; "=" indicates phosphorothioate (PS) bond; "-" indicates phosphodiester (PO) bond; "5C" indicates 5-methylcytosine; "ag" indicates GalNAc; "tg" indicates Teg-GalNAc; and "^" indicates FANA.

[0127] For the avoidance of ambiguity, this LNA has the following formula: TIFF2024534214000017.tif50128 where B is a specific designated base.

[0128] Exemplary written descriptions of selected ASOs are shown in Tables 3 and 4, including corresponding Figures 18D and 18E, which provide visual representations of the modifications.

[0129] In some embodiments, the ASO comprises a sequence and / or a chemical modification selected from the group consisting of SEQ ID NOs: 6-14, 18-35, 39, 41, 75, 76, 77, 78, 87-124, or 143-892. In some embodiments, the ASO comprises a sequence and / or a chemical modification selected from the group consisting of SEQ ID NOs: 15-17, 36-38, 64-74, 125-142, or 893-1029. In some embodiments, the ASO comprises a sequence and a chemical modification selected from the group consisting of SEQ ID NOs: 87-124. In some embodiments, the ASO comprises a sequence and a chemical modification selected from the group consisting of SEQ ID NOs: 125-142. In some embodiments, the ASO comprises a sequence and a chemical modification selected from the group consisting of SEQ ID NOs: 1030-1072.

[0130] High-affinity modified nucleosides A high affinity modified nucleoside is a modified nucleotide that, when incorporated into an oligonucleotide, enhances the affinity of the oligonucleotide for its complementary target, e.g., as measured by melting temperature (Tm). The high affinity modified nucleosides of the present invention preferably provide an increase in melting temperature of +0.5 to +12°C, e.g., +1.5 to +10°C or +3 to +8°C per modified nucleoside. Many high affinity modified nucleosides are known in the art, including, for example, many 2'-substituted nucleosides as well as locked nucleic acids (LNAs) (see, e.g., Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213), each of which is incorporated herein by reference.

[0131] sugar modification The ASOs described herein may include one or more nucleosides with modified sugar moieties, i.e., modifications of the sugar moiety as compared to the ribose sugar moiety found in DNA and RNA. A number of nucleosides with modifications of the ribose sugar moiety have been made primarily to improve certain properties of oligonucleotides, such as affinity and / or nuclease resistance. Such modifications include those in which the ribose ring structure is modified, for example, by substitution with a hexose ring (HNA) or a bicyclic ring (which typically has a two-radius bridge between the C2 and C4 carbons on the ribose ring (LNA)), or a non-linked ribose ring (which typically lacks a bond between the C2 and C3 carbons) (e.g., UNA). Other sugar-modified nucleosides include, for example, bicyclohexose nucleic acids (WO2011 / 017521) or tricyclic nucleic acids (WO2013 / 154798), both of which are incorporated herein by reference. Modified nucleosides also include nucleosides in which the sugar moiety is replaced with a non-sugar moiety, such as in peptide nucleic acids (PNAs) or morpholino nucleic acids.

[0132] Sugar modifications also include modifications made by changing the substituents on the ribose ring to groups other than hydrogen or to the 2'-OH group found naturally in DNA and RNA nucleosides. Substituents may be introduced, for example, at the 2', 3', 4' or 5' position.

[0133] In some embodiments, the oligonucleotide may be a 2'-O-methyl (2'OMe) moiety, a 2'-O-methoxyethyl moiety, a bicyclic sugar moiety, a PNA (e.g., an oligonucleotide that contains one or more N-(2-aminoethyl)-glycine units linked by amide or carbonyl methylene bonds as repeating units instead of a sugar phosphate backbone), a locked nucleoside (LNA) (e.g., an oligonucleotide that contains one or more locked ribose and may be a mixture of 2'-deoxynucleotides or 2'OMe nucleotides), a c-ET (e.g., an oligonucleotide that contains one or more cET sugars), a cMOE (e.g., an oligonucleotide that contains one or more cMOE sugars), a morpholino oligomer (e.g., one or more a phosphorodiamidate morpholino oligomer of 2'-diaminodiphenyl phosphate; a 2'-deoxy-2'-fluoronucleoside (e.g., an oligonucleotide comprising one or more 2'-fluoro-β-D-arabinonucleosides); a tcDNA (e.g., an oligonucleotide comprising one or more tcDNA modified sugars); a constrained ethyl 2'-4' bridged nucleic acid (cEt), an S-cEt, an ethylene bridged nucleic acid (ENA) (e.g., an oligonucleotide comprising one or more ENA modified sugars), a hexitol nucleic acid (HNA) (e.g., an oligonucleotide comprising one or more HNA modified sugars), or a tricyclic analog (tcDNA) (e.g., an oligonucleotide comprising one or more tcDNA modified sugars).

[0134] In some embodiments, the oligonucleotide comprises 2-thiouracil ("2-thioU"), 2-thiocytosine ("2-thioC"), 4-thiouracil ("4-thioU"), 6-thioguanine ("6-thioG"), 2-aminoadenine ("2-aminoA"), 2-aminopurine, pseudouracil, hypoxanthine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5-methylcytosine ("5-methylC"), 5-methyluracil ("5-methylU"), 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5,6-dehydrouracil, 5 nucleobase modifications selected from the group consisting of 5-propynylcytosine, 5-propynyluracil, 5-ethynylcytosine, 5-ethynyluracil, 5-allyluracil ("5-allylU"), 5-allylcytosine ("5-allylC"), 5-aminoallyluracil ("5-aminoallylU"), 5-aminoallyl-cytosine ("5-aminoallylC"), abasic nucleotides, Z bases, P bases, unstructured nucleic acids ("UNA"), isoguanine ("isoG"), and isocytosine ("isoC"), glycerol nucleic acid (GNA), thiomorpholino (C4H9NS) or thiophosphoramidate morpholino (TMO). The synthesis of glycerol nucleic acid (GNA) (also known as glycol nucleic acid) is described in Zhang et al, Current Protocols in Nucleic Acid Chemistry 4.40.1-4.40.18, September 2010, which is incorporated by reference herein. The synthesis of thiophosphoramidate morpholino oligonucleotides is described in Langer et al, J. Am. Chem. Soc. 2020, 142, 38, 16240-16253.

[0135] 2' sugar modified nucleosides 2' sugar modified nucleosides are nucleosides having a substituent other than H or -OH at the 2' position (2' substituted nucleosides) or include 2' linked bicyclic nucleosides that can form a bridge between the 2' carbon and the second carbon in the ribose ring, such as LNA (2'-4' bridged bicyclic) nucleosides.

[0136] Without wishing to be bound by theory, 2' modified sugars may provide enhanced binding affinity to oligonucleotides and / or increased nuclease resistance. Examples of 2' substituted modified nucleosides are 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, and 2'-F-ANA nucleosides. For further examples, see, for example, Freier & Altmann; Nucl.Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr.Opinion in Drug Development, 2000, 3(2), 293-213, and Deleavey and Damha, Chemistry and Biology 2012, 19, 937 (each of which is incorporated herein by reference).

[0137] Locked Nucleoside (LNA) Nucleoside An "LNA nucleoside" is a 2'-sugar modified nucleoside that contains a biradical (also called a "2'-4' bridge") linking the C2' and C4' of the ribose sugar ring of the nucleoside, which restricts or locks the conformation of the ribose ring. In other words, a locked nucleoside is a 4'-CH 2LNAs are nucleosides that contain a bicyclic sugar moiety that includes an -O-2' bridge. This structure effectively "locks" the ribose in a 3'-endo conformation. The addition of locked nucleosides to oligonucleotides has been shown to increase the stability of oligonucleotides in serum and reduce off-target effects (Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). These nucleosides are sometimes also called bridged nucleic acids or bicyclic nucleic acids (BNAs). Locking the ribose conformation is associated with enhanced affinity of hybridization (duplex stabilization) when LNAs are incorporated into oligonucleotides to complementary RNA or DNA molecules. This can be routinely determined by measuring the melting temperature of the oligonucleotide / complement duplex. Exemplary LNA nucleosides include beta-D-oxy-LNA, 6'-methyl-beta-D-oxy-LNA, such as (S)-6'-methyl-beta-D-oxy-LNA (ScET), and ENA.

[0138] Examples of bicyclic nucleosides for use in the polynucleotides of the invention include, but are not limited to, nucleosides that include a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the polynucleotide agents of the invention include one or more bicyclic nucleosides that include a 4' to 2' bridge. Examples of such 4' to 2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH 2 )-O-2'(LNA);4'-(CH 2 )-S-2';4'-(CH 2 ) 2 -O-2'(ENA);4'-CH(CH 3 )-O-2' (also called "constrained ethyl" or "cEt") and 4'-CH(CH 2 OCH 3 )-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4'-C(CH 3 )(CH 3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,283); 4'-CH 2 -N(OCH 3 )-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,425); 4'-CH 2 -ON(CH 3 ) 2 -2' (see, e.g., U.S. Patent Publication No. 2004 / 0171570); 4'-CH 2 -N(R)-O-2' (wherein R is H, C 1 ~C 12 alkyl, or a protecting group] (see, e.g., U.S. Pat. No. 7,427,672); 2 -C(H)(CH 3 )-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH 2 -C(=CH 2 )-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The contents of each of the foregoing are incorporated herein by reference in their entirety.

[0139] Additional representative U.S. patents and published U.S. patent applications that teach the preparation of locked nucleic acid nucleotides include U.S. Pat. Nos. 6,268,490, 6,525,191, 6,670,461, 6,770,748, 6,794,499, 6,998,484, 7,053,207, 7,034,13 ... Nos. 7,084,125, 7,399,845, 7,427,672, 7,569,686, 7,741,457, 8,022,193, 8,030,467, 8,278,425, 8,278,426, 8,278,283, US2008 / 0039618, and US2009 / 0012281.

[0140] Any of the foregoing bicyclic nucleosides may be prepared with one or more stereochemical sugar configurations, such as, for example, α-L-ribofuranose and β-D-ribofuranose (see International Publication WO 99 / 14226, the contents of which are incorporated herein by reference).

[0141] The oligonucleotides of the invention can also be modified to include one or more constrained ethyl nucleosides. As used herein, "constrained ethyl nucleosides" or "cEt" refers to 4'-CH(CH 3 )-O-2' bridge. In one embodiment, the constrained ethyl nucleoside is in the S conformation, referred to herein as "S-cEt."

[0142] The oligonucleotides of the invention may also contain one or more "conformationally restricted nucleosides" ("CRNs"). CRNs are nucleoside analogs with a linker connecting the C2' and C4' carbons of ribose, or the C3 and --C5' carbons of ribose. The CRNs lock the ribose ring into a stable conformation, increasing hybridization affinity to mRNA. The linker is long enough to place the oxygen in an optimal position for stability and affinity, reducing ribose ring puckering.

[0143] Representative publications that teach the preparation of the specific CRNs mentioned above include, but are not limited to, U.S. Patent Application Publication No. 2013 / 0190383, and PCT publication WO2013 / 036868, the contents of each of which are incorporated by reference in their entirety herein.

[0144] In some embodiments, the oligonucleotide of the present invention comprises one or more monomers that are UNA (unlocked nucleoside) nucleosides. UNA is an unlocked acyclic nucleoside, in which the sugar bond is removed to form an unlocked "sugar" residue. In one example, UNA also encompasses monomers in which the C1'-C4' bond (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) is removed. In another example, the C2'-C3' bond (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar is removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008), and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference).

[0145] Representative U.S. patent publications that teach the preparation of UNAs include, but are not limited to, U.S. Patent No. 8,314,227, and U.S. Patent Application Publication Nos. 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020, the entire contents of each of which are incorporated herein by reference.

[0146] The ribose molecule may also be modified with a cyclopropane ring to produce tricyclodeoxynucleic acid (tricycloDNA). The ribose moiety may be replaced with another sugar, such as 1,5-anhydrohexitol, with threose to produce threose nucleosides (TNA), or with arabinose to produce arabinonucleosides. The ribose molecule may also be replaced with a non-sugar, such as cyclohexene to produce cyclohexene nucleosides, or with glycol to produce glycol nucleosides.

[0147] Potentially stabilizing modifications to the termini of nucleoside molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"-phosphate, inverted base dT (idT), and the like. Disclosure of this modification can be found in PCT Publication No. WO2011 / 005861.

[0148] Other alternative chemical compositions of the oligonucleotide of the present invention include 5' phosphate or 5' phosphate mimic of oligonucleotide, for example 5' terminal phosphate or phosphate mimic.Suitable phosphate mimic is disclosed, for example, in US Patent Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0149] Additional non-limiting exemplary LNA nucleosides are described in WO99 / 014226, WO00 / 66604, WO98 / 039352, WO2004 / 046160, WO00 / 047599, WO2007 / 134181, WO2010 / 077578, WO2010 / 036698, WO2007 / 090071, WO2009 / 006478, WO2011 / 156202, WO2008 / 154401, WO2009 / 067647, WO2008 / 150729, Morita et al., Bioorganic & Med. Chem. Lett. 12, 73-76, Seth et al. al. J. Org. Chem. 2010, Vol 75(5) pp. 1569-81; Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238; and Wan and Seth, J. Medical Chemistry 2016, 59, 9645-9667, each of which is incorporated herein by reference.

[0150] In some embodiments, the length of the ASO is 5×n+5 nucleotides (where n is an integer greater than or equal to 3), the nucleotide at position 5×m is an LNA-modified ribonucleotide (where m is an integer from 1 to n), and the nucleotides at the remaining positions are 2'-O-methoxyethyl-modified ribonucleotides.

[0151] In some embodiments, the nucleotide sugar modification is 2'-O-Ci-4 alkyl, e.g., 2'-O-methyl (2'-OMe), 2'-deoxy (2'-H), 2'-O-Ci-3 alkyl-O-Ci-3 alkyl, e.g., 2'-methoxyethyl ("2'-MOE"), 2'-fluoro ("2'-F"), 2'-amino ("2'-NH2"), 2'-arabinosyl ("2'-arabino") nucleotides, 2'-F-arabinosyl ("2'-F-arabino") nucleotides, 2'-locked nucleic acid ("LNA") nucleotides, 2'-amide bridged nucleic acid (AmNA), 2'-unlocked nucleic acid ("ULNA") nucleotides, L-form sugars ("L-sugars"), or 4'-thioribosyl nucleotides.

[0152] Mixmers and Gapmers The ASO may have a mixmer and / or gapmer structure, for example, in the pattern disclosed by the ASO of Figure 18A, Figure 18B, Figure 18C, or Figure 19.

[0153] In certain embodiments, the ASO is a mixmer. As used herein, the term "mixmer" refers to an oligonucleotide that contains an alternating composition of DNA monomers and nucleoside analog monomers over at least a portion of the oligonucleotide sequence. In certain embodiments, the ASO is a mixmer based on a gapmer structure, containing a mixture of DNA and 2'-MOE nucleotides in the gap and flanked by RNA sequences in the wings. The mixmer can be designed to contain a mixture of affinity enhancing nucleotide analogs, such as, in non-limiting examples, 2'-O-alkyl-RNA monomers, 2'-amino-DNA monomers, 2'-fluoro-DNA monomers, LNA monomers, arabinonucleic acid (ANA) monomers, 2'-fluoro-ANA monomers, HNA monomers, INA monomers, 2'-MOE-RNA (2'-O-methoxyethyl-RNA), 2'fluoro-DNA and LNA. In some embodiments, the mixmer cannot recruit RNase H. In some embodiments, the mixmer contains one type of affinity enhancing nucleotide analog along with DNA and / or RNA.

[0154] The multiple different modifications may be spaced apart within the mixmer.For example, the ASO may comprise LNA modifications at multiple nucleotides, and may comprise different modifications at some or all of the remaining nucleotides.In some embodiments, any two adjacent LNA modified nucleotides are separated by at least 1, 2, 3, 4, or 5 nucleotides.The distance between adjacent LNA modified nucleotides throughout the ASO may be constant (for example, any two adjacent LNA modified nucleotides are separated by 1, 2, 3, 4, or 5 nucleotides) or may be variable. In some embodiments, the ASO is 3xn, 3xn-1, or 3xn-2 nucleotides in length (n is an integer 6 or greater), where (a) (i) the nucleotide at position 3xm-2 (m is an integer from 1 to n) is a ribonucleotide that includes a first modification (e.g., LNA), (ii) the nucleotide at position 3xm-1 (m is an integer from 1 to n) is a ribonucleotide that includes a first modification (e.g., LNA), or (iii) the nucleotide at position 3xm (m is an integer from 1 to n) is a ribonucleotide that includes a first modification (e.g., LNA); and (b) the nucleotides at the remaining positions include a second, different modification (e.g., 2'-O-methoxyethyl). An ASO referred to herein as hOTC-ASOe1-1d has such a structure. In some embodiments, the length of the ASO is 2xn or 2xn-1 nucleotides (n is an integer 9 or greater), where (a) (i) the nucleotide at position 2xm-1 (m is an integer from 1 to n) is a ribonucleotide containing a first modification (e.g., LNA), or (ii) the nucleotide at position 2xm (m is an integer from 1 to n) is a ribonucleotide containing a first modification (e.g., LNA); and (b) the nucleotides at the remaining positions contain a second, different modification (e.g., 2'-O-methoxyethyl). The ASO referred to herein as hOTC-ASOe1-1e has such a structure. Similar modification patterns are also contemplated, for example, where the first modification is repeated at exactly 4, 5, or more nucleotides.In some embodiments, the ASO is 4×n, 4×n-1, or 4×n-2 nucleotides in length (n is an integer greater than or equal to 6), wherein (a) (i) the nucleotide at position 4×m-2 (m is an integer from 1 to n) is a ribonucleotide that includes a first modification (e.g., LNA), (ii) the nucleotide at position 4×m-1 (m is an integer from 1 to n) is a ribonucleotide that includes a first modification (e.g., LNA), or (iii) the nucleotide at position 3×m (m is an integer from 1 to n) is a ribonucleotide that includes a first modification (e.g., LNA); and (b) the nucleotides at the remaining positions include a second, different modification (e.g., 2'-O-methoxyethyl). In some embodiments, the ASO is 5×n, 5×n-1, or 5×n-2 nucleotides in length (n is an integer greater than or equal to 6), wherein (a) (i) the nucleotide at position 5×m-2 (m is an integer from 1 to n) is a ribonucleotide that includes a first modification (e.g., LNA), (ii) the nucleotide at position 5×m-1 (m is an integer from 1 to n) is a ribonucleotide that includes a first modification (e.g., LNA), or (iii) the nucleotide at position 5×m (m is an integer from 1 to n) is a ribonucleotide that includes a first modification (e.g., LNA); and (b) the nucleotides at the remaining positions include a second, different modification (e.g., 2'-O-methoxyethyl).

[0155] In some embodiments, the ASO further comprises a GalNAc or Teg-GalNAc moiety at the 5' or 3' end of the ASO.

[0156] In certain embodiments, the ASO comprises a DNA sequence (e.g., having at least 8, 9, 10, 11, 12, 13, 14, or 15 consecutive nucleotides of unmodified DNA) flanking an RNA sequence. Such structures are known as "gapmers", with the internal DNA region referred to as the "gap" and the external RNA region referred to as the "wing" (see, e.g., PCT Publication No. WO2013 / 177248). Gapmers are known to promote degradation of target RNA by recruiting nuclear RNAses (e.g., RNase H). Surprisingly, in the present disclosure, it has been discovered that gapmers can increase target gene expression when bound to regRNA (e.g., hOTC-ASOe1-1a), as can regRNAs with the same sequence but different chemical modifications (e.g., hOTC-ASOe1-1d and hOTC-ASOe1-1h). In certain embodiments, the ASO contains DNA sequences adjacent to an RNA sequence and does not induce RNAse or RNAse H-mediated degradation.

[0157] In certain embodiments, the gapmer is about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length. In certain embodiments, the gap is about 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleotides in length. In certain embodiments, one or both wings are about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides in length. In certain embodiments, one or both wings are modified with an RNA modification, e.g., β-D-ribonucleosides, 2'-modified nucleosides (e.g., 2'-O-(2-methoxyethyl) (2'-MOE), 2'-O-CH 3, or 2'-fluoro-arabino (FANA)), and bicyclic sugar modified nucleosides (e.g., with constrained ethyl or locked nucleic acid (LNA)). In certain embodiments, each ribonucleotide in a gapmer is modified with 2'-MOE. In certain embodiments, a gapmer contains one or more modified internucleotide linkages, e.g., phosphorothioate (PS) internucleotide linkages. In certain embodiments, every two adjacent nucleotides in a gapmer are linked by a phosphorothioate internucleotide linkage.

[0158] In certain embodiments, the ASO does not contain 7 or more, 8 or more, 9 or more, 10 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more consecutive nucleotides of unmodified DNA. In some embodiments, such DNA sequences are disrupted every 2, 3, 4, 5, or more nucleotides by modified (e.g., 2'-MOE modified) ribonucleotides. The ASO referred to herein as hOTC-ASOe1-1f has such a structure. In some embodiments, the ASO contains only ribonucleotides and does not contain deoxyribonucleotides.

[0159] The structural features of mixmers and gapmers may be combined. In certain embodiments, the ASO has a structure similar to that of a mixmer disclosed herein (e.g., a structure with intervening modifications), except that in the gap the second modification is changed to a third modification (e.g., a deoxyribonucleotide). The ASOs referred to herein as hOTC-ASOe1-1c, hOTC-ASOe1-2b, hOTC-ASOe1-5a, and hOTC-ASOe1-6a have such a structure. In certain embodiments, the ASO has a structure similar to that of a gapmer disclosed herein, except that in the gap the nucleotides are modified in a mixmer pattern. The ASO referred to herein as hOTC-ASOe1-1b has such a structure.

[0160] In certain embodiments, the ASO further comprises a ligand moiety, for example, a ligand moiety that specifically targets a tissue or organ of interest. For example, N-acetylgalactosamine (GalNAc) specifically targets the liver. In certain embodiments, the ligand moiety comprises GalNAc. In certain embodiments, the ligand moiety comprises a 3-cluster GalNAc moiety (commonly designated GAlNAc3). Other types of GalNAc moieties are 1-cluster, 2-cluster, or 4-cluster GAlNAc, designated GAlNAc1, GALNAc2, or GAlNAc4. In certain embodiments, the ligand moiety comprises GalNAc1, GALNAc2, GAlNAc3, or GalNAc4.

[0161] Pharmaceutical Compositions In certain embodiments, the ASO disclosed herein can be present in a pharmaceutical composition.The pharmaceutical composition can be formulated for use in various drug delivery systems.One or more pharma- ceutically acceptable excipients or carriers can also be included in the composition for proper formulation.The formulation suitable for use in the present disclosure can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985.For a brief review of methods for drug delivery, see, for example, Langer (Science 249:1527-1533, 1990).

[0162] Exemplary carriers and pharmaceutical formulations suitable for delivering nucleic acids are described in Darymanov and Reineke (2018) Front. Pharmacol. 9:971; Barba et al. (2019) Pharmaceutics 11(8):360; Ni et al. (2019) Life (Basel) 9(3):59. It will be appreciated that the presence of a ligand moiety conjugated to the ASO may obviate the need for a carrier for delivery to the tissue or organ targeted by the ligand moiety.

[0163] The delivery of the oligonucleotide of the present invention to cells, for example, cells in a subject, for example, a human subject, for example, a subject in need thereof, for example, a subject having OTC-related disorder, can be achieved in many different ways.For example, delivery can be carried out by contacting the oligonucleotide of the present invention with cells, either in vitro or in vivo.In vivo delivery can also be carried out directly by administering a composition comprising oligonucleotide to a subject.These alternatives will be further discussed below.

[0164] In general, any method of delivering nucleic acid molecules (in vitro or in vivo) may be adapted for use with the oligonucleotides of the present invention (see, for example, Akhtar S. and Julian R L., (1992) Trends Cell. Biol. 2(5):139-144 and WO94 / 02595, which are incorporated herein by reference in their entirety). For in vivo delivery, factors to be considered for delivering oligonucleotide molecules include, for example, the biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. Non-specific effects of oligonucleotides may be minimized by local administration, for example, by direct injection or implantation into tissue, or by local administration of the preparation. Local administration to the treatment site maximizes the local concentration of the drug, limits exposure of the drug to systemic tissues that may otherwise be harmed by the drug or that may degrade the drug, and allows for administration of a lower total dose of the oligonucleotide molecule.

[0165] To administer oligonucleotides systemically for the treatment of disease, oligonucleotides may contain alternative nucleobases, alternative sugar moieties, and / or alternative internucleoside linkages, or alternatively be delivered using a drug delivery system; both methods act to prevent rapid degradation of oligonucleotides by endo- and exonucleases in vivo. Modification of the oligonucleotide or pharmaceutical carrier may also allow targeting of the oligonucleotide composition to target tissues and avoid undesirable off-target effects. Oligonucleotide molecules may be modified by chemical conjugation to lipophilic groups, such as cholesterol, to promote cellular uptake and prevent degradation. In alternative embodiments, oligonucleotides may be delivered using drug delivery systems, such as nanoparticles, lipid nanoparticles, polyplex nanoparticles, lipoplex nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems promote binding of oligonucleotide molecules (negatively charged) and also enhance interactions with negatively charged cell membranes, allowing efficient uptake of oligonucleotides by cells. Cationic lipids, dendrimers, or polymers can be induced to bind to oligonucleotides or form vesicles or micelles that encase the oligonucleotides. The formation of vesicles or micelles further prevents the degradation of oligonucleotides when administered systemically. In general, any method of delivery of nucleic acids known in the art can be adapted to deliver the oligonucleotides of the present invention. Methods for making and administering cationic oligonucleotide complexes are within the capabilities of those skilled in the art (see, for example, Sorensen, D R., et al. (2003) J. Mol. Biol 327:761-766; Verma, U N. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S 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 oligonucleotides include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N. et al., (2003), supra), oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, T S. et al., (2006) Nature 441:111-114), cardiolipin (Chien, P Y. et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091), polyethylenimine (Bonnet M E. 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, D A. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In some embodiments, the oligonucleotides are complexed with cyclodextrins for systemic administration. Methods of administration and pharmaceutical compositions of oligonucleotides and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is incorporated herein by reference in its entirety. In some embodiments, the oligonucleotides of the present invention are delivered by polyplex or lipoplex nanoparticles.Methods of administration and pharmaceutical compositions for oligonucleotide and polyplex nanoparticles and lipoplex nanoparticles may be found in U.S. Patent Application Nos. 2017 / 0121454; 2016 / 0369269; 2016 / 0279256; 2016 / 0251478; 2016 / 0230189; 2015 / 0335764; 2015 / 0307554; 2015 / 0174549; 2014 / 0342003; 2014 / 0135376; and 2013 / 0317086, which are incorporated by reference in their entireties.

[0166] In some embodiments, the compounds described herein may be administered in combination with additional therapeutic agents. Examples of additional therapeutic agents include standard of care antiepileptic drugs, such as quinidine and / or sodium channel blockers. Additionally, the compounds described herein may be administered in combination with recommended lifestyle changes, such as a ketogenic diet.

[0167] Methods for delivery of membrane molecular assemblies The oligonucleotides of the present invention may also be delivered using various membrane molecular assembly delivery methods, including polymer, biodegradable microparticle, or microcapsule delivery devices known in the art. For example, colloidal dispersion systems may be used for targeted delivery of the oligonucleotide agents described herein. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Liposomes are artificial membrane vesicles that are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUVs), ranging in size from 0.2 to 4.0 μm, can encapsulate a significant percentage of an aqueous buffer solution containing large macromolecules. Liposomes are useful for the introduction and delivery of active ingredients to the site of action. Because liposomal membranes are structurally similar to biological membranes, when liposomes are applied to tissues, the liposomal bilayer fuses with the bilayer of the cell membrane. As fusion of the liposome with the cell proceeds, the internal aqueous contents, including the oligonucleotide, are delivered to the cell, where the oligonucleotide may specifically bind to the target RNA. In some cases, liposomes are also specifically targeted, for example to deliver oligonucleotides to specific cell types.The composition of liposomes is usually a combination of phospholipids, usually in combination with steroids, especially cholesterol.Other phospholipids or other lipids can also be used.The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0168] Liposomes containing oligonucleotides can be prepared in a variety of ways. In one example, the lipid components of the liposome are dissolved in a detergent to form a micelle with the lipid components. For example, the lipid components can be amphipathic cationic lipids or lipid conjugates. The detergent can have a high critical micelle concentration and be non-ionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The oligonucleotide formulation is then added to the micelles containing the lipid components. The cationic groups on the lipids interact with the oligonucleotides and condense around the oligonucleotides to form liposomes. After condensation, the detergent is removed, for example by dialysis, to obtain a liposome formulation of oligonucleotides.

[0169] If necessary, a carrier compound that aids in condensation may be added during the condensation reaction, for example, by controlled addition. For example, the carrier compound may be a polymer other than a nucleic acid (e.g., spermine or spermidine). The pH may be adjusted to promote condensation.

[0170] Methods for generating stable polynucleotide delivery vehicles incorporating polynucleotide / cationic lipid complexes as a structural component of the delivery vehicle are further described, for example, in WO 96 / 37194, the entire contents of which are incorporated herein by reference. Liposome formation has also been described by Feigner, PLet al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417; U.S. Pat. No. 4,897,355; U.S. Pat. al.,(1979)Biochim. Biophys. Acta 557:9;Szoka et al.,(1978)Proc.Natl.Acad.Sci.75:4194;Mayhew et al.,(1984)Biochim. Biophys. Acta 775:169;Kim et al.,(1983)Biochim. Biophys. Acta 728:339; and Fukunaga et al., (1984) Endocrinol. 115:757. 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., (1986) Biochim. Biophys. Acta 858:161.). Microfluidization can be used when consistently small (50-200 nm) and relatively uniform aggregates are required (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169). These methods are readily adapted to packaging oligonucleotide formulations into liposomes.

[0171] Liposomes are broadly divided into two classes. Cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes. The positively charged nucleic acid / liposome complex binds to the negatively charged cell surface and is taken up into endosomes. The acidic pH within the endosome causes the liposome to rupture, releasing its contents into the cytoplasm (Wang et al. (1987) Biochem. Biophys. Res. Commun., 147:980-985).

[0172] pH-sensitive or negatively charged liposomes entrap nucleic acids rather than complexing with them. Because both the nucleic acid and the lipid are similarly charged, repulsion rather than complexation occurs. Nevertheless, some nucleic acid is trapped in 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. Expression of the exogenous gene was detected in the target cells (Zhou et al. (1992) Journal of Controlled Release, 19:269-274).

[0173] One major type of liposome composition includes phospholipids other than naturally occurring phosphatidylcholine. Neutral liposome composition can be formed from, for example, dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome composition is generally formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are mainly formed from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0174] Other examples of methods for introducing 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, (1994) J. Biol. Chem. 269:2550; Nabel, (1993) Proc. Natl. Acad. Sci. 90:11307; Nabel, (1992) Human Gene Ther. 3:649; Gershon, (1993) Biochem. 32:7143; and Strauss, (1992) EMBO J. 11:417.

[0175] Non-ionic liposomal systems, especially those containing non-ionic surfactants and cholesterol, have also been investigated to determine their usefulness in delivering drugs to the skin.Non-ionic liposomal formulations containing NOVASOME™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and NOVASOME™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver cyclosporine-A to the dermis of mouse skin.The results showed that such non-ionic liposomal systems are effective in promoting the deposition of cyclosporine-A into different layers of the skin (Hu et al., (1994) STP Pharma. Sci., 4(6): 466).

[0176] The liposomes may also be sterically stabilized liposomes, which contain one or more specialized lipids that provide an increased circulation life compared to liposomes lacking such specialized lipids. An example of a sterically stabilized liposome is one in which a portion of the vesicle-forming lipid portion of the liposome is monosialoganglioside G M1or (B) one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Without wishing to be bound by any particular theory, it is believed in the art that the enhanced circulation half-life of these sterically stabilized liposomes, at least for those sterically stabilized liposomes that contain gangliosides, sphingomyelin, or PEG-derivatized lipids, is due to reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., (1987) FEBS Letters, 223:42; Wu et al., (1993) Cancer Research, 53:3765).

[0177] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., (1987), 507:64) report that monosialoganglioside G improves the blood half-life of liposomes. M1 reported the ability of (1) sphingomyelin and (2) ganglioside G to bind sphingomyelin. These findings were expanded upon by Gabizon et al. (Proc. Natl. Acad. Sci. USA, (1988), 85:6949). U.S. Patent No. 4,837,028 and WO 88 / 04924, both to Allen et al., reported the ability of (1) sphingomyelin and (2) ganglioside G to bind sphingomyelin. M1 or galactocerebroside sulfate. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97 / 13499 (Lim et al.).

[0178] In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage that they can fuse with cell membranes. Non-cationic liposomes cannot fuse efficiently with plasma membranes, but they can be taken up by macrophages in vivo and used to deliver oligonucleotides to macrophages.

[0179] Additional advantages of liposomes include that liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water-soluble and lipid-soluble drugs; and liposomes can protect oligonucleotides encapsulated in the internal compartment 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 surface charge of the lipids, the size of the vesicles, and the water content of the liposomes.

[0180] 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 spontaneously interact with nucleic acids, forming lipid-nucleic acid complexes that can fuse with negatively charged lipids in the plasma membrane of tissue culture cells to deliver oligonucleotides (see, e.g., Feigner, PL et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417, and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

[0181] The DOTMA analog 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with phospholipids to form DNA-complexed vesicles. LIPOFECTIN™ (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivering highly anionic nucleic acids to living tissue culture cells, containing positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. If sufficient positively charged liposomes are used, the net charge of the resulting complex will also be positive. The positively charged complexes thus prepared spontaneously attach to the negatively charged cell surface and fuse with the plasma membrane, efficiently delivering functional nucleic acids to, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Ind.), differs from DOTMA in that the oleoyl moieties are linked by ester rather than ether bonds.

[0182] Other reported cationic lipid compounds include those conjugated to various moieties, including, for example, carboxyspermine conjugated to either of two types of lipids, including compounds such as 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (TRANSFECTAM™, Promega, Madison, Wis.), and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide ("DPPES") (see, e.g., U.S. Pat. No. 5,171,678).

[0183] Another cationic lipid conjugate involves derivatization of lipids with cholesterol ("DC-Chol") formulated into liposomes in combination with DOPE (see Gao, X. and Huang, L., (1991) Biochim. Biophys. Res. Commun. 179:280). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., (1991) Biochim. Biophys. Acta 1065:8). In certain cell lines, these liposomes containing conjugated cationic lipids are said to be less toxic and provide more efficient transfection than compositions containing DOTMA. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for delivery of oligonucleotides are described in WO 98 / 39359 and WO 96 / 37194.

[0184] Liposomal formulations are particularly suitable for topical administration, and liposomes exhibit several advantages over other formulations.These advantages include reduced side effects associated with high systemic absorption of administered drugs, increased accumulation of administered drugs at desired targets, and the ability to administer oligonucleotides to skin.In some implementations, liposomes are used to deliver oligonucleotides to epidermal cells and also to enhance the penetration of oligonucleotides into skin tissues, such as skin.For example, liposomes may be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (e.g., Weiner et al., (1992) Journal of Drug Targeting, vol. 2, 405-410 and du Plessis et al., (1992) Antiviral Research, 18:259-265; Mannino, RJ and Fould-Fogerite, S., (1998) Biotechniques 6:682-690; Itani, T. et al., (1987) Gene 56:267-276; Nicolau, C. et al. (1987) Meth. Enzymol. 149:157-176; Straubinger, RM and Papahadjopoulos, D. (1983) Meth. Enzymol. 101:512-527; Wang, C.Y and See Huang, L., (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855).

[0185] Non-ionic liposomal systems, particularly those containing non-ionic surfactants and cholesterol, have also been investigated to determine their usefulness in delivering drugs to the skin.Non-ionic liposomal formulations containing NOVASOME I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and NOVASOME II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) have been used to deliver drugs to the dermis of mouse skin.Such formulations containing oligonucleotides are useful for treating skin disorders.

[0186] Liposome targeting can also be based on, for example, organ specificity, cell specificity, and organelle specificity, and is known in the art.For liposome targeted delivery system, lipid groups can be incorporated into the lipid bilayer of liposome to maintain targeting ligand in stable association with the liposome bilayer.Various linking groups can be used to connect lipid chains to targeting ligand.Additional methods are known in the art, and are described, for example, in US Patent Publication No. 20060058255, whose linking groups are incorporated herein by reference.

[0187] Liposomes containing oligonucleotides can be highly deformable. Such deformability can allow liposomes to penetrate pores smaller than the average radius of the liposome. For example, transfersomes are yet another type of liposome, highly deformable lipid aggregates that are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets that are so easily deformable that they can easily penetrate pores smaller than a liquid droplet. Transfersomes may be made by adding surface edge activators, usually surfactants, to standard liposome compositions. Transfersomes containing oligonucleotides may be delivered subcutaneously, for example, by infection, to deliver oligonucleotides to keratinocytes in the skin. To pass through intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter of less than 50 nm, under the influence of a suitable transdermal gradient. Furthermore, due to the properties of lipids, these transfersomes can often self-optimize (e.g., in the skin, adapting to the shape of the pores), self-repair, and reach their target without fragmentation, often self-loading. Transfersomes have been used to deliver serum albumin to the skin, and transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0188] Other formulations suitable for the present invention are described in PCT Publication Nos. WO2009 / 088891, WO2009 / 132131, and WO2008 / 042973, which are incorporated by reference in their entireties.

[0189] Surfactants are widely used in formulations such as emulsions (including microemulsions) and liposomes. The most common way to classify and rank the properties of the many different types of surfactants, both natural and synthetic, is by using the hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for classifying the various surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0190] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants are widely used in pharmaceuticals and cosmetics and are usable at a wide range of pH values. In general, their HLB values ​​range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers, are also included in this class. Polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0191] If the surfactant molecule is negatively charged when dissolved or dispersed in water, the surfactant is classified as anionic.Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyltaurates and sulfosuccinates, and phosphates.The most important members of the anionic surfactant class are alkyl sulfates and soaps.

[0192] If the surfactant molecule carries a positive charge when dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most used members of this class.

[0193] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phosphatides.

[0194] The use of surfactants in pharmaceutical preparations, formulations, and emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0195] The oligonucleotide for use in the method of the present invention can also be provided as a micelle formulation.Micelle is a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure so that all the hydrophobic parts of the molecule are directed inward and the hydrophilic parts remain in contact with the surrounding aqueous phase.When the environment is hydrophobic, the reverse arrangement exists.

[0196] Lipid nanoparticle-based delivery methods The oligonucleotides in the present invention may be fully encapsulated in lipid formulations, such as lipid nanoparticles (LNPs), or other nucleic acid-lipid particles. LNPs are useful for systemic administration because they have an extended circulatory lifetime after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically distant from the site of administration). LNPs include "pSPLPs," which include encapsulated condensing agent-nucleic acid complexes described in PCT Publication No. WO00 / 03683. The particles of the present invention typically have an average diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially non-toxic. Furthermore, the nucleic acid, when present in the nucleic acid-lipid particles of the present invention, is resistant in aqueous solution to degradation by nucleases. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in 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.

[0197] Non-limiting examples of cationic lipids include 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-dilinole ... Noleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleoyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleoyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 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-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or The analogues, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienietetrahydro-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-ylethylazanediedidodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid may, for example, comprise about 20 mol % to about 50 mol %, or about 40 mol % of the total lipid present in the particle.

[0198] The ionizable / non-cationic lipids may be anionic lipids or neutral lipids, including distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DOPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPG), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE ... Non-cationic lipids include, but are not limited to, oleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or mixtures thereof. The non-cationic lipid, for example, when cholesterol is included, can be about 5 mol% to about 90 mol%, about 10 mol%, or about 60 mol% of the total lipid present in the particle.

[0199] The conjugated lipid that inhibits particle aggregation can be, for example, a polyethylene glycol (PEG)-lipid, including, but not limited to, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or mixtures thereof. PEG-DAA conjugates can be, for example, PEG dilauryloxypropyl (C 12 ), PEG dimyristyloxypropyl (C 14 ), PEG-dipalmityloxypropyl (C 16 ), or PEG-distearyloxypropyl (C 18 The conjugated lipid that prevents particle aggregation can be, for example, from 0 mol % to about 20 mol %, or about 2 mol % of the total lipid present in the particle.

[0200] In some embodiments, the nucleic acid-lipid particles further comprise cholesterol, for example, from about 10 mol % to about 60 mol %, or about 50 mol % of the total lipid present in the particle.

[0201] ASO can also be delivered in lipidoids. The synthesis of lipidoids has been widely described, and formulations containing these compounds are particularly suitable for the delivery of modified nucleic acid molecules or ASOs (see Mahon et al, Bioconjug Chem. 2010 21:1448-1454; Schroeder et al, J Intern Med. 2010 267:9-21; Akinc et al, Nat Biotechnol. 2008:26:561-569; Love et al, Proc Natl Acad Sci U A. 2010 107:1864-1869; Siegwart et al, Proc Natl Acad Sci US A. 2011 108:12996-3001, all of which are incorporated herein in their entirety).

[0202] Lipid compositions for RNA delivery are disclosed in WO2012170930A1, WO2013149141A1, and WO2014152211A1, each of which is incorporated herein by reference.

[0203] Therapeutic Applications The present invention provides a method for treating diseases and disorders associated with decreased gene expression (e.g., decreased OTC gene expression). The method uses an ASO or a pharmaceutical composition comprising an ASO that hybridizes with a regulatory RNA transcribed from a regulatory element of a target gene (e.g., OTC). The oligonucleotide compositions described herein are useful in the methods of the present invention, and, without being bound by theory, are believed to exert their desired effects through their ability to regulate the level, condition and / or activity of OTC in cells of a subject (e.g., a mammal, a primate, or a human), for example, by increasing the level of OTC protein protein.

[0204] One aspect of the present invention relates to a method for treating disorders related to OTC (e.g., urea cycle disorder) in a subject in need thereof. Another aspect of the present invention includes increasing the level of OTC in the cells of a subject identified as having an OTC-related disorder. Yet another aspect includes a method for inhibiting the expression of OTC in a cell of a subject. This method can include contacting a cell with an oligonucleotide or ASO in an amount effective to increase the expression of OTC in the cell, thereby increasing the expression of OTC in the cell.

[0205] Based on the above-mentioned method, further aspects of the present invention include the oligonucleotide of the present invention, or a composition comprising such oligonucleotide, for use in treatment, or for use as medicine, or for use in treating OTC or urea cycle related disorders in a subject in need thereof, or for use in increasing the level of OTC in the cells of a subject identified as having OTC related disorders, or for use in increasing the expression of OTC in the cells of a subject.This use comprises contacting a cell with the oligonucleotide in an amount effective to increase the expression of OTC in the cell, thereby increasing the expression of OTC in the cell.The embodiments described below with respect to the method of the present invention are also applicable to these further aspects.

[0206] The contacting of the cell with the oligonucleotide may be in vitro, ex vivo, or in vivo. Contacting the cell with the oligonucleotide in vivo includes contacting the oligonucleotide with a cell or cell population in a subject, for example, a human subject. A combination of in vitro and in vivo cell contacting methods is also possible. As described above, contacting the cell may be direct or indirect. Furthermore, contacting the cell may be achieved via a targeting ligand, including any ligand described herein or known in the art. In some embodiments, the targeting ligand is a carbohydrate moiety, for example, a GalNAc3 ligand, or other ligand that directs the oligonucleotide to a site of interest. The cell may be a liver cell (e.g., a stem cell).

[0207] The administration of the ASO or pharmaceutical composition disclosed herein may be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, perfusion through a catheter, or direct lesion injection. In certain embodiments, the ASO or pharmaceutical composition is administered systemically. In certain embodiments, the ASO or pharmaceutical composition is administered by a parenteral route. For example, in certain embodiments, the ASO or pharmaceutical composition is administered intravenously (e.g., by intravenous infusion), for example, using a prefilled bag, prefilled pen, or prefilled syringe. In other embodiments, the ASO or pharmaceutical composition is administered locally to an organ or tissue in which increased target gene expression is desired (e.g., the liver).

[0208] In some embodiments, the oligonucleotide is administered to the subject so that the oligonucleotide is delivered to a specific site in the subject.Such targeted delivery can be achieved by either systemic or local administration.The increase in the expression of OTC can be evaluated using the measurement of the level or change in the level of OTC mRNA or OTC protein in a sample from a specific site in the subject.In certain embodiments, the method includes the clinically relevant increase in the expression of OTC, for example, as shown by the clinically relevant results after treating the subject with an agent that reduces the expression of OTC.

[0209] In other embodiments, the oligonucleotide is administered in an amount and for a duration effective to result in a reduction (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of one or more symptoms of an OTC disorder, such as high blood ammonia levels.

[0210] Increased OTC expression levels The therapeutic method disclosed herein, using ASO targeting OTC, is designed to increase the expression level of OTC in a subject. The increase in expression of OTC gene includes any increase in the level of OTC gene, for example, at least partial increase in the expression of OTC gene. The increase can be evaluated by the increase in absolute or relative levels of one or more of these variables compared to a control level. The control level can be any type of control that is a control level utilized in the art, for example, a baseline level before administration, or a level determined from a similar subject, cell, or a sample that is untreated or treated with a control (for example, a buffer only control or a non-active agent control). In certain embodiments, the method produces a clinically relevant increase in the expression of OTC, for example, as shown by clinically relevant results after treating a subject with an agent that increases the expression of OTC.

[0211] In certain embodiments, the methods disclosed herein increase OTC gene expression by at least about 1%, at least about 2%, 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% relative to baseline levels prior to administration. In certain embodiments, the methods disclosed herein increase OTC gene expression by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold compared to baseline levels prior to administration. In certain embodiments, the subject has a deficiency in OTC expression, and the methods disclosed herein restore the OTC expression level or activity to at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the average OTC expression level or activity in subjects of a similar age and sex species.

[0212] The expression of OTC gene can be evaluated based on the level of any variable associated with OTC gene expression, such as OTC mRNA level or OTC protein level.It is understood that OTC is an X-chromosomal gene in certain mammals (e.g., humans and mice), and female subjects show a mosaic pattern of X-chromosome inactivation.In certain embodiments, the expression level or activity of OTC herein refers to the average expression level or activity in liver.

[0213] In certain embodiments, surrogate markers may be used to detect increased levels of OTC expression. For example, effective treatment of an OTC-related disorder, as indicated by acceptable diagnostic and monitoring criteria, with an agent that increases OTC expression may be understood to indicate a clinically relevant reduction in OTC.

[0214] Increased expression of the OTC gene may be evident by an increase in the amount of mRNA in which the OTC gene is transcribed and expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) that have been treated (e.g., by contacting the cell(s) with an oligonucleotide of the present invention or by administering an oligonucleotide of the present invention to a subject in which the cells are or were present), such that expression of the OTC gene is increased when compared to a second cell or group of cells that has not been so treated or is substantially identical to the first cell or group of cells that has not been treated (control cell(s) that have not been treated with the oligonucleotide or that have not been treated with an oligonucleotide targeting the gene of interest).

[0215] In other embodiments, increased expression of the OTC gene can be assessed with respect to a parameter functionally related to OTC gene expression, such as increased OTC protein expression or OTC activity. OTC increase can be determined in any cell expressing OTC, either endogenously or heterologously from an expression construct, by any assay known in the art.

[0216] Increased expression of OTC may be evidenced by an increase in the level of OTC protein expressed by a cell or group of cells (e.g., the level of protein expressed in a sample from a subject) compared to a control cell or group of cells. Increased expression of OTC may also be evidenced by an increase in the mRNA level of OTC in a treated cell or group of cells compared to a control cell or group of cells.

[0217] The control cell or cell group that can be used to evaluate the increase in the expression of OTC gene includes a cell or cell group that has not yet been contacted with the oligonucleotide of the present invention.For example, the control cell or cell group can be derived from an individual subject (e.g., a human or animal subject) before the subject is treated with oligonucleotide.

[0218] The level of OTC mRNA expressed by a cell or group of cells can be determined using any method known in the art for assessing mRNA expression. In one embodiment, the expression level of OTC in a sample is determined by detecting a transcribed polynucleotide, or a portion thereof, such as the mRNA of the OTC gene. RNA can be extracted from cells using RNA extraction techniques, including, for example, using acid phenol / guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNEASY™ RNA preparation kit (Qiagen) or PAXgene (PreAnalytix, Switzerland). Exemplary assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays, Northern blotting, in situ hybridization, and microarray analysis. Circulating OTC mRNA can be detected using the methods described in PCT Publication WO2012 / 177906, the entire contents of which are incorporated herein by reference. In some embodiments, the level of expression of OTC is determined using a nucleic acid probe. As used herein, the term "probe" refers to any molecule that can selectively bind to a specific OTC sequence, for example, to an mRNA or polypeptide. Probes can be synthesized by those skilled in the art or derived from appropriate biological preparations. Probes can also be specifically designed to be labeled. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0219] The isolated mRNA can be used in hybridization or amplification assays, including but not limited to Southern or Northern analysis, polymerase chain reaction (PCR) analysis, and probe arrays. One method for determining mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the OTC mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with the 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 an alternative embodiment, 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. One skilled in the art can easily adapt known mRNA detection methods for use in determining the level of OTC mRNA.

[0220] Alternative methods for determining the expression level of OTC in a sample include, for example, processes of nucleic acid amplification and / or reverse transcription (to prepare cDNA) of mRNA in the sample, such as RT-PCR (Mullis, 1987, experimental embodiment described in 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), transcription amplification systems (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 detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are particularly useful for the detection of nucleic acid molecules when such molecules are present in very low numbers. In certain embodiments of the present invention, the level of expression of OTC is determined by quantitative fluorogenic RT-PCR (i.e., the TAQMAN™ system) or the DUAL-GLO® luciferase assay.

[0221] The expression level of OTC mRNA can be monitored using membrane blots (such as those used in hybridization analyses such as Northern, Southern, dot, etc.), or microwells, sample tubes, gels, beads, or fibers (or any solid support containing bound nucleic acid). See U.S. Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195, and 5,445,934, which are incorporated herein by reference. Determining the OTC expression level can also include using a nucleic acid probe in solution.

[0222] In some embodiments, the level of mRNA expression is evaluated using branched DNA (bDNA) assay, quantitative PCR (qPCR), RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, or microarray analysis. Such methods can also be used to detect OTC nucleic acid.

[0223] The level of OTC protein expression can be determined using any method known in the art for measuring protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), superdiffusion chromatography, fluid or gel precipitation reaction, absorption spectroscopy, colorimetry, spectrophotometry, flow cytometry, FACS, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, electrochemiluminescence assay, Luminex, MSD, FISH, etc. Such assays can also be used to detect proteins that indicate the presence or replication of OTC protein. EXAMPLES

[0224] Below are examples of specific embodiments for carrying out the present invention. These examples are presented for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0225] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, within the skill of the art. Such techniques are explained fully in the literature, see, for example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Edition (Easton, Pennsylvania: Mack Publishing Company, 1992); rd Ed. (Plenum Press) Vols A and B (1992).

[0226] Example 1: Modulation of OTC expression using regRNA-targeted ASO Two human OTC regRNA targets (RR1 and RR2) were identified for human OTC. 69 ASOs targeting RR1 and 133 ASOs targeting RR2 were synthesized. These 202 initial ASOs were screened in primary human hepatocytes at 5 μM for efficacy in increasing OTC mRNA. ASOs that showed efficacy were further tested for dose-dependent efficacy at 1.25 μM, 2.5 μM, and 5 μM in primary human hepatocytes and primary human donor hepatocytes. Positive ASOs that showed dose-dependent efficacy were selected for ASO base walking and tiling around the regRNA hit regions. Based on the initial screening, 31 RR1 ASOs and 35 RR2 ASOs were selected for base walking and tiling around the initial ASO hits. These additional ASOs were further tested for dose-dependent efficacy. ASOs were selected for chemistry fine tuning by altering the chemistry, type, and location of chemical modifications of the selected ASOs. We identified 71 ASOs targeting RR1 and 67 ASOs targeting RR2.

[0227] This process was repeated for mouse OTC regRNA to identify ASOs that alter mouse OTC expression. For mouse OTC, four mouse OTC regRNA targets were identified. 126 ASOs targeting regRNA were synthesized. These 126 initial ASOs were screened in primary mouse hepatocytes for efficacy in increasing OTC mRNA. Positive ASOs that showed dose-dependent efficacy were selected for ASO base walking and tiling around the regRNA hit region. Based on the initial screening, 24 ASOs were selected for base walking and tiling around the initial ASO hit. These additional ASOs were further tested for dose-dependent efficacy. Four ASOs were selected for chemistry fine tuning by changing the chemistry, type, and location of the chemical modifications of the selected ASOs.

[0228] The selection and chemical modifications of human and mouse ASOs are shown in Tables 2, 3, 4 and Figures 18A, 18B, 18C, 18D, and 18E.

[0229] This example was designed to evaluate the regulation of OTC expression in human hepatocytes using an ASO targeting eRNA transcribed from the enhancer of human OTC.

[0230] Hepatocytes from four donors (HUM4178, HUM181511A, HUM190171, HUM181371) were cultured in vitro. Cells were plated in growth medium and 4-6 hours after plating, treated with hOTC-ASOe1-1d, hOTC-ASOe1-1h, hOTC-ASOe2-1, or hOTC-ASOe1-1a at final concentrations of 1.25 μM, 2.5 μM, 5 μM, or 10 μM (see Figures 18A, 18B, 18D, and 18E for human OTC sequence and chemical modifications of selected ASOs, and Figure 18C for mouse OTC sequence and chemical modifications of selected ASOs). Cells were harvested 48 hours after treatment and processed for RNA isolation, cDNA synthesis, and QPCR analysis. For OTC expression, the Taqman probe Hs00166892_m1(OTC)60X was used. OTC levels were normalized to B2M expression.

[0231] Figure 2A shows OTC mRNA after treatment with hOTC-ASOe1-11. Figure 2B shows OTC mRNA after treatment with hOTC-ASOe1-8. Figure 2C shows OTC mRNA after treatment with hOTC-ASOe2-1. Figure 2D shows OTC mRNA after treatment with hOTC-ASOe1-1. Treatment with each ASO resulted in a dose-dependent increase in OTC expression in each donor. Thus, four different RNA actuators targeting the same regRNA increased human OTC mRNA dose-dependent substance.

[0232] Hepatocytes from OTC-deficient donors were cultured in vitro. Cells were plated in growth medium and treated with ASOs hOTC-ASOe1-10 and hOTC-ASOe1-2c at a final concentration of 5uM 4 hours after plating. Non-targeting control (NTC) ASOs containing random sequences were used as negative controls. Supernatants were collected for urea production analysis and cell lysates were collected for mRNA 2 days after treatment. For mRNA analysis, taqman probe Hs00166892_m1 was used for OTC expression. OTC levels were normalized to B2M expression. For urea production, collected supernatants were measured by Urea Nitrogen (BUN) colorimetric detection kit (Thermofisher, Cat. No.: EIABUN) and normalized by Albumin ELISA (Bethyl, Cat. No.: E88-129). Statistics were performed using one-way ANOVA in Prism (GraphPad).

[0233] The urea assay was also repeated in a dose study in wild-type hepatocytes using hOTC-ASOe1-2a. Cells were plated in growth medium and treated with ASO hOTC-ASOe1-2a at final concentrations of 1.25uM, 2.5uM, 5uM, and 10uM for 4 hours after plating. A non-targeting control (NTC) ASO containing a random sequence was used as a negative control. Supernatants were collected for urea production analysis and cell lysates were collected for mRNA 6 days after treatment. Samples were processed as described above.

[0234] As shown in Figure 3A and Figure 3B, treatment with both ASOs resulted in increased autogenism in patient cells (Figure 3B), which correlated with OTC mRNA upregulation (Figure 3A). The normal range in the urea production assay is 18-30ug urea per mg albumin. One ASO increased the average concentration to approximately 13ug urea / mg albumin, almost double the 7ug urea / mg albumin of the negative control sample, which was almost within the normal urea production range. In addition, hOTC-ASOe1-2a induced a dose-dependent increase in OTC mRNA (Figure 3C) and urea (Figure 3D) in WT hepatocytes.

[0235] The majority of regRNAs have no large sequence regions that are conserved between the human and mouse genomes. For in vivo proof of concept, we identified regRNAs surrounding the mouse Otc region, designed ASOs targeting these mouse regRNAs (promoter and enhancer), and expressed them in wild-type (B6EiC3SnF1 / J, [WT]) primary mouse hepatocytes and in Otc-deficient donors (B6EiC3Sna / A-Otc spf-ash / J, [OTCD]) primary mouse hepatocytes were screened.

[0236] Primary hepatocytes were cultured from male mouse strains B6EiC3SnF1 / J (control WT) and Otc-deficient donors (B6EiC3Sna / A-Otc spf-ash / J, Catalog: 001811) (JAX lab). spf ash Mice carry the variant c.386G>A, p.Arg129His in the Otc gene that affects splicing, resulting in reduced OTC mRNA levels in spf / ash liver (5-12% of wild-type controls). Thus, male spf ash The mice have a mild biochemical phenotype with low OTC activity (5%-10% of wild type).

[0237] Primary hepatocytes were seeded at 20,000 cells per well on day 0. On day 2, cells were treated with mouse ASO at a final concentration of 5 μM. Cells were incubated for 2 days and lysates were harvested for mRNA analysis on day 2 after treatment. Taqman probe Mm01288053_m1 was used for mouse OTC expression. Ppia and Hprt were used as housekeeper genes for gene expression normalization. Statistics were performed using one-way ANOVA in Prism (GraphPad).

[0238] Five of the six ASOs increased OTC mRNA in WT hepatocytes in vitro (One-way ANOVA*: p0.05-0.005; **: p<0.005) (Figure 4). Four of the six ASOs increased OTC mRNA in OTCD hepatocytes in vitro (One-way ANOVA*: p0.05-0.005; **: p<0.005) (Figure 5). Thus, ASO-targeting regRNA may be used to increase OTC expression in diseased mouse liver cells. ASO-mediated OTC upregulation in OTC-deficient mouse cells allows these to be tested in disease models, providing an in vivo phenotypic readout.

[0239] Additional chemical modifications were made to hOTC-ASOe1-1. The modifications are shown in Table 3 and FIG. 18D. The new ASOs were evaluated in hepatocytes as previously described at 5uM, 9uM, or 10uM concentrations. Table 5 shows the fold change and standard deviation of OTC mRNA for the indicated ASOs.

[0240] (Table 5) TIFF2024534214000018.tif217136TIFF2024534214000019.tif29136

[0241] Additional chemical modifications were made to hOTC-ASOe2-2. The modifications are shown in Table 4 and FIG. 18E. The new ASOs were evaluated in hepatocytes as previously described at 5uM, 9uM, or 10uM concentrations. Table 6 shows the fold change and standard deviation of OTC mRNA for the indicated ASOs.

[0242] (Table 6) TIFF2024534214000020.tif137136

[0243] The dose response of two ASOs, hOTC-ASOe1-1d and hOTC-ASOe2-2e, was also evaluated. Cells were incubated with increasing concentrations of each ASO as described above. OCT mRNA was determined by qRT-PCR.

[0244] As shown in Table 7, treatment of hepatocytes with increasing amounts of hOTC-ASOe1-1d resulted in a dose-dependent increase in OTC mRNA.

[0245] (Table 7) hOTC-ASOe1-1d TIFF2024534214000021.tif41128

[0246] As shown in Table 8, treatment of hepatocytes with increasing amounts of hOTC-ASOe2-2e resulted in a dose-dependent increase in OTC mRNA.

[0247] (Table 8) hOTC-ASOe2-2e TIFF2024534214000022.tif41128

[0248] Additional ASOs were generated and tested in hepatocytes as described above. The ASO sequences, start and end positions on chromosome X, and fold change (FC) and standard deviation (SD) of OTC mRNA are shown in Table 9.

[0249] SEQ ID NO:143-892 ASO targeting human OTC eRNA-1 (SEQ ID NO:1). All bases are 2'-O-methoxyethyl and all cytidines have a 5-methyl (5-methyl on cytidine).

[0250] ASOs of SEQ ID NOs: 893-1029 target human OTC eRNA-2 (SEQ ID NO: 2). The ASOs are 2'-O-methoxyethyl with LNA at bases 6, 11, and 16. Such ASOs may be described as 5xn+5 nucleotides (n is an integer ≥ 3) where the nucleotide at position 5xm is a ribonucleotide modified with LNA (m is an integer ≥ 1) and the nucleotides at the remaining positions are ribonucleotides modified with 2'-O-methoxyethyl and all cytidines have a 5-methyl (5-methyl on cytidine).

[0251] The ASOs of SEQ ID NOs: 1030-1072 target the ASO of human OTC paRNA-1 (SEQ ID NO: 1077). All bases are 2'-O-methoxyethyl and all cytidines have a 5-methyl (5-methyl on cytidine).

[0252] (Table 9) TIFF2024534214000023.tif169160TIFF2024534214000024.tif213160TIFF20245342140000 25.tif213160TIFF2024534214000026.tif213160TIFF2024534214000027.tif213160TIFF202 4534214000028.tif213160TIFF2024534214000029.tif211160TIFF2024534214000030.tif21 3160TIFF2024534214000031.tif213160TIFF2024534214000032.tif213160TIFF20245342140 00033.tif213160TIFF2024534214000034.tif213160TIFF2024534214000035.tif213160TIF F2024534214000036.tif213160TIFF2024534214000037.tif213160TIFF2024534214000038.t if213160TIFF2024534214000039.tif213160TIFF2024534214000040.tif213160TIFF2024534 214000041.tif213160TIFF2024534214000042.tif213160TIFF2024534214000043.tif107160

[0253] Example 2: Modulation of SERPING1 expression using paRNA- or eRNa-targeted ASOs This example was designed to evaluate the regulation of SERPING1 expression in mouse hepatocytes using an ASO targeting paRNA transcribed from the mouse SERPING1 promoter.

[0254] See Figure 19 for the sequences and chemical modifications of selected mouse Serping1 ASOs.

[0255] Female C57Bl / 6 mice (approximately 6-7 weeks old) were treated with a single 5 mg / kg IP dose of IFNy (125 μg per mouse) or PBS as a negative control and sacrificed 30 min, 1 h, 2 h, 6 h, 10 h, and 24 h after treatment. Male C57Bl / 6 mice (7 weeks old) were treated twice (12 h apart) with a 15 mg / kg IP dose of tofacitinib and sacrificed 2 h and 6 h after treatment. Livers from mice in both experiments were harvested at the enumeration time points and processed for RNA isolation and cDNA synthesis to perform relative RNA measurements (Taqman qPCR (Mm00437835_m1)).

[0256] Serping1 mRNA was upregulated in a time-dependent manner with IFNy, with the highest fold change (approximately 3-fold induction) occurring at 24 h after administration (Figure 6A). Serping1 mRNA was downregulated with the Jak1 inhibitor tofacitinib, with a 50% reduction occurring at 6 h (Figure 6B). Thus, Serping1 is likely regulated by the IFNy-Jak pathway.

[0257] Female C57Bl / 6 mice (approximately 6-7 weeks old) were treated with a single 5mg / kg IP dose of IFNg (125ug per mouse) and sacrificed 24, 48, and 72 hours after treatment. Serum was collected for mRNA and protein analysis by Western Blot. The serping1 antibody used was rabbit monoclonal [EPR8015] against SERPING1 (ab134918). Protein levels were normalized to transferrin protein (rabbit Abcam 82411). Serping1 mRNA was normalized to Hmbs as a housekeeping gene.

[0258] A sustained increase in serum Serping1 mRNA and protein was observed from 24 to 48 hours (FIG. 7). Serum mRNA levels after IFNg treatment are shown in Table 10.

[0259] (Table 10) TIFF2024534214000044.tif32128

[0260] Female C57Bl / 6 mice (approximately 6-7 weeks old) were then treated with a single 5 mg / kg IP dose of IFNy (125 μg per mouse) or PBS as a negative control and sacrificed 6 and 24 hours after treatment. Livers from the 6 and 24 hour time points were processed by Qiagen Trizol method and measured by SYBR green PCR. Serping1 mRNA and regRNA expression levels were determined using PCR.

[0261] As shown in FIG. 8, regRNA levels increase initially, followed by an increase in mRNA after induction with IFNg.

[0262] Cryopreserved mouse hepatocytes (Lonza) were then plated onto collagen-coated plates, allowed to attach for 24 hours, stimulated with 1000ng / ml IFNy, and harvested at 0.5, 2, 4, 8, 24, and 30 hours post-treatment. Cells were lysed in Qiagen RLT buffer, processed using the Quick-RNA Zymo kit, and mRNA was measured by SYBR green qPCR using regRNA-specific primers.

[0263] As shown in Figures 9A and 9B, IFNy stimulation increased Serping1 regRNA before upregulating Serping1 mRNA. Serping1 regRNA levels peaked at 2 hours, whereas Serping1 mRNA peaked at 30 hours. Thus, IFNy treatment results in a time-dependent increase in Serping1 mRNA in mouse hepatocytes.

[0264] Female C57Bl / 6 mice (approximately 6-7 weeks old) were treated with a single 5mg / kg IP dose of IFNg (125ug per mouse) and harvested 6 and 24 hours after treatment. Liver powder from the 6 and 24 hour time points was processed for ATAC-seq. Epigenomic data indicated two hotspots, enhancer 2 and promoter 2, as ideal regions for targeting and upregulation (Figure 10).

[0265] Cryopreserved mouse hepatocytes (Lonza) were then treated with selected ASOs (mSerping1pa-ASO-1, mSerping1pa-ASO-2, and mSerping1pa-ASO-3) in power medium in a dose-response manner by free uptake method on day 1 (24 hours after plating) and harvested on day 3. Scrambled ASO (NTC-3S) was used as a control. Cells were lysed in RLT Qiagen buffer, processed by RNAeasy Plus 96 Kit, and mRNA was measured by Taqman qPCR. mSerping1pa-ASO-1 is the optimized sequence of mSerping1pa-ASO-2.

[0266] Serping1 mRNA was upregulated in a dose-dependent manner using the selected ASOs targeting paRNA (Figure 11B), whereas the neighboring genes Irf1 and Ubel26 were not upregulated. A schematic diagram of the Serping1 chromosomal vicinity is shown in Figure 11A.

[0267] Next, an optimized version of the lead ASO sequence was designed and tested in freshly isolated mouse hepatocytes. Cells were treated with ASO in a dose-response manner by free uptake on day 1 (24 h after plating) in power medium and harvested on day 3. A scrambled ASO (NTC-3S) was used as a control. mSERPING1-ASOpa-6 is an IONIS mouse sequence targeting Serping1 (Bhattacharjee et al., 2013).

[0268] As shown in Figure 12, Serping1 was upregulated in a dose-dependent manner using ASOs targeting regRNA. The fold change in Serping1 mRNA after treatment with ASOs is shown in Table 11.

[0269] (Table 11) TIFF2024534214000045.tif41128

[0270] Longer assays were also performed. Freshly isolated mouse hepatocytes were treated with 10 μM of selected ASOs (mSerping1pa-ASO-2, mSerping1pa-ASO-3, mSerping1pa-ASO-4) in power medium by the free uptake method on day 1 (24 h after plating) and harvested at 8 h, 24 h, 48 h, and 72 h for RNA processing. Scrambled ASO (NTC-Scr3S) was used as a control. mRNA was normalized to NTC.

[0271] The selected ASOs increased Serping1 by approximately 1.5–2× at 24 h (Figure 13).

[0272] Next, an in vivo assay was performed. Male C57 / Bl6 mice (approximately 8 weeks old) were treated with the selected ASO conjugated to GalNAc (mSerping1 ASO-2 GalNAc) via SC injection on days 1 and 4, and serum was collected on day 6. PBS and scrambled ASO NTC were used as controls. Serum bleeds were used to measure Serping1 protein by Western blot. A schematic diagram of the study design is shown in Figure 14A.

[0273] After two doses of GalNAc-conjugated ASO, Serping1 protein levels increase approximately 1.5-fold compared to the negative control (Figure 14B).

[0274] We next evaluated the additive effect of IFNg+ASO treatment on Serping1 mRNA expression. Cryopreserved mouse hepatocytes were treated with 5 μM mSerping1 ASO-2+100 ng / ml IFNg in Power Medium by free uptake method on day 2. Cells were harvested on day 4 for mRNA analysis. Untreated mice and scrambled ASO NTC were used as controls. 5 μM mSerping1 ASO-2 in combination with IFNg led to the highest fold change, about 2.75-fold, compared to the negative control (Figure 15A).

[0275] A time course assay for combination therapy was also performed. Freshly isolated mouse hepatocytes were treated with 10 μM mSerping1 ASO-2, mSerping1 ASO-3, or mSerping1 ASO-4 + 1000 ng / ml IFNg in Power Medium by free uptake method on day 1 (24 h after plating) and harvested at 8 h, 24 h, 48 h, and 72 h for RNA processing. Scrambled ASO NTC + IFNg was used as a control.

[0276] Higher concentrations of ASO also led to an approximately three-fold increase in Serping1 mRNA over control mice (Figure 15B).

[0277] We next assessed the rescued effect of Jak1 inhibitor + ASO on Serping1 mRNA. Cryopreserved mouse hepatocytes were treated with 5 μM mSerping1 ASO-2 + 3 μM Jak1 inhibitor tofacitinib in Power Medium by free uptake method on day 2. Cells were harvested on day 4 for mRNA analysis.

[0278] mSerping1 ASO-2 in combination with Jak1 inhibition resulted in the restoration of Serping1 mRNA to normal levels (Figure 16).

[0279] Similar rescue experiments were performed in a Serping1 knockdown (KD) line using 1 μM Jak1 inhibitor tofacitinib. This line mimics HAE disease because in HAE there is only one healthy copy of Serping1, and therefore the absolute levels are 50% of those in WT individuals.

[0280] Freshly isolated mouse hepatocytes were treated with 10 μM and 5 μM mSerping1 ASO-2 and mSerping1 ASO-3 in power medium containing 1 μM Jak1 by free uptake method on day 1. Cells were harvested on day 4 for mRNA analysis.

[0281] Jak1 inhibitors reduced Serping1 to 50% of normal expression, similar to HAE disease. After treatment with selected ASOs, Serping1 levels were restored by more than 1.5-fold, close to WT levels (Figure 17).

[0282] Additional ASOs were tiled around mSERPING1-ASOpa-1 (CO-3149), mSERPING1-ASOpa-2 (CO-2043), and mSERPING1-ASOpa-3 (CO-2051). The new sequences are shown below: TIFF2024534214000046.tif208158

[0283] ASOs were tested as previously described. Briefly, mouse hepatocytes were plated and treated with ASOs 24 hours after plating on day 1. Cells were harvested 48 hours after treatment. As shown in Figure 34A, ASOs CO-3265, CO-3279, CO-2043, and CO-2051 increased Serping1 mRNA expression in a dose-dependent manner.

[0284] Next, the selected Serping1 ASOs were tested in C1NH+ / - hepatocytes derived from C57BL / 6J mice. C1NH+ / - hepatocytes lack Serping1 expression. As shown in Figure 34B, ASOs CO-2043, CO-2051, CO-3265, CO-3419, CO-4069, and CO-3279 increased Serping1 gene expression in C1NH+ / - hepatocytes in a dose-dependent manner.

[0285] GAlNAc-ASOs were also tested in C1NH-deficient mice. Mice were bled and administered ASOs CO-2051 and CO-3265 on days 1 and 3, and sacrificed on day 6. As shown in Figure 35, both ASOs increased Serping1 mRNA in mice.

[0286] Next, a vascular permeability assay was performed. C57 Bl6 mice were subcutaneously injected with ASO at a dose of 260 mg / kg / week. Evan Blue treatment was performed on days 6 and 8, IP injected at 150 mg / kg, based on reference J Clin Invest. 2002;109(8):1057-1063. Quantification of dye was performed on mice terminated on day 8. At necropsy, tissues were dried, weighed, and added to 1 mL of formamide. Dye was extracted from tissues and measured at OD 620 nm. As shown in Figure 36, CO-2051 reduced the amount of dye extravasation in both ears and colons of CINH+ / - mice.

[0287] CO-2051 also increased Serping1 mRNA in both WT and C1NH+ / - mice. WT or C1NH+ / - mice were treated with 260mg / kg ASO. Blood was collected and processed for serum on days 1, 3, 5, and 7 for protein measurement by Western blot with a fixed loading volume (e.g., 1uL serum). Serping1 and transferrin abcam antibodies were added using standard methods. Respective bands were imaged using a LiCOR scanner and quantified using ImageStudio Analysis software. As shown in Figures 37A and 3B, Serping1 upregulation was observed with naked ASO in WT and diseased mice.

[0288] This assay was repeated and sustained protein upregulation was observed using a lower dose of GalNAc-ASO CO-2051 (15 mg / kg) (Figures 37C and 37D).

[0289] Example 3: Tiling and optimization of human OTC regRNA-targeting ASOs Additional ASOs created by base walking and extension around hOTC-ASOe1-2a were synthesized and characterized. In addition, the ASOs were fine-tuned by varying the chemistry, type, and location of chemical modifications. The ASOs synthesized and further characterized were the ASO sequences hOTC-ASOe1-1a, hOTC-ASOe1-3a, hOTC-ASOe1-4a, hOTC-ASOe1-1h, and hOTC-ASOe1-1d.

[0290] Additional ASOs made by base walking and extension around hOTC-ASOe2-2a were also synthesized and characterized. In addition, the ASOs were fine-tuned by varying the chemistry, type, and location of chemical modifications. The ASOs synthesized and further characterized were ASO sequences hOTC-ASO-e2-2a, hOTC-ASO-e2-2b, hOTC-ASO-e2-2c, hOTC-ASO-e2-2d, hOTC-ASO-e2-2e, hOTC-ASO-e2-4, hOTC-ASO-e2-5, hOTC-ASO-e2-6, and hOTC-ASO-e2-7. For the human OTC sequences and chemical modifications of selected ASOs, see Tables 2, 3, 4, and Figures 18A, 18B, 18D, and 18E.

[0291] Hepatocytes from a single donor were cultured in vitro. Cells were plated in growth medium and treated 4–6 hours after plating with final concentrations of 1 μM, 3 μM, or 9 μM hOTC-ASO-e1-4a (Figure 20A, base walking ASO), or 1.25 μM, 2.5 μM, 5 μM, or 10 μM hOTC-ASOe1-1d, hOTC-ASOe1-1h, or hOTC-ASOe1-1a (Figure 20B, fine-tuning ASO).

[0292] Hepatocytes from a single donor were cultured in vitro. Cells were plated in growth medium and 4-6 hours after plating, treated with hOTC-ASO-e2-2a, hOTC-ASO-e2-2b, hOTC-ASO-e2-2c, hOTC-ASO-e2-2d, hOTC-ASO-e2-2e, hOTC-ASO-e2-4, hOTC-ASO-e2-5, hOTC-ASO-e2-6, and hOTC-ASO-e2-7 at final concentrations of 1 μM, 3 μM, or 9 μM.

[0293] Cells were harvested 48 hours after treatment and processed for RNA isolation, cDNA synthesis and QPCR analysis. For OTC expression, Taqman probe Hs00166892_m1(OTC)60X was used. OTC levels were normalized to B2M expression.

[0294] Base walking and extension around hOTC-ASOe1-2a led to a 3-fold improvement in potency compared to the parent sequence hOTC-ASOe1-2a (Figure 20A). Further fine-tuning by altering the type, chemistry, and location of the modification resulted in increased efficacy compared to the parent sequence hOTC-ASOe1-2a, as shown by a dose-dependent increase in OTC mRNA in primary hepatocytes (Figure 20B).

[0295] Fine-tuning the hOTC-ASOe2-2a-based modifications by altering their type, chemistry, and location resulted in increased efficacy compared to the parental sequence, as shown by a dose-dependent increase in OTC mRNA in primary hepatocytes (Figure 21).

[0296] Next, the selected ASOs were characterized in OTC-deficient donor cell lines. Hepatocytes from OTC-deficient donors were cultured in vitro. Cells were plated in growth medium and 4 hours after plating, treated with ASOs hOTC-ASOe1-10, hOTC-ASOe1-2a, hOTC-ASOe1-12, hOTC-ASOe1-11, and hOTC-ASOe1-1a at final concentrations of 1 μM, 3 μM, and 9 μM. A non-targeting control (NTC) ASO containing a random sequence was used as a negative control (SRC3). Supernatants were collected for urea production analysis and cell lysates were collected for mRNA at 2 and 6 days after treatment. For mRNA analysis, taqman probe Hs00166892_m1 was used for OTC expression. OTC levels were normalized to B2M expression. Urea production was measured in the collected supernatant using a Urea Nitrogen (BUN) colorimetric detection kit (Thermofisher, Catalog No.: EIABUN) and normalized by Albumin ELISA (Bethyl, Catalog No.: E88-129). Statistics were performed using one-way ANOVA in Prism (GraphPad).

[0297] As shown in Figure 22, a dose-dependent increase in OTC mRNA was observed after treatment with multiple ASOs on days 2 and 6.

[0298] Next, an in vitro PBMC assay was performed to assess ASO toxicity.

[0299] Peripheral blood mononuclear cells (PBMCs) were isolated from fresh human whole blood (provided by Research Blood Component LLC). A volume of 15 ml of whole blood was mixed with 15 ml of PBS+2% FBS, added to a SepMate isolation tube (STEMCELL Technologies) containing 15 ml of Ficoll, and centrifuged at 800g for 20 min. The resulting top layer was removed and the remaining mononuclear cell layer was washed with 20 ml of PBS+2% FBS followed by centrifugation at 300g for 8 min. Two further washes with PBS+2% FBS were performed. After the third wash, the cell pellet was resuspended in red blood cell lysis buffer (Abcam, ab204733) for 10 min followed by centrifugation at 400g for 5 min. The pellet was then resuspended in 10 ml of PBS+2% FBS and centrifuged at 120g for 10 min, and the final PBMC pellet was resuspended in RPMI 1640 (Sigma Aldrich). Isolated PBMCs were seeded at a density of 100,000 cells per well in V-bottom 96-well plates and treated with 0.7 μM or 1.4 μM hOTC-ASOe1-1a or NTC. After 24 hours, plates were centrifuged at 1200 rpm for 5 min and supernatants were collected for cytokine analysis. Human TNFα, IL6, IL1β, IFNα and IFNβ were quantified using the Luminex platform in collaboration with Dana Farber Cancer Institute.

[0300] As shown in FIG. 23, treatment of cells with hOTC-ASOe1-1a did not induce cytokine release by PBMCs.

[0301] Example 4: Tiling and optimization of mouse OTC regRNA-targeting ASOs Mouse ASOs targeting additional mouse regRNAs were generated and tested. The ASOs synthesized and characterized were mOTC-ASOe-3, mOTC-ASOe-4, mOTC-ASOe-5, and mOTC-ASOe-6.

[0302] Newly synthesized mouse ASOs were tested in mouse primary hepatocytes as described above. Briefly, primary hepatocytes were seeded at 20,000 cells per well on day 0. On day 2, cells were treated with 10, 5, 2.5, 1.25 or 0.625 μM mouse ASOs. Cells were incubated for 2 days and lysates were harvested for mRNA analysis on day 2 after treatment. Taqman probe Mm01288053_m1 was used for mouse OTC expression. Ppia and Hprt were used as housekeeper genes for gene expression normalization. Statistics were performed using one-way ANOVA in Prism (GraphPad).

[0303] As shown in Figure 24A, the new mouse ASO increased mouse Otc expression in a dose-dependent manner.

[0304] Terminal GalNAc was conjugated to mOTC-ASOe-3 to obtain ASO CO-4474. This ASO was then administered to OTC-deficient mice (OTC def Briefly, ten male B6EiC3Sn a / A-Otc mice were tested in an ammonium challenge assay. spf-ash 10 / J mice (homozygous) and 10 C57 WT mice were treated with ammonium once a week for 4 weeks and ASO was administered on days 1, 3, 5, 8, 10, 12, 15, and 17. Mice were administered either 100 mg / kg / week ASO or 50 mg / week ASO. Samples were collected at the end of the study for OTC mRNA quantification as previously described.

[0305] As shown in Figure 24B, Otc regRNA-targeting ASO CO-4474 inhibited Otc defIn mice, CO-4474 did not increase the mRNA of mouse OTC. Furthermore, Otc regRNA targeting ASO CO-4474 did not change other mouse UCD gene expression. However, as shown in Figure 24C, CO-4474 reduced ammonia to WT levels. Thus, mouse Otc ASO could rescue the Otc-deficient phenotype.

[0306] Example 5: regRNA-targeted ASOs lead to increased epigenomic H3K27 acetylation We next assessed the relative enhancer activity in human hepatocytes following ASO treatment.

[0307] Primary human hepatocytes from a single donor (HUM181371, Lonza) were cultured in vitro. 6 cells, 10cm 2 Collagen-coated plates were used to plate in plating medium, and plates were agitated every 15 min to ensure that cell density was uniform across the plate. Plating medium was changed to growth medium 4 h after plating, and growth medium was changed every 48 h for 6 days. At the day 4 medium change, 2 μM ASOs were diluted in growth medium. 7.5 × 10 6 Hepatocytes were treated with either non-targeting control (NTC) ASO or hOTC-ASOe1-10, which targets a (negative-strand) non-coding RNA (regRNA) transcribed from the OTC enhancer. Hepatocytes were treated with ASO for 48 h and crosslinked for 15 min by adding 11% formaldehyde (1% final) to the culture medium on day 6. Formaldehyde was quenched for 5 min by the addition of 200 mM glycine, and cells were scraped and washed three times with ice-cold 1x PBS.

[0308] Prior to crosslinking, small peripheral cell scrapings were collected for RNA isolation, cDNA synthesis (random hexamers) and qPCR analysis (OTC mRNA and PPIA TaqMan probes #Hs00166892_m1 and #Hs04194521_s1, respectively) to verify OTC mRNA upregulation in hOTC-ASOe1-10 treated hepatocytes compared to NTC ASO treatment. Cycle threshold (CT) values ​​were normalized to the endogenous control gene (i.e., PPIA) CT value (=dCT) and relative fold change was calculated by subtracting hOTC-ASOe1-10 dCT from NTC ASO dCT values ​​(Figure 25A).

[0309] H3K27ac chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) was performed on crosslinked hepatocyte samples treated with either NTC ASO or hOTC-ASOe1-10.Cell pellets were lysed for 10 min at 4°C with ice-cold LB1 (50 mM Hepes-KOH, pH 7.5, 140 mM NaCl, 140 mM NaCl, 1 mM EDTA, pH 8.0, 10% glycerol solution, 0.5% NP-40, 0.25% Triton X-100) and fresh protease inhibitors, followed by incubation for 10 min at 4°C with LB2 (10 mM Tris-HCL pH 8.0, 200 mM NaCl, 1 mM EDTA, pH 8.0, 1 mM EGTA, pH 8.0) and fresh protease inhibitors. Nuclei were centrifuged at 1350 rcf, 5 min, 4°C and resuspended in 1 mL sonication buffer (50 mM Hepes-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, pH 8.0, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS) + fresh protease inhibitors. Chromatin was sheared using a Covaris focused ultrasonicator and conditions (10 min time, fill level 5, duty cycle 5, peak input power 140, cycles / burst 200). Sheared chromatin was centrifuged at 20,000 rcf, 5 min, 4°C and the supernatant was transferred to a DNA low binding tube. 50 μL was saved for input. 5μg of anti-H3K27ac (abcam#ab4729) was pre-incubated with blocked (0.5% BSA / 1XPBS) Protein A conjugated magnetic beads the day before. Chromatin and bead-antibody bound complexes were combined and incubated overnight at 4°C with rotation.The next day, the bound chromatin-beads were washed 2X, 5 min, 4°C with 1 mL of each of the following buffers: sonication buffer, wash buffer 2 (50 mM Hepes-KOH pH 7.5, 350 mM NaCl, 1 mM EDTA pH 8.0, 1% Triton X-100, 0.2% Na-deoxycholate, 0.1% SDS), and wash buffer 3 (20 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCl, 0.5% Na-deoxycholate). Samples were washed 1X with TE + 0.2% Triton X-100 followed by 2X with TE. Chromatin was eluted and reverse crosslinked overnight at 65°C in SDS elution buffer (50 mM Tri-HCl pH 8.0, 10 mM EDTA pH 8.0, 1% SDS). ChIP samples were placed on a magnet and the eluted (reverse crosslinked chromatin) was transferred to a new tube. Samples (ChIP and input) were treated with RNase A for 30 min at 37°C, followed by proteinase K (20mg / mL) for 90 min at 55°C. DNA was purified by adding 600uL of phenol / chloroform / isoamyl alcohol to each sample and centrifuged at 16,000rcf, 5 min, 4°C using MaXtract high density gel tubes (Qiagen#129056). Supernatants were precipitated with Na acetate and ethanol overnight at -20°C, centrifuged at 20,000rcf, 4°C, washed with 1mL of 75% ethanol, and eluted in 25μL of nuclease-free water. chIP DNA and input DNA were subjected to library synthesis for high throughput sequencing using the NEBNext DNA Library Prep Kit according to the manufacturer's guidelines. Two biological replicates (four samples for each ASO treatment), each consisting of two technical replicates, were subjected to this assay.

[0310] ChIP-seq libraries were paired-end sequenced using Novoseq SP (150bp) and aligned to the human hg38 genome using Bowtie2, and the alignment files were processed with peaks called using SamTools and MACS2 (Figure 25B). Differential peaks in the OTC enhancer, OTC promoter and control regions (GAPDH, RPGR, TSPAN7) were identified between hOTC-ASOe1-10 and NTC ASO treatments through normalization methods and using DESeq2 (Figure 25C).

[0311] Figure 25A shows the upregulation of OTC mRNA by hOTC-ASOe1-10 compared to NTC ASO and untreated hepatocytes after 48 hours of treatment, indicating that ASO treatment was successful in upregulating OTC mRNA. These samples were used for subsequent H3K27ac ChIP-seq experiments.

[0312] Figure 25B shows genome browser track images for the OTC enhancer, OTC promoter and the adjacent gene RPGR. The enhancer and promoter regions are marked by histone H3K27 acetylation in both experimental hOTC-ASOe1-10 and NTC ASO-treated hepatocytes, indicating that the OTC enhancer is active in cultured human hepatocytes.

[0313] Figure 25C shows fold change (FC) quantification of H3K27ac epigenetic marks for hOTC-ASOe1-10 treatment compared to non-targeted ASO treatment and false discovery rate (FDR). The data show that 48 hours of treatment with hOTC-ASOe1-10 significantly increases histone acetylation (FC, 1.72-1.93) in the OTC enhancer compared to non-targeted ASO treatment. Negative control regions such as GAPDH, adjacent genes, RPGR, and TSPAN7 promoter do not show significant increased H3K27ac deposition. Without wishing to be bound by theory, the results suggest that the observed epigenetic effect from hOTC-ASOe1-10 (increased H3K27ac) is specific to the target region (OTC enhancer) predicted to regulate the OTC gene. Thus, without wishing to be bound by theory, the ASOs described herein regulate OTC gene expression by altering the epigenomic signature in the OTC enhancer.

[0314] Example 6: ASO treatment does not alter chromatin accessibility at the OTC enhancer We next assessed whether hOTC-ASOe1-10 binding of regRNA directly or indirectly increased chromatin accessibility at enhancers targeted by the ASO.

[0315] Primary human hepatocytes from a single donor (HUM181371, Lonza) were cultured in vitro. Plating medium was changed to growth medium 4 h after plating and growth medium was changed every 48 h for 6 days. On day 5, medium was changed and 2 μM ASOs were diluted in growth medium. Hepatocytes were treated with either non-targeting ASOs or hOTC-ASOe1-10 for 24 h. ATAC-seq optimized for primary human hepatocytes in monoculture was performed using the Omni-ATAC protocol. Following on-plate DNase treatment, hepatocytes were detached and enriched for viable cells using a magnetic Dead Cell Removal Kit (Miltenyi #130-090-101). Approximately 50,000 viable cells per replicate were used for the Omni-ATAC protocol. Three technical replicates were created per treatment.

[0316] ATAC-seq libraries were paired-end sequenced using Novoseq SP (150 bp) and aligned to the human genome hg38. Aligned reads were processed accordingly to identify accessible chromatin regions using the MACS2 pipeline described in the methods used in Corces et al., 2017.

[0317] Figure 26 shows accessible chromatin regions in the OTC promoter and the enhancer and adjacent RPGR promoter (shown as boxed regions). These results indicate that hOTC-ASOe1-10 does not cause changes in chromatin accessibility in the OTC enhancer or promoter, suggesting that the ASO acts downstream of transcription factor (activator) binding.

[0318] Example 7: ASO effect on regRNA precedes OTC mRNA transcription burst We next assessed the temporal response of regRNA activation upon ASO treatment with induction of OTC mRNA, as well as activation of the enhancer histone modification “memory.”

[0319] Primary human hepatocytes from a single donor (HUM181371, Lonza) were cultured in vitro. Plating medium was changed to growth medium 4 h after plating and growth medium was changed every 48 h for 6 days. On days 4-5, cells were treated at various time points (noted in Figure 27A-C) with 5 μM ASOs diluted in growth medium before harvesting. All wells were harvested simultaneously using cell lysis buffer for subsequent RNA isolation (MagMax MirVana, ThermoFisher), cDNA synthesis (random hexamer), crystal digital PCR (cdPCR; regRNA detection) and quantitative-PCR (qPCR; mRNA detection). Experiments were performed in biological triplicates, each with technical triplicates.

[0320] cdPCR was performed using the naica® Crystal Digital PCR™ System from Stilla Technologies. The concentration of regRNA was determined using a custom TaqMan assay and normalized to the endogenous control HPRT1 (TaqMan assay #4326321E, ThermoFisher). Relative fold changes were calculated by normalizing to NTC ASO-treated cells at each time point.

[0321] Quantitative PCR was performed using TaqMan probe #Hs00166892_m1 specific for OTC mRNA, and each value was normalized to the endogenous control PPIA (TaqMan endogenous control assay #4326316E, Thermofisher). Relative fold changes were calculated by normalizing to NTC ASO-treated cells at each time point. Technical triplicates were averaged for each biological triplicate, and the values ​​were plotted in a bar graph (n=2 biological). Each error bar represents the standard deviation.

[0322] H3K27ac ChIP followed by qPCR was performed on cultured primary hepatocytes as described in Example 5 above, with the difference that 5 μM ASO was used, treated on either day 4 or day 5, and harvested on day 6. ChIP-qPCR experiments were performed in biological series and duplicates (24 and 48 hours, respectively). qPCR was performed using SYBR and primers designed to amplify the genomic region of the OTC enhancer. Values ​​plotted are the relative fold change of hOTC-ASOe1-10 treated hepatocytes normalized to NTC ASO treated hepatocytes.

[0323] It has been shown that eRNA generated at the enhancer (regRNA) is transcribed bidirectionally and enhancer activity correlates with the amount of transcribed eRNA. The relative expression levels of both regRNAs transcribed from the OTC enhancer were obtained over time after ASO treatment (Figures 27A and 27B).

[0324] Figure 27A shows the relative levels of target (negative strand) regRNA over time following ASO treatment. Effects are measured as early as 2 hours after treatment, with the effect size decreasing by 8-16 hours. Upregulation is observed again at 18 hours (red arrowheads), confirming that these loci are upregulated in a manner consistent with previous studies in the literature. 6 These results suggest that the IL-16 receptor agonist undergoes a transcriptional burst as previously described.

[0325] FIG. 27B shows a similar effect on the non-targeted (plus) strand with a bimodal upregulation at early (1 hr) and late (18 hr) times (red arrowheads).

[0326] Figure 27C shows the effect of OTC mRNA over time after hOTC-ASOe1-10 ASO treatment. OTC mRNA is upregulated at 12 hours and again after 24 / 48 hours (arrowhead). This effect mimics a similar transcriptional "burst" phenomenon observed with regRNA (negative and positive). As expected, regRNA upregulation precedes OTC mRNA upregulation, indicating that the effect of increasing regRNA concentration results in increased OTC mRNA levels.

[0327] H3K27ac ChIP-qPCR results (Figure 27D) show that H3K27ac is deposited after hOTC-ASOe1-10 ASO treatment (24 hours), indicating that the effect on RNA precedes epigenetic changes at this enhancer. hOTC-ASOe1-10 treatment results in an increase in H3K27ac ChIP signal from 24 to 48 hours. Without wishing to be bound by theory, these results suggest that acetylation of residue K27 on histone H3 may be important for maintaining enhancer activity after initial regRNA / mRNA induction. A temporal model of the transcriptional and chromatin response to OTC ASO is shown in Figure 27E.

[0328] Example 8: Negative regulator protein binding is reduced by ASO treatment Next, we evaluated the perturbation of repressor protein complex interactions at the OTC enhancer after treatment with hOTC-ASOe1-10 in human hepatocytes. No significant changes in chromatin accessibility were observed with hOTC-ASOe1-10 treatment (Figure 26). Without wishing to be bound by theory, this indicated that any effects could be due to the replacement of other proteins, such as negative regulators.

[0329] Candidate negative regulators were selected using publicly available ENCODE ChIP-seq data from HepG2 cells. Briefly, ENCODE transcription factor (TF) data in HepG2 cells was filtered for TF occupancy at the OTC enhancer, and further filtering criteria eliminated all TFs not related to negative control mechanisms. A total of five negative regulator proteins were found to bind to the OTC enhancer in HepG2 cells (ARID1, BCL6, HDAC1, HDAC5, and NCOR1). SP1 is a transcription factor involved in general transcriptional activation, and was found bound to the OTC enhancer and used as a control.

[0330] Primary human hepatocytes from a single donor (HUM181371, Lonza) were cultured in vitro. 6 cells, 10cm 2 Collagen-coated plates were used to plate in plating medium, and plates were agitated every 15 min to ensure that cell density was uniform across the plate. Plating medium was changed to growth medium 4 h after plating, and growth medium was changed every 48 h for 6 days. On day 5, medium was changed and 5 μM ASOs were diluted in growth medium. 1.5 × 10 7 100 hepatocytes were cultured in a 2x10cm plate containing OTC enhancer. 2 Hepatocytes were treated with either NTC ASOs or hOTC-ASOe1-10 targeting (negative-strand) non-coding RNAs (regRNAs) transcribed from 100-well plate (100-well plate). Hepatocytes were treated with specific ASOs for 24 h and crosslinked for 15 min by adding 11% formaldehyde (1% final) to the culture medium on day 6. Formaldehyde was quenched for 5 min by addition of 200 mM glycine, and cells were scraped and washed 3x with ice-cold 1x PBS.

[0331] Prior to crosslinking, small peripheral cell scrapings were collected for RNA isolation to confirm upregulation of OTC mRNA in hOTC-ASOe1-10 treated hepatocytes compared to NTC ASO treatment as described in Example 1 (Figure 1A).

[0332] Hepatocytes were treated with ASOs for 24 h followed by ChIP qPCR performed in biological triplicates for each repressor TF ChIP'd.

[0333] ChIP and subsequent qPCR for each respective negative regulator was performed on cultured primary hepatocytes as described in Example 1 (Figure 1B) using specific antibodies against ARID1, BCL6, HDAC1, HDAC5, NCOR1 and SP1 (sc-32761X, PA527390, 40967ACTMOTIF, 40970ACTMOTIF, #A301145A, sc-17824X, respectively). ChIP-qPCR experiments were performed in biological triplies. qPCR was performed using SYBR and primers designed to amplify the genomic region of the OTC enhancer. Values ​​plotted are the relative fold change of hOTC-ASOe1-10 treated hepatocytes normalized to NTC ASO treated hepatocytes.

[0334] Values ​​plotted in Figures 28A and 28B are the relative fold change of hOTC-ASOe1-10 compared to NTC ASO (n=3) and error bars indicate standard deviation.

[0335] We performed rChIP-qPCR to assess the requirement of RNA for target protein-chromatin interactions. The assay was performed using a standard ChIP protocol with an added Rnase A treatment step after immunopurification of chromatin-protein complexes.

[0336] Figure 28A shows the relative loss of binding for the indicated negative regulators. Of the five negative regulators, only HDAC5 and NCOR showed reduced binding at the OTC enhancer in hepatocytes treated with hOTC-ASOe1-10 compared to NTC ASO treatment. This suggests that hOTC-ASOe1-10 binding to regRNAs inhibits (directly or indirectly) these regRNAs and associated chromatin (enhancers) from interacting with repressor complexes that include HDAC5 and NCOR.

[0337] Figure 28B shows that negative regulators do not require RNA molecules to bind to their targets.When crosslinked chromatin is treated with RNase to degrade RNA, the binding of HDAC5 and NCOR1 is not reduced at OTC enhancer in hepatocytes (no treatment).This result does not suggest that regRNA does not interact with repressor proteins, but rather that interaction is not essential for their recruitment to OTC enhancer.

[0338] Example 9: Knockdown of the repressor complex reduces the effect of ASO treatment on OTC mRNA upregulation We next assessed knockdown of the bound repressor complex at the OTC enhancer, which reduces the effects observed with ASO treatment.

[0339] Primary human hepatocytes from a single donor (HUM181371, Lonza) were cultured in vitro using 48-well collagen-coated tissue culture plates. Plating medium was changed to growth medium 4 hours after plating and growth medium was changed every 48 hours for 6 days. On day 3, cells were transfected with 10 nM siRNA targeting HDAC5, NCOR1 (Dharmacon M-003498-02-0005 and M-003518-01-0005, respectively) for 18 hours using Lipofectamine RNAiMax and the manufacturer's recommended protocol (Thermofisher, 13778150). The medium was changed the next day (day 4), either 5 μM NTC ASO or hOTC-ASOe1-10 was diluted in growth medium and cultured for 48 h, and hepatocytes were collected for RNA isolation (MagMax MirVana kit, ThermoFisher #A27828), cDNA synthesis using random hexamers, and qPCR analysis to assess knockdown efficiency and effect on OTC mRNA (TaqMan probes #Hs01094541_m1, Hs00608351_m1, Hs00166892_m1). Knockdown experiments combined with ASO treatment were performed in biological triplicates, each with three technical replicates (per treatment). Values ​​plotted on the graphs are the average of technical replicates for each biological experiment (n=3).

[0340] Knockdown efficiency of HDAC5 or NCOR1 siRNA treatment was determined by normalizing the relative CT value of each sample to the endogenous control (PPIA) and calculating the fold change based on samples without siRNA treatment.

[0341] To understand the effect of siHDAC5 or siNCOR1 on hOTC-ASOe1-10 activity, all treatments were normalized to NTC ASO within each siRNA experiment to reduce confounding effects of knockdown.

[0342] Values ​​plotted in Figures 29A-C are the average of technical triplicates from three biological replicate experiments. Each error bar represents the standard deviation. P-values ​​are calculated by unpaired Student's t-test using the average of each biological replicate (n=3).

[0343] Treatment with siHDAC5 or siNCOR1 resulted in at least a 50% reduction in target mRNA levels compared to untreated hepatocytes, as shown in Figure 29A.

[0344] Figure 29B shows the effect of HDAC5 or NCOR1 knockdown on OTC mRNA.The siRNA treatment of either of these factors leads to the increase of OTC mRNA expression in hepatocytes, demonstrating that these complexes are involved in the repression of OTC mRNA.The relaxation of this repression mechanism in OTC enhancer causes the marginal increase of basal OTC level.

[0345] The effect of knockdown on hOTC-ASOe1-10 is shown in Figure 29C. hOTC-ASOe1-10 significantly (p-value=0.0154) upregulated OTC mRNA (FC=1.81, without siRNA treatment). Hepatocytes treated with siHDAC5 or siNCOR1 showed significant upregulation of OTC mRNA with hOTC-ASOe1-10 compared to NTC ASO (FC=1.41 and 1.28, respectively). This experiment shows that hOTC-ASOe1-10 has an effect on OTC mRNA when repressor complex protein is knocked down, since OTC mRNA level is already slightly increased.

[0346] Without being bound by theory, under normal homeostatic cellular conditions, the levels of regRNA and mRNA transcribed from the OTC enhancer and gene body, respectively, are low. Negative regulators such as HDAC5 and NCOR1 likely bind to the enhancer and regulate its low activity, as well as transcriptional activators are found priming the locus. hOTC-ASOe1-10 treatment leads to an increase in regRNA levels, likely through inhibition of repressor complex binding. This activation of the OTC enhancer promotes a positive transcriptional response in the OTC gene, thus resulting in a transcriptional burst in the OTC enhancer and promoter (Figure 30).

[0347] Example 10: Characterization of ASOs in Non-Human Primates Materials and Methods 15N-Ammonium chloride was obtained from Cambridge isotope (Tewksbury, Mass.).

[0348] Ammonia measurements and urea production in NHPs Ammonia challenge and urea production assays in cynomolgus monkeys [NHPs] were performed in the fasted state, i.e., with overnight food withdrawal, prior to ammonia challenge. 15N-ammonium chloride solution was injected subcutaneously into NHPs, multiple blood draws were performed over 0-120 min, and plasma was immediately obtained by centrifugation. An aliquot of plasma was shipped at 4°C to IDEXX for ammonia level measurement. Another aliquot was snap frozen and shipped to NovaBioAssay (Woburn, MA) for 15N-urea / total urea level measurement.

[0349] ASO treatment of NHPs Male cynomolgus monkeys (2-4 years old) were given a single subcutaneous injection of 50 mg / kg ASO on day 0 and a second dose on day 21. PBS as negative control.

[0350] result CO-5318 (hOTC-ASOe1-1as) and CO-5319 (hOTC-ASOe2-2w) reduced ammonia and increased urea in NHPs (FIG. 31). Thus, ASOs demonstrate therapeutic efficacy in NHPs.

[0351] Example 11: Characterization of ASOs in humanized mice Materials and Methods Ammonia measurements and urea production for the study of humanized Yecuris FRG mice Ammonia challenge and urea production assays in female liver humanized Fah- / -Rag2- / -Il2rg- / -[FRG] mice on a C57Bl / 6 background were repopulated with healthy human hepatocytes and performed in fasting state, i.e., overnight food withdrawal, before ammonia challenge. After overnight fasting on days 1, 8, 15, and 22 (after the last collection), animals were challenged with 15NH4Cl (15N-ammonia) by intraperitoneal injection. Thirty minutes later, urine and blood (processed to plasma) were collected. An aliquot of plasma was shipped at 4 degrees to IDEXX (North Grafton, MA) to measure ammonia levels. Another aliquot was snap frozen and shipped to NovaBioAssay (Woburn, MA) to measure 15N-urea / total urea levels.

[0352] ASO treatment for mouse studies Five-month-old female humanized Yecuris FRG mice were subcutaneously injected with 50 mg / kg / week of ASO on days 8, 12, 15, and 19. PBS was used as a control.

[0353] OTC spf / ash Ammonia measurement and urea production for mouse studies Wild type C57BL / 6J[WT] and a / A-Otc spf-ashAmmonia challenge and urea production assays in both / J,[OTCD] were performed in fasting state, i.e., overnight food withdrawal, before ammonia challenge. 15N-ammonium chloride solution was subcutaneously injected into WT and OTOCD mice, blood was collected 30 min after ammonium chloride injection, and plasma was immediately obtained by centrifugation. An aliquot of plasma was shipped at 4 degrees to IDEXX to measure ammonia levels. Another aliquot was snap frozen and shipped to NovaBioAssay (Woburn, MA) to measure 15N-urea / total urea levels.

[0354] ASO treatment for mouse studies Male C57BL / 6J[WT] and a / A-Otc spf-ash / J,[OTCD] (6–7 weeks old) were injected subcutaneously with ASO at either 50 or 100 mg / kg / week on days 1, 3, 5, 8, 10, 12, 15, and 17. PBS served as a negative control.

[0355] Taqman probes (all from Thermofisher) TIFF2024534214000047.tif87128

[0356] result NH4Cl challenge was given to humanized mice to measure the effect of ASO on urea production. As shown in Figure 32, both CO-5318 and CO-5319 alter OTC and CPS1 mRNA expression. However, as shown in Figure 33, CO-5318 and CO-5319 treatment in humanized mice showed a decrease in ammonia and a corresponding increase in urea over time. Two-way ANOVA, *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001.

[0357] Incorporation by Reference Unless otherwise indicated, the entire disclosures of each of the patent documents and scientific articles referred to herein are incorporated by reference for all purposes.

[0358] Equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are therefore to be considered in all respects as illustrative and not limiting of the invention described herein. The scope of the invention is therefore indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. An antisense oligonucleotide (ASO) targeting a regulatory RNA (regRNA) of human ornithine transcarbamylase (OTC), the ASO comprising a nucleotide sequence complementary to at least 8 consecutive nucleotides of any one of SEQ ID NOs: 1-4 or 1077.

2. 2. The ASO of claim 1, wherein the regRNA has the nucleotide sequence of SEQ ID NO: 1, and the ASO comprises the nucleotide sequence of any one of SEQ ID NOs: 18, 26, 39, 6-14, 19-25, 27-35, 41, 75-78, 87-124, or 143-892.

3. 2. The ASO of claim 1, wherein the regRNA has the nucleotide sequence of SEQ ID NO: 2, and the ASO comprises the nucleotide sequence of SEQ ID NO: 70, 15-17, 36-38, 64-69, 71-74, 125-142, or 893-1029.

4. The ASO described in claim 1, wherein the ASO comprises a nucleotide sequence of SEQ ID NO: 70 or 17.

5. The ASO of claim 1, wherein the ASO is 50, 40, 30, or 25 nucleotides in length or less.

6. The ASO of claim 1, wherein the ASO comprises a nucleotide containing one or more chemical modifications.

7. The ASO of claim 6, wherein at least 3, 4, or 5 nucleotides at the 5' end and at least 3, 4, or 5 nucleotides at the 3' end of the ASO comprise ribonucleotides having one or more chemical modifications.

8. 7. The ASO of claim 6, wherein the one or more chemical modifications comprise a nucleotide sugar modification comprising one or more of 2'-O-C1-4 alkyl, e.g., 2'-O-methyl (2'-OMe), 2'-deoxy (2'-H), 2'-O-C1-3 alkyl-O-C1-3 alkyl, e.g., 2'-methoxyethyl (2'-MOE), 2'-fluoro (2'-F), 2'-amino (2'-NH2), 2'-arabinosyl (2'-arabino) nucleotide, 2'-F-arabinosyl (2'-F-arabino) nucleotide, 2'-locked nucleic acid (LNA) nucleotide, 2'-amide bridged nucleic acid (AmNA), 2'-unlocked nucleic acid (ULNA) nucleotide, L-sugar (L-sugar), 4'-thioribosyl nucleotide, constrained ethyl (cET), 2'-fluoro-arabino (FANA), or thiomorpholino.

9. 7. The ASO of claim 6, wherein the one or more chemical modifications comprise an internucleotide linkage modification comprising one or more of phosphorothioate (PS or (P(S))), phosphoramidate (P(NR1R2), e.g., dimethylaminophosphoramidate (P(N(CH3)2)), phosphonocarboxylate (P(CH2)nCOOR), e.g., phosphonoacetate (PACE or (P(CH2COO-)), thiophosphonocarboxylate ((S)P(CH2)nCOOR), e.g., thiophosphonoacetate (thioPACE or ((S)P(CH2COO-)), alkylphosphonate (P(C1-3 alkyl), e.g., methylphosphonate-P(CH3), boranophosphonate (P(BH3)), or phosphorodithioate (P(S)2).

10. The one or more chemical modifications may be 2-thiouracil (2-thioU), 2-thiocytosine (2-thioC), 4-thiouracil (4-thioU), 6-thioguanine (6-thioG), 2-aminoadenine (2-aminoA), 2-aminopurine, pseudouracil, hypoxanthine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5-methylcytosine (5-methylC), 5-methyluracil (5-methylU), 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5,6-dehydrouracil, 5-propynylcytosine ...

7. The ASO of claim 6, comprising nucleobase modifications comprising one or more of 5-aminoallyluracil (5-allylU), 5-propynyluracil, 5-ethynylcytosine, 5-ethynyluracil, 5-allyluracil (5-allylU), 5-allylcytosine (5-allylC), 5-aminoallyluracil (5-aminoallylU), 5-aminoallyl-cytosine (5-aminoallylC), abasic nucleotides, Z bases, P bases, unstructured nucleic acids (UNA), isoguanine (isoG), isocytosine (isoC), glycerol nucleic acid (GNA), or thiophosphoramidate morpholino (TMO).

11. 7. The ASO of claim 6, wherein the one or more chemical modifications comprise 2'-O-methoxyethyl, 5-methyl on cytidine, locked nucleic acid (LNA), phosphodiester (PO) internucleotide linkage, or phosphorothioate (PS) internucleotide linkage.

12. The ASO of claim 6, which does not contain more than 10 consecutive nucleotides of unmodified DNA.

13. The ASO of claim 12, which does not contain deoxyribonucleotides.

14. The ASO of claim 6, which does not contain unmodified ribonucleotides.

15. An ASO described in claim 1, further comprising a ligand portion.

16. 7. The ASO of claim 6, comprising 10 or more contiguous nucleotides of unmodified DNA flanked by at least three nucleotides of modified ribonucleotides at each of the 5' and 3' ends.

17. The ASO of claim 6, wherein each cytidine in the ASO is modified by 5-methyl.

18. A pharmaceutical composition comprising the ASO of claim 1 and a pharmaceutically acceptable carrier or excipient carrier.

19. A method for increasing transcription of OTC in human cells, the method comprising contacting the cells with the ASO described in claim 1.

20. A pharmaceutical composition comprising an effective amount of the ASO of claim 1 for treating a urea cycle disorder in a subject in need of such treatment.

21. An antisense oligonucleotide (ASO) targeting a regulatory RNA (regRNA) of mouse ornithine transcarbamylase (OTC), the ASO comprising a nucleotide sequence complementary to at least eight consecutive nucleotides of any one of SEQ ID NOs: 1073 to 1076.

22. An antisense oligonucleotide (ASO) targeting a regulatory RNA (regRNA) of mouse Serping1, the ASO comprising a nucleotide sequence complementary to at least eight consecutive nucleotides of either SEQ ID NO: 5 or 1078.

Images