Methods for regulating gene transcription using antisense oligonucleotides targeted to regulatory RNAs - Patents.com

Abstract

Described herein are methods for regulating gene transcription using antisense oligonucleotides (ASOs) that target regulatory RNAs, such as promoter-associated RNAs and enhancer RNAs. These methods are useful for regulating the levels of gene products, for example, increasing the expression of carbamoyl phosphatase synthase 1 (CPS1), 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 / 292,920, filed December 22, 2021, and U.S. Provisional Application No. 63 / 308,373, filed February 9, 2022, both of which are incorporated by reference in their entireties 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 on December 21, 2022, is named CTC-028WO_140628340009.xml and is 925,586 bytes in size.

[0003] FIELD OF THEINVENTION The present disclosure relates to methods of upregulating or downregulating gene transcription using antisense oligonucleotides (ASOs) that target regulatory RNAs, such as promoter-associated RNAs and enhancer RNAs (e.g., CPS1 regulatory RNA). [Background technology]

[0004] background 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 undruggable 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] overview In one aspect, provided herein is an antisense oligonucleotide (ASO) complementary to at least 8 consecutive nucleotides of a regulatory RNA (regRNA) of carbamoyl phosphate synthase 1 (CPS1), wherein the regRNA has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 49, 50, 69-79, and 89-90.

[0008] 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.

[0009] 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.

[0010] In some embodiments, the regulatory RNA has a nucleotide sequence of SEQ ID NO:49, and the ASO comprises the nucleotide sequence of SEQ ID NO:1.

[0011] In some embodiments, the regulatory RNA has a nucleotide sequence of SEQ ID NO:49 and the ASO comprises the nucleotide sequence of SEQ ID NO:2.

[0012] In some embodiments, the regulatory RNA has a nucleotide sequence of SEQ ID NO:49 and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:1-3.

[0013] In some embodiments, the regulatory RNA has a nucleotide sequence of SEQ ID NO:50, and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:4-15.

[0014] In some embodiments, the regulatory RNA has a nucleotide sequence of SEQ ID NO: 89 and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 487-574.

[0015] In some embodiments, the regulatory RNA has a nucleotide sequence of SEQ ID NO: 90 and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 575-662.

[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 a polynucleotide that comprises one or more chemical modifications. In some embodiments, the ASO comprises an RNA polynucleotide that comprises one or more chemical modifications. In some embodiments, the ASO comprises RNA and DNA polynucleotides that comprise 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-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") 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 bond 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-methylcytidine, locked nucleic acid (LNA), and phosphorothioate internucleotide linkages.

[0023] In some embodiments, the ASO comprises eight or more contiguous nucleotides of unmodified DNA flanked by at least three nucleotides of modified ribonucleotides on each of the 5' and 3' ends.

[0024] In some embodiments, the ASO comprises the nucleotide sequence and / or chemical modifications of any one of SEQ ID NOs: 16, 22-27, 31-39, 403-412, 414-415, 417-432, 435-480, or 482-486.

[0025] In some embodiments, the ASO comprises the nucleotide sequence of SEQ ID NO:409 and / or chemical modifications.

[0026] In some embodiments, the ASO further comprises at least one phosphorothioate internucleotide linkage.

[0027] In some embodiments, the ASO comprises the nucleotide sequence of SEQ ID NO:403-406, 409-415, 417-424, 470, 473, 477, 480 or 486.

[0028] In some embodiments, the chemical modification is cET.

[0029] In some embodiments, the ASO comprises the nucleotide sequence of SEQ ID NO:425-432 or 435-442.

[0030] In some embodiments, the chemical modification is LNA.

[0031] In some embodiments, the ASO comprises the nucleotide sequence of SEQ ID NO:443-458.

[0032] In some embodiments, the chemical modifications are LNA and 2'-O-methoxyethyl.

[0033] In some embodiments, the ASO comprises the nucleotide sequence of SEQ ID NO: 400, 408, 460, 462, 464-466, 468, 469, 471, 472, 475, 476, or 479.

[0034] In some embodiments, the ASO does not contain 8 or more contiguous nucleotides of unmodified DNA.

[0035] In some embodiments, the ASO does not include unmodified ribonucleotides.

[0036] In some embodiments, the ASO does not contain deoxyribonucleotides.

[0037] In some embodiments, the length of the ASO is 2×n+4 nucleotides (where n is an integer greater than or equal to 8), the nucleotide at position 2×m is an LNA-modified ribonucleotide (where m is an integer from 1 to n), and the remaining nucleotides are 2'-O-methoxyethyl-modified ribonucleotides.

[0038] In some embodiments, the ASO comprises the nucleotide sequence of SEQ ID NO: 393 or 394.

[0039] In some embodiments, the length of the ASO is 3×n+2 nucleotides (where n is an integer greater than or equal to 4), 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.

[0040] In some embodiments, the ASO comprises the nucleotide sequence of SEQ ID NO: 392 or 395.

[0041] 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 a ribonucleotide modified with LNA (where m is an integer from 1 to n), and the five nucleotides at the 3' and 5' positions are ribonucleotides modified with 2'-O-methoxyethyl.

[0042] In some embodiments, the ASO comprises the nucleotide sequence of SEQ ID NO: 19 or 20.

[0043] In some embodiments, the length of the ASO is 4 x n nucleotides (n is an integer greater than or equal to 3), the nucleotide at the 4 x m position is an LNA-modified ribonucleotide (m is an integer from 1 to n), and the five nucleotides at the 3' and 5' positions are 2'-O-methoxyethyl-modified ribonucleotides.

[0044] In some embodiments, the ASO comprises the nucleotide sequence of SEQ ID NO:21.

[0045] In some embodiments, the length of the ASO is 4×n+4 nucleotides (where n is an integer greater than or equal to 3), the nucleotide at position 4×m is an LNA-modified ribonucleotide (where m is an integer from 1 to n), and the remaining nucleotides are 2'-O-methoxyethyl-modified ribonucleotides.

[0046] In some embodiments, the ASO comprises the nucleotide sequence of SEQ ID NO: 396 or 397.

[0047] In some embodiments, each ribonucleotide of the ASO is modified with 2'-O-methoxyethyl.

[0048] In some embodiments, the ASO comprises the nucleotide sequence of any one of SEQ ID NOs: 17, 28-30, and 40-48.

[0049] In some embodiments, each nucleotide of the ASO is a 2'-O-methoxyethyl modified ribonucleotide.

[0050] In some embodiments, the ASO comprises the nucleotide sequence of any one of SEQ ID NOs: 17, 28-30, and 40-48.

[0051] In some embodiments, each cytidine in the ASO is modified with 5-methyl.

[0052] 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.

[0053] In some embodiments, the ASO comprises the nucleotide sequence of SEQ ID NO: 398 or 399.

[0054] In some embodiments, the ASO further comprises a GalNAc moiety, optionally a GalNAc3 moiety.

[0055] In some embodiments, the ASO further comprises a biotin or cholesterol moiety.

[0056] In some embodiments, each cytidine in the ASO is modified with 5-methyl.

[0057] In some embodiments, the regRNA is an enhancer RNA (eRNA).

[0058] In another aspect, provided herein is a pharmaceutical composition comprising an ASO disclosed herein and a pharma- ceutically acceptable carrier or excipient carrier.

[0059] In another aspect, provided herein is a method for increasing transcription of CPS1 in a human cell, the method comprising contacting the cell with an ASO disclosed herein or a pharmaceutical composition disclosed herein.

[0060] In some embodiments, the cell is a hepatocyte.

[0061] In some embodiments, the ASO increases the amount of regulatory RNA in a cell compared to a cell that has not been contacted with the ASO or pharmaceutical composition.

[0062] In some embodiments, the ASO increases the stability of a regulatory RNA in a cell compared to a cell that has not been contacted with the ASO or pharmaceutical composition.

[0063] In another 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 disclosed herein or a pharmaceutical composition disclosed herein.

[0064] In some embodiments, the urea cycle disorder is CPS1 deficiency.

[0065] In some embodiments, the urea cycle disorder is hyperammonemia.

[0066] In some embodiments, the ASO increases the amount of regulatory RNA in a cell of the subject (e.g., compared to a cell (e.g., a similar cell derived from the subject) that has not been contacted with the ASO or pharmaceutical composition).

[0067] In some embodiments, the ASO increases the stability of a regulatory RNA in a cell of a subject (e.g., compared to a cell (e.g., a similar cell derived from the subject) that has not been contacted with the ASO or pharmaceutical composition).

[0068] In some embodiments, the cell is a hepatocyte. [Brief description of the drawings]

[0069] [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 in the antisense direction from its own downstream promoter, so that the transcript at least partially overlaps with the mRNA. In general, eRNA and paRNA upregulate gene expression, while NAT downregulates gene expression.

[0070] [Diagram 2] Analysis of mouse and human CPS1 ATAC-seq and H3K27Ac ChIP-SEQ for identification of regRNAs.

[0071] [Diagram 3] Relative CPS1 mRNA expression in hepatocytes treated with ASO and non-targeting control (NTC) is shown.

[0072] [Figure 4A] Schematic diagram of various human CPS1 ASOs with chemical modifications. Light grey indicates 2'-O-(2-Methoxyethyl) (2'-MOE) modifications. Dark grey indicates Locked Nucleic Acid (LNA) modifications. *C indicates 5-methyl on cytidine. [Figure 4B] Relative CPS1 mRNA expression in ASO-treated hepatocytes is shown.

[0073] [Figure 5A-1] Schematic diagrams of various human CPS1 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. Nucleotide sequences with specific chemical modifications shown in this diagram are assigned unique sequence identifiers. [Figure 5A-2] See description of Figure 5A-1. [Figure 5B] Schematic diagram of various mouse CPS1 ASOs with chemical modifications.

[0074] [Figure 6A] 1 shows CPS1 mRNA levels in wild-type and OTC-deficient hepatocytes treated with CPS1 ASO. [Figure 6B] Heatmaps of CPS1 and OTC mRNA levels are shown. [Figure 6C] Heatmaps of CPS1 and OTC mRNA levels are shown.

[0075] [Figure 7A] 1 shows OTC mRNA levels in wild-type and OTC-deficient hepatocytes treated with CPS1 ASO. [Figure 7B]Heatmaps of CPS1 and OTC mRNA levels are shown. [Figure 7C] Heatmaps of CPS1 and OTC mRNA levels are shown.

[0076] [Figure 8] FIG. 1 is a schematic diagram of in vivo ammonia challenge in CPS1 ASO-treated mice.

[0077] [Figure 9A] Relative CPS1 mRNA expression in mouse liver is shown. [Figure 9B] Plasma ammonia levels in treated mice are shown. [Figure 9C] The relative mRNA expression of other urea cycle genes in treated mice is shown. [Figure 9D] Shows mRNA expression of genes adjacent to the CPS1 locus after ASO treatment.

[0078] [Figure 10] 1 shows ammonia levels in OTC-deficient mice treated with CPS1 ASO.

[0079] [Figure 11] A shows the dose-dependent effect on plasma ammonia in vivo in male OTC-D mice (Otcspf / ash) after ASO treatment. B shows that there was a dose-dependent effect on urea in vivo in male OTC-D mice (Otcspf / ash) after ASO treatment. C shows that the reduction in ammonia correlated with the total amount of ASO administered.

[0080] [Figure 12]A shows that ASO treatment of CPS1 increased mouse OTC and CPS1 mRNA expression in OTC-deficient mice (1.26 FC for CPS1 and 1.43 FC for OTC). B shows that ASO treatment increased additional urea cycle genes such as Nags, Ass1, Asl, and Arg1. In both figures, WT C57 mouse mRNA is shown in the left bar, PBS-treated Otcspf-ash / J is shown in the middle bar, and ASO-treated Otcspf-ash / J is shown on the right.

[0081] [Figure 13] Figure 1 shows that mouse Otc-deficient livers exhibited high ammonia levels 30 min after ammonia treatment. Treatment with mouse CPS1 ASO reduced ammonia levels to WT levels.

[0082] [Figure 14-1] Schematic diagram of various human CPS1 ASOs with chemical modifications. Unique sequence identifiers are assigned to nucleotide sequences with specific chemical modifications shown in this figure. [Figure 14-2] See description of Figure 14-1. [Figure 14-3] See description of Figure 14-1.

[0083] [Figure 15] (A) shows that hCPS1-ASO-1x reduced plasma ammonia in vivo in OTCdef mice with humanized livers. (B) shows that hCPS1-ASO-1x increased urea in vivo in OTCdef mice with humanized livers. (C) shows that hCPS1-ASO-1g also increased OTC protein levels after treatment with 5 mg / kg and 20 mg / kg ASO.

[0084] [Figure 16] The indicated ASOs increased CPS1 mRNA and urea production in both healthy and OTCdef primary human hepatocytes.

[0085] [Figure 17] We show that hCPS1-ASO-1x reduced ammonia production in NHPs by a statistically significant amount compared to PBS treatment alone by day 27. We also show the AUC of ammonia both over time and per dose in the NHP experiments.

[0086] [Figure 18] It is shown that hCPS1-ASO-1x reduced ammonia in NHPs after administration of a single dose of 5 mg / kg for up to 5 weeks.

[0087] [Figure 19] Quantification of ammonia AUC on days 22, 29, and 36 in the NHP study is shown.

[0088] [Figure 20] 1 shows that the combination of hCPS1-ASO-1x and 13C-sodium acetate treatment enhanced urine production in NHPs on day 43 compared to hCPS1-ASO-1x alone. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0089] Detailed Description The present disclosure 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., carbamoyl phosphate synthase 1 (CPS1)), thereby treating diseases associated with abnormal gene expression (e.g., urea cycle disorders). Urea cycle disorders are reviewed in Haberle et al., 2012, Orphanet J. Rare Dis. 7:32, which is incorporated herein by reference in its entirety. Urea cycle disorders result in the accumulation of ammonia and other precursor metabolites, causing the development of hyperammonia and associated symptoms, such as cerebral edema, and associated signs of lethargy, anorexia, hyperventilation or hypoventilation, hypothermia, seizures, neurological posturing, and coma. Mild or partial deficiencies of urea cycle enzymes cause ammonia accumulation and elevated plasma ammonia concentrations. CPS1 deficiency is the most severe of the urea cycle disorders. Individuals with complete CPS1 deficiency rapidly develop hyperammonemia and are at risk for further hyperammonemic events.

[0090] I. Definition To facilitate the understanding of this application, a number of terms and phrases are defined below.

[0091] As used herein, the terms "a" and "an" mean "one or more" and include pluralities unless the context is inappropriate.

[0092] As used herein, the term "carbamoyl phosphate synthase 1" or "CPS1" refers to the protein of UniProt Accession No. P31327, and related isoforms and orthologs, as described in the applicable databases as of the priority date of this application, to the gene encoding said protein (e.g., NCBI Entrez Gene:1373), or to the mRNA encoding said protein (e.g., Accession Nos. NM_001122633.3, NM_001369256.1, NM_001369257.1, and NM_001875.5).

[0093] 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.

[0094] 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."

[0095] 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.

[0096] As used herein, the stability of regRNA is inversely correlated with the degradation rate of regRNA.If ASO increases the stability of regRNA, it will decrease the degradation rate of regRNA.If ASO reduces the stability of regRNA, it will increase 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.

[0097] 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.

[0098] 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, ameliorating, or eliminating, or ameliorating a symptom thereof.

[0099] 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.

[0100] 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).

[0101] 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 contemplated that there are compositions as 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.

[0102] 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.

[0103] II. 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. 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). paRNAs are RNAs transcribed from the promoter of a target gene in the antisense orientation (the transcript in the sense orientation is the mRNA of the target gene). They can be identified by similar methods, taking into account their specific location and orientation. In the human CPS1 gene, multiple separate eRNAs have been identified that transcribe from the same enhancer region. The nucleotide sequences of exemplary regRNAs are shown in Table 1 below. Any of these regRNAs are contemplated as the target regRNA of the ASO disclosed herein.

[0104] Table 1. Exemplary regRNAs TIFF2025500409000002.tif210165TIFF2025500409000003.tif225165TIFF2025500409000004.tif169165TIFF2025500409000005.tif115165TIFF2025500409000006.tif228165TIFF2025500409000007.tif187165TIFF2025500409000008.tif208165TIFF2025500409000009.tif203165TIFF2025500409000010.tif218165TIFF2025500409000011.tif213165TIFF2025500409000012.tif239165TIFF2025500409000013.tif187165TIFF2025500409000014.tif223165TIFF2025500409000015.tif225165TIFF2025500409000016.tif228165TIFF2025500409000017.tif203165TIFF2025500409000018.tif157165TIFF2025500409000019.tif193165TIFF2025500409000020.tif223165TIFF2025500409000021.tif230165TIFF2025500409000022.tif218165TIFF2025500409000023.tif198165TIFF2025500409000024.tif223165TIFF2025500409000025.tif115165TIFF2025500409000026.tif213165TIFF2025500409000027.tif223165TIFF2025500409000028.tif223165TIFF2025500409000029.tif189165TIFF2025500409000030.tif198165TIFF2025500409000031.tif223165TIFF2025500409000032.tif43165

[0105] The present disclosure describes ASOs that increase the amount or stability of target regRNA, thereby increasing the expression of target genes. This is different from previously described ASOs designed to inhibit eRNA (see, for example, PCT Application Publication No. WO2013 / 177248 and PCT Application Publication No. WO2017 / 075406). Without wishing to be bound by theory, it is hypothesized that the ability of ASOs to upregulate regRNA is due to the selection of target sequences in regRNA and / or chemical modification of ASOs.

[0106] ASO sequence As disclosed herein, ASOs that bind to sequences closer to the 5' or 3' end of target CPS1 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 target CPS1 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).

[0107] In certain embodiments, the ASO is no more than 25, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides in length. In certain embodiments, the ASO is no more than 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides in length. In certain embodiments, the ASO is at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0108] In certain embodiments, the ASO is designed to lack stable secondary structures formed within itself or between other identical ASO molecules, thereby increasing the amount of ASO in single-stranded form available for hybridization 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.

[0109] For example, ASOs have been designed to target human CPS1 eRNA. The nucleotide sequences of these ASOs are shown in Table 2 below.

[0110] Table 2. Exemplary ASO sequences targeting regRNA TIFF2025500409000033.tif207165TIFF2025500409000034.tif234165TIFF2025500409000035.tif234165 TIFF2025500409000036.tif234165TIFF2025500409000037.tif234165TIFF2025500409000038.tif234165 TIFF2025500409000039.tif234165TIFF2025500409000040.tif234165TIFF2025500409000041.tif234165 TIFF2025500409000042.tif234165TIFF2025500409000043.tif234165TIFF2025500409000044.tif192165

[0111] Table 3 provides additional chemical modifications of selected hCPS1-ASOs.

[0112] Table 3. Further chemical modifications of selected hCPS1-ASOs TIFF2025500409000045.tif187165TIFF2025500409000046.tif198165TIFF202 5500409000047.tif198165TIFF2025500409000048.tif198165TIFF20255004090 00049.tif198165TIFF2025500409000050.tif198165TIFF2025500409000051.t if198165TIFF2025500409000052.tif198165TIFF2025500409000053.tif151165

[0113] In some embodiments, hCPS1-ASO-317-492 (SEQ ID NOs: 487-662) contains pS bond, 5-methylcytosine, and a complete MOE nucleotide.

[0114] Hybridization and ΔG As used herein, the term "hybridizing" or "hybridizing" should be understood as the formation of hydrogen bonds between base pairs on opposing strands of two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid), 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), 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 m is 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. To have the potential to modulate its intended nucleic acid target by hybridization, the oligonucleotides of the present disclosure 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, e.g., less than −20 kcal / mol, e.g., 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.

[0115] 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, still with only 19 bases on each strand that are 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 would result in 94.7% complementarity, making the duplex region fully complementary. 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.

[0116] dsRNA comprises two RNA strands that are complementary and hybridize to form a duplex structure under the conditions in which the dsRNA is used. One strand of the dsRNA (antisense strand) comprises a region of complementarity that is substantially complementary to the target sequence, generally completely complementary. The target sequence may be derived from the sequence of a CPS1 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 the dsRNA may be included as a self-complementary region of a single nucleic acid molecule, as opposed to being on a separate oligonucleotide. In general, 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, 18-35, 1 8~34, 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, 22-49, 2 2~48, 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 , 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 disclosure.

[0117] 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 disclosure.

[0118] 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, hCPS1-ASO-1 can be chemically modified to include any one of the modifications hCPS1-ASO1-1a to hCPS1-ASO1-1g as shown in FIG. 5A. Such modifications can be, but are not limited to, 2'-O-(2-methoxyethyl) (2'-MOE, MOE), locked nucleic acid (LNA), 5-methyl on cytidine, 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 5-methyl. Exemplary ASOs that include chemical modifications are shown in Figure 5A and Figure 14.

[0119] 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.

[0120] 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). In some embodiments, all nucleotide bases are modified in the ASO. In certain embodiments, the chemical modification comprises a β-D-ribonucleoside, a 2'-modified nucleoside (e.g., 2'-O-(2-methoxyethyl) (2'-MOE), 2'-O-CH3, or 2'-fluoro-arabino (FANA)), a bicyclic sugar modified nucleoside (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 modification comprises a 2'-MOE and a phosphorothioate internucleotide linkage. 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 by 2'-MOE. In certain embodiments, each nucleotide of the ASO is modified by 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 phosphorothioate internucleotide linkage.

[0121] 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)).

[0122] Chemical structures may also be described by letters, where "M" indicates MOE, "d" indicates DNA, "L" indicates LNA, "=" indicates a phosphorothioate (PS) bond, "-" indicates a phosphodiester (PO) bond; "5C" indicates 5-methylcytosine, "ag" indicates GalNAc, "tg" or "TEG" indicates Teg-GalNAc, "ag" or "AlGal" indicates GalNAc, "BioTEG" indicates biotin, "CholTEG" indicates cholesterol, "c" indicates cET, and "^" indicates FANA.

[0123] For the avoidance of ambiguity, this LNA has the following formula: TIFF2025500409000054.tif64165 where B is a specific designated base.

[0124] Exemplary textual descriptions of selected ASOs are shown in Table 3, including corresponding Figures 5A and 14, which provide a visual representation of the modifications.

[0125] In some embodiments, the ASO comprises a sequence selected from the group consisting of SEQ ID NOs: 1-15, 91-391, or 487-662. In some embodiments, the ASO comprises a sequence and a chemical modification selected from the group consisting of SEQ ID NOs: 16-48, 392-480, or 482-486. In some embodiments, the ASO comprises a sequence and / or a chemical modification selected from the group consisting of SEQ ID NOs: 1-48, or 91-662.

[0126] In some embodiments, the regulatory RNA comprises a sequence selected from the group consisting of SEQ ID NOs: 80-88. In some embodiments, the ASO comprises a sequence selected from the group consisting of SEQ ID NOs: 51-68.

[0127] In some embodiments, the ASO comprises a sequence set forth in Table 2. In some embodiments, the ASO comprises a sequence and / or chemical modification set forth in Table 3 and Figures 4A, 5A, 5B, and 14.

[0128] 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.

[0129] High-affinity modified nucleosides A high affinity modified nucleoside is a modified nucleoside 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 disclosure 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.

[0130] sugar modification The ASOs described herein may include one or more nucleosides having 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 having modifications of the ribose sugar moiety have been made primarily for the purpose of improving 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 biradical 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 (see, for example, PCT Publication No. WO2011 / 017521) or tricyclic nucleic acids (see, for example, PCT Publication No. 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, for example, in the case of peptide nucleic acids (PNAs) or morpholino nucleic acids.

[0131] 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.

[0132] 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).

[0133] 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 herein by reference. The synthesis of thiophosphoramidate morpholino oligonucleotides is described in Langer et al, J. Am. Chem. Soc. 2020, 142, 38, 16240-16253.

[0134] 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 biradicals that can form a bridge between the 2' carbon and the second carbon in the ribose ring, such as LNA (2'-4' biradical bridged) nucleosides.

[0135] 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).

[0136] 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 nucleoside that contains a bicyclic sugar moiety that contains a 4'-CH2-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 may also be called bridged nucleic acids or bicyclic nucleic acids (BNA). Ribose conformation locking is associated with enhanced hybridization affinity (duplex stabilization) when LNA is incorporated into an oligonucleotide to a complementary RNA or DNA molecule. 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.

[0137] Examples of bicyclic nucleosides for use in the polynucleotides of the invention include, but are not limited to, nucleosides that contain 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 contain a 4' to 2' bridge. Examples of such 4' to 2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (also known as "constrained ethyl" or "cEt") and 4'-CH(CHOCH3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845). ; 4'-C(CH3)(CH3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,283); 4'-CH2-N(OCH3)-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,425); 4'-CH2-ON(CH3)2-2' (see, e.g., U.S. Pat. Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2' (wherein R is H, C1-C 12 alkyl, or a protecting group] (see, e.g., U.S. Pat. No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing are incorporated herein by reference.

[0138] 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.

[0139] Any of the foregoing bicyclic nucleosides can be prepared with one or more stereochemical sugar configurations, such as, for example, α-L-ribofuranose and β-D-ribofuranose (see PCT Publication No. WO 99 / 14226, the contents of which are incorporated herein by reference).

[0140] The oligonucleotides of the present invention may also be modified to include one or more constrained ethyl nucleosides. As used herein, a "constrained ethyl nucleoside" or "cEt" is a locked nucleoside that includes a bicyclic sugar moiety that includes a 4'-CH(CH3)-O-2' bridge. In some embodiments, the constrained ethyl nucleoside is in the S conformation, referred to herein as "S-cEt."

[0141] 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.

[0142] 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 No. WO2013 / 036868, the contents of each of which are incorporated by reference in their entirety herein.

[0143] 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).

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] Additional non-limiting exemplary LNA nucleosides are described in PCT Publication Nos. 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. 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.

[0149] 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.

[0150] In some embodiments, the nucleotide sugar modification is 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") 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-type sugars ("L-sugars"), or 4'-thioribosyl nucleotides.

[0151] Mixmers and Gapmers The ASO may have a mixmer and / or gapmer structure, for example, in the patterns disclosed by the ASOs of FIG. 5A, FIG. 5B, or FIG.

[0152] 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.

[0153] 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 hCPS1-ASO-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 hCPS1-ASO-1e has such a structure. Similar modification patterns are also contemplated herein, for example, where the first modification is repeated every 4, 5, or more nucleotides.

[0154] In some embodiments, the ASO further comprises a GalNAc or Teg-GalNAc moiety at the 5' or 3' end of the ASO, hi some embodiments, the ASO further comprises a cholesterol or biotin moiety at the 5' or 3' end of the ASO.

[0155] In certain embodiments, the ASO comprises a DNA sequence (e.g., having at least 5, 6, 7, 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., hCPS1-ASO-1a), as can regRNA with the same sequence but different chemical modifications (e.g., hCPS1-ASO-1g).

[0156] In certain embodiments, the gapmer is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length. In certain embodiments, the gap is about 5, 6, 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 comprise RNA modifications, such as β-D-ribonucleosides, 2'-modified nucleosides (e.g., 2'-O-(2-methoxyethyl) (2'-MOE), 2'-O-CH3, 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 the gapmer is modified with a 2'-MOE. In certain embodiments, the gapmer comprises one or more modified internucleotide linkages, such as phosphorothioate (PS) internucleotide linkages. In certain embodiments, every two adjacent nucleotides in a gapmer are linked by a phosphorothioate internucleotide linkage.

[0157] 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. In some embodiments, the ASO contains only ribonucleotides and no deoxyribonucleotides.

[0158] 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). 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.

[0159] 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.

[0160] III. 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 suitable formulation.Formulations 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, which is incorporated herein by reference in its entirety.

[0161] 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.

[0162] Delivery of the oligonucleotide of the present disclosure to cells, for example, cells in a subject, for example, a human subject, for example, a subject in need thereof, for example, a subject with CPS1-related disorder or other urea cycle disorder, can be achieved in many different ways.For example, delivery can be performed by contacting the oligonucleotide of the present disclosure with cells, either in vitro or in vivo.In vivo delivery can also be performed directly by administering a composition comprising the oligonucleotide to a subject.These alternatives are further discussed below.

[0163] 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 disclosure (see, for example, Akhtar S. and Julian R L., (1992) Trends Cell. Biol. 2(5):139-144 and PCT Publication No. 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 administering the preparation locally. Local administration to the treatment site maximizes the local concentration of the agent, limits exposure of the agent to systemic tissues that may otherwise be harmed by the agent or that may degrade the agent, and allows for administration of a lower total dose of the oligonucleotide molecule.

[0164] 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 the negatively charged cell membrane, allowing efficient uptake of oligonucleotides by cells. Cationic lipids, dendrimers, or polymers can be induced to form vesicles or micelles that bind to or encase 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 disclosure. 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 disclosure are delivered by polyplex or lipoplex nanoparticles.Methods of administration and pharmaceutical compositions for oligonucleotide and polyplex nanoparticles and lipoplex nanoparticles can be found in U.S. Patent Publication 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.

[0165] In some embodiments, the compounds described herein may be administered in combination with additional therapies, such as a low protein diet or standard urea cycle disorder treatments, such as nitrogen scavengers, including, but not limited to, glycerol phenylbutyrate, sodium benzoate, phenyl butyrate, or phenyl acetate.

[0166] Methods for delivery of membrane molecular assemblies The oligonucleotides of the present disclosure may also be delivered using various membrane molecular assembly delivery methods including liquid nanoparticles (LNPs), polymers, biodegradable microparticles, 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) with diameters ranging from 0.2 to 4.0 μm can encapsulate a significant fraction 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 the fusion of the liposome with the cell proceeds, the aqueous contents inside, including the oligonucleotide, are delivered to the cell, where the oligonucleotide can specifically bind to the target RNA.In some cases, the liposome is also specifically targeted, for example, to direct the oligonucleotide to a specific cell type.The composition of the liposome 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 the liposome depend on pH, ionic strength, and the presence of divalent cations.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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. Due to the acidic pH within the endosome, the liposome ruptures and releases its contents into the cytoplasm (Wang et al. (1987) Biochem. Biophys. Res. Commun., 147:980-985).

[0171] 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).

[0172] 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.

[0173] 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.

[0174] 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).

[0175] 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).

[0176] 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 galactocerebroside sulfate, galactocerebroside sulfate, and phosphatidylinositol to form liposomes. 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., disclose liposomes containing (1) sphingomyelin and (2) ganglioside GM1 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.).

[0177] 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.

[0178] 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.

[0179] 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).

[0180] 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.

[0181] 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).

[0182] 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.

[0183] 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).

[0184] 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.

[0185] 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.

[0186] 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 delivery of serum albumin via transfersomes has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0187] Other formulations suitable for the present disclosure are described in PCT Publication Nos. WO2009 / 088891, WO2009 / 132131, and WO2008 / 042973, which are incorporated by reference in their entireties.

[0188] 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).

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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).

[0194] The oligonucleotide for use in the method of the present disclosure 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.

[0195] Lipid nanoparticle-based delivery methods The oligonucleotides of the present disclosure 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 disclosure 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 disclosure, 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.

[0196] 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 ... DLin-C-DAP, 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-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.

[0197] The ionizable / non-cationic lipids may be anionic lipids or neutral lipids, including distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DPPG), dioleoylphosphatidylethanolamine (D ... 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.

[0198] 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.

[0199] 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.

[0200] ASOs may also be delivered in lipidoids. The synthesis of lipidoids has been extensively 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).

[0201] Lipid compositions for RNA delivery are disclosed in PCT Publication Nos. WO2012 / 170930A1, WO2013 / 149141A1, and WO2014 / 152211A1, each of which is incorporated herein by reference.

[0202] IV. Therapeutic Applications The present disclosure provides a method for treating diseases and disorders associated with decreased gene expression (e.g., decreased CPS1 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., CPS1). The oligonucleotide compositions described herein are useful in the methods of the present disclosure, and are believed to exert their desired effects through their ability to regulate the level, state and / or activity of CPS1 in cells of a subject (e.g., a mammal, a primate, or a human), for example, by increasing the level of CPS1 protein.

[0203] Also provided herein is a method of treating or preventing hyperammonemia in a subject, comprising administering to the subject an ASO described herein. The present disclosure further provides a method of treating or preventing hyperammonemia in a subject having or at risk of developing hyperammonemia, comprising administering to the subject an ASO described herein. In some embodiments, the subject having or at risk of developing hyperammonemia has one or more of the following: urea cycle disorder, hepatic encephalopathy, acute liver failure, cirrhosis, nonalcoholic fatty liver disease, renal dysfunction and / or failure, propionic acidemia, methylmalonic acidemia, isovaleric acidemia, urinary tract infection, intestinal bacterial overgrowth, fatty acid oxidation disorder, and systemic infection. In some embodiments, the subject has or is at risk of developing hyperammonemia associated with the ingestion of a drug (e.g., valproic acid, carbamazepine, fluorouracil, sulfadiazine, ribavirin, salicylate, and glycine). In some embodiments, the method comprises administering to the subject an ASO described herein. In certain embodiments, treatment with an ASO as described herein results in a reduction in ammonia levels (e.g., blood ammonia levels, plasma ammonia levels, systemic ammonia levels, or cerebrospinal fluid ammonia levels) in a subject compared to a subject not treated with an ASO (e.g., the same subject before the treatment). In some embodiments, the reduction in ammonia levels (e.g., blood ammonia levels, systemic ammonia levels, or cerebrospinal fluid ammonia levels) is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% reduction in ammonia levels (e.g., blood ammonia levels, systemic ammonia levels, or cerebrospinal fluid ammonia levels) in a subject (e.g., compared to the same subject before administration of the ASO).

[0204] Methods for measuring ammonia levels (e.g., in a plasma sample) are known in the art and include the steps of: ammonia reacts with α-ketoglutarate and reduced nicotinamide adenine dinucleotide phosphate (NADPH) in the presence of L-glutamate dehydrogenase to produce glutamate and NADP + The amount of oxidized NADPH is then measured (e.g., photometrically) and equates to the amount of ammonia.

[0205] One aspect of the disclosure relates to a method of treating a disorder, such as a urea cycle disorder, in a subject, such as a human subject, in need thereof. In some embodiments, provided herein is a method of treating a urea cycle disorder in a subject, such as a human subject, in need thereof, comprising administering to the subject an ASO provided herein. In some embodiments, the urea cycle disorder is carbamoyl phosphate synthase 1 (CPS1) deficiency, ornithine transcarbamylase (OTC) deficiency, citrullinemia type I (argininosuccinate synthase (ASS) deficiency), argininosuccinate lyase (ASL) deficiency, N-acetylglutamate synthase (NAGS) deficiency, arginase deficiency (hyperargininemia, ARG1 deficiency), ornithine translocase deficiency (ORNT1 deficiency, hyperornithinemic-hyperammonemia-homocitrullinuria syndrome (HHH)), or citrin deficiency. In some embodiments, the urea cycle disorder is not N-acetylglutamate synthase (NAGS) deficiency. In some embodiments, administration of an ASO provided herein to a subject having a urea cycle disorder reduces the risk, frequency, and / or severity of hyperammonemia in the subject (e.g., compared to a subject not administered the ASO (e.g., the same subject prior to administration of the ASO)).

[0206] In some embodiments, the urea cycle disorder is carbamoyl phosphate synthase I deficiency (CPS1 deficiency). In some embodiments, the urea cycle disorder is hyperammonemia. In some embodiments, the urea cycle disorder is ornithine transcarbamylase deficiency (OTC deficiency). In some embodiments, the urea cycle disorder is citrullinemia type I (ASS1 deficiency). In some embodiments, the urea cycle disorder is arginosuccinic aciduria (ASL deficiency). In some embodiments, the urea cycle disorder is arginase deficiency (hyperargininemia, ARG1 deficiency). In some embodiments, the urea cycle disorder is ornithine translocase deficiency (ORNT1 deficiency, hyperornithinemic-hyperammonemia-homocitrullinuria syndrome). In some embodiments, the urea cycle disorder is citrin deficiency.

[0207] Another aspect of the disclosure includes a method of increasing the level of CPS1 in a cell of a subject identified as having a CPS1-related disorder (e.g., CPS1 deficiency) or other urea cycle disorder. Yet another aspect includes a method of inhibiting expression of CPS1 in a cell of a subject. The method may include contacting a cell with an ASO described herein in an amount effective to increase expression of CPS1 in the cell, thereby increasing expression of CPS1 in the cell. In certain embodiments, the method includes contacting a cell with an ASO described herein in an amount effective to increase expression of CPS1 in the cell, thereby increasing expression of OTC in the cell. In certain embodiments, the method includes contacting a cell with an ASO described herein in an amount effective to increase expression of CPS1 in the cell, thereby increasing expression of other urea cycle genes, such as OTC, ASS1, ASL, ARG1, or ORNT1 in the cell.

[0208] Based on the above-mentioned method, further aspects of the present disclosure include the ASO or oligonucleotide of the present disclosure, or a composition comprising such ASO or oligonucleotide, for use in treatment or as a medicine, or for use in treating CPS1-related disorders (e.g., CPS1 deficiency) or other urea cycle disorders in a subject in need thereof, or for use in increasing the level of CPS1 in a cell of a subject identified as having a CPS1-related disorder or other urea cycle disorder, or for use in increasing the expression of CPS1 in a cell of a subject. This use includes contacting a cell with an ASO or oligonucleotide in an amount effective to increase the expression of CPS1 in the cell, thereby increasing the expression of CPS1 in the cell. The embodiments described below with respect to the method of the present disclosure are also applicable to these further aspects.

[0209] The contacting of cells with ASO or oligonucleotide may be in vitro, ex vivo, or in vivo. Contacting cells with oligonucleotide in vivo includes contacting cells or cell populations in a subject, for example, a human subject, with ASO or oligonucleotide. A combination of in vitro and in vivo cell contacting methods is also possible. As described above, contacting cells may be direct or indirect. Furthermore, contacting cells 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 any other ligand that directs the oligonucleotide to a site of interest. The cell may be a liver cell (e.g., a hepatocyte).

[0210] The administration of the ASO or oligonucleotide 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 oligonucleotide or pharmaceutical composition is administered systemically. In certain embodiments, the ASO or oligonucleotide or pharmaceutical composition is administered by a parenteral route. For example, in certain embodiments, the ASO or oligonucleotide 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 oligonucleotide or pharmaceutical composition is administered locally to an organ or tissue in which increased target gene expression is desired (e.g., the liver).

[0211] In some embodiments, the ASO or oligonucleotide is administered to the subject so that the ASO or oligonucleotide is delivered to a specific site in the subject. Such targeted delivery can be achieved by either systemic or local administration. The increased expression of CPS1 can be evaluated using the measurement of the level or change in the level of CPS1 mRNA or CPS1 protein in a sample derived from a specific site in the subject. In some embodiments, the method comprises measuring the level of ammonia in a sample (e.g., blood, urine, plasma, or cerebrospinal fluid) derived from the subject. Methods for measuring ammonia levels are well known in the art and are also described above. In certain embodiments, the method comprises a clinically relevant increase in the expression of CPS1, for example, as indicated by a clinically relevant outcome after treating the subject with an agent that increases the expression of CPS1.

[0212] 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 a CPS1-related disorder or urea cycle disorder, such as high blood ammonia levels.

[0213] Increased CPS1 expression levels The therapeutic methods disclosed herein are designed to use ASOs targeting CPS1 to increase CPS1 expression levels in a subject compared to untreated subjects (e.g., the same subject before administration of ASOs or pharmaceutical compositions). Increased expression of the CPS1 gene includes any increase in the level of the CPS1 gene, e.g., at least a partial increase in the expression of the CPS1 gene. Increase can be assessed by an increase in the absolute or relative level 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, e.g., 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 (e.g., a buffer only control or a non-active agent control). In certain embodiments, the method results in a clinically relevant increase in the expression of CPS1, e.g., as shown by clinically relevant results after treating the subject with an agent that increases the expression of CPS1.

[0214] In certain embodiments, the methods disclosed herein increase CPS1 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 CPS1 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 CPS1 expression, and the methods disclosed herein restore CPS1 expression levels 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 CPS1 expression level or activity in subjects of the same age and sex species.

[0215] The expression of CPS1 gene can be evaluated based on the level of any variable associated with CPS1 gene expression, such as CPS1 mRNA level or CPS1 protein level. It is understood that CPS1 is a chromosome-2 gene in certain mammals (e.g., humans and mice). In certain embodiments, the expression level or activity of CPS1 herein refers to the average expression level or activity in liver.

[0216] In certain embodiments, surrogate markers may be used to detect increased CPS1 expression levels. For example, effective treatment of a CPS1-related disorder, as indicated by acceptable diagnostic and monitoring criteria, with an agent that increases CPS1 expression may be understood to indicate a clinically relevant decrease in CPS1.

[0217] Increased expression of the CPS1 gene can be evident by an increase in the amount of mRNA in which the CPS1 gene is transcribed and expressed by a first cell or group of cells (such cells can 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 disclosure or by administering an oligonucleotide of the present disclosure to a subject in which the cells are or were present), such that expression of the CPS1 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).

[0218] In other embodiments, increased expression of the CPS1 gene can be assessed with respect to a parameter functionally related to CPS1 gene expression, such as increased CPS1 protein expression or CPS1 activity. CPS1 increase can be determined by any assay known in the art in any cell that expresses CPS1, either endogenously or heterologously from an expression construct.

[0219] Increased expression of CPS1 can be revealed by an increase in the level of CPS1 protein expressed by a cell or cell group (e.g., the level of protein expressed in a sample from a subject) compared to a control cell or a control group of cells. Increased expression of CPS1 can also be revealed by an increase in the mRNA level of CPS1 in treated cells or cell groups compared to a control cell or cell group.

[0220] The control cell or cell group that can be used to evaluate the increase in the expression of CPS1 gene includes the cell or cell group that has not yet been contacted with the oligonucleotide of the present disclosure.For example, the control cell or cell group can be derived from an individual subject (e.g., human or animal subject) before the subject is treated with oligonucleotide.

[0221] The level of CPS1 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 CPS1 in a sample is determined by detecting a transcribed polynucleotide, or a portion thereof, such as the mRNA of the CPS1 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 CPS1 mRNA can be detected using the methods described in PCT Publication No. WO2012 / 177906, the entire contents of which are incorporated herein by reference. In some embodiments, the level of expression of CPS1 is determined using a nucleic acid probe. As used herein, the term "probe" refers to any molecule that can selectively bind to a specific CPS1 sequence, for example, to an mRNA or polypeptide. Probes may be synthesized by those skilled in the art or derived from appropriate biological preparations. Probes may 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.

[0222] 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 includes contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the CPS1 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 CPS1 mRNA.

[0223] Alternative methods for determining the expression level of CPS1 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, for example, 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 system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-beta replicase (Lizardi et al. (1988) Bio / Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033), or any other nucleic acid amplification method, followed by detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are particularly useful for detecting nucleic acid molecules when such molecules are present in very low numbers. In certain embodiments of the present invention, the level of expression of CPS1 is determined by quantitative fluorogenic RT-PCR (i.e., the TAQMAN™ system) or DUAL-GLO® luciferase assay.

[0224] The expression level of CPS1 mRNA can be monitored using membrane blots (such as those used in hybridization analyses such as Northern, Southern, and dot), 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 CPS1 expression level can also include using a nucleic acid probe in solution.

[0225] In some embodiments, the level of mRNA expression is evaluated using branched DNA (bDNA) assay or real-time RT PCR or qPCR. Such methods can also be used to detect CPS1 nucleic acid.

[0226] The level of CPS1 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, spectrophotometric analysis, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, electrochemiluminescence assay, etc. Such assays can also be used to detect proteins that indicate the presence or replication of CPS1 protein.

[0227] V. Additional Embodiments Embodiment 1. An ASO that is complementary to at least 8 consecutive nucleotides of a regulatory RNA (regRNA) for carbamoyl phosphate synthase 1 (CPS1), wherein the ASO is complementary to a sequence in the regRNA that is not more than 200 nucleotides from an end of the regRNA. Embodiment 2. The ASO of embodiment 1, which is complementary to a sequence in the regRNA that is no more than 200 nucleotides from the 3' end of the regRNA. Embodiment 3. The ASO of embodiment 1, which is complementary to a sequence in the regRNA that is no more than 200 nucleotides from the 5' end of the regRNA. Embodiment 4 The ASO of embodiment 1 or 2, wherein the ASO comprises an RNA polynucleotide comprising one or more chemical modifications. Embodiment 5. The ASO of embodiment 1, 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. Embodiment 6. The ASO of embodiment 1 or 2, wherein the one or more chemical modifications include 2'-O-methoxyethyl, 5-methylcytidine, locked nucleic acid (LNA), and phosphorothioate internucleotide linkages. Embodiment 7 The ASO of embodiments 1 to 3, wherein the ASO does not contain 8 or more consecutive nucleotides of unmodified DNA. Embodiment 8. An ASO that is complementary to at least 8 consecutive nucleotides of a regulatory RNA of the CPS1 gene, said ASO not containing 8 or more consecutive nucleotides of unmodified DNA. Embodiment 9. The ASO of embodiments 1 to 8, wherein the ASO does not contain deoxyribonucleotides. Embodiment 10. The ASO of embodiments 1 to 9, wherein the ASO does not contain unmodified ribonucleotides. Embodiment 11. The ASO of embodiments 1 to 10, wherein the length of the ASO is 3×n+2 nucleotides (n is an integer equal to or greater than 6), the nucleotide at position 3×m is a ribonucleotide modified with LNA (m is an integer from 1 to n), and the nucleotides at the remaining positions are ribonucleotides modified with 2′-O-methoxyethyl. Embodiment 12 The ASO of any one of embodiments 1 to 11, wherein each nucleotide of the ASO is a 2'-O-methoxyethyl modified ribonucleotide. Embodiment 13 The ASO of any one of embodiments 1 to 12, wherein each cytidine in the ASO is modified with 5-methyl. Embodiment 14 The ASO of embodiments 1-13, wherein the ASO is no more than 50, 40, 30, or 25 nucleotides in length. Embodiment 15. The ASO of embodiments 1 to 14, wherein the regRNA is eRNA. Embodiment 16. A pharmaceutical composition comprising the ASO of any one of embodiments 1 to 15, and a pharma- ceutically acceptable carrier or excipient. Embodiment 17. A method for increasing the amount and / or stability of a regulatory RNA in a cell, comprising contacting the cell with an ASO of any one of embodiments 1 to 15 that hybridizes to the regulatory RNA. Embodiment 18. A method for increasing transcription of a target gene in a cell, comprising contacting the cell with an ASO of any one of embodiments 1 to 15 that hybridizes with a regulatory RNA of the target gene. Embodiment 19. The method of embodiment 17 or 18, wherein the cell is a mammalian cell.

[0026] Embodiment 20. The method of embodiment 19, wherein the cell is a human cell. Embodiment 21. The method of embodiments 17-20, wherein the target gene is CPS1. EXAMPLES

[0228] Example 1: Modulation of CPS1 expression using regRNA-targeted ASO This example was designed to evaluate the regulation of CPS1 expression in human hepatocytes using an ASO targeting eRNA transcribed from the enhancer of human CPS1.

[0229] Regulatory RNAs (regRNAS) within the enhancer of CPS1 were bioinformatically identified using ATAC-seq and H3K27Ac ChIP-SEQ data from human and mouse data. As shown in Figure 2, two regRNAs conserved between human and mouse were identified (RR44_v1 and RR44_v2).

[0230] We designed 330 unique antisense oligonucleotides (ASOs) targeting CPS1, 94 of which target the CPS1 regRNA (RR44) described above.

[0231] Donor-derived hepatocytes were cultured in vitro. Cells were plated in growth medium and treated 4–6 h after plating with final concentrations of 1.25 μM, 2.5 μM, or 5 μM, or with hCPS1-ASO-1a or control (see Figure 5A for human CPS1 sequence and chemical modifications of selected ASOs). Cells were harvested 48 h after treatment and processed for RNA isolation, cDNA synthesis, and QPCR analysis. A 60x Taqman probe was used for CPS1 expression. CPS1 levels were normalized to B2M expression. Figure 3 shows CPS1 mRNA after treatment with hCPS1-ASO-1a, demonstrating that ASOs targeting regRNA RR44 increase human CPS1 mRNA in a dose-dependent manner.

[0232] Using hCPS1-ASO-1a as a starting point, additional ASOs were designed by base walking. These ASOs are shown in Figure 4A. Using the methods described above, these additional ASOs were assessed for their effect on CPS1 mRNA, and the results are shown in Figure 4B. Additional ASOs are shown in Figure 5A.

[0233] The majority of regRNAs do not have large sequence regions that are conserved between the human and mouse genomes. For in vivo proof of concept, we identified regRNAs surrounding the mouse CPS1 region, designed ASOs targeting these mouse regRNAs (promoter and enhancer), and expressed them in wild-type (B6EiC3SnF1 / J, [WT]) primary mouse hepatocytes and CPS1-deficient donors (B6EiC3Sn a / A-CPS1 spf-ash / J,[CPS1D]) primary mouse hepatocytes.

[0234] Primary hepatocytes were cultured from mouse strains B6EiC3SnF1 / J (control WT) and OTC-deficient donors (B6EiC3Sn a / A-OTC1 spf-ash / J, Catalog: 001811) (JAX lab). spf ashMice carry a variant in the OTC gene, c.386G>A, p.Arg129His, that affects splicing, resulting in reduced mRNA levels of OTC 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).

[0235] Primary hepatocytes were seeded at 20,000 cells per well on day 0. Cells were treated with ASOs at concentrations of 1.25 μM, 2.5 μM, and 5 μM on day 2. Cells were incubated for 2 days and lysates were collected for mRNA analysis on the second day of treatment. Mouse CPS1 and OTC expression was determined using Taqman probes. Ppia and Hprt were used as housekeeper genes for gene expression normalization. Statistics were performed using one-way ANOVA in Prism (GraphPad).

[0236] CPS1 mRNA levels in wild-type and OTC-deficient hepatocytes treated in vitro are shown in Figure 6A, and heat maps are shown in Figures 6B and 6C. As shown, a subset of identified ASOs upregulates CPS1 mRNA by 2-fold in treated hepatocytes. Furthermore, it was observed that ASOs that upregulate CPS1 can also increase the expression of OTC (OTC mRNA levels are shown in Figure 7A, and heat maps are shown in Figures 7B and 7C). Thus, ASO-targeted regRNA can be used to increase CPS1 expression in diseased mouse liver cells. ASO-mediated CPS1 upregulation in OTC-deficient mouse cells allows them to be tested in disease models, with in vivo phenotypic readouts.

[0237] In vivo regulation of CPS1 MaleB6EiC3Sn a / A-Otc spf-ash / JMice (homozygous) (4-5 mice / group) were fasted and then challenged with 0.75 M NH4Cl injected intraperitoneally. Two hours later, a subcutaneous injection of GalNAc-conjugated ASO at a dose of 200 mg / kg / week was administered. Approximately 48 hours later, mice were again administered GalNAc-conjugated ASO. Approximately 24 hours later, mice were challenged with 0.75 M NH4Cl injected intraperitoneally and approximately 24 hours later, GalNAc-conjugated ASO was again administered subcutaneously. 72 hours later, mice were injected intraperitoneally with 0.75 M NH4Cl and administered GalNAc-conjugated ASO. 48 hours later, mice were injected intraperitoneally with 0.75 M NH4Cl and 2 hours later, mice were sacrificed. The timeline is shown in Figure 8. Serum was collected 30-60 min after NH4Cl injection and terminal plasma samples were analyzed for ammonia levels by IDEXX. Liver, serum, and urine were collected after sacrifice.

[0238] As shown in Figure 9A, ASO treatment increased CPS1 mRNA in mouse liver by about 1.4-fold. Furthermore, plasma ammonia levels were reduced after treatment, indicating increased urine production (Figure 9B). ASO targeting CPS1 regRNA also increased a number of other urea cycle genes, including ASS1, ASL, and ARG1, in OTC-deficient mice (Figure 9C). Importantly, ASO targeting CPS1 regRNA did not affect the expression levels of neighboring genes (Figure 9D).

[0239] In additional experiments, male B6EiC3Sn a / A-Otc spf-ash / J Mice (homozygous) (4-5 mice / group) were fasted and then challenged with 0.75 M NH4Cl via intraperitoneal injection and GalNAc-conjugated ASOs were injected subcutaneously. Final plasma samples collected 60 min after NH4Cl injection were analyzed by IDEXX for ammonia levels, with an upper limit of detection of 1020 μg / dL. As shown in Figure 10, CPS1 regRNA targeting mCPS1-ASO-2 reduced ammonia to wild-type levels in OTC-deficient mice.

[0240] As shown in Figures 11A and 11B, male OTCD mice (Otc spf / ash ) had a dose-dependent effect on plasma ammonia (FIG. 11A) and urea (FIG. 11B) in vivo. Mice were fasted overnight before the ammonia challenge. Briefly, mice were given 0.75 M ammonium chloride solution via IP injection. Intermediate plasma was collected 30 min (±5%) after each ammonium challenge. Ammonia levels were measured in fresh plasma samples by a Beckman Coulter AU® chemistry analyzer. Furthermore, the reduction in ammonia correlated with the total amount of ASO administered (FIG. 11C). Without wishing to be bound by theory, this data suggests dose accumulation of ASO, thus supporting a long duration of action of ASO. Furthermore, this data suggests that efficacy of ASO may be achieved by splitting the dose over time, which allows for a higher margin of safety during dosing. For example, a dosing interval of at least one month may be used.

[0241] A second in vivo study was performed to assess the duration of action of the ASO. spf-ash / JMice (homozygous) or wild type (C57BL / 6) mice (5 mice / group) were fasted and then challenged with 0.75 M NH4Cl injected intraperitoneally. Two hours later, a subcutaneous injection of GalNAc-conjugated ASO was administered at a dose of 100 mg / kg / week (day 1). One group of WT and OTC-deficient mice received PBS as a control group. Mice were administered GalNAc-conjugated ASO on days 3 and 5 again. Ammonia was administered intraperitoneally to mice on days 8, 11, 15, 19, 22, and 26. Circulating ammonia was measured after IP ammonia challenge. Mice were sacrificed on day 26 and livers were harvested and processed for mRNA and protein expression. Geometric means of mRNA were quantified by Gusb, Gapdh, Actb, Ppia, and Hprt genes. Statistics were performed by two-way ANOVA. Two-way ANOVA, *:P<0.05, **:P<0.01, ***:P<0.001, ****:P<0.0001.

[0242] As shown in FIG. 12A, Cps1 ASO treatment increased mouse Otc and Cps1 mRNA expression in OTC-deficient mice (1.26 FC for Cps1 and 1.43 FC for Otc). WT C57 mouse mRNA is shown in the left bar, and PBS-treated Otc spf-ash / J The central bar shows the Otc spf-ash / J In addition, ASO treatment increased additional urea cycle genes, such as Nags, Ass1, Asl, and Arg1 (Figure 12B). WT C57 mouse mRNA is shown in the left bar, and PBS-treated Otc spf-ash / J The central bar shows the Otc spf-ash / J is shown on the right.

[0243] In the PBS control group, mouse Otc-deficient livers showed high ammonia 30 minutes after ammonia treatment (see FIG. 13, 1.0 filled triangles). Ammonia levels were consistent throughout the study. Treatment with mouse Cps1 ASO (inverted triangles) reduced ammonia to WT levels (squares) and maintained wild-type (WT) levels throughout the study (FIG. 13). ASO was administered on days 1, 3, and 5, with duration of action of approximately 4 weeks.

[0244] Yecuris mice (Fah mice with OTC-deficient humanized liver) - / - , Rag2 - / - , Il2rg - / - The in vivo assay was repeated with hCPS1-ASO-1x in male humanized Yecuris mice (FRG mice). Male humanized Yecuris mice were repopulated with human ornithine transcarbamylase-deficient hepatocytes (OTCD) or healthy wild-type hepatocytes (WT) (5 mice / group). After overnight fasting, 0.75 M 15 NH4Cl was injected intraperitoneally. Two hours later, GalNAc conjugate hCPS1-ASO-1x was injected subcutaneously multiple times on days 1, 8, and 15. In some groups, single injections were performed at doses of 5, 20, and 50 mg / kg / week. One group of WT and OTCD Yecuris FRG mice received PBS as a control group. Ammonia challenge and urine production assays in humanized Yecuris mice were performed in fasting state, i.e., with food withheld overnight, prior to ammonia challenge. Animals were immunized by intraperitoneal injection after an overnight fast on days 1, 8, 15, and 22 (final collection). 15 The mice were challenged with NH4Cl. Thirty minutes later, urine and plasma were collected.

[0245] As shown in Figures 15A and 15B, hCPS1-ASO-1x inhibited OTC with humanized liver. def hCPS1-ASO-1g reduced plasma ammonia and increased urea in mice in vivo. hCPS1-ASO-1g also increased OTC protein levels after treatment at 5 mg / kg and 20 mg / kg (FIG. 15C).

[0246] In vitro regulation of urine production in primary human hepatocytes ASOs were also tested for their ability to agonize urine production in human cells in vitro. Primary human hepatocytes from healthy donors and OTC def Primary human hepatocytes were plated and 1.25uM hCPS1-ASO-1a or hCPS1-ASO-1g was incubated with the cells for 2 days. On day 2, 2M NH4Cl was added to the cell mixture. Cells and cell media were collected on day 3 for quantification of mRNA and urea. As shown in Figure 16, both ASOs were significantly associated with hepatocyte proliferation in healthy donors and OTC donors. def Both increased CPS1 mRNA and urea production in primary human hepatocytes.

[0247] Further synthesis and characterization of ASOs Further ASOs were synthesized and characterized. Hepatocytes were treated with 5 uM ASOs as above and the fold change in CPS1 mRNA was determined.

[0248] Table 4 demonstrates the mRNA FC and standard deviation for CPS1 mRNA.

[0249] (Table 4) TIFF2025500409000055.tif195165TIFF2025500409000056.tif209165TIFF202 5500409000057.tif214165TIFF2025500409000058.tif214165TIFF20255004090 00059.tif214165TIFF2025500409000060.tif214165TIFF2025500409000061.t if214165TIFF2025500409000062.tif214165TIFF2025500409000063.tif209165

[0250] hCPS1-ASO-1a and hCPS1-ASO1g were tested at increasing concentrations in hepatocytes. ASOs from 0.156 to 5 uM were incubated with hepatocytes as described above. As shown in Table 5, both hCPS1-ASO-1a and hCPS1-ASO1g induced a dose-dependent increase in CPS1 mRNA.

[0251] (Table 5) TIFF2025500409000064.tif74165

[0252] Selected ASOs were subjected to additional chemical modifications. The chemical modifications are shown in Figure 14. Hepatocytes were incubated with 1.25-5 uM of selected ASOs as described above. The highest CSP1 mRNA fold change (FC) and standard deviation (SD) are shown in Table 6.

[0253] (Table 6) TIFF2025500409000065.tif210165TIFF2025500409000066.tif237165TIFF2025500409000067.tif84165

[0254] In total, 109 initial ASOs targeting CPS1 RR44 were generated, of which 76 ASOs passed the first-pass screening, including 69 gapmers and 7 conformational ASOs. These 76 ASOs were selected for tiling, and 9 ASOs were selected from this screening, including 4 gapmers and 5 conformations. An additional 24 ASOs were generated by fine-tuning, including 12 gapmers and 12 mixmers, with additional chemistry. hCPS1-ASO-1g (SEQ ID NO: 22) was selected for further characterization. PO linkages, GalNAc moieties, and an additional 5meC moiety were added for in vivo studies, resulting in hCPS1-ASO-1x (SEQ ID NO: 409).

[0255] Example 2: Synthesis and characterization of additional CPS1 regRNA-targeting ASOs Two additional regRNAs, RR89 and RR90, were identified. ASOs targeting the novel regRNAs were synthesized and tested at 5uM in hepatocytes as described above. The CPS1 mRNA fold increase after treatment with these ASOs is shown in Table 7 below.

[0256] (Table 7) TIFF2025500409000068.tif211165TIFF2025500409000069.tif220165TIFF20255004090 00070.tif220165TIFF2025500409000071.tif220165TIFF2025500409000072.tif105165

[0257] Example 3: In vivo characterization of selected ASOs in non-human primates On day 0, 2-4 year old male cynomolgus monkeys (NHPs) were subcutaneously injected with either 20 mg / kg or 50 mg / kg hCPS1-ASO-1x, with some groups receiving a repeat dose on day 21. Three NHPs were randomly assigned to each group. One group received PBS as a negative control. Ammonia challenge and urea production assays in NHPs were performed in an overnight fasting state prior to ammonia challenge. 15 NH4Cl solution was subcutaneously injected into NHPs, and multiple blood samples were taken over a period of 0-120 min, and plasma was immediately obtained by centrifugation.

[0258] hCPS1-ASO-1x reduced ammonia production in NHPs by a statistically significant amount compared to PBS treatment alone by day 27. Figure 17 shows the AUC of ammonia over time and the AUC of ammonia per dose.

[0259] A second in vivo NHP study was conducted to evaluate efficacy with lower doses of hCPS1-ASO-1x. Female cynomolgus monkeys (NHPs) aged 2-4 years were subcutaneously injected with either 5 mg / kg or 15 mg / kg hCPS1-ASO-1x on day 1, with a repeat dose on day 36. Each group consisted of three NHPs. One group received PBS as a negative control. Ammonia challenge and urine production assays in NHPs were performed in an overnight fasting state prior to ammonia challenge. Ammonia challenge of 200 mg / kg was administered subcutaneously on day 1, with a repeat dose on day 36. 15 NHPs were subcutaneously injected with NH4Cl solution, and blood was collected several times between 0 and 120 min, and plasma was obtained immediately by centrifugation. 13 C-urea production assay was performed. 13 C-sodium acetate was provided by oral gavage, multiple blood draws were performed from 0 to 240 min, and plasma was immediately obtained by centrifugation. Figure 18 shows that hCPS1-ASO-1x reduced ammonia in NHPs after a single dose of 5 mg / kg for up to 5 weeks (ammonia levels on days 22 and 36 are shown). Furthermore, urea levels were increased in ASO-treated NHPs up to 29 days after dosing. Figure 19 provides quantification of ammonia AUC on days 22, 29, and 36.

[0260] hCPS1-ASO-1x and 13 Combination with C-sodium acetate treatment enhanced urine production in NHPs on day 43 compared to hCPS1-ASO-1x alone (Figure 20).

[0261] In summary, upregulation of CPS1 increased urea cycle output in mouse, NHP, and human hepatocytes both in vitro and in vivo. def Increasing CPS1 mRNA in the model also increases the metabolism of ammonia to urea, and the NHP data and humanized mouse liver data support this finding.

[0262] Incorporation by Reference Unless otherwise stated, the entire disclosures of each of the patent documents and scientific articles referred to herein are incorporated by reference for all purposes.

[0263] 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. 1. An antisense oligonucleotide (ASO) complementary to at least 8 consecutive nucleotides of a regulatory RNA (regRNA) of carbamoyl phosphate synthase 1 (CPS1), wherein the regRNA has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 49, 50, 69-79 and 89-90.

2. The ASO of claim 1, which is complementary to a sequence in the regRNA that is no more than 200 nucleotides from the 3' end of the regRNA.

3. The ASO of claim 1, which is complementary to a sequence in the regRNA that is no more than 200 nucleotides from the 5' end of the regRNA.

4. The ASO of claim 1 or 2, wherein the regulatory RNA has a nucleotide sequence of SEQ ID NO:49 and the ASO comprises a nucleotide sequence of SEQ ID NO:

1.

5. The ASO of any one of claims 1, 2, or 4, wherein the regulatory RNA has a nucleotide sequence of SEQ ID NO:49 and the ASO comprises a nucleotide sequence of SEQ ID NO:

2.

6. The ASO of any one of claims 1, 2, or 4, wherein the regulatory RNA has a nucleotide sequence of SEQ ID NO:49 and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:1-3.

7. The ASO of claim 1 or 2, wherein the regulatory RNA has a nucleotide sequence of SEQ ID NO:50, and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:4-15.

8. The ASO of claims 1 to 3, wherein the regulatory RNA has the nucleotide sequence of SEQ ID NO:89 and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:487 to 574.

9. The ASO of claims 1 to 3, wherein the regulatory RNA has the nucleotide sequence of SEQ ID NO: 90 and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 575 to 662.

10. The ASO of any one of claims 1 to 9, which is no more than 50, 40, 30, or 25 nucleotides in length.

11. The ASO of any one of claims 1 to 10, comprising a polynucleotide comprising one or more chemical modifications.

12. The ASO of claim 11, 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.

13. The one or more chemical modifications may be 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") nucleotides, 2'-F-arabinosyl ("2'-F"), 2'-F-F"), 2' ...

13. The ASO of claim 11 or 12, comprising a nucleotide sugar modification comprising one or more of: 2'-locked nucleic acid ("LNA") nucleotides, 2'-amide bridged nucleic acid (AmNA), 2'-unlocked nucleic acid ("ULNA") nucleotides, L-sugars ("L-sugars"), 4'-thioribosyl nucleotides, constrained ethyl (cET), 2'-fluoro-arabino (FANA), or thiomorpholino.

14. 14. The ASO of any one of claims 11-13, 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., 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(C1-3alkyl), e.g., methylphosphonate-P(CH3), boranophosphonate (P(BH3)), or phosphorodithioate (P(S)2).

15. The one or more chemical modifications can 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 ...

15. The ASO of any one of claims 11-14, comprising nucleobase modifications comprising one or more of ropinyluracil, 5-ethynylcytosine, 5-ethynyluracil, 5-allyluracil ("5-allyl U"), 5-allylcytosine ("5-allyl C"), 5-aminoallyluracil ("5-aminoallyl U"), 5-aminoallyl-cytosine ("5-aminoallyl C"), abasic nucleotides, Z bases, P bases, unstructured nucleic acid ("UNA"), isoguanine ("isoG"), isocytosine ("isoC") glycerol nucleic acid (GNA), glycerol nucleic acid (GNA), or thiophosphoramidate morpholino (TMO).

16. The ASO of any one of claims 11 to 15, wherein the one or more chemical modifications include 2'-O-methoxyethyl, 5-methylcytidine, locked nucleic acid (LNA), and phosphorothioate internucleotide linkages.

17. The ASO of any one of claims 11 to 16, comprising 8 or more contiguous nucleotides of unmodified DNA flanked by at least 3 nucleotides of modified ribonucleotides at each of the 5' and 3' ends.

18. The ASO of claim 17, comprising the nucleotide sequence and / or chemical modifications of any one of SEQ ID NOs: 16, 22-27, 31-39, 400, 403-415, 417-432, 435-480, or 482-486.

19. The ASO of claim 17 or 18, comprising the nucleotide sequence of SEQ ID NO: 409 and / or chemical modifications.

20. The ASO of claim 17 or 18, further comprising at least one phosphorothioate internucleotide linkage.

21. The ASO of claim 20, comprising the nucleotide sequence of SEQ ID NO: 403-406, 409-415, 417-424, 470, 473, 477, 480 or 486.

22. The ASO of claim 17, wherein the chemical modification is cET.

23. The ASO of claim 22, comprising a nucleotide sequence of SEQ ID NO: 425-432 or 435-442.

24. The ASO of claim 17, wherein the chemical modification is LNA.

25. The ASO of claim 24, comprising a nucleotide sequence of SEQ ID NO: 443-458.

26. The ASO of claim 17, wherein the chemical modifications are LNA and 2'-O-methoxyethyl.

27. The ASO of claim 26, comprising the nucleotide sequence of SEQ ID NO: 400, 408, 460, 462, 464-466, 468, 469, 471, 472, 475, 476, or 479.

28. The ASO of any one of claims 11 to 16, which does not contain 8 or more consecutive nucleotides of unmodified DNA.

29. The ASO of any one of claims 11 to 16, which does not contain unmodified ribonucleotides.

30. 30. The ASO of any one of claims 11 to 16 or 29, which does not contain deoxyribonucleotides.

31. The ASO of any one of claims 11 to 16, 29, or 30, wherein the length of the ASO is 2xn+4 nucleotides (where n is an integer equal to or greater than 8), the nucleotide at position 2xm is a ribonucleotide modified by LNA (where m is an integer from 1 to n), and the remaining nucleotides are ribonucleotides modified by 2'-O-methoxyethyl.

32. The ASO of claim 35, comprising the nucleotide sequence of SEQ ID NO: 393 or 394.

33. The ASO of any one of claims 11 to 16, 29, or 30, wherein the length of the ASO is 3xn+2 nucleotides (n is an integer equal to or greater than 4), the nucleotide at position 3xm is a ribonucleotide modified by LNA (m is an integer from 1 to n), and the remaining nucleotides are ribonucleotides modified by 2'-O-methoxyethyl.

34. The ASO of claim 33, comprising the nucleotide sequence of SEQ ID NO: 392 or 395.

35. 31. The ASO of any one of claims 11 to 16, 29, or 30, wherein the length of the ASO is 3xn+2 nucleotides (n is an integer equal to or greater than 6), the nucleotide at position 3xm is a ribonucleotide modified by LNA (m is an integer from 1 to n), and the five nucleotides at the 3' and 5' positions are ribonucleotides modified by 2'-O-methoxyethyl.

36. The ASO of claim 35, comprising the nucleotide sequence of SEQ ID NO: 19 or 20.

37. 31. The ASO of any one of claims 11 to 16, 29 or 30, wherein the ASO has a length of 4xn nucleotides (n is an integer equal to or greater than 3), the nucleotide at the 4xm position is a ribonucleotide modified by LNA (m is an integer from 1 to n), and the five nucleotides at the 3' and 5' positions are ribonucleotides modified by 2'-O-methoxyethyl.

38. The ASO described in claim 37, comprising the nucleotide sequence of SEQ ID NO:

21.

39. The ASO of any one of claims 11 to 16, 29, or 30, wherein the length of the ASO is 4xn+4 nucleotides (n is an integer equal to or greater than 3), the nucleotide at position 4xm is a ribonucleotide modified by LNA (m is an integer from 1 to n), and the remaining nucleotides are ribonucleotides modified by 2'-O-methoxyethyl.

40. The ASO of claim 39, comprising the nucleotide sequence of SEQ ID NO: 396 or 397.

41. The ASO of any one of claims 11 to 16, 29, or 30, wherein each ribonucleotide of the ASO is modified with 2'-O-methoxyethyl.

42. The ASO of claim 41, comprising any one of the nucleotide sequences of SEQ ID NOs: 17, 28-30, and 40-48.

43. The ASO of any one of claims 11 to 16, 29, or 30, wherein each nucleotide of the ASO is a 2'-O-methoxyethyl modified ribonucleotide.

44. The ASO of claim 43, comprising any one of the nucleotide sequences of SEQ ID NOs: 17, 28-30, and 40-48.

45. The ASO of any one of claims 11 to 44, wherein each cytidine in the ASO is modified by 5-methyl.

46. The ASO of any one of claims 11 to 16, wherein the length of the ASO is 5xn+5 nucleotides (n is an integer equal to or greater than 3), the nucleotide at position 5xm is a ribonucleotide modified by LNA (m is an integer from 1 to n), and the nucleotides at the remaining positions are ribonucleotides modified by 2'-O-methoxyethyl.

47. The ASO of claim 46, comprising the nucleotide sequence of SEQ ID NO: 398 or 399.

48. The ASO of any one of claims 11 to 47, further comprising a GalNAc moiety, and optionally a GalNAc3 moiety.

49. The ASO of any one of claims 11 to 48, further comprising a biotin or cholesterol moiety.

50. 50. The ASO of any one of claims 11 to 49, wherein each cytidine in the ASO is modified by 5-methyl.

51. The ASO of any one of claims 1 to 50, wherein the regRNA is an enhancer RNA (eRNA).

52. A pharmaceutical composition comprising an ASO according to any one of claims 1 to 51 and a pharma- ceutically acceptable carrier or excipient carrier.

53. A method for increasing transcription of CPS1 in a human cell, the method comprising contacting the cell with an ASO described in any one of claims 1 to 51 or a pharmaceutical composition described in claim 52.

54. 54. The method of claim 53, wherein the cell is a hepatocyte.

55. The method of claim 53 or 54, wherein the ASO increases the amount of the regulatory RNA in the cell compared to a cell not contacted with the ASO or pharmaceutical composition.

56. The method of any one of claims 53 to 55, wherein the ASO increases the stability of the regulatory RNA in the cell compared to a cell not contacted with the ASO or pharmaceutical composition.

57. A method for treating a urea cycle disorder, comprising administering to a subject in need thereof an effective amount of an ASO according to any one of claims 1 to 51 or a pharmaceutical composition according to claim 52.

58. 58. The method of claim 57, wherein the urea cycle disorder is CPS1 deficiency.

59. 59. The method of claim 57 or 58, wherein the urea cycle disorder is hyperammonemia.

60. The method of any one of claims 57 to 59, wherein the ASO increases the amount of the regulatory RNA in a cell of the subject compared to a cell not contacted with the ASO or pharmaceutical composition.

61. The method of any one of claims 57 to 60, wherein the ASO increases the stability of the regulatory RNA in a cell of the subject compared to a cell not contacted with the ASO or pharmaceutical composition.

62. 62. The method of claim 60 or 61, wherein the cell is a hepatocyte.

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