Oral delivery of antisense conjugates targeting PCSK9

Abstract

The present disclosure provides pharmaceutical compositions for oral delivery comprising an antisense oligonucleotide (e.g., CIVI008) and an oral delivery agent (such as 5-CNAC). In some aspects, the present disclosure provides a capsule comprising a dry mixture of CIVI008 and 5-CNAC, and optionally a statin.

Description

[Technical Field]

[0001] CROSS-REFERENCE AND INCORPORATION BY REFERENCE TO RELATED APPLICATIONS This PCT application claims priority to U.S. Provisional Application No. 63 / 124,581, filed December 11, 2020, U.S. Provisional Application No. 63 / 134,884, filed January 7, 2021, U.S. Provisional Application No. 63 / 178,340, filed April 22, 2021, and U.S. Provisional Application No. 63 / 261,505, filed September 22, 2021, the disclosures of which are incorporated herein by reference in their entireties.

[0002] Reference to electronically submitted sequence listings The contents of the electronically submitted Sequence Listing ASCII text file submitted with this application (filename: 4009-021PC04_Sequence_listing_ST25.txt; size: 26,230 bytes; and creation date: December 9, 2021) are hereby incorporated by reference in their entirety. [Technical field]

[0003] The present disclosure relates to oral formulations of antisense oligonucleotide conjugates that target proprotein convertase subtilisin / kexin type 9 (PCSK9) mRNA in cells and reduce PCSK9 expression, which is beneficial for various medical disorders, such as hypercholesterolemia and related disorders. [Background technology]

[0004] Proprotein convertase subtilisin / kexin type 9 (PCSK9) has emerged as a promising therapeutic target for reducing low-density lipoprotein cholesterol (LDL-C). PCSK9 promotes the degradation of LDL receptors, resulting in elevated LDL-C in individuals with high PCSK9 activity. Lindholm et al. (Molecular Therapy (2012); 202, 376-381) reported a PCSK9-targeted LNA antisense oligonucleotide that sustainably lowers LDL-C in nonhuman primates. The compound used was a 14-mer compound called SPC5001, also disclosed in International Publication WO 2011 / 009697. This compound was discontinued during clinical trials due to severe nephrotoxicity and acute kidney injury. See, for example, van Poelgeest et al., American Journal of Kidney Disease 62(4):796-800 (2013).

[0005] Oral delivery of pharmacologically active substances is generally the route of choice; it is convenient, relatively easy, and generally painless, resulting in higher patient compliance than other delivery methods. However, biological, chemical, and physical barriers, such as variable pH in the gastrointestinal tract, strong digestive enzymes, and gastrointestinal membranes that are impermeable to active substances, can make oral delivery of some pharmacologically active substances problematic in mammals. For example, oral delivery of therapeutic nucleic acids (e.g., antisense oligonucleotides) is needed. Therefore, the development of systems that enable oral delivery of therapeutic nucleic acids (e.g., antisense oligonucleotides) is necessary. Summary of the Invention

[0006] The present disclosure provides pharmaceutical compositions comprising an antisense oligomer (e.g., antisense oligonucleotide, ASO) and an oral delivery agent (e.g., SNAC, C10, 5-CNAC, or salts thereof, hydrates thereof, or solvates thereof, or combinations thereof), wherein the antisense oligomer is 16 to 22 contiguous nucleotides in length, wherein the sequence of the antisense oligomer comprises a 16-nucleotide contiguous sequence that is 100% complementary to the sequence of SEQ ID NO: 31, wherein the antisense oligomer is a gapmer comprising at least one LNA unit, and wherein the antisense oligomer targets an RNA encoding PCSK9. In some aspects, the RNA is a pre-mRNA, a splice variant of the pre-mRNA, a mature mRNA, or an allelic variant or mutant thereof. In some aspects, the antisense oligomer targets a sequence within an exon. In some aspects, the antisense oligomer targets a sequence within an intron. In some aspects, the antisense oligomer targets a sequence comprising the boundary between exon and intron. In some aspects, the antisense oligomer targets the upstream sequence (e.g., 5'UTR) of the 5' end of the open reading frame encoding PCSK9. In some aspects, the antisense oligomer targets the downstream sequence (e.g., 3'UTR) of the 3' end of the open reading frame encoding PCSK9.

[0007] In some aspects, the LNA is oxy-LNA, thio-LNA, amino-5LNA, 5'-methyl-LNA, ENA, cET, cMOE, or a combination thereof. In some aspects, the LNA is a stereoisomer of β-D configuration or α-L configuration. In some aspects, the antisense oligomer comprises at least one cET unit. In some aspects, the antisense oligomer comprises 2, 3, 4, 5, 6, or 7 LNA units. In some aspects, all LNA units in the antisense oligomer are stereoisomers of the same configuration. In some aspects, all LNA units in the antisense oligomer are β-D-oxy LNA units. In some aspects, all LNA units in the antisense oligomer are α-L-oxy LNA units.

[0008] In some aspects, the sequence of the antisense oligomer comprises at least one internucleoside linkage that is phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, or boranophosphate. In some aspects, all internucleoside linkages are phosphorothioate. In some aspects, one or more of the internucleoside linkages comprise a chiral center in the R conformation and / or a chiral center in the S conformation. In some aspects, all chiral centers are in the R conformation. In some aspects, all chiral centers are in the S conformation. In some aspects, the antisense oligomer and the oligomer of SEQ ID NO: 31 can form a duplex with improved thermal stability relative to the corresponding duplex containing the corresponding LNA-free antisense oligomer.

[0009] The present disclosure also provides a pharmaceutical composition comprising an antisense oligonucleotide conjugate and an oral delivery agent (e.g., SNAC, C10, 5-CNAC, or a salt thereof, a hydrate thereof, or a solvate thereof, or a combination thereof), wherein the antisense oligonucleotide conjugate comprises the following (i) and (ii): (i) antisense oligomers consisting of 16–22 consecutive nucleotides; wherein the sequence of the antisense oligomer comprises a contiguous sequence of 16 nucleotides in length that is 100% complementary to the sequence of SEQ ID NO: 31; and wherein the antisense oligomer is a gapmer comprising at least one LNA unit; and (ii) at least one non-nucleotide or non-polynucleotide moiety covalently attached to the antisense oligomer, either directly or via a linker disposed between the contiguous oligomer sequences; wherein the antisense oligonucleotide conjugate targets RNA (eg, mRNA) encoding PCSK9.

[0010] In some aspects, the RNA is a pre-mRNA, a splice variant of a pre-mRNA, a mature mRNA, or an allelic variant or mutant thereof, as disclosed above.

[0011] In some aspects, the non-nucleotide moiety or non-polynucleotide moiety is a liver targeting moiety that is linked to the 5'-end or 3'-end of the antisense oligomer.In some aspects, the liver targeting moiety is linked to the antisense oligomer via a linker.In some aspects, the liver targeting moiety comprises a carbohydrate conjugate moiety that comprises a carbohydrate selected from the group consisting of galactose, lactose, N-acetylgalactosamine (GalNAc), mannose, mannose-6-phosphate, and combinations thereof.

[0012] In some aspects, the carbohydrate-conjugate moiety is not a linear carbohydrate polymer. In some aspects, the carbohydrate-conjugate moiety is a carbohydrate group comprising one, two, three, or four carbohydrate moieties. In some aspects, all of the carbohydrate moieties are identical. In some aspects, at least one carbohydrate moiety is different (non-identical) from the remaining carbohydrate moieties. In some aspects, the carbohydrate-conjugate moiety comprises at least one conjugate moiety that targets the asialoglycoprotein receptor. In some aspects, the conjugate moiety that targets the asialoglycoprotein receptor comprises a monovalent, divalent, trivalent, or tetravalent GalNAc cluster. In some aspects, each GalNAc in the GalNAc cluster is linked to a branch point group via a spacer. In some aspects, the branch point group comprises a peptide, e.g., dilysine. In some aspects, the spacer comprises a PEG spacer. In some aspects, the linker comprises a biocleavable phosphate nucleotide linker comprising a C6-C12 aminoalkyl group or 1-6 nucleotides. In some aspects, the trivalent GalNAc cluster comprises a Conj1, Conj2, Conj1a, or Conj2a GalNAc conjugate moiety. In some aspects, the non-nucleotide or non-polynucleotide moiety is covalently attached to the antisense oligomer via a covalent bond.

[0013] The present disclosure also provides pharmaceutical compositions comprising an antisense oligonucleotide conjugate of SEQ ID NO: 18 or SEQ ID NO: 19, as shown in FIG. 18A, and an oral delivery agent (e.g., SNAC, C10, 5-CNAC, or a salt thereof, a hydrate thereof, or a solvate thereof, or a combination thereof), wherein the antisense oligonucleotide conjugate targets RNA encoding PCSK9. In some aspects, the RNA is pre-mRNA, a splice variant of pre-mRNA, a mature mRNA, or an allelic variant or mutant thereof. In some aspects, the antisense oligomer targets a sequence within an exon. In some aspects, the antisense oligomer targets a sequence within an intron. In some aspects, the antisense oligomer targets a sequence including a boundary between an exon and an intron. In some aspects, the antisense oligomer targets a sequence upstream of the 5' end of an open reading frame encoding PCSK9. In some aspects, the antisense oligomer targets a sequence downstream of the 3' end of the open reading frame encoding PCSK9.

[0014] In some aspects, the pharmaceutical compositions disclosed herein further comprise at least one pharmaceutically acceptable additive or a combination thereof. In some aspects, the at least one pharmaceutically acceptable additive or a combination thereof is selected from the group consisting of: pH adjusters, preservatives, flavorings, taste masking agents, fragrances, humectants, tonicity agents, colorants, surfactants, plasticizers, lubricants, fluidization aids, compression aids, solubilizers, excipients, diluents, phosphate buffer salts, citric acid, glycols, dispersing agents, crospovidone, povidone, or any combination thereof. In some aspects, the pharmaceutical compositions disclosed herein further comprise a therapeutic agent selected from the group consisting of: statins, bile-sorbing resins, nicotinic acid, fibric acid derivatives, probucol, neomycin, dextrothyroxine, plant stanol esters, cholesterol absorption inhibitors, implitapide, inhibitors of bile acid transporters, regulators of hepatic CYP7a, estrogen replacement therapy agents, anti-inflammatory agents, or combinations thereof. In some aspects, the statin is selected from the group consisting of: Lovastatin, cerivastatin, pravastatin, atorvastatin, simvastatin, rosuvastatin, fluvastatin, and combinations thereof.

[0015] The present disclosure also provides a method for treating a disease or condition caused by an abnormal expression level and / or activity of PCSK9 in a subject, wherein the method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition disclosed herein, for example, a composition comprising an antisense oligonucleotide conjugate of the present disclosure (e.g., CIVI008) and an oral delivery agent (e.g., SNAC, C10, 5-CNAC, or a salt thereof, a hydrate thereof, or a solvate thereof, or a combination thereof), wherein administration of the pharmaceutical composition reduces the subject's serum PCSK9 level and / or serum LDL cholesterol level. In some aspects, the PCSK9 is (i) a PCSK9 allelic variant; (ii) a PCSK9 mutant; or (iii) a PCSK9 splice variant. In some aspects, the PCSK9 mutant is a gain-of-function mutant of PCSK9.

[0016] In some aspects, the disease or condition is selected from the group consisting of atherosclerosis, hypercholesterolemia, HDL / LDL cholesterol imbalance, dyslipidemia, coronary artery disease (CAD), and coronary heart disease (CHD). In some aspects, the dyslipidemia is familial hyperlipidemia (FCHL) or acquired hyperlipidemia. In some aspects, the hypercholesterolemia is familial hypercholesterolemia or statin-resistant hypercholesterolemia.

[0017] In some aspects, the methods of treatment disclosed herein further comprise administration of a therapeutic agent selected from the group consisting of: statins, bile-sorbing resins, nicotinic acid, fibric acid derivatives, probucol, neomycin, dextrothyroxine, plant stanol esters, cholesterol absorption inhibitors, implitapide, inhibitors of bile acid transporters, regulators of hepatic CYP7a, estrogen replacement therapy agents, anti-inflammatory agents, or combinations thereof. In some aspects, the statin is selected from the group consisting of: Lovastatin, cerivastatin, pravastatin, atorvastatin, simvastatin, rosuvastatin, fluvastatin, and combinations thereof.

[0018] In some aspects, the pharmaceutical compositions disclosed herein are administered orally. In some aspects, the pharmaceutical compositions are administered in a single dose. In some aspects, the pharmaceutical compositions are administered in multiple doses.

[0019] The present disclosure also provides a method of treating a disorder selected from the group consisting of atherosclerosis, hyperlipidemia, hypercholesterolemia, HDL / LDL cholesterol imbalance, coronary artery disease (CAD), or coronary heart disease (CHD) in a subject, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition disclosed herein, e.g., a composition comprising an antisense oligonucleotide conjugate of the present disclosure (e.g., CIVI008) and an oral delivery-enhancing agent (e.g., SNAC, C10, 5-CNAC, or salts thereof, hydrates thereof, or solvates thereof, or combinations thereof).

[0020] The present disclosure also provides a method for reducing the expression level of PCSK9 and / or the activity of PCSK9 in a cell in vitro or in vivo, comprising administering to the cell an effective amount of a pharmaceutical composition disclosed herein, e.g., a composition comprising an antisense oligonucleotide conjugate of the present disclosure (e.g., CIVI008) and an oral delivery-enhancing agent (e.g., SNAC, C10, 5-CNAC, or salts thereof, hydrates thereof, or solvates thereof, or combinations thereof).

[0021] Also provided is a method for reducing PCSK9 expression levels and / or PCSK9 activity in a subject, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition disclosed herein, e.g., a composition comprising an antisense oligonucleotide conjugate of the present disclosure (e.g., CIVI008) and an oral delivery-enhancing agent (e.g., SNAC, C10, 5-CNAC, or salts thereof, hydrates thereof, or solvates thereof, or combinations thereof).

[0022] The present disclosure also provides a method for lowering cholesterol levels in a subject, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition disclosed herein, e.g., a composition comprising an antisense oligonucleotide conjugate of the present disclosure (e.g., CIVI008) and an oral delivery-enhancing agent (e.g., SNAC, C10, 5-CNAC, or salts thereof, hydrates thereof, or solvates thereof, or combinations thereof).

[0023] Also provided is a method for producing a pharmaceutical composition, the method comprising mixing: (i) and (ii) (i) antisense oligomers of 16–22 contiguous nucleotides in length; wherein the sequence of the antisense oligomer comprises a contiguous sequence of 16 nucleotides in length that is 100% complementary to the sequence of SEQ ID NO: 31; wherein the antisense oligomer is a gapmer comprising at least one LNA unit; and wherein the antisense oligomer targets RNA encoding PCSK9; and (ii) an oral delivery agent (e.g., SNAC, C10, 5-CNAC, or a salt thereof, a hydrate thereof, or a solvate thereof, or a combination thereof).

[0024] The present disclosure also provides a method of making a pharmaceutical composition, the method comprising mixing: (i) and (ii) (i) a conjugate comprising (a) and (b) of the following: (a) Antisense oligomers consisting of 16–22 consecutive nucleotides; wherein the sequence of the antisense oligomer comprises a contiguous sequence of 16 nucleotides in length that is 100% complementary to the sequence of SEQ ID NO: 31; and wherein the antisense oligomer is a gapmer comprising at least one LNA unit; and (b) at least one non-nucleotide or non-polynucleotide moiety covalently attached to the antisense oligomer, either directly or via a linker disposed between the contiguous oligomer sequences; wherein the antisense oligonucleotide conjugate targets RNA encoding PCSK9; and (ii) an oral delivery agent (e.g., SNAC, C10, 5-CNAC, or a salt thereof, a hydrate thereof, or a solvate thereof, or a combination thereof).

[0025] In some aspects, the sequence of the antisense oligomer comprises or consists of SEQ ID NO:26 (i.e., a sequence 100% complementary to SEQ ID NO:31, having the same length as SEQ ID NO:31). In some aspects, the sequence of the antisense oligomer comprises or consists of SEQ ID NO:2 or SEQ ID NO:3 (i.e., a sequence having the base sequence of SEQ ID NO:26, but containing a particular pattern of base and backbone modifications). In some aspects, the antisense oligomer conjugate comprises or consists of SEQ ID NO:18 or SEQ ID NO:19; i.e., SEQ ID NO:2 or SEQ ID NO:3 conjugated to a GalNAc2 moiety.

[0026] In some aspects of the pharmaceutical compositions and methods of the present disclosure, the oral delivery agent comprises, for example, caprylic acid (C8), capric acid (C10), its derivatives, its pharmaceutically acceptable hydrates, solvates, or salts, or any combination thereof.In some aspects, the pharmaceutically acceptable salts are sodium salts, potassium salts, and combinations thereof.In some aspects, the pharmaceutical composition is solid.

[0027] In some aspects, the pharmaceutical composition is in the form of a tablet or capsule. In some aspects, the capsule is a liquid capsule. In some aspects, the pharmaceutical composition is enteric coated. In some aspects, the tablet or capsule is enteric coated. In some aspects, the tablet or capsule weighs 5 mg to 1000 mg, 10 mg to 500 mg, 10 mg to 250 mg, 100 mg to 200 mg, or 250 mg to 500 mg. In some aspects, the amount of antisense oligomer or antisense oligonucleotide conjugate in the tablet or capsule is in the range of 1 mg to 100 mg, 5 mg to 100 mg, 10 mg to 100 mg, 20 mg to 100 mg, or 20 mg to 50 mg.

[0028] In some aspects, the oral delivery agent is a salt, hydrate, or solvate of N-(8-(2-hydroxybenzoyl)amino)caprylic acid, or a combination thereof. In some aspects, the salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid is selected from the group consisting of sodium salt, potassium salt, calcium salt, and any combination thereof of N-(8-(2-hydroxybenzoyl)amino)caprylic acid. In some aspects, the salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid is the sodium salt. In some aspects, the salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid is the monosodium salt (salcaprozate sodium 203787-91-1, SNAC, sodium 8-(2-hydroxybenzamido)octanoate). In some aspects, the salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid is the disodium salt.

[0029] In some aspects, the oral delivery agent is a salt, hydrate, or solvate of N-(5-chlorosalicyloyl)-8-aminocaprylic acid, or a combination thereof. In some aspects, the salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid is selected from the group consisting of sodium salt, potassium salt, calcium salt, and any combination thereof of N-(5-chlorosalicyloyl)-8-aminocaprylic acid. In some aspects, the salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid is a sodium salt. In some aspects, the salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid is a monosodium salt (5-SNAC). In some aspects, the salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid is a disodium salt.

[0030] In some aspects, the oral delivery agent is a salt, hydrate, or solvate of capric acid (C10), or a combination thereof. In some aspects, the salt of capric acid (C10) is selected from the group consisting of sodium, potassium, and calcium salts of capric acid (C10), and any combination thereof. In some aspects, the salt of capric acid (C10) is sodium caprate.

[0031] In some aspects, the pharmaceutical composition is administered about 5 to about 60 minutes before a meal, hi some aspects, the pharmaceutical composition is administered at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, or at least about 60 minutes before a meal.

[0032] In some aspects, the pharmaceutical composition further comprises a pH-sensitive coating agent. In some aspects, the pH-sensitive coating agent is a pH-sensitive polymer. In some aspects, the pH-sensitive polymer comprises cellulose, acrylic acid, a derivative thereof, or a combination thereof. In some aspects, the pH-sensitive coating agent comprises a pH-sensitive hydrogel, a pH-activated drug delivery system, a pH-sensitive liposome, micelle or lipid nanoparticle, a pH-sensitive microsphere, a pH-sensitive nanoparticle, or any combination thereof.

[0033] The present disclosure also provides (GalNAc)3-amino-hexamethylene-5'phosphorothioyl)-2'-O,4'-C-methyleneadenosilyl-(3'>5'O,O-phosphorothioyl)-2'-O,4'-C-methyleneadenosilyl-(3'>5'O,O-phosphorothioyl)-2'-O,4'-C-methylenethimidylyl-(3'>5'O,O-phosphorothioyl)-2'-deoxyguanosilyl-(3 '>5'O,O-phosphorothioyl)-2'-deoxycytidinylyl-(3'>5'O,O-phosphorothioyl)-2'-deoxythymidylyl-(3'>5'O,O-phosphorothioyl)-2'-deoxyadenosilyl-(3'>5'O,O-phosphorothioyl)-2'-deoxycytidinylyl-(3'>5'O,O-phosphorothioyl)-2'-deoxyadenosilyl-(3'>5'O,O-phosphorothioyl) O-phosphorothioyl)-2'-deoxyadenosilyl-(3'>5'O,O-phosphorothioyl)-2'-deoxyadenosilyl-(3'>5'O,O-phosphorothioyl)-2'-deoxyadenosilyl-(3'>5'O,O-phosphorothioyl)-2'-deoxycytidinylyl-(3'>5'O,O-phosphorothioyl)-2'-O,4'-C-methylene(5-methyl-cytidinyl)-(3'>5'O, and a pill or capsule comprising an oral delivery agent selected from the group consisting of SNAC, C10,5-CNAC, a hydrate thereof, a solvate thereof, or a salt thereof, and a combination thereof.

[0034] The present disclosure also provides a pill or capsule comprising CIVI008 as shown in Figure 18B, and comprising an oral delivery agent selected from the group consisting of SNAC, C10, 5-CNAC, a hydrate thereof, a solvate thereof, or a salt thereof, and combinations thereof. In some aspects, the pill or capsule comprises CIVI008 and SNAC. In some aspects, the pill or capsule comprises CIVI008 and C10. In some aspects, the pill or capsule comprises CIVI008 and 5-CNAC.

[0035] In some aspects of the compositions and methods disclosed herein, the oral delivery agent comprises: [ka] and; where R 1 , R 2 , R 3 , and R 4 are independently hydrogen, -OH, -NR 6 R 7 , halogen, C1-C4 alkyl, or C1-C4 alkoxy; R 5 substituted or unsubstituted C2 to C 16 Alkylene, substituted or unsubstituted C2-C 16 Alkenylene, substituted or unsubstituted C1-C 12 alkyl (arylene), or substituted or unsubstituted aryl (C1-C4 alkylene); and R 6 and R 7 are independently hydrogen, oxygen, or C1-C4 alkyl.

[0036] In some aspects of the compositions and methods disclosed herein, the oral delivery agent is selected from the group consisting of N-(8-[2-hydroxybenzoyl]amino)caprylic acid (SNAC), N-(5-chlorosalicyloyl)-8-aminocaprylic acid (5-CNAC), N-(10-[2-hydroxybenzoyl]amino)decanoic acid (SNAD), 4-[(4-chloro-2-hydroxybenzoyl)amino]butanoic acid (4-CNAB), N-(8-[4-methoxy-chloro-2-hydroxybenzoyl-amino)octanoic acid (4-MOAC), 8-(4-hydroxyphenoxy)octanoic acid (4-HPO), 4-m-tosyl The compound is selected from the group consisting of 4-isopropylbenzyloxybutyric acid (3-TBA), 4-(3-hydroxyphenylsulfanyl)butyric acid (3-HPSB), 5-phenylpentanoic acid (5-PPA), 8-(2-hydroxyphenoxy)octyldiethanolamine (2-HPOD), (4-isopropylbenzyloxy)acetic acid (4-IBOA), 2-(5-pentanoic acid)-5-(2-hydroxyphenyl)-1,3,4-oxadiazole (2-PHOD), 7-oxo-7-phenylheptanoic acid (7-OPHA), 4-(3-fluorophenylsulfanyl)butyric acid (3-FPSB), or any combination thereof.

[0037] In some aspects, the oral delivery agent is the disodium salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid.

[0038] In some aspects, the oral delivery agent is a salt, hydrate, or solvate of N-(5-chlorosalicyloyl)-8-aminocaprylic acid, or a combination thereof. In some aspects, the salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid is selected from the group consisting of sodium salt, potassium salt, calcium salt, and any combination thereof of N-(5-chlorosalicyloyl)-8-aminocaprylic acid. In some aspects, the salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid is a sodium salt. In some aspects, the salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid is a monosodium salt. In some aspects, the salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid is a disodium salt.

[0039] The present disclosure also provides a capsule comprising (i) a composition comprising CIVI008 and (ii) a composition comprising 5-CNAC. In some aspects, the composition comprising CIVI008 and the composition comprising 5-CNAC are a dry blend. In some aspects, the capsule is a gelatin capsule. In some aspects, the gel capsule is a hard-shell gelatin capsule. In some aspects, the capsule is enteric coated. In some aspects, the capsule contains 5 mg to 30 mg of CIVI008 and 100 mg to 200 mg of 5-CNAC. In some aspects, the capsule contains about 5 mg, about 10 mg, about 20 mg, about 25 mg, or about 30 mg of CIVI008. In some aspects, the capsule contains 10 mg of CIVI008 and 100 mg of 5-CNAC; 20 mg of CIVI008 and 200 mg of 5-CNAC; 5 mg of CIVI008 and 200 mg of 5-CNAC; 10 mg of CIVI008 and 200 mg of 5-CNAC; 25 mg of CIVI008 and 200 mg of 5-CNAC; or 30 mg of CIVI008 and 200 mg of 5-CNAC. In some aspects, the capsule contains a statin.

[0040] In some aspects, the present disclosure provides a method for treating a disorder selected from the group consisting of atherosclerosis, hyperlipidemia, hypercholesterolemia, HDL / LDL cholesterol imbalance, coronary artery disease (CAD), or coronary heart disease (CHD) in a subject, comprising administering to a subject in need thereof a capsule containing (i) a composition comprising CIVI008 and (ii) a composition comprising 5-CNAC. The present disclosure also provides a method for reducing PCSK9 expression levels and / or PCSK9 activity in a subject, comprising administering to a subject in need thereof a capsule containing (i) a composition comprising CIVI008 and (ii) a composition comprising 5-CNAC. Also provided is a method for lowering cholesterol levels in a subject, comprising administering to a subject in need thereof a capsule containing (i) a composition comprising CIVI008 and (ii) a composition comprising 5-CNAC. Also provided is a capsule comprising (i) a composition comprising CIVI008 and (ii) a composition comprising 5-CNAC for use in treating a disorder selected from the group consisting of atherosclerosis, hyperlipidemia, hypercholesterolemia, HDL / LDL cholesterol imbalance, coronary artery disease (CAD), or coronary heart disease (CHD). Also provided is a capsule comprising (i) a composition comprising CIVI008 and (ii) a composition comprising 5-CNAC for use as a therapeutic agent for reducing PCSK9 expression levels and / or PCSK9 activity in a subject. The present disclosure also provides a capsule comprising (i) a composition comprising CIVI008 and (ii) a composition comprising 5-CNAC for use as a therapeutic agent for lowering cholesterol levels in a subject. Also provided is a method for producing a capsule comprising a composition comprising CIVI008 and a composition comprising 5-CNAC, the method comprising: (i) dry-blending a first composition comprising CIVI008 and a second composition comprising 5-CNAC; and (ii) encapsulating the dry-blended mixture obtained in step (i). [Brief explanation of the drawings]

[0041] [Figure 1]1A, 1B, and 1C show examples of tri-GalNAc conjugates that can be used in the oral compositions disclosed herein. Conjugates 1-4 represent four suitable GalNAc conjugate moieties, and conjugates 1a-4a represent the same conjugates with an additional linker moiety (Y) used to link the conjugate to an oligomer (region A) or a biocleavable linker (e.g., region B). The wavy line represents a covalent bond to the oligomer. [Figure 2]

[0033] Figures 1A and 1B show examples of cholesterol- and tocopherol-conjugated moieties that can be used in the oral compositions disclosed herein. Conjugates 5a and 6a represent the same conjugate with an additional linker moiety (Y) that is used to link the conjugate to an oligomer (region A) or a biocleavable linker (region B, etc.). The wavy line represents a covalent bond to the oligomer. [Figure 3] Specific LNA compounds are indicated: beta-D-oxy LNAs are identified by a superscript L following the letter; the subscript s represents a phosphorothioate linkage; the superscript Me preceding a capital C indicates a 5-methylcytosine LNA; non-LNA nucleotides are DNA nucleotides (no superscript L). [Figure 4] Examples of cholesterol conjugates of LNA compounds that can be used in the oral compositions disclosed herein are shown below: beta-D-oxy LNAs are identified by the superscript L following the letter; the subscript s represents a phosphorothioate bond; the subscript o represents a phosphodiester bond; the superscript Me preceding the capital C represents 5-methylcytosine LNA; and non-LNA nucleotides are DNA nucleotides (no superscript L). [Figure 5A]

[0033] Figure 1 shows an example of a GalNAc conjugate of an LNA compound that can be used in the oral compositions disclosed herein. This conjugate essentially corresponds to Conj2a in Figure 1, where the LNA oligomer is replaced by a wavy line. Beta-D-oxy LNAs are identified by the superscript L following the letter; the subscript s represents a phosphorothioate bond; the superscript Me preceding a capital C indicates 5-methylcytosine LNA; and non-LNA nucleotides are DNA nucleotides (no superscript L). [Figure 5B] The detailed structure of SEQ ID NO:18 is shown. [Figure 5C] The detailed structure of SEQ ID NO:19 is shown. [Figure 6] An example of a FAM conjugate group is shown. [Figure 7] LNA-FAM conjugates with and without a cleavable phosphodiester bond are shown. Beta-D-oxy LNAs are identified by a superscript L following the letter; subscript s represents a phosphorothioate bond; subscript o represents a phosphodiester bond; superscript Me preceding a capital C represents a 5-methylcytosine LNA; non-LNA nucleotides are DNA nucleotides (no superscript L). [Figure 8] Anti-PCSK9 gapmers ranked based on in vitro potency are shown. [Figure 9] A selection of anti-PCSK9 gapmers ranked based on in vitro potency is shown. [Figure 10] 1 shows in vitro potency and IC50 data for selected anti-PCSK9 compounds. [Figure 11] 1 shows in vivo levels of alanine aminotransferase (ALT) for selected anti-PCSK9 conjugates. [Figure 12]It is shown that the internucleoside linkage L can be, for example, phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate, or methylphosphonate (e.g., phosphodiester). PO is a phosphodiester linkage. Compound (a) has a region B containing a single DNA (or RNA), and the linkage between the second and first regions is PO. Compound (b) has two DNA / RNA (e.g., DNA) nucleosides linked by phosphodiester bonds. Compound (c) has three DNA / RNA (e.g., DNA) nucleosides linked by phosphodiester bonds. In some aspects, region B can be further extended with additional phosphodiester DNA / RNA (e.g., DNA nucleosides). The conjugate group (denoted X, or herein referred to as region C) is shown on the left side of each compound (e.g., cholesterol, GalNAc, Conj1-4, 1a-4a, and 5 or 6) and can optionally be covalently attached to the terminal nucleoside of region B via a phosphonucleoside linking group (such as phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate, phosphoramidate, or methylphosphonate) or can be linked via another alternative bond, for example, via a triazole bond (see L in compounds d, e, and f). [Figure 13]12 shows compounds containing an optional linker (Y) between the third (conjugate) region (X) and the second region (region B); again, the terminology shown in FIG. 12 is used. Suitable linkers are disclosed herein, including, for example, alkyl linkers (e.g., C6 linkers). In compounds a), b), and c), the linker between X and region B is linked to region B via a phosphonucleoside linking group (e.g., phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate, phosphoramidate, or methylphosphonate), or alternatively via another alternative bond, e.g., a triazole bond (Li). In these compounds, Li represents the internucleoside linkage between the first region (A) and the second region (B). Compounds d), e), and f) further comprise a linker (Y) between region B and the conjugate group, where region Y can be linked to region B, for example, via a phosphonucleoside linking group (phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, boranophosphate, or methylphosphonate) or, in some aspects, via a triazole bond. Additionally or alternatively, X can be an activating or reactive group. X can be covalently linked to region B via a phosphonucleoside linking group (phosphodiester, phosphorothioate, phosphoroamidate, phosphorodithioate, boranophosphate, or methylphosphonate), or can be linked via another alternative bond, for example, a triazole bond. [Figure 14]Figure 1 shows in vivo silencing of PCSK9 mRNA after cholesterol conjugate administration. Mice were injected with a single 10 mg / kg dose of unconjugated LNA-antisense oligonucleotide (#40) or an equimolar amount of LNA antisense oligonucleotide conjugated to cholesterol with different linkers (conjugates), and sacrificed on days 1, 3, 7, and 10 after administration. RNA was isolated from liver and kidney and subjected to PCSK9-specific RT-qPCR. Panel A shows PCSK9 mRNA quantification in liver samples normalized to BACT and expressed as a percentage of the mean of the equivalent saline control. Panel B shows PCSK9 mRNA quantification in kidney samples normalized to BACT and expressed as a percentage of the mean of the equivalent saline control. [Figure 15] 1 shows Kim-1 expression in a rat safety study (see Example 5). [Figure 16] 1 shows serum PCSK9 and LDL cholesterol in samples from cynomolgus monkeys given four injections (one injection per week) of SEQ ID NO:2 or SEQ ID NO:18 at 0.5 mg / kg / week or 1.5 mg / kg / week. [Figure 17] 1 shows serum PCSK9 and LDL cholesterol in samples from cynomolgus monkeys given four injections (one injection per week) of SEQ ID NO:3 and SEQ ID NO:19 at 0.5 or 1.5 mg / kg / week. [Figure 18A] Schematic structures of antisense oligonucleotide conjugates of SEQ ID NO:18 and SEQ ID NO:19 are shown. [Figure 18B] The detailed chemical structure and formal name of the antisense oligonucleotide conjugate CIVI008 (sepadaclusen) are shown. [Figure 19] 1 shows the chemical structures of exemplary oral delivery agents. [Figure 20] 1 shows the design of the clinical trial shown in Example 9. [Figure 21] Plasma concentration levels of CIVI008 after administration with and without SNAC are shown. [Figure 22]1 shows plasma AUC levels for the subcutaneous (SQ) groups (P0001, P0002, P0901, and P0902) and oral (PO) groups (P0301, P0302, P0303, P1201, P1202, P1203, P0401, P0402, P0403, P1301, P1302, P1303, P0501, and P1401) in the study of Example 9. [Figure 23] 1 shows the liver concentration levels for CIVI008 in the SQ and PO groups of the study shown in Example 9. [Figure 24] The mean change in PCSK9 expression levels relative to baseline levels at days 35 and 42 after oral administration of CIVI008 is shown. [Figures 25A-25B] Changes in plasma LDL cholesterol levels relative to baseline after administration of one or two capsules of CIVI008 (Figure 25A) or under control conditions (Figure 25B) are shown. [Figure 26] 1 shows the change from baseline in plasma LDL cholesterol levels over the course of the study shown in Example 9. [Figure 27] 1 shows a schematic illustration of the study shown in Example 11. [Figure 28] 1 shows plasma concentration levels of CIVI008 after dosing with 5-CNAC. [Figure 29] 1 shows a comparison between the pharmacokinetic parameters (mean AUC0-5 and mean Cmax) corresponding to administration of CIVI008 capsules containing either SNAC or 5-CNAC. [Figure 30] Plasma concentration levels and pharmacokinetic parameters (mean AUC0-5 and mean Cmax) corresponding to administration of CIVI008 in size 4 capsules (Group A) or size 0 capsules (Group B) containing 5-CNAC are shown. [Figure 31] 1 shows the mean AUC and mean Cmax on days 1 and 3 in monkeys administered 5 mg to 30 mg of CIVI008 formulated with 5-CNAC. [Figure 32A-32B]Figure 32 shows a comparison of mean pharmacokinetic parameters on days 1 and 3 in monkeys administered similar doses of CIVI008 in capsules prepared either by dry blending (Figure 32A) or lyophilization (Figure 32B). [Figure 33] After 22 days of treatment, PCSK9 and plasma LDL were reduced from baseline. [Figure 34] 1 shows the % LDL reduction from baseline in monkeys administered CIVI008 formulated with either SNAC or 5-CNAC. DETAILED DESCRIPTION OF THE INVENTION

[0042] The present disclosure provides pharmaceutical compositions for oral delivery comprising an antisense oligomer (e.g., an antisense oligonucleotide) or an antisense oligonucleotide conjugate (e.g., CIVI008) and an oral delivery agent (e.g., C10, SNAC, or 5-CNAC). In some aspects, the antisense oligomer is 16 to 22 contiguous nucleotides in length, and comprises a 16-nucleotide sequence that is 100% complementary to the PCSK9 target sequence of SEQ ID NO: 31; wherein the antisense oligomer is a gapmer containing at least one LNA unit; and wherein the antisense oligomer targets an RNA encoding PCSK9.

[0043] In some aspects, the oligomer comprises the sequence of SEQ ID NO: 18 or SEQ ID NO: 19. In some aspects, the antisense oligonucleotide conjugate comprises (i) and (ii) below: (i) an antisense oligomer portion of 16 to 22 consecutive nucleotides in length; wherein the sequence comprises a contiguous sequence of 16 nucleotides in length that is 100% complementary to the PCSK9 target sequence of SEQ ID NO: 31; wherein the antisense oligomer is a gapmer comprising at least one LNA unit; and wherein the antisense oligomer targets RNA encoding PCSK9; and (ii) a GalNAc moiety attached to the 5' end of the oligomer. In some aspects, the antisense oligonucleotide conjugate is CIVI008, as shown in Figure 18B.

[0044] Oral pharmaceutical compositions disclosed herein, e.g., compositions comprising CIVI008, e.g., in the form of a pill or capsule, include at least one oral delivery agent that protects the payload (e.g., CIVI008) during passage through the gastrointestinal tract (e.g., passage through the stomach and upper small intestine). In some aspects, the oral delivery agent is a salt (e.g., a sodium salt) of a fatty acid such as caprylic acid (octanoic acid, C8), capric acid (decanoic acid or decylic acid, C10), a derivative thereof (e.g., 8-[2-hydroxybenzoyl]amino)caprylic acid, SNAC, or 5-CNAC), or a combination thereof. In a particular aspect, the oral delivery agent is a salt, e.g., the sodium salt of SNAC or the sodium salt of 5-CNAC.

[0045] Also provided are methods of making the pharmaceutical compositions disclosed herein.The present disclosure also provides methods of treating a subject in need thereof, comprising administering to a subject an oral pharmaceutical composition disclosed herein.

[0046] definition In order to make the description herein more readily understandable, certain terms are first defined, with further definitions provided throughout the Detailed Description section of the specification.

[0047] It should be noted that the terms "a" or "an" item refer to one or more of that item; for example, "a nucleotide sequence" should be understood to refer to a sequence of one or more nucleotides. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used synonymously herein.

[0048] Furthermore, "and / or" herein should be considered a specific disclosure of each of the two specified characteristics or components in the presence or absence of the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0049] Whenever aspects described herein are expressed using the phrase "comprising," it should be understood that this also refers to similar aspects described using "consisting of" and / or "consisting essentially of."

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in the technical fields relevant to this disclosure. For example, the Concise Dictionary of Biomedicine and Molecular Biology, 2nd Edition, Juo, Pei-Show, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd Edition, 1999, Academic Press Publishers; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press; these provide a general dictionary for those skilled in the art for many of the terms used in this disclosure.

[0051] Units, prefixes, and symbols are expressed in the form accepted by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. Unless otherwise expressly stated, the 5' to 3' direction of nucleotide sequences is expressed in left to right direction. The amino to carboxy direction of amino acid sequences is expressed in left to right direction. The headings provided herein are not intended to limit the various aspects of this disclosure, which may be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more particularly defined by reference to the specification as a whole.

[0052] As used herein, the term "about" means "approximately," "roughly," "on the order of," or "in the range of." When the term "about" is used in conjunction with a numerical range, it modifies the range by extending it above or below the stated numerical value. Generally, the term "about" allows the recited numerical value to be modified above or below that value, for example, by a variance of 10 percent. For example, when an antisense oligomer or antisense oligonucleotide conjugate (e.g., CIVI008) is said to reduce PCSK9 protein expression by at least about 60% in cells following administration of the oligomer or conjugate, it means that PCSK9 expression levels are reduced in the range of 50% to 70%.

[0053] For purposes of this disclosure, the terms "oligomer" and "oligonucleotide" are used synonymously and refer to a molecule formed by the covalent linkage of two or more nucleotides. As used herein, a single nucleotide (unit) may be referred to as a monomer or unit.

[0054] As used herein, the term "derivative" refers to a compound that is structurally related to a compound disclosed herein (e.g., C10, SNAC, or 5-CNAC), e.g., a compound having the same carbon skeleton, e.g., a chemical modification introduced into a side chain or group disclosed herein at one or more positions, wherein the derivative has a biological activity substantially similar to that of the substance or molecule, e.g., the ability to function as an oral delivery agent; it is a derivative.

[0055] The term "nucleotide" as used herein refers to a glycoside comprising a sugar moiety, a base moiety, and a covalent linking group (e.g., a phosphate or phosphorothioate internucleoside linking group), and includes both naturally occurring nucleotides, such as DNA or RNA, and non-naturally occurring nucleotides comprising modified sugar and / or base moieties. Non-naturally occurring nucleotides are also referred to herein as "nucleotide analogs." A single nucleotide may also be referred to herein as a monomer or unit. In certain aspects, the term "nucleotide analog" refers to a nucleotide having a modified sugar moiety. Non-limiting examples of nucleotides having modified sugar moieties (e.g., LNA) are disclosed herein. In other aspects, the term "nucleotide analog" refers to a nucleotide having a modified nucleobase moiety. Nucleotides having modified nucleobase moieties include, but are not limited to, 5-methyl-cytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine. In some aspects, the terms "nucleotide," "unit," and "monomer" are used synonymously. When referring to a sequence of nucleotides or monomers, it should be understood that reference is made to a sequence of bases such as A, T, G, C, or U, and analogs thereof.

[0056] The term "nucleoside" is used herein to refer to a glycoside comprising a sugar moiety and a base moiety, and can therefore refer to a nucleotide unit covalently linked by an internucleoside bond between nucleotides of an oligomer (e.g., antisense oligomer, ASO) or antisense oligonucleotide conjugate (e.g., CIVI008) disclosed herein. In the field of biotechnology, the term "nucleotide" is also often used to refer to a nucleic acid monomer or unit. With respect to an oligomer (e.g., antisense oligomer, ASO) or antisense oligonucleotide conjugate (e.g., CIVI008) disclosed herein, the term "nucleotide" can potentially refer to a nucleobase sequence comprising a single base, i.e., cytosine (DNA and RNA), guanine (DNA and RNA), adenine (DNA and RNA), thymine (DNA), and uracil (RNA), with a sugar backbone and an internucleoside linkage present. Similarly, the term "nucleotide" refers to a "nucleoside," particularly in the case of oligonucleotides in which one or more of the internucleoside linkage groups are modified. For example, the term "nucleotide" can be used to specify the presence or nature of an internucleoside linkage.

[0057] In some aspects, the terms "nucleoside," "nucleotide," "unit," and "monomer" are used synonymously. Note that when referring to a sequence of nucleotides or monomers, what is being referenced is a sequence of bases such as A, T, G, C, or U.

[0058] The plural form of the term "nucleic acids" or "nucleotides" is intended to include multiple nucleic acids. In some aspects, the term "nucleic acids" or "nucleotides" refers to an in vivo or in vitro target sequence, e.g., pre-mRNAs, mRNAs, or DNAs. When the term refers to a nucleic acid or nucleotide in a target sequence, the nucleic acid or nucleotide may be a naturally occurring sequence within a cell. In other aspects, "nucleic acids" or "nucleotides" refers to a sequence within a disclosed oligomer or conjugate. When the term refers to a sequence within an oligomer (e.g., antisense oligomer, ASO) or antisense oligonucleotide conjugate (e.g., CIVI008) disclosed herein, the nucleic acid or nucleotide may be non-naturally occurring, i.e., chemically synthesized, enzymatically produced, recombinantly produced, or any combination thereof. In some aspects, the nucleic acid or nucleotide in the oligomer (e.g., antisense oligomer, ASO) or antisense oligonucleotide conjugate (e.g., CIVI008) disclosed herein is produced synthetically or recombinantly and is not a naturally occurring sequence or a fragment thereof. In some aspects, the nucleic acid or nucleotide in the oligomer (e.g., antisense oligomer, ASO) or antisense oligonucleotide conjugate (e.g., CIVI008) disclosed herein is not naturally occurring because it contains at least one nucleoside analog that is not naturally occurring in nature.

[0059] As used herein, the terms "reverse complement," "reverse complementary," and "reverse complementarity" are synonymous with the terms "complement," "complementary," and "complementarity."

[0060] The term "complementary" means that two sequences are complementary when one sequence binds to the other in an antiparallel manner, where the 3' end of each sequence binds to the 5' end of the other sequence, and each A, T(U), G, and C of one sequence is juxtaposed with a T(U), A, C, and G of the other sequence, respectively. Typically, complementary sequences of oligonucleotides are at least 90%, preferably 95%, and most preferably 100% complementary to a given sequence.

[0061] The terms "corresponding nucleotide analog" and "corresponding nucleotide" are used to indicate that the nucleotide analog and the nucleotide of a natural nucleotide are identical. For example, if the 2-deoxyribose unit of a nucleotide is linked to adenine, the "corresponding nucleotide analog" includes a pentose unit (different from 2-deoxyribose) linked to adenine. Examples of nucleobases include, but are not limited to, adenine, guanine, cytosine, thymidine, uracil, xanthine, hypoxanthine, 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine. In some aspects, the nucleobases are independently selected from the group consisting of adenine, guanine, cytosine, thymidine, uracil, and 5-methylcytosine. In some aspects, the nucleobases are independently selected from the group consisting of adenine, guanine, cytosine, thymidine, and 5-methylcytosine. In some aspects, at least one of the nucleobases present in an oligomer of the present disclosure is a modified nucleobase selected from the group consisting of 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine.

[0062] " Nucleotide analogue " is a variant of natural nucleotide, such as the DNA or RNA nucleotide of modified sugar and / or base portion.In principle, analogue can simply be " silent " or " equivalent " to the natural nucleotide in the context of the oligonucleotide; that is, it does not have any functional effect on the activity of oligonucleotide inhibiting target gene expression.Such " equivalent " analogue can still be useful; for example, it can be useful when the analogue is more convenient or cheaper to produce, or is more stable under storage or production conditions, or can be used as tag or label, etc.

[0063] As used herein, the term "PCSK9" refers to proprotein convertase subtilisin / kexin type 9, an enzyme encoded by the PCSK9 gene located on human chromosome 1. The PCSK9 gene is located on chromosome 1 band 1p32.3. It is the ninth member of the proprotein convertase family of proteins that activate other proteins. Similar genes (orthologs) are found in many species. The solved structure of PCSK9 reveals four major components of the unprocessed protein: a signal peptide (residues 1-30); an N-terminal prodomain (residues 31-152); a catalytic domain (residues 153-425); and a C-terminal domain (residues 426-692; this domain is further divided into three modules). The N-terminal prodomain has a flexible crystalline structure and is important for regulating PCSK9 function by interacting with and inhibiting the catalytic domain; without inhibition, the catalytic domain binds to the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR. Previous studies have shown that the C-terminal domain is not involved in binding to the LDLR, but recent studies have demonstrated that the C-terminal domain binds to the LDLR. PCSK9 secretion is highly dependent on the self-cleavage of the signal peptide and N-terminal prodomain, whereas the N-terminal prodomain retains its binding to the catalytic domain even after cleavage. In particular, residues 61–70 in the N-terminal prodomain are important for its self-processing.

[0064] The term "natural variant thereof" refers to a PCSK9 polypeptide variant of a nucleic acid sequence that occurs naturally in a specified taxonomic group, such as a mammal (such as a mouse, a monkey, and preferably a human). Typically, when referring to a "natural variant" of a polynucleotide, the term can also include any allelic variant of the genomic DNA encoding PCSK9 on chromosome 4, any allelic variant in the 4C7 region due to chromosomal translocation or chromosomal duplication, and any allelic variant of RNA (such as mRNA derived therefrom). A "natural variant" can also include variants resulting from alternative splicing of PCSK9 mRNA. When referring to a specific polypeptide sequence, for example, the term also includes naturally occurring forms of the protein that may be processed, for example, by co-translational or post-translational modification, such as signal peptide cleavage, proteolytic cleavage, or glycosylation.

[0065] As used herein, "2'-F" refers to a sugar containing a fluoro group at the 2' position.

[0066] As used herein, "2'-OMe" or "2'-OCH3" or "2'-O-methyl" each refer to a nucleoside containing a sugar containing an -OCH3 group at the 2' position of the sugar ring.

[0067] As used herein, the term "nucleotide length" refers to the total number of nucleotides (monomers) in a given sequence. As will be understood by those skilled in the art, the 5' terminal nucleotide of an oligonucleotide includes the 5' terminal group, but does not include the 5' internucleoside linkage group.

[0068] The compounds described herein may contain multiple asymmetric centers and may exist in the form of optically pure optical isomers, mixtures of optical isomers (e.g., racemates, mixtures of diastereoisomers, diastereoisomeric racemates, or mixtures of diastereoisomeric racemates, etc.). In some aspects, the asymmetric center may be an asymmetric carbon atom. The term "asymmetric carbon atom" refers to a carbon atom bearing four different substituents. According to the Cahn-Ingold-Prelog transformation, the asymmetric carbon atom may be in the "R" or "S" configuration.

[0069] As used herein, the term "bicyclic sugar" refers to a modified sugar moiety comprising a 4- to 7-membered ring that includes a bridge connecting two atoms of the ring, thereby forming a second ring, resulting in a bicyclic structure. In some aspects, such as in LNA nucleosides, the bridge connects C2' and C4' of the ribose sugar ring of the nucleoside (i.e., a 2'-4' bridge).

[0070] As used herein, a "coding region," "coding sequence," or "open reading frame" refers to a portion of a polynucleotide that consists of codons translatable into amino acids. A "stop codon" (TAG, TGA, or TAA) is typically not translated into amino acids but can be considered part of the coding region; on the other hand, any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, untranslated regions ("UTRs"), etc., are not part of the coding region. The boundaries of a coding region are typically determined by a 5'-terminal start codon that encodes the amino terminus of the resulting polypeptide and a 3'-terminal translation stop codon that encodes the carboxyl terminus of the resulting polypeptide. In some aspects, an oligomer (e.g., an antisense oligomer, ASO) or antisense oligonucleotide conjugate (e.g., CIVI008) disclosed herein can target the PCSK9 coding region of a nucleic acid, e.g., RNA, encoding a PCSK9 protein.

[0071] The term "non-coding region" as used herein refers to a nucleotide sequence that is not a coding region. Examples of non-coding regions include, but are not limited to, promoters, ribosome binding sites, transcription terminators, introns, untranslated regions ("UTRs"), non-coding exons, etc. Some exons may be the entire 5' untranslated region (5'UTR) or 3' untranslated region (3'UTR) of each transcript. Untranslated regions are important for efficient translation of transcripts and for controlling the translation rate and half-life of the transcripts. In some aspects, the oligomers (e.g., antisense oligomers, ASOs) or antisense oligonucleotide conjugates (e.g., CIVI008) disclosed herein may target PCSK9 non-coding regions of nucleic acids (e.g., RNA) encoding PCSK9 protein.

[0072] The term "region" when used in reference to a nucleotide sequence refers to a portion of that sequence. For example, the phrases "region within a nucleotide sequence" or "region within the complement of a nucleotide sequence" refer to a sequence that is shorter than the nucleotide sequence but is at least 10 nucleotides longer, respectively, located within the specified nucleotide sequence or its complement. The terms "sub-sequence" or "subsequence" can also refer to a region of a nucleotide sequence.

[0073] When referring to a nucleotide sequence, the term "downstream" means that the nucleic acid or nucleotide sequence is located 3' to a reference nucleotide sequence. In certain aspects, the downstream nucleotide sequence refers to the sequence following the transcription start site. For example, the translation start codon of a gene is located downstream of the transcription start site. In some aspects, the oligomer (e.g., antisense oligomer, ASO) or antisense oligonucleotide conjugate (e.g., CIVI008) disclosed herein can target a region downstream of the PCSK9 open reading frame (ORF) of a nucleic acid, e.g., RNA, encoding the PCSK9 protein.

[0074] The term "upstream" refers to a nucleotide sequence located 5' to a reference nucleotide sequence. In some aspects, an oligomer (e.g., an antisense oligomer, ASO) or antisense oligonucleotide conjugate (e.g., CIVI008) disclosed herein can target a region upstream of the PCSK9 open reading frame (ORF) of a nucleic acid, e.g., RNA, encoding the PCSK9 protein.

[0075] As used herein, the term "regulatory region" refers to a nucleotide sequence located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region that influences the transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, UTRs, and stem-loop structures. If the coding region is intended for expression in eukaryotic cells, a polyA signal and transcription termination sequence are typically located 3' of the coding sequence. In some aspects, oligomers (e.g., antisense oligomers, ASOs) or antisense oligonucleotide conjugates (e.g., CIVI008) disclosed herein can target regulatory regions.

[0076] As used herein, the term "transcript" refers to the primary transcript synthesized by transcription of DNA and processed into messenger RNA (mRNA), i.e., precursor messenger RNA (pre-mRNA), and the processed mRNA itself. The term "transcript" can be used synonymously with "pre-mRNA" and "mRNA." After a DNA strand is transcribed as a primary transcript, the newly synthesized primary transcript undergoes multiple modifications to mature and functional forms, producing different proteins and RNAs (mRNA, tRNA, rRNA, lncRNA, miRNA, etc.). Thus, the term "transcript" includes exons, introns, 5'UTR, and 3'UTR.

[0077] As used herein, the term "expression" refers to the process by which a gene product, e.g., RNA or polypeptide, is produced from a polynucleotide. This includes, but is not limited to, transcription of a polynucleotide into messenger RNA (mRNA) and translation of mRNA into a polypeptide. Expression produces a "gene product." A gene product herein can be either a nucleic acid, e.g., messenger RNA produced by transcription of a gene, or a polypeptide translated from a transcript. Gene products as described herein further include nucleic acids that have undergone post-transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides that have undergone post-translational modifications, e.g., methylation, glycosylation, lipid addition, association with other protein subunits, or proteolytic cleavage.

[0078] The term "identical" or percent "identity" with respect to two or more nucleic acids refers to two or more sequences that are identical or have a specified percentage (%) of identical nucleotides or amino acid residues when compared and aligned for maximum correspondence (introducing gaps as necessary), without taking into account any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are known in the art.

[0079] The term "percent sequence identity" or "percent identity" used between two polynucleotide or polypeptide sequences refers to the number of perfectly matched positions shared by the sequences of the comparison portion, taking into account the additions or deletions (i.e., gaps) that must be introduced to obtain optimal alignment of the two sequences. A matched position is any position where the same nucleotide or amino acid exists in both the target sequence and the reference sequence. Gaps present in the target sequence are not counted, because gaps are not nucleotides or amino acids. Similarly, gaps present in the reference sequence are not counted, because they are counted in the nucleotides or amino acids of the target sequence, but not in the nucleotides or amino acids of the reference sequence.

[0080] One non-limiting example of such a sequence alignment algorithm is the algorithm described in Karlin et al., 1990, Proc. Natl. Acad. Sci., 87:2264-2268, as modified as described in Karlin et al., 1993, Proc. Natl. Acad. Sci., 90:5873-5877, and incorporated into the NBLAST and XBLAST programs (Altschul et al., 1991, Nucleic Acids Res., 25:3389-3402). In certain aspects, gapped BLAST can be used, as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods in Enzymology, 266:460-480), ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or Megalign (DNASTAR) are additional publicly available software programs for sequence alignment. In certain aspects, the GAP program from the GCG software package is used to determine the percent identity between two nucleotide sequences (e.g., using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6). In another specific aspect, the GAP program of the GCG software package incorporating the Needleman and Wunsch algorithm (J. Mol. Biol. (48): 444-453 (1970)) can be used to determine the percent identity between two amino acid sequences (e.g., using either a BLOSUM62 matrix or a PAM250 matrix, and using gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, or 5). Alternatively, in specific aspects, the Myers and Miller (CABIOS, 4: 11-17 (1989)) algorithm is used to determine the percent identity between nucleotide or amino acid sequences.For example, percent identity can be determined using the ALIGN program (version 2.0) and using PAM 120 with a residue table, a gap length penalty of 12, and a gap penalty of 4. One of skill in the art can determine appropriate parameters for maximal alignment with a particular alignment software. In certain aspects, the default parameters of the alignment software are used.

[0081] Those skilled in the art will understand that generating sequence alignments for calculating percent sequence identity is not limited to pairwise comparison of sequences, which is performed exclusively by primary sequence data. Sequence alignments can also be by multiple sequence alignments. One suitable program for generating multiple sequence alignments is ClustalW2, available at www.clustal.org. Another suitable program is MUSCLE, available at www.drive5.com / muscle / . ClustalW2 and MUSCLE are alternatively available, for example, at EBI (European Bioinformatics Institute).

[0082] It will be appreciated that sequence alignments can be generated by integrating sequence data with data from different types of sources, such as structural data (e.g., protein crystal structures), functional data (e.g., mutation locations), or phylogenetic data. Suitable programs for integrating heterogeneous data to generate multiple sequence alignments include T-Coffee, available at www.tcoffee.org, or alternatively, for example, at EBI. It will also be appreciated that the final alignment used to calculate percent sequence identity can be adjusted either automatically or manually.

[0083] In certain aspects, the percent identity "X" of a first nucleotide sequence to a second nucleotide sequence is calculated by 100 x (Y / Z), where Y is the number of amino acid residues scored as perfect matches in an alignment of the first and second sequences (as aligned by visual inspection or by a particular sequence alignment program); and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

[0084] Different regions in a single polynucleotide target sequence that aligns with a polynucleotide reference sequence can have their own percent sequence identity.Please note that sequence identity% value is rounded to one decimal place.For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2.Please also note that length value is always an integer.

[0085] When determining the degree of "complementarity" between an oligomer (or a region thereof) of the present disclosure and a target region (such as a target region disclosed herein), the degree of "complementarity" (also expressed as "homology" or "identity") is expressed as the % identity (or % homology) between the sequence of the oligomer (or a region thereof) and the sequence of the target region (or the reverse complement of the target region) that best aligns therewith. The percentage (%) is calculated by counting the number of aligned bases that are identical between the two sequences, dividing by the total number of consecutive monomers in the oligomer, and multiplying by 100. In such a comparison, if gaps exist, it is preferable to consider such gaps to be mere mismatches rather than regions where the number of monomers in the gap differs between the oligomer of the present disclosure and the target region.

[0086] As used herein, the terms "inhibit" and "reduce" refer to, for example, when "inhibiting" and "reducing" PCSK9 gene transcript expression and / or PCSK9 protein levels or PCSK9 activity, an oligomer (e.g., antisense oligomer, ASO) or antisense oligonucleotide conjugate (e.g., CIVI008) disclosed herein reduces PCSK9 gene transcript expression and / or PCSK9 protein levels and / or activity in a cell, tissue, or subject.

[0087] In some aspects, the terms "inhibit" and "reducing" refer to complete inhibition (100% inhibition or undetectable levels) of PCSK9 gene transcript or PCSK9 protein levels and / or activity of PCSK9. In other aspects, the terms "inhibit" and "reducing" refer to, for example, at least about 5% inhibition, 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 90%, at least 95%, or at least 99% inhibition of PCSK9 gene transcript and / or PCSK9 protein expression and / or activity of PCSK9 in a cell, tissue, or subject.

[0088] Polynucleotide variants may have alterations in coding regions, non-coding regions, or both. In one aspect, the polynucleotide variants contain alterations caused by silent substitutions, additions, or deletions, but do not result in changes to the properties or activity of the encoded polypeptide. In another aspect, nucleotide variants are caused by silent substitutions due to the degeneracy of the genetic code. In other aspects, variants contain substitutions, deletions, or additions of 5 to 10 amino acids, 1 to 5 amino acids, or 1 to 2 amino acids in any combination. Polynucleotide variants can be created for various purposes; for example, they can be created to optimize codon expression in a particular host (e.g., converting codons in human mRNA to those in a bacterial host such as E. coli).

[0089] Naturally occurring variants are called "allelic variants" and refer to one of multiple alternative forms occupying a given genetic locus on the chromosome of an organism (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can vary at either the polynucleotide and / or polypeptide level and are included in the present disclosure. Alternatively, non-naturally occurring variants can be generated by mutagenesis techniques or direct synthesis.

[0090] As used herein, the terms "linked to" and "conjugated to" are used synonymously and refer to a covalent or non-covalent bond formed between a first moiety and a second moiety, e.g., between an oligomer of the disclosure (e.g., an antisense oligonucleotide) and a conjugate moiety (e.g., GalNAc).

[0091] The terms "individual," "subject," "host," and "patient" are used synonymously herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired. The compositions and methods described herein are applicable to both human treatment and veterinary applications. In some aspects, the subject is a mammal, and in other aspects, the subject is a human. "Mammalian subject" herein includes all mammals, including, but not limited to, humans, companion animals (e.g., dogs, cats, etc.), farm animals (e.g., cows, sheep, pigs, horses, etc.), and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.).

[0092] The term "pharmaceutical composition" refers to a formulation in which the biological activity of the active ingredient can be effective, without any additional ingredients that would cause intolerable toxicity to the subject to which the composition is administered. Such compositions can be sterile. The term "oral pharmaceutical composition" refers to a pharmaceutical composition that can be administered orally. Oral administration is a route of administration in which a substance is taken through the mouth. The abbreviation "Per os" for oral (PO) is also sometimes used to indicate oral drug administration. Many therapeutic drugs are taken orally because they are intended to have a systemic effect, for example, by reaching different parts of the body via the bloodstream.

[0093] The term "delivery agent" herein refers to a carrier compound or molecule useful in the oral delivery of a therapeutic agent of the present disclosure. The term "oral delivery agent" herein refers to a carrier compound or molecule useful in the oral delivery of a therapeutic agent of the present disclosure.

[0094] In some aspects, the pharmaceutical composition of the present disclosure is orally administered.The term "oral" and its grammatical variants (for example, oral) herein include any kind of oral delivery route (including buccal, sublabial, and sublingual).Orally administered drugs can be in various forms; such forms include oral solid dosage (OSD) forms (for example, tablets that are swallowed, chewed in water or under the tongue, or dissolved; capsules and chewable capsules, for example, those with coatings that dissolve in the stomach or intestine and release drug there; sustained-release or sustained-release tablets and capsules that gradually release drug; powders; or granules), and oral liquid dosage forms (for example, tea, drops, liquid, suspension, or syrup).

[0095] As used herein, "administering" means providing a composition, e.g., an oral pharmaceutical composition comprising an oligomer (e.g., antisense oligomer, ASO) or antisense oligonucleotide conjugate (e.g., CIVI008) disclosed herein, to a subject via a pharmaceutically acceptable route, e.g., orally. An "effective amount" refers to, for example, an amount of an oral pharmaceutical composition comprising an oligomer (e.g., antisense oligomer, ASO) or antisense oligonucleotide conjugate (e.g., CIVI008) disclosed herein sufficient to achieve a specifically stated purpose. An "effective amount" can be determined empirically by routine methods in relation to the stated purpose.

[0096] As used herein, "treat," "treatment," or "treating" refers to, for example, lessening the severity of a disease or condition, but not necessarily curing the disease or condition; shortening the duration of the disease course; ameliorating or eliminating one or more symptoms associated with a disease or condition; or providing a beneficial effect to a subject with a disease or condition. The term also includes prophylaxis or prevention of a disease or condition or its symptoms.

[0097] As used herein, "prevent" or "preventing" refers to reducing the occurrence or severity of a particular outcome. In some aspects, preventing an outcome is achieved by prophylactic treatment. In some aspects, an oral pharmaceutical composition comprising an oligomer (e.g., an antisense oligomer, ASO) or antisense oligonucleotide conjugate (e.g., CIVI008) disclosed herein is prophylactically administered to a subject. In some aspects, the subject is at risk of developing, for example, hypercholesterolemia or heart disease.

[0098] Section 1: Oral Pharmaceutical Compositions The present disclosure provides pharmaceutical compositions for oral administration, i.e., oral pharmaceutical compositions or formulations comprising an antisense oligomer and an oral delivery agent. In some aspects, the antisense oligomer is 16 to 22 contiguous nucleotides in length; wherein the sequence of the antisense oligomer comprises a contiguous sequence of 16 nucleotides in length that is 100% complementary to the sequence of SEQ ID NO: 31; wherein the antisense oligomer is a gapmer comprising at least one LNA unit; And wherein the antisense oligomer targets the RNA encoding PCSK9.In some aspects, the antisense oligomer can be conjugated with a moiety that can target specific tissue, for example, the liver.Therefore, in some aspects, the present disclosure provides a pharmaceutical composition for oral administration, i.e., an oral pharmaceutical composition or oral formulation, comprising an antisense oligonucleotide conjugate and an oral delivery agent; wherein the antisense oligonucleotide conjugate comprises, for example, the following (i) and (ii): (i) antisense oligomers consisting of 16–22 consecutive nucleotides; wherein the sequence of the antisense oligomer comprises a contiguous sequence of 16 nucleotides in length that is 100% complementary to the sequence of SEQ ID NO: 31; Also wherein the antisense oligomer is a gapmer comprising at least one LNA unit; and (ii) at least one non-nucleotide or non-polynucleotide moiety covalently attached to the antisense oligomer, either directly or via a linker disposed between the contiguous oligomer sequences; wherein the antisense oligonucleotide conjugate targets RNA encoding PCSK9. In some aspects, the oligomer can be any of the oligomers disclosed in Section 2 of this disclosure. In some aspects, the antisense oligonucleotide conjugate can be any of the oligonucleotide conjugates disclosed in Section 3 of this disclosure.

[0099] In some aspects, the antisense oligonucleotide conjugate is the molecule set forth in SEQ ID NO: 18 or SEQ ID NO: 19 presented in Figure 18A. In some aspects, the antisense oligonucleotide conjugate is (GalNAc)3-amino-hexamethylene-5'phosphorothioyl)-2'-O,4'-C-methyleneadenosilyl-(3'>5'O,O-phosphorothioyl)-2'-O,4'-C-methyleneadenosilyl-(3'>5'O,O-phosphorothioyl)-2'-O,4'-C-methylenetrenithiyl. thymidylyl-(3'>5'O,O-phosphorothioyl)-2'-deoxyguanosilyl-(3'>5'O,O-phosphorothioyl)-2'-deoxycytidinylyl-(3'>5'O,O-phosphorothioyl)-2'-deoxythymidylyl-(3'>5'O,O-phosphorothioyl)-2'-deoxyadenosilyl-(3'>5'O,O-phosphorothioyl)-2'-deoxycytidinylyl Diphenylyl-(3'>5'O,O-phosphorothioyl)-2'-deoxyadenosilyl-(3'>5'O,O-phosphorothioyl)-2'-deoxyadenosilyl-(3'>5'O,O-phosphorothioyl)-2'-deoxyadenosilyl-(3'>5'O,O-phosphorothioyl)-2'-deoxyadenosilyl-(3'>5'O,O-phosphorothioyl)-2'-deoxyadenosilyl-(3'>5'O,O-phosphorothioyl)-2'-deoxy cytidinylyl-(3'>5'O,O-phosphorothioyl)-2'-O,4'C-methylene(5-methyl-cytidinyl)-(3'>5'O,O-phosphorothioyl)-2'-O,4'-C-methylene(5-methyl-cytidinyl)-(3'>5'O,O-phosphorothioyl)-2'-O,4'-C-methyleneadenosilyl hexadeca sodium salt, i.e., CIVI008. See also the detailed chemical formula of CIVI008 in Figure 18B.

[0100] In some aspects, the oral pharmaceutical composition of the present disclosure comprises an oligomer or oligonucleotide conjugate (e.g., CIVI008) disclosed herein and an oral delivery agent selected from the group consisting of SNAC, C10, 5-CNAC, hydrates thereof, solvates thereof, or salts thereof, and combinations thereof. In some aspects, the oral delivery agent useful in the present disclosure may comprise, for example, any of the 123 modified amino acids disclosed in U.S. Patent No. 5,866,536, or any of the 193 modified amino acids disclosed in U.S. Patent No. 5,773,647, or any combination thereof. The entire contents of U.S. Patent Nos. 5,773,647 and 5,866,536 are incorporated herein by reference.

[0101] In some aspects, the oral delivery agent comprises a single compound (e.g., C8, C10, SNAC, or 5-CNAC). In other aspects, the oral delivery agent comprises a combination of compounds (e.g., any combination of C8, C10, SNAC, or 5-CNAC). In some aspects, the oral delivery agent comprises C8, C10, SNAC, 5-CNAC, 4-CNAB, 4-MOAC, SNAD, 4-HPO (8-(4-hydroxyphenoxyoctanoic acid), 5-PPA (5-phenyl pentanoate), 2-PHOD (8-(2-hydroxyphenoxy)octyldiethanolamine), 3-TBA (4-m-tosyloxybutyric acid, 2-HPOD (2-(5-pentanoic acid)-5-(2-hydroxyphenyl)-1,3,4-oxadiazole), 7-OPHA (7-oxo-7-phenylheptanoic acid), 3-HPSB (4-(3-hydroxyphenylsulfanyl)butyric acid), 4-IBOA ((4-isopropylbenzyloxy)acetic acid), 3-FPSB (4-(3-fluorophenylsulfanyl)butyric acid), or any combination thereof. See, e.g., FIG. 22.

[0102] In some aspects, the SNAC may optionally be replaced with a similar compound, such as SNAD (sodium 10-N-(2-hydroxybenzoyl)aminodecanoate). The structure of SNAD differs from that of SNAC only in the length of the fatty acid moiety. In some aspects of the present disclosure, the SNAC may optionally be replaced with a similar compound, in which the caprylic acid moiety of SNAC is replaced with another fatty acid moiety at least 6 carbon atoms in length, e.g., 6-20 carbon atoms in length, optionally 6-18 carbon atoms in length, optionally 6-16 carbon atoms in length, optionally 6-14 carbon atoms in length, optionally 6-12 carbon atoms in length, and optionally 6-10 carbon atoms in length. The fatty acid moiety may be saturated (e.g., like caprylic acid in SNAC or decanoic acid in SNAD) or unsaturated (i.e., containing at least one unsaturated carbon-carbon bond).

[0103] In some aspects, the oral delivery agent comprises N-(8-(2-hydroxybenzoyl)amino)caprylic acid, as shown below. [ka]

[0104] In some aspects, the oral delivery agent comprises a salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid. In some aspects, the oral delivery agent comprises a solvate of N-(8-(2-hydroxybenzoyl)amino)caprylic acid. In some aspects, the oral delivery agent comprises a hydrate of N-(8-(2-hydroxybenzoyl)amino)caprylic acid. In some aspects, the oral delivery agent comprises a salt, hydrate, or solvate of N-(8-(2-hydroxybenzoyl)amino)caprylic acid, or any combination thereof.

[0105] In some aspects, the salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid is selected from the group consisting of sodium, potassium, calcium salts of N-(8-(2-hydroxybenzoyl)amino)caprylic acid, and any combination thereof. In some aspects, the salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid is the sodium salt. In some aspects, the salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid is the disodium salt. In some aspects, the salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid is the monosodium salt (salcaprozate sodium 203787-91-1, SNAC, sodium 8-(2-hydroxybenzamido)octanoate) as shown below. [ka]

[0106] In some aspects, the oral delivery agent comprises 5-CNAC (N-(5-chlorosalicyloyl)-8-aminocaprylic acid), as shown below. [ka]

[0107] In some aspects, the oral delivery agent comprises a salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid. In some aspects, the oral delivery agent comprises a solvate of N-(5-chlorosalicyloyl)-8-aminocaprylic acid. In some aspects, the oral delivery agent comprises a hydrate of N-(5-chlorosalicyloyl)-8-aminocaprylic acid. In some aspects, the oral delivery agent comprises a salt, hydrate, or solvate of N-(5-chlorosalicyloyl)-8-aminocaprylic acid, or any combination thereof.

[0108] In some aspects, the salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid is selected from the group consisting of sodium salt, potassium salt, calcium salt, and any combination thereof of N-(5-chlorosalicyloyl)-8-aminocaprylic acid. In some aspects, the salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid is the sodium salt. In some aspects, the salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid is the disodium salt. In some aspects, the salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid is the monosodium salt.

[0109] In some aspects, the oral delivery agent comprises 4-CNAB (4-[(4-chloro-2-hydroxy-benzoyl)amino]butanoic acid; salclobudate), as shown below. [ka]

[0110] In some aspects, the oral delivery agent comprises a salt of 4-[(4-chloro-2-hydroxy-benzoyl)amino]butanoic acid. In some aspects, the oral delivery agent comprises a solvate of 4-[(4-chloro-2-hydroxy-benzoyl)amino]butanoic acid. In some aspects, the oral delivery agent comprises a hydrate of 4-[(4-chloro-2-hydroxy-benzoyl)amino]butanoic acid. In some aspects, the oral delivery agent comprises a salt, hydrate, or solvate of 4-[(4-chloro-2-hydroxy-benzoyl)amino]butanoic acid, or any combination thereof.

[0111] In some aspects, the salt of 4-[(4-chloro-2-hydroxy-benzoyl)amino]butanoic acid is selected from the group consisting of sodium salt, potassium salt, calcium salt of 4-[(4-chloro-2-hydroxy-benzoyl)amino]butanoic acid, and any combination thereof. In some aspects, the salt of 4-[(4-chloro-2-hydroxy-benzoyl)amino]butanoic acid is the sodium salt. In some aspects, the salt of 4-[(4-chloro-2-hydroxy-benzoyl)amino]butanoic acid is the disodium salt. In some aspects, the salt of 4-[(4-chloro-2-hydroxy-benzoyl)amino]butanoic acid is the monosodium salt.

[0112] In some aspects, the oral delivery agent comprises 4-MOAC (N-(8-[4-methoxy-chloro-2-hydroxybenzoyl-amino)octanoic acid), as shown below. [ka]

[0113] In some aspects, the oral delivery agent comprises a salt of N-(8-[4-methoxy-chloro-2-hydroxybenzoyl-amino)octanoic acid. In some aspects, the oral delivery agent comprises a solvate of N-(8-[4-methoxy-chloro-2-hydroxybenzoyl-amino)octanoic acid. In some aspects, the oral delivery agent comprises a hydrate of N-(8-[4-methoxy-chloro-2-hydroxybenzoyl-amino)octanoic acid. In some aspects, the oral delivery agent comprises a salt, hydrate, or solvate of N-(8-[4-methoxy-chloro-2-hydroxybenzoyl-amino)octanoic acid, or any combination thereof.

[0114] In some aspects, the salt of N-(8-[4-methoxy-chloro-2-hydroxybenzoyl-amino)octanoic acid is selected from the group consisting of sodium salt, potassium salt, calcium salt of N-(8-[4-methoxy-chloro-2-hydroxybenzoyl-amino)octanoic acid, and any combination thereof. In some aspects, the salt of N-(8-[4-methoxy-chloro-2-hydroxybenzoyl-amino)octanoic acid is the sodium salt. In some aspects, the salt of N-(8-[4-methoxy-chloro-2-hydroxybenzoyl-amino)octanoic acid is the disodium salt. In some aspects, the salt of N-(8-[4-methoxy-chloro-2-hydroxybenzoyl-amino)octanoic acid is the monosodium salt.

[0115] In some aspects, the oral delivery agent comprises SNAD (N-(10-[2-hydroxybenzoyl]-amino)decanoic acid), as shown below. [ka]

[0116] In some aspects, the oral delivery agent comprises a salt of N-(10-[2-hydroxybenzoyl]-amino)decanoic acid. In some aspects, the oral delivery agent comprises a solvate of N-(10-[2-hydroxybenzoyl]-amino)decanoic acid. In some aspects, the oral delivery agent comprises a hydrate of N-(10-[2-hydroxybenzoyl]-amino)decanoic acid. In some aspects, the oral delivery agent comprises a salt, hydrate, or solvate of N-(10-[2-hydroxybenzoyl]-amino)decanoic acid, or any combination thereof.

[0117] In some aspects, the salt of N-(10-[2-hydroxybenzoyl]-amino)decanoic acid is selected from the group consisting of sodium salt, potassium salt, calcium salt of N-(10-[2-hydroxybenzoyl]-amino)decanoic acid, and any combination thereof. In some aspects, the salt of N-(10-[2-hydroxybenzoyl]-amino)decanoic acid is a sodium salt. In some aspects, the salt of N-(10-[2-hydroxybenzoyl]-amino)decanoic acid is a disodium salt. In some aspects, the salt of N-(10-[2-hydroxybenzoyl]-amino)decanoic acid is a monosodium salt.

[0118] In some aspects, the oral delivery agent comprises a compound presented in Figure 22. In some aspects, the oral delivery agent comprises a salt of a compound presented in Figure 22. In some aspects, the oral delivery agent comprises a solvate of a compound presented in Figure 22. In some aspects, the oral delivery agent comprises a hydrate of a compound presented in Figure 22. In some aspects, the oral delivery agent comprises a salt, hydrate, or solvate of a compound presented in Figure 22, or any combination thereof.

[0119] In some aspects, the salt of the compound presented in Figure 22 is selected from the group consisting of a sodium salt, a potassium salt, a calcium salt, or any combination thereof, of the compound presented in Figure 22. In some aspects, the salt of the compound presented in Figure 22 is a sodium salt. In some aspects, the salt of the compound presented in Figure 22 is a disodium salt. In some aspects, the salt of the compound presented in Figure 22 is a monosodium salt.

[0120] U.S. Patent Publication No. 5650386A, U.S. Patent Publication No. 6399798 B2, U.S. Patent Publication No. 7384982 B2, U.S. Patent Publication No. 7659311 B2, U.S. Patent Publication No. 8003697 B2, U.S. Patent Publication No. 8207227 No. B2, U.S. Patent Publication No. 8658695 B2, U.S. Patent Publication No. 7544833 B2, U.S. Patent No. 7659311, U.S. Patent No. 8003697, U.S. Patent No. 8207227, U.S. Patent No. 8658695, U.S. Patent No. 73849 82, U.S. Patent Publication No. 9,278,123 B2, U.S. Patent Publication No. 10,086,047 B2, U.S. Patent Publication No. 20180360918 A1, U.S. Patent Publication No. 20110092426 A1, U.S. Patent Publication No. 20150283212 A1, U.S. Patent Publication No. 8,435,946 B2, U.S. Patent Publication No. 8,748,383 B2, U.S. Patent Publication No. 7,569,539 B2; the contents of all of these references are incorporated herein by reference in their entirety.

[0121] In some aspects, the oral delivery agent comprises a compound of the formula shown below: [ka] where: R 1 , R 2 , R 3 , and R 4 are independently hydrogen, -OH, -NR 6 R 7 , halogen, C1-C4 alkyl, or C1-C4 alkoxy; R 5 substituted or unsubstituted C2 to C 16 Alkylene, substituted or unsubstituted C2-C 16 Alkenylene, substituted or unsubstituted C1-C 12 alkyl (arylene), or substituted or unsubstituted aryl (C1-C4 alkylene); and R 6 and R 7 are independently hydrogen, oxygen, or C1-C4 alkyl.

[0122] In some aspects, the oral delivery agent has the formula: [ka] Although including compounds of; wherein the compound is a free acid or a sodium salt (e.g., a monosodium or disodium salt). In some aspects, the compound is a hydrate or solvate. In some aspects, the compound is an alcohol solvate. In some aspects, the alcohol solvate is an ethanol solvate. In some aspects, the ethanol solvate is a solvate of a salt. In some aspects, the ethanol solvate is an ethanol solvate of a monosodium salt. In some aspects, the ethanol solvate is an ethanol solvate of a disodium salt. In some aspects, the compound is a hydrate. In some aspects, the hydrate is a hydrate of a salt. In some aspects, the hydrate is a hydrate of a monosodium salt. In some aspects, the hydrate is a hydrate of a disodium salt.

[0123] In some aspects, oral delivery agents useful in the present disclosure can include medium chain fatty acids (MCFAs), such as C8 (caprylic acid), C10 (capric acid), or C12 (lauric acid), a derivative thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, or any combination thereof. [ka]

[0124] In some aspects, the oral delivery agent comprises a solvate of a salt of C8, C10, SNAC, or 5-CNAC. As used herein, the term "solvate" includes, but is not limited to, a molecular or ionic complex between a solvent molecule or ion and a delivery agent compound molecule or ion, or a salt thereof, or a hydrate thereof, or a solvate thereof.

[0125] In some aspects, the oral delivery agent comprises a hydrate of a salt of C8, C10, SNAC, or 5-CNAC. As used herein, the term "hydrate" includes: (i) A substance containing water combined in molecular form; and (ii) a crystalline substance containing one or more water molecules of crystallization or a crystalline substance containing free water; These include, but are not limited to, the following:

[0126] In some aspects, the oral delivery agent comprises a solvate of a salt of C8, C10, SNAC, or 5-CNAC, wherein the salt is a sodium salt, a potassium salt, a calcium salt, or a combination thereof. In some aspects, the oral delivery agent comprises a solvate of a salt of C8, C10, SNAC, or 5-CNAC, wherein the salt is a sodium salt.

[0127] In some aspects, the oral delivery agent comprises a solvate of a salt of C8, C10, SNAC, or 5-CAN, wherein the salt is a monosodium salt. In some aspects, the oral delivery agent comprises a sodium salt of C8, such as monosodium caprate. In some aspects, the oral delivery agent comprises a sodium salt of C10, such as monosodium caprylate. In some aspects, the oral delivery agent comprises a sodium salt of SNAC, such as monosodium SNAC. In some aspects, the oral delivery agent comprises a sodium salt of 5-CNAC, such as monosodium 5-CNAC.

[0128] In some aspects, the oral delivery agent comprises an alcohol solvate of a salt of C8, C10, SNAC, or 5-CNAC, wherein the salt is a sodium salt. In some aspects, the oral delivery agent comprises an alcohol solvate of a salt of C8, C10, SNAC, or 5-CNAC, wherein the salt is a monosodium salt. In some aspects, the oral delivery agent comprises a hydrate of a salt of C8, C10, SNAC, or 5-CNAC, wherein the salt is a sodium salt. In some aspects, the oral delivery agent comprises a hydrate of a salt of C8, C10, SNAC, or 5-CNAC, wherein the salt is a monosodium salt. In some aspects, the oral delivery agent comprises a hydrate of a salt of C8, C10, SNAC, or 5-CNAC, wherein the salt is a sodium salt and the hydrate is a monohydrate.

[0129] Methods for preparing sodium salts, alcohol solvates, and hydrates are described, for example, in International Patent Publication WO 00 / 059863; the entire contents of this reference are incorporated herein by reference. For example, the sodium salt may be prepared from an ethanol solvate by evaporating or drying the ethanol solvate by methods known in the art to form an anhydrous sodium salt. Drying is typically carried out at a temperature of about 80°C to about 120°C, e.g., about 85°C to about 90°C. In some aspects, drying is carried out at about 85°C. The drying step is typically carried out at a pressure of about 660 mmHg (8.8 kPa) or greater. The anhydrous sodium salt typically contains less than about 5% by weight of ethanol, and preferably less than about 2% by weight of ethanol, based on 100% total weight of the anhydrous sodium salt.

[0130] Sodium salts of the oral delivery agents disclosed herein can also be prepared by forming a slurry of the delivery agent in water and adding aqueous sodium hydroxide, sodium alkoxide, etc. Suitable sodium alkoxides include, but are not limited to, sodium methoxide, sodium ethoxide, and combinations thereof. Another method for preparing sodium salts is to react the delivery agent with sodium hydroxide to obtain the sodium salt.

[0131] The sodium salt can be isolated as a solid by concentrating the solution containing the sodium salt into a thick paste by vacuum distillation.This paste can be dried in a vacuum oven to obtain the sodium salt of the delivery agent as a solid.The solid can also be isolated by spray-drying the aqueous solution of disodium salt.The oral delivery agent disclosed herein can be prepared by methods known in the art, for example, as described above in U.S. Patent No. 5,773,647 and U.S. Patent No. 5,866,536; these references are incorporated herein by reference in their entirety.

[0132] Ethanol solvates of oral delivery agent molecules (e.g., C8, C10, SNAC, 5-CNAC, or any combination thereof) disclosed herein include, but are not limited to, molecular or ionic complexes of ethanol solvent molecules or ions with molecules or ions of the sodium salt of the delivery agent. Typically, the ethanol solvates contain about one ethanol molecule or ion for each molecule of the sodium salt of the delivery agent.

[0133] The ethanol solvate of the sodium salt of the delivery agent can be prepared by dissolving the delivery agent in ethanol.Then, the delivery agent / ethanol solution is reacted with a molar excess of sodium salt (such as monosodium salt) relative to the delivery agent (i.e., for each mole of delivery agent, there is more than one mole of sodium cation), to obtain the ethanol solvate.Suitable monosodium salts include, but are not limited to, sodium hydroxide; sodium alkoxides (such as sodium methoxide and sodium ethoxide); and any combination thereof.Generally, the reaction is carried out at the reflux temperature of the mixture or lower (such as ambient temperature).Then, the resulting slurry is concentrated by atmospheric distillation, and the concentrated slurry is cooled, and the ethanol solvate is recovered by methods known in the art, such as by filtering the solid matter.The recovered solid matter can then be vacuum dried to obtain the ethanol solvate.

[0134] Hydrates of the sodium salt of the delivery agent may be prepared by drying the ethanol solvate to form an anhydrous disodium salt, as described above, and then hydrating the anhydrous sodium salt. In some aspects, monohydrates of the sodium salt are formed. Anhydrous sodium salts are highly hygroscopic, so hydrate formation occurs upon exposure to moisture in the air. Generally, the hydration step is carried out in an environment with a relative humidity of at least 50% at about ambient temperature to about 50°C, preferably ambient temperature to about 30°C. Alternatively, the anhydrous sodium salt may be hydrated with steam.

[0135] Oral pharmaceutical compositions of the present disclosure typically contain an effective amount of one or more delivery agents (e.g., C8, C10, SNAC, 5-CNAC, or any combination thereof) disclosed herein; i.e., an amount sufficient to deliver an active agent (e.g., CIVI008) to achieve a desired effect. Generally, the oral delivery agent (e.g., C8, C10, SNAC, 5-CNAC, or any combination thereof) is present in an amount of about 2.5% to about 99.4% by weight. In some aspects, the oral delivery agent (e.g., C8, C10, SNAC, 5-CNAC, or any combination thereof) is present in an amount of about 15% to about 75% by weight. In some aspects, the oral delivery agent (e.g., C8, C10, SNAC, 5-CNAC, or any combination thereof) is present in an amount of at least about 25%, at least about 30%, or at least about 35% by weight, and not more than about 60% or about 70%. Thus, in some aspects, the oral delivery agent (e.g., C8, C10, SNAC, 5-CNAC, or any combination thereof) is present in an amount of 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%, or at least about 70% by weight. In some aspects, the oral delivery agent (e.g., C8, C10, SNAC, 5-CNAC, or any combination thereof) is present in an amount of about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70% by weight.

[0136] In some aspects, the oral pharmaceutical composition of the present disclosure is administered in a single dose. The term "single dose" refers to the administration of a single dose of an oral pharmaceutical composition according to the present invention in the form of an oral unit dose, for example, comprising an oligomer or oligonucleotide conjugate disclosed herein (e.g., CIVI008) and an oral delivery agent selected from the group consisting of SNAC, C10, or 5-CNAC, their hydrates, solvates, or salts thereof, and combinations thereof, to a human or animal patient.

[0137] In some aspects, the oral pharmaceutical composition of the present disclosure is administered multiple times. The term "multiple doses" refers to administering at least two doses of an oral pharmaceutical composition according to the present invention in the form of an oral unit dose, for example, comprising an oligomer or oligonucleotide conjugate disclosed herein (e.g., CIVI008) and an oral delivery agent selected from the group consisting of SNAC, C10, or 5-CNAC, their hydrates, solvates, or salts, and combinations thereof, to a human or animal patient according to a dosing interval appropriate for the composition.

[0138] The term "oral unit dose" herein refers to an individually packaged, physically discrete unit suitable for human and animal ingestion, as known in the art. For purposes of this disclosure, it is understood that a dosage form comprising a therapeutically effective amount of an oligomer or oligonucleotide conjugate disclosed herein (e.g., CIVI008) and an oral delivery agent may contain one or more unit doses (e.g., tablets, capsules) to achieve a therapeutic effect.

[0139] Oral dosage forms (e.g., tablets or capsules) of oral pharmaceutical compositions of the present disclosure (e.g., antisense oligonucleotide conjugates such as CIVI008 combined with an oral delivery agent such as SNAC, C10, or 5-CNAC) can be administered about 5 to about 60 minutes before a meal. Oral dosage forms (e.g., tablets or capsules) of oral pharmaceutical compositions of the present disclosure (e.g., antisense oligonucleotide conjugates such as CIVI008 combined with an oral delivery agent such as SNAC, C10, or 5-CNAC) can be administered about 30 to about 60 minutes before a meal. Oral dosage forms (e.g., tablets or capsules) of oral pharmaceutical compositions of the present disclosure (e.g., antisense oligonucleotide conjugates such as CIVI008 combined with an oral delivery agent such as SNAC, C10, or 5-CNAC) can be administered about 45 to about 90 minutes before a meal. An oral dosage form (e.g., tablet or capsule) of an oral pharmaceutical composition of the present disclosure (e.g., an antisense oligonucleotide conjugate such as CIVI008 in combination with an oral delivery agent such as SNAC, C10, or 5-CNAC) can be administered about 60 minutes (1 hour) to about 120 minutes (2 hours) before a meal.

[0140] In some aspects, an oral pharmaceutical composition of the present disclosure (e.g., an antisense oligonucleotide conjugate such as CIVI008 in combination with an oral delivery agent such as SNAC, C10, or 5-CNAC) can be administered at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 65 minutes, at least about 70 minutes, at least about 75 minutes, at least about 80 minutes, at least about 85 minutes, at least about 90 minutes, at least about 95 minutes, at least about 100 minutes, at least about 105 minutes, at least about 110 minutes, at least about 115 minutes, or at least about 120 minutes before a meal.

[0141] In some aspects, an oral pharmaceutical composition of the present disclosure (e.g., an antisense oligonucleotide conjugate such as CIVI008 in combination with an oral delivery agent such as SNAC, C10, or 5-CNAC) is administered about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, or about 120 minutes before a meal.

[0142] In some aspects, an oral pharmaceutical composition of the present disclosure (e.g., an antisense oligonucleotide conjugate such as CIVI008 in combination with an oral delivery agent such as SNAC, C10, or 5-CNAC) is administered at least about 30 minutes before food ingestion. In some aspects, an oral pharmaceutical composition of the present disclosure (e.g., an antisense oligonucleotide conjugate such as CIVI008 in combination with an oral delivery agent such as SNAC, C10, or 5-CNAC) is administered at least about 45 minutes before food ingestion. In some aspects, an oral pharmaceutical composition of the present disclosure (e.g., an antisense oligonucleotide conjugate such as CIVI008 in combination with an oral delivery agent such as SNAC, C10, or 5-CNAC) is administered at least about 60 minutes before food ingestion. In some aspects, an oral pharmaceutical composition of the present disclosure (e.g., an antisense oligonucleotide conjugate such as CIVI008 in combination with an oral delivery agent such as SNAC, C10, or 5-CNAC) is administered at least about 2 hours before food ingestion.

[0143] The oral pharmaceutical composition of the present disclosure (e.g., an antisense oligonucleotide conjugate such as CIVI008 combined with an oral delivery agent such as SNAC, C10, or 5-CNAC) can be provided in a solid form. In some aspects, the solid form is a capsule, such as a soft gel capsule or a liquid-filled capsule (liquid capsule). The oral pharmaceutical composition of the present disclosure can also be provided as a tablet, caplet, or other solid oral dosage form, all of which can be prepared by methods known in the art.

[0144] In some aspects, the oral dosage form (e.g., tablet or capsule) can have a weight of about 5 mg to about 1000 mg, about 10 mg to about 500 mg, about 10 mg to about 250 mg, about 100 mg to about 200 mg, or about 250 mg to about 500 mg. In some aspects, the weight of the oral dosage form (e.g., tablet or capsule) is about 5 mg, about 10 mg, about 20 mg, about 25 mg, about 50 mg, about 100 mg, about 200 mg, about 250 mg, about 375 mg, about 500 mg, about 750 mg, or about 1000 mg.

[0145] In some aspects, the amount of antisense oligomer or antisense oligonucleotide conjugate (e.g., CIVI008) in an oral dosage form (e.g., tablet or capsule) ranges from about 1 mg to about 100 mg, about 5 mg to about 100 mg, about 10 mg to about 100 mg, about 20 mg to about 100 mg, or about 20 mg to about 50 mg.

[0146] In some aspects of the present disclosure, CIVI008 is formulated in capsule form, wherein the capsule is a hard-shell gelatin capsule. In some aspects, the capsule is a size 0 capsule (sealed length: 21.7 mm x outer diameter: 7.6 mm). In some aspects, the capsule is a size 4 capsule (sealed length: 14.3 mm x outer diameter: 5.05 mm). In some aspects, the capsule contains about 5 mg to about 30 mg of CIVI008 (sepadaclusen sodium), e.g., about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, or about 30 mg of CIVI008. In some aspects, the capsule contains about 100 mg or about 200 mg of 5-CNAC, e.g., about 100 mg, about 125 mg, about 150 mg, about 175 mg, or about 200 mg of 5-CNAC. In some aspects, the capsule fill is prepared by dry blending the ingredients (i.e., dried CIVI008 and dried 5-CNAC). In some aspects, the capsule fill is prepared by lyophilizing a co-melt mixture of the ingredients (i.e., CIVI008 and 5-CNAC). In some aspects, the capsule contains about 10 mg of CIVI008 and about 100 mg of 5-CNAC. In some aspects, the capsule contains about 20 mg of CIVI008 and about 200 mg of 5-CNAC. In some aspects, the capsule contains about 5 mg of CIVI008 and about 200 mg of 5-CNAC. In some aspects, the capsule contains about 25 mg of CIVI008 and about 200 mg of 5-CNAC. In some aspects, the capsule contains about 30 mg of CIVI008 and about 200 mg of 5-CNAC.

[0147] In some cases, the disclosure provides pharmaceutical compositions, e.g., pharmaceutical compositions in capsule form, comprising, for example, about 10 mg CIVI008 and about 100 mg 5-CNAC, about 20 mg CIVI008 and about 200 mg 5-CNAC, about 5 mg CIVI008 and about 200 mg 5-CNAC, about 25 mg CIVI008 and about 200 mg 5-CNAC, or about 30 mg CIVI008 and about 200 mg 5-CNAC, wherein administration of the pharmaceutical composition to a subject results in a mean AUC 0-50 Compared to the mean AUC 0-50 is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%. In a particular aspect, the mean AUC measured when a corresponding pharmaceutical composition containing SNAC instead of 5-CNAC is administered to a subject 0-50 Compared to the mean AUC 0-50 The increase is about 80%.

[0148] As used herein, the term "corresponding pharmaceutical composition containing SNAC instead of 5-CNAC" refers to a reference pharmaceutical composition containing the same ingredients as the test pharmaceutical composition, where the only difference between the reference pharmaceutical composition and the test composition is the presence of SNAC instead of 5-CNAC in the reference pharmaceutical composition. For example, if the test pharmaceutical composition is contained in a size 4 capsule containing 10 mg of CIVI008 and 100 mg of 5-CNAC, the corresponding reference pharmaceutical composition will also be filled into a size 4 capsule and contain 10 mg of CIVI008 and 100 mg of SNAC.

[0149] In some aspects, the disclosure provides pharmaceutical compositions, e.g., in capsule form, comprising, e.g., about 10 mg CIVI008 and about 100 mg 5-CNAC, about 20 mg CIVI008 and about 200 mg 5-CNAC, about 5 mg CIVI008 and about 200 mg 5-CNAC, about 25 mg CIVI008 and about 200 mg 5-CNAC, or about 30 mg CIVI008 and about 200 mg 5-CNAC, wherein administration of the pharmaceutical composition to a subject results in a mean C measured when a corresponding pharmaceutical composition comprising SNAC instead of 5-CNAC is administered to the subject. max Compared to average C max is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, or at least about 150%. In a particular aspect, the mean C measured when a subject is administered a corresponding pharmaceutical composition comprising SNAC instead of 5-CNAC is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, or at least about 150%. max Compared to average C max The increase is about 110%.

[0150] In some aspects, the amount of antisense oligomer or antisense oligonucleotide conjugate (e.g., CIVI008) in an oral dosage form (e.g., tablet or capsule) is about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg, about 44 mg, about 45 mg, about 46 mg, about 47 mg, about 48 mg, about 49 mg, about 50 mg, about 51 mg, about 52 mg, about 53 mg, about 54 mg, about 55 mg, about 56 mg, about 57 mg, about 58 mg, about 59 mg, about 60 mg, about 61 mg, about 62 mg, about 63 mg, about 64 mg, about 65 mg, about 66 mg, about 67 mg, about 68 mg, about 69 mg, about 70 mg, about 71 mg, about 72 mg, about 73 mg, about 74 mg, about 75 mg, about 76 mg, about 77 mg, about 78 mg, about 79 6mg, about 17mg, about 18mg, about 19mg, about 20mg, about 21mg, about 22mg, about 23mg, about 24mg, about 25mg, about 26mg, about 27mg, about 28mg, about 29mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg, about 44 mg , about 45 mg, about 46 mg, about 47 mg, about 48 mg, about 49 mg, about 50 mg, about 51 mg, about 52 mg, about 53 mg, about 54 mg, about 55 mg, about 56 mg, about 57 mg, about 58 mg, about 59 mg, about 60 mg, about 61 mg, about 62 mg, about 63 mg, about 64 mg, about 65 mg, about 66 mg, about 67 mg, about 68 mg, about 69 mg, about 70 mg, about 71 mg, about 72 mg, about 7 3 mg, about 73 mg, about 75 mg, about 76 mg, about 77 mg, about 78 mg, about 79 mg, about 80 mg, about 81 mg, about 82 mg, about 83 mg, about 84 mg, about 85 mg, about 86 mg, about 87 mg, about 88 mg, about 89 mg, about 90 mg, about 91 mg, about 92 mg, about 93 mg, about 94 mg, about 95 mg, about 96 mg, about 97 mg, about 98 mg, about 99, or about 100 mg.

[0151] In some aspects, the oral pharmaceutical compositions of the present disclosure may contain at least one pharmaceutically acceptable excipient or a combination thereof in addition to the antisense oligonucleotide conjugate (e.g., CIVI008) disclosed herein and an oral delivery agent (e.g., SNAC, C10, or 5-CNAC). In some aspects, for example, the at least one pharmaceutically acceptable excipient or a combination thereof in a commonly used amount is selected from the group consisting of, but not limited to, a pH adjusting agent, a preservative, a flavoring, a taste masking agent, a fragrance, a humectant, a tonicity agent, a colorant, a surfactant, a plasticizer, a lubricant (e.g., magnesium stearate), a flow aid, a compression aid, a solubilizer, an excipient, a diluent (e.g., microcrystalline cellulose (e.g., Avicel PH102)), or any combination thereof. In some aspects, oral pharmaceutical compositions of the present disclosure (e.g., antisense oligonucleotide conjugates such as CIVI008 in combination with an oral delivery agent such as SNAC, C10, or 5-CNAC) comprise microcrystalline cellulose. In some aspects, oral pharmaceutical compositions of the present disclosure (e.g., antisense oligonucleotide conjugates such as CIVI008 in combination with an oral delivery agent such as SNAC, C10, or 5-CNAC) comprise phosphate buffer salts, citric acid, glycols, other dispersing agents, or any combination thereof.

[0152] In some aspects, oral pharmaceutical compositions of the present disclosure (e.g., antisense oligonucleotide conjugates such as CIVI008 combined with oral delivery agents such as SNAC, C10, or 5-CNAC) can include a diluent (e.g., microcrystalline cellulose (e.g., Avicel)) and a lubricant (e.g., magnesium stearate). In some aspects, oral pharmaceutical compositions of the present disclosure (e.g., antisense oligonucleotide conjugates such as CIVI008 combined with oral delivery agents such as SNAC, C10, or 5-CNAC) can include povidone and / or crospovidone. The crospovidone can be any crospovidone. Crospovidone is a synthetic crosslinked homopolymer of N-vinyl-2-pyrrolidone, also known as 1-ethenyl-2-pyrrolidinone, having a molecular weight of 1,000,000 or greater. Commercially available crospovidones include Polyplasdone XL, Polyplasdone XL-10, Polyplasdone INF-10 available from ISP, and Kollidon CL available from BASF. In some aspects, the crospovidone is Polyplasdone XL. Povidone is a synthetic polymer consisting of linear 1-vinyl-2-pyrrolidone groups, typically having a molecular weight of 2,500 to 3,000,000. Commercially available povidones include Kollidon K-30, Kollidon K-90F available from BASF, and Plasdone K-30 and Plasdone K-29 / 32 available from ISP. As noted above, crospovidones and povidones are commercially available. Alternatively, they may be synthesized using known processes. The crospovidone, povidone, or combination thereof may be present in the oral pharmaceutical compositions of the present disclosure in an amount of 0.5 to 50 percent by weight, based on the total weight of the entire oral pharmaceutical composition (e.g., an amount of about 2 to about 25 percent, or about 5 to about 20 percent by weight, based on the total weight of the entire oral pharmaceutical composition).

[0153] In some aspects, an oral dosage form (e.g., a tablet or capsule) comprising an oral pharmaceutical composition of the present disclosure (e.g., an antisense oligonucleotide conjugate (such as CIVI008) and an oral delivery agent (such as NAP or C10), and optionally a statin) can include a coating, e.g., an enteric coating and / or a pH-sensitive coating, and can optionally include an enzyme inhibitor. Thus, in some aspects, the solid oral dosage form does not substantially disintegrate or dissolve in the stomach, but does substantially disintegrate or dissolve in the intestine. In some aspects, an oral pharmaceutical composition of the present disclosure (e.g., an antisense oligonucleotide conjugate (such as CIVI008) and an oral delivery agent (such as SNAC, C10, or 5-CNAC), and optionally a statin) can further include one or more enzyme inhibitors that prevent enzymatic degradation of the active agents in the pharmaceutical formulation (e.g., the antisense oligonucleotide conjugate (e.g., CIVI008) and / or the optional therapeutic agent (such as a statin) in the stomach or upper intestinal tract.

[0154] In some aspects, the oral pharmaceutical composition of the present disclosure or the oral dosage form (e.g., tablet or capsule) disclosed herein is enteric-coated to delay disintegration in the stomach.Enteric coating agents include, but are not limited to, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, polyvinyl acetate phthalate, cellulose acetate trimellitate, cellulose acetate phthalate, poly(methacrylic acid-ethyl acrylic acid), poly(methacrylic acid-methyl methacrylic acid), and combinations thereof.In yet another aspect, the oral pharmaceutical preparation may be formulated so that erosion occurs on the surface of the oral dosage form rather than disintegration.

[0155] In some aspects, the oral pharmaceutical composition of the present disclosure or the oral dosage form (e.g., tablet or capsule) disclosed herein further comprises a pH-sensitive coating agent, such as a pH-sensitive polymer, that protects the oral pharmaceutical composition or the oral dosage form from the acidic environment in the stomach.In some aspects, the pH-sensitive polymer includes cellulose, acrylic acid, or its derivatives.In some aspects, the pH-sensitive coating agent includes a pH-sensitive hydrogel, a pH-activated drug delivery system, a pH-sensitive liposome, micelle, or lipid nanoparticle, a pH-sensitive microsphere, a pH-sensitive nanoparticle, or any combination thereof.

[0156] For example, enteric (poorly soluble in the stomach) coatings for liquid or gel capsules, pH-sensitive coatings, enzyme inhibitors, and gelatin-based formulations are described in more detail below.

[0157] In some aspects, the oral pharmaceutical compositions of the present disclosure may comprise, in addition to the antisense oligonucleotide conjugate (e.g., CIVI008) and oral delivery agent (e.g., SNAC, C10, or 5-CNAC) disclosed herein, a second therapeutically active compound (therapeutic agent) selected from the group consisting of a statin (e.g., lovastatin, cerivastatin, pravastatin, atorvastatin, simvastatin, rosuvastatin, fluvastatin, or a combination thereof), ezetimibe, a bile-sorbing resin, nicotinic acid, a fibric acid derivative, probucol, neomycin, dextrothyroxine, a plant stanol ester, a cholesterol absorption inhibitor, implitapide, an inhibitor of bile acid transporters, a regulator of hepatic CYP7a, an estrogen replacement therapy agent, and an anti-inflammatory agent. Generally, the oral pharmaceutical compositions of the present disclosure may include, in addition to the antisense oligonucleotide conjugate (e.g., CIVI008) and oral delivery agent (e.g., SNAC, C10, or 5-CNAC) disclosed herein, a second therapeutically active compound used in the art to treat a disease or condition associated with increased PCSK9 expression and / or activity.

[0158] In some aspects, the present disclosure provides in vitro methods for reducing PCSK9 expression levels and / or PCSK9 activity in a cell, the methods comprising administering an effective amount of an oral pharmaceutical composition of the present disclosure comprising, for example, an antisense oligonucleotide conjugate disclosed herein (e.g., CIVI008), an oral delivery agent (e.g., SNAC, C10, or 5-CNAC), and optionally a second therapeutic agent, such as a statin.

[0159] The present disclosure also provides a method of reducing PCSK9 expression levels and / or PCSK9 activity levels in a subject in need thereof, comprising administering an effective amount of an oral pharmaceutical composition of the present disclosure (e.g., an antisense oligonucleotide conjugate disclosed herein (e.g., CIVI008), an oral delivery agent (e.g., SNAC, C10, or 5-CNAC), and optionally a second therapeutic agent such as a statin). Also provided is a method of reducing cholesterol levels in a subject in need thereof, comprising administering to the subject an effective amount of an oral pharmaceutical composition of the present disclosure, comprising, for example, an antisense oligonucleotide conjugate disclosed herein (e.g., CIVI008), an oral delivery agent (e.g., SNAC, C10, or 5-CNAC), and optionally a second therapeutic agent such as a statin.

[0160] The oral pharmaceutical composition of the present disclosure can be prepared by conventional methods, for example, by mixing and kneading the active substance mixture or active substance, the oral delivery agent, and other ingredients, filling them into capsules, or molding them instead of filling them into capsules, and then tableting or compressing them to obtain tablets.In addition, a solid dispersion can be formed by known methods, and then further processed to form tablets or capsules.In some aspects, the components of the oral pharmaceutical composition of the present disclosure are mixed uniformly or evenly throughout the solid dosage form.

[0161] The term "capsule" as used herein is intended to mean a pharmaceutical preparation that typically includes a hard or soft shell (e.g., a gelatin shell) containing a single dose of an active agent (e.g., CIVI008). In one aspect, the capsule is intended for oral administration. In some aspects, the capsule shell (also known as the capsule body) disintegrates in the stomach after ingestion (e.g., swallowing), releasing the capsule contents (e.g., a dry mixture disclosed herein that includes CIVI008 and 5-CNAC).

[0162] As used herein, the term "dry blending" refers to combining and thoroughly blending multiple components (e.g., CIVI800 and 5-CNAC) in the absence of a liquid medium. In some aspects, the components of the dry blend (e.g., CIVI800, 5-CNAC, or both) can be in powder form. In some aspects, the components of the dry blend (e.g., CIVI800 and 5-CNAC) can be in particulate form, e.g., granular.

[0163] In some aspects, the present disclosure provides a pharmaceutical composition comprising 10 mg of CIVI008 (sepadaclusen) and 100 mg of 5-CNAC, wherein both components are dry blended. In some aspects, the present disclosure provides a pharmaceutical composition comprising 20 mg of CIVI008 (sepadaclusen) and 200 mg of 5-CNAC, wherein both components are dry blended. In some aspects, the present disclosure provides a pharmaceutical composition comprising 5 mg of CIVI008 (sepadaclusen) and 200 mg of 5-CNAC, wherein both components are dry blended. In some aspects, the present disclosure provides a pharmaceutical composition comprising 10 mg of CIVI008 (sepadaclusen) and 200 mg of 5-CNAC, wherein both components are dry blended. In some aspects, the present disclosure provides a pharmaceutical composition comprising 25 mg of CIVI008 (sepadaclusen) and 200 mg of 5-CNAC, wherein both components are dry blended. In some aspects, the present disclosure provides a pharmaceutical composition comprising 30 mg of CIVI008 (cepadaclusen) and 200 mg of 5-CNAC, wherein both components are dry blended.

[0164] In some aspects, the present disclosure provides a capsule (e.g., an enteric-coated hard-shell gelatin capsule) containing 10 mg of CIVI008 (sepadaclusen) and 100 mg of 5-CNAC, wherein both components are dry blended. In some aspects, the present disclosure provides a capsule containing 20 mg of CIVI008 (sepadaclusen) and 200 mg of 5-CNAC, wherein both components are dry blended. In some aspects, the present disclosure provides a capsule containing 5 mg of CIVI008 (sepadaclusen) and 200 mg of 5-CNAC, wherein both components are dry blended. In some aspects, the present disclosure provides a capsule containing 10 mg of CIVI008 (sepadaclusen) and 200 mg of 5-CNAC, wherein both components are dry blended. In some aspects, the present disclosure provides a capsule containing 25 mg of CIVI008 (sepadaclusen) and 200 mg of 5-CNAC, wherein both components are dry blended. In some aspects, the present disclosure provides a capsule containing 30 mg of CIVI008 (sepadaclusen) and 200 mg of 5-CNAC, wherein both components are dry blended.

[0165] In some aspects, the present disclosure provides a method for treating a disease or condition caused by high PCSK9 expression levels and / or PCSK9 activity in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition comprising 10 mg of CIVI008 (sepadacrusen) and 100 mg of 5-CNAC, wherein both components are dry-blended and optionally contained in a capsule (e.g., an enteric-coated hard-shell gelatin capsule). In some aspects, the present disclosure provides a method for treating a disease or condition caused by high PCSK9 expression levels and / or PCSK9 activity in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition comprising 20 mg of CIVI008 (sepadacrusen) and 200 mg of 5-CNAC, wherein both components are dry-blended and optionally contained in a capsule. In some aspects, the present disclosure provides a method for treating a disease or condition caused by high PCSK9 expression levels and / or PCSK9 activity in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition comprising 5 mg of CIVI008 (sepadacrusen) and 200 mg of 5-CNAC, wherein both components are dry-blended and optionally contained in a capsule. In some aspects, the present disclosure provides a method for treating a disease or condition caused by high PCSK9 expression levels and / or PCSK9 activity in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition comprising 10 mg of CIVI008 (sepadacrusen) and 200 mg of 5-CNAC, wherein both components are dry-blended and optionally contained in a capsule.In some aspects, the present disclosure provides a method for treating a disease or condition caused by high PCSK9 expression levels and / or PCSK9 activity in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition comprising 25 mg of CIVI008 (sepadacrusen) and 200 mg of 5-CNAC, wherein both components are dry-blended and optionally contained in a capsule. In some aspects, the present disclosure provides a method for treating a disease or condition caused by high PCSK9 expression levels and / or PCSK9 activity in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition comprising 30 mg of CIVI008 (sepadacrusen) and 200 mg of 5-CNAC, wherein both components are dry-blended and optionally contained in a capsule.

[0166] In some aspects, the present disclosure provides pharmaceutical compositions comprising CIVI008 (sepadaclusen) and 5-CNAC in a ratio of 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:30, 1:40, or 1:50, wherein both components are dry blended. In some aspects, the present disclosure provides pharmaceutical compositions comprising CIVI008 (sepadaclusen) and 5-CNAC in a ratio of 1:5, wherein both components are dry blended. In some aspects, the present disclosure provides pharmaceutical compositions comprising CIVI008 (sepadaclusen) and 5-CNAC in a ratio of 1:10, wherein both components are dry blended. In some aspects, the present disclosure provides pharmaceutical compositions comprising CIVI008 (sepadaclusen) and 5-CNAC in a ratio of 1:20, wherein both components are dry blended. In some aspects, the present disclosure provides a pharmaceutical composition comprising CIVI008 (cepadaclusen) and 5-CNAC in a ratio of 1:40, wherein both components are dry blended.

[0167] In some aspects, the present disclosure provides capsules containing CIVI008 (sepadaclusene) and 5-CNAC in a ratio of 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:30, 1:40, or 1:50, where both components are dry blended. In some aspects, the present disclosure provides capsules containing CIVI008 (sepadaclusene) and 5-CNAC in a ratio of 1:5, where both components are dry blended. In some aspects, the present disclosure provides capsules containing CIVI008 (sepadaclusene) and 5-CNAC in a ratio of 1:10, where both components are dry blended. In some aspects, the present disclosure provides capsules containing CIVI008 (sepadaclusene) and 5-CNAC in a ratio of 1:20, where both components are dry blended. In some aspects, the present disclosure provides capsules containing CIVI008 (sepadaclusen) and 5-CNAC in a 1:40 ratio, where both components are dry blended.

[0168] In some aspects, the present disclosure provides a method for treating a disease or condition caused by high PCSK9 expression levels and / or PCSK9 activity in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition comprising CIVI008 (sepadacrusen) and 5-CNAC in a ratio of 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:30, 1:40, or 1:50, wherein both components are dry blended, and optionally the components are contained in a capsule. In some aspects, the present disclosure provides a method for treating a disease or condition caused by high PCSK9 expression levels and / or PCSK9 activity in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition comprising CIVI008 (sepadacrusen) and 5-CNAC in a ratio of 1:5, wherein both components are dry blended, and optionally the components are contained in a capsule. In some aspects, the present disclosure provides a method for treating a disease or condition caused by high PCSK9 expression levels and / or PCSK9 activity in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition comprising CIVI008 (sepadacrusen) and 5-CNAC in a ratio of 1:10, wherein both components are dry mixed, and optionally the components are contained in a capsule. In some aspects, the present disclosure provides a method for treating a disease or condition caused by high PCSK9 expression levels and / or PCSK9 activity in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition comprising CIVI008 (sepadacrusen) and 5-CNAC in a ratio of 1:20, wherein both components are dry mixed, and optionally the components are contained in a capsule. In some aspects, the present disclosure provides methods for treating a disease or condition caused by high PCSK9 expression levels and / or PCSK9 activity in a subject in need thereof, comprising administering an effective amount of a pharmaceutical composition comprising CIVI008 (cepadacrusen) and 5-CNAC in a ratio of 1:40, wherein both components are dry blended, and optionally the components are contained in a capsule.

[0169] The present disclosure also provides kits comprising an oral pharmaceutical composition of the present disclosure (e.g., comprising CIVI008 and an oral delivery agent such as SNAC or 5-CNAC) and instructions indicating that the oral pharmaceutical composition may be taken, for example, before food intake.

[0170] In some aspects, oral pharmaceutical compositions of the present disclosure comprise an antisense oligonucleotide conjugate comprising an antisense oligomer that is 16-22 contiguous nucleotides, wherein the sequence of the antisense oligomer comprises a sequence of 16 contiguous nucleotides in length that is 100% complementary to the sequence of SEQ ID NO:31 (or another suitable PCSK9 target region disclosed herein); and wherein the antisense oligomer is a gapmer (e.g., a 3-10-3 gapmer), the gapmer comprising at least one LNA unit and at least one non-nucleotide or non-polynucleotide moiety (e.g., a liver-targeting moiety such as GalNAc), wherein the at least one non-nucleotide or non-polynucleotide moiety is covalently attached to the antisense oligomer either directly or via a linker disposed between the contiguous oligomer sequence and the non-nucleotide or non-polynucleotide moiety; wherein the antisense oligonucleotide conjugate targets the RNA encoding PCSK9.

[0171] In some aspects, the antisense oligonucleotide conjugate comprises or consists of SEQ ID NO:18 or SEQ ID NO:19. In some aspects, the antisense oligonucleotide conjugate is a pharmaceutically acceptable salt of an antisense oligonucleotide conjugate disclosed herein, for example, an antisense oligonucleotide conjugate of SEQ ID NO:18 or SEQ ID NO:19. In some aspects, the salt is a sodium salt. In some aspects, the antisense oligonucleotide conjugate comprises one sodium ion per nucleotide unit. Thus, in one particular aspect, the antisense oligonucleotide of the present disclosure is the sodium salt of SEQ ID NO:19, designated CIVI008, which is the hexadeca sodium salt of the antisense oligonucleotide conjugate of SEQ ID NO:19, as shown in Figure 18B.

[0172] The type of oligonucleotide conjugate in the oral pharmaceutical composition of the present disclosure that can be used to target a nucleic acid, e.g., RNA, encoding PCSK9 protein is not limited to antisense oligonucleotides (ASOs). In some aspects, as described in more detail below, the oligomer in the oligonucleotide conjugate of the present disclosure can be an siRNA, shRNA, aptamer, or any nucleic acid capable of regulating PCSK9 expression and / or PCSK9 activity. In some aspects, the oligomer in the oligonucleotide conjugate of the present disclosure can be a monomer or a multimer, e.g., a plurality of concatenated linking units of an oligomer of the present disclosure (e.g., an oligomer derived from SEQ ID NO:26, e.g., SEQ ID NO:2 or SEQ ID NO:3). Thus, in some aspects, the oligomer comprises multiple concatenated copies of SEQ ID NO:2 or SEQ ID NO:3, which are cleavable linkers inserted between each ASO unit in the multimeric oligomer. In some aspects, the oligomer of the present disclosure can be single-stranded or double-stranded.

[0173] In some aspects, the oligomer or oligonucleotide conjugate disclosed herein can target RNA, for example, pre-mRNA, splice variant of pre-mRNA, or mature mRNA. In some aspects, the RNA, for example, pre-mRNA, splice variant of pre-mRNA, or mature mRNA corresponds to an allelic variant of normal PCSK9 gene. In some aspects, the RNA, for example, pre-mRNA, splice variant of pre-mRNA, or mature mRNA corresponds to a mutant of normal PCSK9 gene. In some aspects, the mutant is a gain-of-function mutant. In some aspects, it may be desirable to inhibit the expression and / or activity of a gain-of-function mutant to reduce PCSK9 activity. However, in some aspects, it may be desirable to inhibit the expression of a loss-of-function mutant when the expression of the mutant is harmful or may cause the accumulation of deposits of the mutant PCSK9 protein or inactive PCSK9 protein.

[0174] In some aspects, the oligomer or oligonucleotide conjugates disclosed herein can target sequences within PCSK9 exons. In some aspects, the oligomer or oligonucleotide conjugates disclosed herein can target sequences within PCSK9 introns. In some aspects, the oligomer or oligonucleotide conjugates disclosed herein can target sequences including the boundaries between exons and introns. In some aspects, the oligomer or oligonucleotide conjugates disclosed herein can target sequences upstream of the 5' end of an open reading frame encoding PCSK9. In some aspects, the oligomer or oligonucleotide conjugates disclosed herein can target sequences downstream of the 3' end of an open reading frame encoding PCSK9.

[0175] Sections 2 (Antisense Oligomers) and 3 (Oligonucleotide Conjugates) describe oligomers (e.g., antisense oligonucleotides, ASOs) and oligonucleotide conjugates (e.g., antisense oligonucleotide conjugates) that can be used in the oral pharmaceutical compositions of the present disclosure. Accordingly, it is understood that any of the antisense oligomers (e.g., oligomers of SEQ ID NO: 2 or 3) and / or antisense oligonucleotide conjugates (e.g., oligonucleotide conjugates of SEQ ID NO: 18 or 19) can be combined with at least one oral delivery agent disclosed herein, e.g., SNAC, C10, or 5-CNAC, to produce an oral pharmaceutical composition of the present disclosure (e.g., a pill or capsule containing CIVI008 and SNAC or 5-CNAC). Such oral pharmaceutical compositions can also include pharmaceutically acceptable diluents, carriers, salts, or adjuvants disclosed herein or known in the art. For example, WO2007 / 031091 provides suitable pharmaceutically acceptable diluents, carriers and adjuvants, suitable doses, formulations, routes of administration, compositions, dosage forms, combinations with other therapeutic agents, and prodrug formulations that can be used in the oral pharmaceutical compositions of the present disclosure.

[0176] The oral antisense pharmaceutical composition disclosed herein can be used to treat the disease or pathology caused by the abnormal expression level and / or activity of PCSK9.Therefore, the present disclosure provides a method for treating the disease or pathology caused by the abnormal expression level and / or activity of PCSK9 in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of the oral pharmaceutical composition disclosed herein (for example, the oral pharmaceutical composition comprising the antisense oligonucleotide conjugate such as CIVI008 and an oral delivery agent such as SNAC, C10 or 5-CNAC), wherein the administration of the pharmaceutical composition reduces the serum PCSK9 level in the subject, and / or reduces the serum LDL cholesterol level.

[0177] In some aspects, the disease or condition is selected from the group consisting of atherosclerosis, hypercholesterolemia (e.g., familial hypercholesterolemia or statin-resistant hypercholesterolemia), HDL / LDL cholesterol imbalance, dyslipidemia (e.g., familial hyperlipidemia (FCHL) or acquired hyperlipidemia), coronary artery disease (CAD), and coronary heart disease (CHD). Accordingly, the present disclosure provides a method of treating a disease or condition selected from the group consisting of atherosclerosis, hypercholesterolemia (e.g., familial hypercholesterolemia or statin-resistant hypercholesterolemia), HDL / LDL cholesterol imbalance, dyslipidemia (e.g., familial hyperlipidemia (FCHL) or acquired hyperlipidemia), coronary artery disease (CAD), and coronary heart disease (CHD) in a subject in need thereof, the method comprising administering an effective amount of an oral pharmaceutical composition disclosed herein (e.g., an oral pharmaceutical composition comprising an antisense oligonucleotide conjugate, such as CIVI008, and an oral delivery agent, such as SNAC, C10, or 5-CNAC).

[0178] The present disclosure also provides a method of making an oral pharmaceutical composition, the method comprising mixing: (i) and (ii) (i) an antisense oligomer or antisense oligonucleotide conjugate disclosed herein (e.g., CIVI008); and (ii) Oral delivery agents (e.g., SNAC, C10, or 5-CNAC).

[0179] In some aspects, the oral delivery agent (e.g., SNAC, C10, or 5-CNAC) is covalently linked to the antisense oligomer or antisense oligonucleotide conjugate (e.g., CIVI008) disclosed herein either directly or through a linker or a combination of linkers, wherein the linker or combination of linkers may include a cleavable linker. In some aspects, the oral delivery agent (e.g., SNAC, C10, or 5-CNAC) is covalently linked to the oligomer moiety directly or through a spacer. In some aspects, the oral delivery agent (e.g., SNAC, C10, or 5-CNAC) is covalently linked to the non-nucleotide or non-polynucleotide moiety of the antisense oligonucleotide conjugate (e.g., the GalNAc moiety, CIVI008) disclosed herein either directly or through a linker, spacer, or a combination thereof. Thus, in some aspects, the oral delivery agent (e.g., SNAC, C10, or 5-CNAC) or a combination thereof is linked to a GalNAc conjugate moiety that includes, for example, a cleavable linker. By cleaving the cleavable linker or a combination thereof, both GalNAc and the oral delivery agent (e.g., SNAC, C10, or 5-CNAC) can be released from the conjugate. In other aspects, the GalNAc moiety and the oral delivery agent are linked via two separate cleavable linkers, which can be the same or different types. In some aspects, both cleavable linkers can be cleaved according to the same mechanism (e.g., two pH-sensitive linkers). In other aspects, each cleavable linker can be cleaved according to a different mechanism (e.g., one linker is a pH-sensitive linker, and the second linker can be enzymatically cleaved (e.g., by esterase)).

[0180] In some aspects, the oral delivery agent (e.g., SNAC, C10, or 5-CNAC) can be covalently attached to the 5'-end of an oligomeric moiety disclosed herein. In some aspects, the oral delivery agent (e.g., SNAC, C10, or 5-CNAC) can be covalently attached to the 3'-end of an oligomeric moiety disclosed herein. In some aspects, the oral delivery agent (e.g., SNAC, C10, or 5-CNAC) can be covalently attached directly (e.g., at the 5'- or 3'-nucleotide) to the 5'- or 3'-end of an oligomeric moiety disclosed herein. In some aspects, the oral delivery agent (e.g., SNAC, C10, or 5-CNAC) can be covalently attached indirectly via a linker, spacer, or combination thereof to the 5'- or 3'-nucleotide at the 5'- or 3'-end of an oligomeric moiety disclosed herein.

[0181] In some aspects, the oral delivery agent (e.g., SNAC, C10, or 5-CNAC) can be covalently bound to the antisense oligonucleotide conjugate disclosed herein. In some aspects, the oral delivery agent (e.g., SNAC, C10, or 5-CNAC) can be covalently bound to the oligomer portion of the antisense oligonucleotide conjugate disclosed herein. In some aspects, the oral delivery agent (e.g., SNAC, C10, or 5-CNAC) can be covalently bound to the non-nucleotide or non-polynucleotide portion (e.g., GalNAc portion) of the antisense oligonucleotide conjugate disclosed herein.

[0182] In some aspects, the oral delivery agent (e.g., SNAC, C10, or 5-CNAC) can be covalently linked to the antisense oligonucleotide conjugate disclosed herein via a linker, a spacer, or a combination thereof. In some aspects, the oral delivery agent (e.g., SNAC, C10, or 5-CNAC) can be covalently linked to the oligomer portion of the antisense oligonucleotide conjugate disclosed herein via a linker, a spacer, or a combination thereof. In some aspects, the oral delivery agent (e.g., SNAC, C10, or 5-CNAC) can be covalently linked to the non-nucleotide or non-polynucleotide portion (e.g., GalNAc portion) of the antisense oligonucleotide conjugate disclosed herein via a linker, a spacer, or a combination thereof.

[0183] In some aspects, an oligomer moiety disclosed herein or an antisense oligonucleotide conjugate disclosed herein can be covalently linked to two or more oral delivery agents disclosed herein (e.g., SNAC, C10, or 5-CNAC). In some aspects, an oligomer moiety disclosed herein or an antisense oligonucleotide conjugate disclosed herein can have two or more oral delivery agents disclosed herein (e.g., SNAC, C10, or 5-CNAC) covalently linked at different positions (e.g., an oral delivery agent linked to the oligomer and an oral delivery agent linked to a GalNAc moiety).

[0184] Section 2: Oligomers In some aspects, the oligomers of the present disclosure are LNA gapmers 16-20 nucleotides in length, comprising a contiguous 16-nucleotide sequence complementary to the corresponding length of SEQ ID NO: 31. Also disclosed are oligomers comprising a contiguous sequence selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29, and 44. The antisense oligomers or conjugates of the present disclosure target PCSK9 and are therefore capable of downregulating expression of and / or inhibiting PCSK9, such as PCSK9 in humans or cells expressing PCSK9. In some aspects, the oligomers of the present disclosure can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 nucleotides in length. In some aspects, the oligomers of the present disclosure are greater than 14 nucleotides in length. In some aspects, the internucleoside linkages of a contiguous sequence of 10 to 16 nucleotides that is complementary to the corresponding length of SEQ ID NO: 33 or 34 or 45 can be phosphorothioate linkages.

[0185] In some aspects, an oligomer of the present disclosure (e.g., an oligomer complementary to SEQ ID NO:31) comprises, consists of, or consists essentially of a contiguous sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:3.

[0186] In some aspects, oligomers of the disclosure (e.g., oligomers complementary to SEQ ID NO:31) contain 10 to 16 nucleosides linked by phosphorothioates. In some aspects, oligomers of the disclosure (e.g., oligomers complementary to SEQ ID NO:31) contain more than 14 nucleosides linked by phosphorothioates. In some aspects, the oligomers (e.g., oligomers complementary to SEQ ID NO:31) are 15, 16, 17, 18, 18, 19, 20, 21, or 22 nucleotides in length.

[0187] In some aspects, an oligomer of the present disclosure (e.g., an oligomer complementary to SEQ ID NO: 31) is an LNA gapmer comprising a contiguous sequence of more than 14 nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, or 22 nucleotides, wherein the contiguous sequence is complementary to the corresponding length of SEQ ID NO: 31, and wherein the contiguous sequence comprises nucleotide analogs. In a specific aspect, the oligomer is 16 nucleotides in length. In one aspect, an oligomer of the present disclosure comprises an affinity-enhancing nucleotide analog. In some aspects, the nucleotide analog is a sugar-modified nucleotide, such as a sugar-modified nucleotide selected independently or dependently from the group consisting of a 2'-O-alkyl-RNA unit, a 2'-OMe-RNA unit, a 2'-amino-DNA unit, and a 2'-fluoro-DNA unit.

[0188] In some aspects, an oligomer of the present disclosure (e.g., an oligomer complementary to SEQ ID NO: 31) is an LNA gapmer oligonucleotide. In some aspects, the LNA gapmer comprises wings on both sides (5' and 3') of 2-4 nucleotide analogs, preferably LNA analogs. In some aspects, an oligomer of the present disclosure (e.g., an oligomer complementary to SEQ ID NO: 31) can optionally comprise an additional 1-6 nucleotides (e.g., 1, 2, 3, 4, 5, or 6 nucleotides), where the additional 1-6 nucleotides can form or encompass an in vivo cleavable nucleotide region, such as a phosphate nucleotide linker. In some aspects, the in vivo cleavable nucleotide region is comprised of a short stretch (e.g., 1, 2, 3, 4, 5, or 6 nucleotides) of physiologically unstable nucleotides. This can be achieved by using phosphodiester linkages in DNA / RNA nucleosides, although other nucleosides can also be utilized as long as physiological instability is maintained. Physiological instability can be measured, for example, using liver extracts, as shown in Example 6.

[0189] In some aspects, an oligomer of the present disclosure comprises a contiguous 16-nucleotide sequence that is complementary to the corresponding length of SEQ ID NO:31 (first region, or region A). The oligomer of the present disclosure may comprise an additional nucleotide region. In some aspects, the additional nucleotide region comprises an in vivo-cleavable nucleotide region, such as a phosphate nucleotide sequence (second region, region B), that can covalently link linkage region A to a non-nucleotide moiety, such as a conjugate group (third region, or region C). In some aspects, the contiguous nucleotide sequence of an oligomer of the present disclosure (region A) is directly covalently linked to region C. In some aspects, region C is in vivo-cleavable.

[0190] In some aspects, the oligomer consists of or comprises a sequence of 16-22 contiguous nucleotides, such as 16, 17, 18, 19, 20, 21, or 22 nucleotides in length (e.g., 16 nucleotides in length). Thus, in some aspects, the oligomer can refer to the combined length of region A and region B, e.g., 16-22 nucleotides in region A (e.g., 16 nucleotides) and, e.g., 1-6 nucleotides in region B.

[0191] In some aspects, the oligomer of the present disclosure does not contain RNA (units); for example, in some aspects, the oligomer may contain only DNA units. In some aspects, the oligomer of the present disclosure contains both DNA and RNA units. In some aspects, the oligomer according to the present disclosure, the first region of the oligomer, or both the first and second regions of the oligomer (e.g., as a single continuous sequence) are linear molecules. In some aspects, the first region of the oligomer, or both the first and second regions of the oligomer (e.g., as a single continuous sequence) are synthesized as linear molecules. Thus, in some aspects, the oligomer can be a single-stranded molecule. In some aspects, the oligomer does not contain a short region, for example, of at least 3, 4, or 5 contiguous nucleotides, that is complementary to a corresponding region within the same oligomer (i.e., double-stranded).

[0192] In some aspects, the oligomer is not a double-stranded nucleic acid.In some aspects, the oligomer of the present disclosure is not an siRNA.In some aspects, the oligomer of the present disclosure is not an shRNA.In some aspects, the oligomer is a double-stranded nucleic acid.In some aspects, the oligomer of the present disclosure is an siRNA.In some aspects, the oligomer of the present disclosure is an shRNA.

[0193] In some aspects, the oligomer of the present disclosure is an antisense oligonucleotide (ASO). In some aspects, the oligomer of the present disclosure is a multimer. In some aspects, the oligomer of the present disclosure is a multimeric ASO, for example, it can contain a plurality of concatenated linked oligomers of the present disclosure. In some aspects, the oligomer of the present disclosure contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more concatenated linked oligomers. In some aspects, the concatenated linked oligomers are linked via a cleavable linker inserted between each ASO unit of the ASO multimer.

[0194] The terms "corresponding to" and "corresponds to," as used to contrast two oligonucleotides disclosed herein, refer to a comparison between the nucleotide sequence (i.e., nucleobase or base sequence) of an oligomer of the present disclosure and the reverse complement of the nucleic acid target or a subregion thereof (e.g., SEQ ID NO: 31, 32, 33, 34, or 45). Nucleotide analogs are compared directly to their equivalent or corresponding nucleotides. In some aspects, the oligomer (or a first region thereof) is complementary (e.g., fully complementary) to the target region or a subregion thereof (e.g., SEQ ID NO: 31, 32, 33, 34, or 45).

[0195] Section 2a: Oligomeric sequences In some aspects, the oligomers of the present disclosure can target the regions disclosed in Table 1A. Table 1A shows the sequences of the target regions in PCSK9 mRNA as well as their locations. In some aspects, the oligomers of the present disclosure comprise a complementary region that is complementary to at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides of one of the target sequences in Table 1A. In some aspects, the oligomers of the present disclosure comprise a complementary region that is complementary to at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides of one of the target sequences in Table 1A, wherein the complementary region is located at the 5' end of the oligomer. In some aspects, oligomers of the present disclosure comprise a region of complementarity that is complementary to at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides of one of the target sequences in Table 1A, wherein the region of complementarity is located at the 3' end of the oligomer. In some aspects, oligomers of the present disclosure comprise a region of complementarity that is complementary to at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides of one of the target sequences in Table 1A, wherein the region of complementarity is located at the 5' end of the oligomer, and wherein the oligomer is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some aspects, an oligomer of the disclosure comprises a region of complementarity that is complementary to at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides of one of the target sequences of Table 1A, wherein the region of complementarity is located at the 3' end of the oligomer, and wherein the oligomer is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0196] [Table 1]

[0197] Table 1B lists SEQ ID NOS: 25-29 and 44, which are base sequences or nucleobase motif sequences. For example, SEQ ID NO: 26 is a 16-mer targeting SEQ ID NO: 31. In some aspects, the nucleobase motif sequence can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides complementary to an extension from the 5' end, 3' end, or both, of the motif sequence presented in Table 1B. The oligomer sequences of the present disclosure are derived from the motif sequences disclosed herein that contain specific patterns of nucleobase analogs and / or internucleoside linkages.

[0198] [Table 2]

[0199] Table 1C shows the different oligomer sequences, their conjugate types, and their targets corresponding to the target region on the PCKS9 gene.

[0200] [Table 3]

[0201] SEQ ID NO: 1 is SPC5001, an antisense oligomer disclosed in WO2011 / 009697 that is known to cause severe nephrotoxicity when administered to human subjects. SEQ ID NOs: 1-24 and 40-43 are oligomers containing nucleotide analogs, such as LNA gapmer oligomers; here, lowercase letters represent DNA units (nucleosides / nucleotides) and uppercase letters represent LNA units. In some aspects, all LNA Cs are 5-methylcytosine. In some aspects, all LNA units are β-D-oxyLNA. In some aspects, all internucleoside linkages of nucleosides in SEQ ID NOs: 1-24 and 40-43 are phosphorothioate linkages. In some aspects, phosphorothioate linkages are present only in the first and / or second 5' phosphorothioate linkages. In some aspects, phosphorothioate linkages are present only in the first and / or second 3' phosphorothioate linkages, hi some aspects, phosphorothioate linkages are present in the first and / or second 5' phosphorothioate linkages and the first and / or second 3' phosphorothioate linkages.

[0202] SEQ ID NOs: 1-24 and 40-43 are gapmers, each wing comprising 2, 3, or 4 LNA units. In some aspects, oligomers of the disclosure correspond to variants of SEQ ID NOs: 1-24 and 40-43, wherein the variants comprise 1, 2, 3, or 4 additional LNA units in the 5' wing of the gapmer. In some aspects, oligomers of the disclosure correspond to variants of SEQ ID NOs: 1-24 and 40-43, wherein the variants comprise 1, 2, 3, or 4 additional LNA units in the 3' wing of the gapmer. In some aspects, oligomers of the disclosure correspond to variants of SEQ ID NOs: 1-24 and 40-43, wherein the variants comprise 1, 2, 3, or 4 additional LNA units in the 5' wing of the gapmer and 1, 2, 3, or 4 additional LNA units in the 3' wing of the gapmer. In some aspects, the LNA unit of a gapmer disclosed herein, for example a gapmer of Table 1C or a variant thereof, may be oxy-LNA, thio-LNA, amino-5LNA, 5'-methyl-LNA, ENA, cET, or cMOE.

[0203] SEQ ID NOs:9-16 and 41-43 include the oligomer (as represented by the SEQ ID NOs) as well as a cholesterol conjugate, wherein the cholesterol conjugate may be covalently attached to the oligomer at the 5'- or 3'-end of the oligomer, optionally via a bio-cleavable linker (such as a phosphate nucleoside linker). In some aspects, the cholesterol conjugate is linked to the 5'-end of the oligomer. SEQ ID NOs:17-24 include the oligomer (as represented by the SEQ ID NOs) as well as a GalNAc conjugate, wherein the GalNAc conjugate may be covalently attached to the oligomer at the 5'- or 3'-end of the oligomer, optionally via a bio-cleavable linker (such as a phosphate nucleoside linker or a cleavable peptide linker). In some aspects, the GalNAc conjugate is linked to the 5'-end of the oligomer. The specific oligomers and conjugates used, as described herein, are specifically shown in Figure 3 (unconjugated oligomers), Figure 4 (cholesterol conjugates), and Figures 5A, 5B, and 5C (GalNAc conjugates). Other examples of conjugate moieties that can be used in the oligomers of the present disclosure are shown in Figures 1A, 1C, and 1C and Figure 2, and are described below in the section "GalNAc conjugate moieties."

[0204] Table 2 shows specific combinations of oligomers and conjugates.

[0205] [Table 4]

[0206] The oligomer conjugates shown in Table 2 correspond to oligomers of SEQ ID NOs: 1-8 conjugated to a conjugate moiety presented in Figure 1A, Figure 1B, Figure 1C, or Figure 2; the conjugation to the conjugate moiety is via the 5' end of each of SEQ ID NOs: 1-8. The oligomers are covalently attached at the site indicated by the wavy line in Figure 1A, Figure 1B, Figure 1C, or Figure 2. In some aspects, the conjugate moiety is linked to the 3' end of the oligomer.

[0207] Figures 5A, 5B, and 5C show the combination of the above SEQ ID NOs with Conj2a. Figures 5B and 5C are two detailed examples of the compound of Figure 5A, corresponding to the conjugates of SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

[0208] In some aspects, an optional bio-cleavable linker (B) is present between the conjugate moiety (C) and the oligomer (A). For Conj1-4 and 1a-4a, the GalNAc conjugate itself is bio-cleavable due to the use of a peptide linker within the GalNAc cluster, with or without the use of such an additional bio-cleavable linker (B). In some aspects, the activity of the GalNAc cluster oligomer conjugate is enhanced by including a bio-cleavable linker (B), such as a phosphate nucleotide linker disclosed herein. Figure 4 shows the combination of the above SEQ ID NOs with Conj5a (cholesterol-C6).

[0209] Section 2b: PCSK9 target region The present disclosure is directed to an orally administered pharmaceutical composition comprising an oligomer (e.g., ASO) of the present disclosure, which can regulate PCSK9 gene expression by specifically targeting a region in PCSK9 RNA, e.g., mRNA. In some aspects, the oligomer can down-regulate PCSK9 gene expression by binding to such a target region. Thus, in some aspects, the oligomer of the present disclosure can affect the expression of PCSK9 in a mammalian subject, such as a human, by binding to a specific target region in PCSK9 RNA, e.g., mRNA. In some aspects, the oligomer of the present disclosure can affect the expression of PCSK9 in human cells, e.g., hepatocytes, by binding to a specific target region in PCSK9 RNA, e.g., mRNA.

[0210] The term "target nucleic acid" or "target region" herein refers to a small sequence of nucleic acid, e.g., RNA, such as mRNA, encoding mammalian PCSK9 or a naturally occurring variant or mutant thereof, e.g., human PCSK9. In some aspects, the nucleic acid encoding mammalian PCSK9 or a naturally occurring variant or mutant thereof is RNA. In some aspects, the RNA is mRNA, such as pre-mRNA. In some aspects, the RNA is mature mRNA. An oligomer according to the present disclosure is preferably hybridizable to the target nucleic acid. It will be understood that SEQ ID NO:46 is a cDNA sequence and therefore corresponds to the mature mRNA target sequence, although uracil is substituted with thymidine in the cDNA sequence.

[0211] In some aspects, the target sequence corresponds to a subsequence of the PCSK9-encoding sequence set forth in SEQ ID NO: 46. Specific target sequences are set forth in Table 1A above, e.g., SEQ ID NO: 30, corresponding to positions 3643-3656 of SEQ ID NO: 46; SEQ ID NO: 31, corresponding to positions 3643-3658 of SEQ ID NO: 46; SEQ ID NO: 32, corresponding to positions 3251-3265 of SEQ ID NO: 46; SEQ ID NO: 33, corresponding to positions 3251-3266 of SEQ ID NO: 46; SEQ ID NO: 34, corresponding to positions 3373-3388 of SEQ ID NO: 46; and SEQ ID NO: 45, corresponding to positions 1005-1017 of SEQ ID NO: 46.

[0212] In some aspects, the target sequence can extend 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides beyond the 5' end of the target region of SEQ ID NO: 30, 31, 32, 33, 34, or 45. In some aspects, the target sequence can extend 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides beyond the 3' end of the target region of SEQ ID NO: 30, 31, 32, 33, 34, or 45. In some aspects, the target sequence can extend 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides beyond the 5' end and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides beyond the 3' end of the target region of SEQ ID NO: 30, 31, 32, 33, 34, or 45. In some aspects, the extended target region overlaps with 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides of the target region of SEQ ID NO: 30, 31, 32, 33, 34, or 45.

[0213] In some aspects, the target region comprises or consists of a corresponding target sequence region derived from the sequence of a mutant or allelic variant of the PCSK9 gene encoding the mRNA of SEQ ID NO: 46. In other aspects, the target region may be a small sequence present in another mRNA transcript variant encoding PCSK9. In some aspects, the target region comprises or consists of a corresponding target sequence region derived from the sequence of a paralogue or ortholog of the PCSK9 gene encoding the mRNA of SEQ ID NO: 46.

[0214] In some aspects, the target region corresponds to NCBI Reference Sequence Accession Number: NM_174936, a subsequence of the sequence of Homo sapiens proprotein convertase subtilisin / kexin type 9-encoding mRNA (PCSK9) transcript variant 1. This NCBI Reference Sequence database entry can be accessed at www.ncbi.nlm.nih.gov / nuccore / NM_174936; the contents of this entry are incorporated herein by reference in its entirety.

[0215] The PCSK9 coding sequence in the NM_174936 mRNA transcript is located at positions 291...2369. Thus, in some aspects, the target region is located between positions 291 and 2369 of the NM_174936 mRNA transcript. In some aspects, the target region is located within the 5' non-coding region. In some aspects, the target region is located within the 3' non-coding region. The NM_174936 mRNA transcript contains exons located at positions 1...497 (exon 1), 498...689 (exon 2), 690...813 (exon 3), 814...947 (exon 4), 948...1089 (exon 5), 1090...1286 (exon 6), 1287...1470 (exon 7), 1471...1644 (exon 8), 1645...1793 (exon 9), 1794...1971 (exon 10), 1972...2153 (exon 11), and 2154...3637 (exon 12). Exons 1-12 are separated by introns 1-11; for example, intron 1 is located between exon 1 and exon 2, intron 2 is located between exon 2 and exon 3, etc.

[0216] In some aspects, the target region is in exon 1. In some aspects, the target region is in exon 2. In some aspects, the target region is in exon 3. In some aspects, the target region is in exon 4. In some aspects, the target region is in exon 5. In some aspects, the target region is in exon 6. In some aspects, the target region is in exon 7. In some aspects, the target region is in exon 8. In some aspects, the target region is in exon 9. In some aspects, the target region is in exon 10. In some aspects, the target region is in exon 11. In some aspects, the target region is in exon 12.

[0217] In some aspects, the target region is present within an intron of the PCSK9 pre-mRNA. In some aspects, the target region is present within intron 1. In some aspects, the target region is present within intron 2. In some aspects, the target region is present within intron 3. In some aspects, the target region is present within intron 4. In some aspects, the target region is present within intron 5. In some aspects, the target region is present within intron 6. In some aspects, the target region is present within intron 7. In some aspects, the target region is present within intron 8. In some aspects, the target region is present within intron 9. In some aspects, the target region is present within intron 10. In some aspects, the target region is present within intron 11. In some aspects, the target region comprises the boundary between exon 1 and intron 1. In some aspects, the target region comprises the boundary between intron 1 and exon 2. In some aspects, the target region comprises the boundary between exon 2 and intron 2. In some aspects, the target region comprises the boundary between intron 2 and exon 3. In some aspects, the target region comprises the boundary between exon 3 and intron 3. In some aspects, the target region comprises the boundary between exon 4 and intron 4. In some aspects, the target region comprises the boundary between intron 4 and exon 5. In some aspects, the target region comprises the boundary between exon 5 and intron 5. In some aspects, the target region comprises the boundary between intron 5 and exon 6. In some aspects, the target region comprises the boundary between exon 6 and intron 6. In some aspects, the target region comprises the boundary between intron 6 and exon 7. In some aspects, the target region comprises the boundary between exon 7 and intron 7. In some aspects, the target region comprises the boundary between intron 7 and exon 8.In some aspects, the target region comprises the boundary between exon 8 and intron 8. In some aspects, the target region comprises the boundary between intron 8 and exon 9. In some aspects, the target region comprises the boundary between exon 9 and intron 9. In some aspects, the target region comprises the boundary between intron 9 and exon 10. In some aspects, the target region comprises the boundary between exon 10 and intron 10. In some aspects, the target region comprises the boundary between intron 10 and exon 11. In some aspects, the target region comprises the boundary between exon 11 and intron 11. In some aspects, the target region comprises the boundary between intron 11 and exon 12.

[0218] In some aspects, the oligomer of the disclosure binds to the target nucleic acid (e.g., SEQ ID NO: 31), and its effect on PCSK9 expression levels and / or PCSK9 activity levels is at least about a 10% to about a 20% decrease in PCSK9 expression levels and / or PCSK9 activity levels compared to normal PCSK9 expression levels (e.g., PCSK9 expression levels in saline-treated cells, animals, or humans) and / or normal activity levels (e.g., expression levels in saline-treated cells, animals, or humans). In some aspects, the reduction in PCSK9 expression and / or activity is 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 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% compared to normal expression and / or activity levels. In some aspects, the decrease in expression and / or activity is about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% compared to normal expression and / or activity levels.

[0219] In some aspects, such modulation (e.g., reduction in expression levels and / or activity) is observed when the oligomers of the present disclosure are used at a concentration of 0.04 nM to 25 nM, such as 0.8 nM to 20 nM. In some aspects, the oligomers may be used at a concentration of at least about 0.04 nM, at least about 0.05 nM, at least about 0.06 nM, at least about 0.07 nM, at least about 0.08 nM, at least about 0.09 nM, at least about 0.1 nM, at least about 0.2 nM, at least about 0.3 nM, at least about 0.4 nM, at least about 0.5 nM, at least about 0.6 nM, at least about 0.7 nM, at least about 0.8 nM, at least about 0.9 nM, at least about 1 nM, at least about 2 nM, at least about 3 nM, at least about 4 nM, at least about 5 nM, at least about 6 nM, or at least about 7 nM. Such modulation (e.g., decreased expression levels and / or activity) is observed when the oligomers of the disclosure are used at a concentration of at least about 7 nM, at least about 8 nM, at least about 9 nM, at least about 10 nM, at least about 11 nM, at least about 12 nM, at least about 13 nM, at least about 14 nM, at least about 15 nM, at least about 16 nM, at least about 17 nM, at least about 18 nM, at least about 19 nM, at least about 20 nM, at least about 21 nM, at least about 22 nM, at least about 23 nM, at least about 24 nM, or at least about 25 nM. In some aspects, the agonist is about 0.04 nM, about 0.05 nM, about 0.06 nM, about 0.07 nM, about 0.08 nM, about 0.09 nM, about 0.1 nM, about 0.2 nM, about 0.3 nM, about 0.4 nM, about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 11 nM, about 12 nM, about 13 nM, about 14 nM, about 15 nM, about 16 nM, about 17 nM, about 18 nM, about 19 nM, about 20 nM, about 21 nM, about 22 nM, about 23 nM, about 24 nM, about 25 nM, about 26 nM, about 27 nM, about 28 nM, about 29 nM, about 30 nM, about 31 nM, about 32 nM, about 33 nM, about 34 nM, about 35 nM, about 36 nM, about 37 nM, about 38 nM, about 39 nM, about 40 nM, about 41 nM, about 42 nM, about 43 nM, about 44 nM, about 45 nM, about 46 nM, about 47 nM, about 48 nM, about 49 nM, about 50 nM, about 51 nM, about 52 nM, about 53 nM, about 54 nM, about 55 nM, Such modulation (e.g., decreased expression levels and / or activity) is observed when using oligomers of the present disclosure at concentrations of about 9 nM, about 10 nM, about 11 nM, about 12 nM, about 13 nM, about 14 nM, about 15 nM, about 16 nM, about 17 nM, about 18 nM, about 19 nM, about 20 nM, about 21 nM, about 22 nM, about 23 nM, about 24 nM, about 25 nM.

[0220] In some aspects, such modulation (e.g., reduction in expression levels and / or activity) is observed when a compound of the present disclosure is used at a concentration of 0.01 to 15 mg / kg (e.g., 0.05 to 10 mg / kg, e.g., 0.1 to 7.5 mg / kg, e.g., 0.25 to 5 mg / kg, e.g., 0.5 to 2.5 mg / kg). In some aspects, the IL-14 receptor agonist is at least about 0.01 mg / kg, at least about 0.02 mg / kg, at least about 0.03 mg / kg, at least about 0.04 mg / kg, at least about 0.05 mg / kg, at least about 0.06 mg / kg, at least about 0.07 mg / kg, at least about 0.08 mg / kg, at least about 0.09 mg / kg, at least about 0.1 mg / kg, at least about 0.2 mg / kg, at least about 0.3 mg / kg, at least about 0.4 mg / kg, at least about 0.5 mg / kg, at least about 0.6 mg / kg, at least about 0.7 mg / kg, at least about 0.8 mg / kg, at least about 0.9 mg / kg, at least about 1 mg / kg, at least about 1.5 mg / kg, at least about 2 mg / kg, at least about 2.5 mg / kg, at least about 3 mg / kg, at least about Such modulation (e.g., decreased expression levels and / or activity) is observed when using a compound of the present disclosure at a concentration of at least about 4 mg / kg, at least about 4.5 mg / kg, at least about 5 mg / kg, at least about 6 mg / kg, at least about 6.5 mg / kg, at least about 7 mg / kg, at least about 7.5 mg / kg, at least about 8 mg / kg, at least about 8.5 mg / kg, at least about 9 mg / kg, at least about 9.5 mg / kg, at least about 10 mg / kg, at least about 10.5 mg / kg, at least about 11 mg / kg, at least about 11.5 mg / kg, at least about 12 mg / kg, 12.5 mg / kg, at least about 13 mg / kg, at least about 13.5 mg / kg, at least about 14 mg / kg, at least about 14.5 mg / kg, or at least about 15 mg / kg.In some aspects, the compound is about 0.01 mg / kg, about 0.02 mg / kg, about 0.03 mg / kg, about 0.04 mg / kg, about 0.05 mg / kg, about 0.06 mg / kg, about 0.07 mg / kg, about 0.08 mg / kg, about 0.09 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 4 mg / kg, about Such modulation (e.g., decreased expression levels and / or activity) is observed when using compounds of the present disclosure at concentrations of 4.5 mg / kg, about 5 mg / kg, about 6 mg / kg, about 6.5 mg / kg, about 7 mg / kg, about 7.5 mg / kg, about 8 mg / kg, about 8.5 mg / kg, about 9 mg / kg, about 9.5 mg / kg, about 10 mg / kg, about 10.5 mg / kg, about 11 mg / kg, about 11.5 mg / kg, about 12 mg / kg, 12.5 mg / kg, about 13 mg / kg, about 13.5 mg / kg, about 14 mg / kg, about 14.5 mg / kg, or about 15 mg / kg.

[0221] In some aspects, the expression level of PCSK9 and / or the activity level of PCSK9 after administration of a compound of the present disclosure is less than about 2%, less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, less than about 75%, or less than about 80% of the expression level of PCSK9 and / or the activity level of PCSK9 before administration of a compound of the present disclosure.

[0222] In some aspects, the PCSK9 expression level and / or PCSK9 activity level after administration of a compound of the present disclosure is about 2% to about 5%, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, or about 75% to about 80% of the PCSK9 expression level and / or PCSK9 activity level before administration of a compound of the present disclosure.

[0223] Modulation of expression levels can be determined by measuring protein levels, for example, by SDS-PAGE followed by Western blotting using a suitable antibody against the target protein. Alternatively, modulation of expression levels can be determined by measuring mRNA levels, for example, by Northern blotting or quantitative RT-PCR. When measuring mRNA levels, the level of downregulation, when using an appropriate formulation at a concentration of 0.04 nM to 25 nM, such as 0.8 to 20 nM, is typically 10 to 20% of the normal level in the absence of a compound of the present disclosure in some aspects.

[0224] Accordingly, the present disclosure provides an in vitro or in vivo method of downregulating or inhibiting expression of PCSK9 protein and / or mRNA in a cell expressing PCSK9 protein and / or mRNA, the method comprising administering to the cell an oligomer or conjugate according to the present disclosure, e.g., a pharmaceutical composition of the present disclosure (e.g., an oral formulation (e.g., an oral formulation comprising CIVI008 and an oral delivery agent (such as SNAC, C10, or 5-CNAC, derivatives thereof, pharmaceutically acceptable hydrates, solvates, or salts thereof; or any combination thereof)), for the purpose of downregulating or inhibiting expression of PCSK9 protein and / or mRNA in the cell. Preferably, the cell is a mammalian cell, such as a human cell.

[0225] Section 2c: Chain length The oligomers of the present disclosure can comprise or consist of a contiguous nucleotide sequence having a total length of 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides. In some aspects, the oligomer is longer than 22 contiguous nucleotides. The chain length includes region A, or region A and region B. In some aspects, the oligomers of the present disclosure can be multimers comprising, for example, two, three, four, five, six, or more of the concatenated ASOs disclosed herein, although it should be understood that the concatenated ASOs can also be linked by spacers or linkers comprising nucleotide or non-nucleotide units, optionally positioned between each ASO in the multimer. Thus, in some aspects, an oligomer of the present disclosure can comprise or consist of a sequence having an overall contiguous length of 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 95, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, or at least about 200 nucleotides.

[0226] In some aspects, the oligomer as a whole comprises or consists of a sequence of 16 to 22 contiguous nucleotides in length (e.g., a contiguous length of 16, 17, 18, 18, 20, 21, or 22 nucleotides). In some specific aspects, the oligomer of region A comprises or consists of a sequence of 16 contiguous nucleotides.

[0227] In some aspects, oligomers according to the present disclosure consist of a contiguous length of 16, 17, 18, 19, 20, 21, or more than 22 nucleotides. In some aspects, oligomers according to the present disclosure comprise a contiguous length of fewer than 22, fewer than 21, fewer than 20, fewer than 19, fewer than 18, or fewer than 17 nucleotides. In some aspects, oligomers according to the present disclosure consist of a contiguous length of 16 or 17, 16-18, 16-19, 16-20, 16-21, or 16-22 nucleotides.

[0228] Section 2d: Nucleotide analogues In some aspects, the oligomers of the present disclosure include non-natural nucleotide analogs, e.g., nucleotides with modified sugar moieties, such as bicyclic nucleotides or 2'-modified nucleotides (e.g., 2'-substituted nucleotides). Substitution of natural nucleotides with non-natural analogs can confer desirable properties or characteristics to the oligomer, such as increased resistance to degradation or stability. In some aspects, the nucleotide analogs exert a functional effect on the expression of the oligomer, for example, by increasing binding affinity to the target (affinity enhancement), and / or increasing resistance to intracellular nucleases, and / or increasing transportability into cells. Specific examples of nucleoside analogs are described, for example, in Freier & Altmann (1997) Nucl. Acid Res. 25:4429-4443 and Uhlmann (2000) Curr. Opinion in Drug Development 3:293-213, and are also shown in Scheme 1 below. [ka]

[0229] The present disclosure provides oligomers comprising or consisting of simple sequences of natural nucleotides, preferably 2'-deoxynucleotides (generally referred to herein as "DNA"), but which may also be ribonucleotides (generally referred to herein as "RNA"), or a combination of such natural nucleotides and one or more non-natural nucleotides, i.e., nucleotide analogs. Such nucleotide analogs may advantageously enhance the affinity of the oligomer for the target sequence. Examples of suitable nucleotide analogs are described in WO2007 / 031091; the entire contents of this reference are incorporated herein by reference.

[0230] The inclusion of affinity-enhancing nucleotide analogs (such as LNA or 2'-substituted sugars) in the oligomer allows the size of the specifically binding oligomer to be reduced, and also lowers the upper limit on the size of the oligomer before non-specific or aberrant binding occurs. In some aspects, the oligomers of the present disclosure contain at least one nucleotide analog. In some aspects, the oligomers of the present disclosure contain at least two nucleotide analogs. In some aspects, the oligomers of the present disclosure contain 3, 4, 5, 6, 7, or 8 nucleotide analogs, for example, 6 or 7 nucleotide analogs. In some aspects, all nucleotide analogs are identical. In some aspects, some nucleotide analogs are different. In some aspects, all nucleotides in the oligomer are nucleotide analogs. In some aspects, when all nucleotides in the oligomer are nucleotide analogs, all nucleotide analogs are identical. In some aspects, when all nucleotides in the oligomer are nucleotide analogs, some of the nucleotide analogs are different. In some aspects, oligomers of the present disclosure include nucleotide analogs; for example, analog A and analog B in a particular pattern, for example, ABABABABABABABABABABA.

[0231] Examples of nucleotide analogs include those modified in the sugar moiety to introduce a 2'-substituent or to form a bicyclic structure which may enhance binding affinity and also enhance nuclease resistance.

[0232] In some aspects, the nucleotide analogs present in the antisense oligomers of the present disclosure (such as regions X' and Y' referred to in the "Gapmer Design" section) are independently selected from, for example: 2'-O-alkyl-RNA units, 2'-OMe-RNA units, 2'-O-alkyl-DNA, 2'-amino-DNA units, 2'-fluoro-DNA units, LNA units, arabinonucleic acid (ANA) units, 2'-fluoro-ANA units, HNA units, INA (intercalating nucleic acid; Christensen (2002) Nucl. Acids. Res. 30:4918-4925) units, and 2'MOE units.

[0233] In some aspects, nucleotide analogue is 2'-O-methoxyethyl-RNA (2'MOE), 2'-fluoro-DNA monomer or LNA nucleotide analogue, and therefore, the antisense oligonucleotide of the present disclosure can comprise nucleotide analogues independently selected from these three types of analogues, or can comprise only one type of analogue selected from these three types.In some aspects, at least one of the nucleotide analogues is 2'-MOE-RNA (2, 3, 4, 5, 6, 7, 8, 9 or 10 units of 2'-MOE-RNA nucleotide unit).In some aspects, at least one of the nucleotide analogues is 2'-fluoro-DNA (2, 3, 4, 5, 6, 7, 8, 9 or 10 units of 2'-fluoro-DNA nucleotide unit).

[0234] In some aspects, the oligomer can be a double-stranded RNA comprising a sense strand and an antisense strand, wherein the sense strand follows the pattern: 3'ABABABABAAABABBBABA5', while the antisense strand follows the pattern: 5'BABABABABABBBABAAABABAB3', where A is a 2'-fluoro analog and B is a 2-OMe analog, and wherein the antisense strand has a two-base overhang compared to the sense strand.In some aspects, the 5' end of the sense strand is linked (conjugated) to a GalNAc moiety.In some aspects, the last two bonds at the 5' end of the antisense strand are PS.

[0235] In some aspects, the oligomer can be a double-stranded RNA comprising a sense strand and an antisense strand, wherein the sense strand follows the pattern: 3'BBBBBBABAAABBBBBBBBBB5', and the antisense strand follows the pattern: 5'BBBBBBBABABBBBABBABBBAB3', where A is a 2'-fluoro analog, while B is a 2-OMe analog, and wherein the antisense strand has a 2-base overhang compared to the sense strand.In some aspects, the 5' end of the sense strand is linked (conjugated) to a GalNAc moiety.In some aspects, the last two bonds at the 5' end and 3' end of the antisense strand are PS, and the last two bonds at the 3' end of the sense strand are PS.

[0236] In some aspects, the oligomer may be a double-stranded RNA comprising a sense strand and an antisense strand, wherein the sense strand comprises at its 5'-end one or more terminal internucleotide linkages that are chirality-modified; the antisense strand comprises at its 5'-end one or more terminal internucleotide linkages that are chirality-modified, and at its 3'-end one or more terminal internucleotide linkages that are chirality-modified.

[0237] In some aspects, the oligomer can be a double-stranded RNA comprising a sense strand and an antisense strand, wherein the sense strand sequence has 4 to 12 asymmetric 2'-O-alkyl modifications, at least four of which are present as the four terminal nucleotides at the 5' end, and the antisense sequence has at least four asymmetric phosphorothioate modifications.

[0238] In some aspects, the oligomer can be a double-stranded RNA comprising a first and a second sequence having a first monomer within the first sequence and a second monomer within the second sequence within the first three, four, five, or six positions of either the 3' or 5' end, wherein the first and second monomers are selected such that: (i) the first and second monomers are natural ribonucleotides or modified ribonucleotides having natural bases, and form a non-canonical Watson-Crick pair when the ribonucleotide of the first monomer and the ribonucleotide of the second monomer occupy complementary positions that are not paired and therefore do not have a substantial level of hydrogen bonding; and (ii) the stability of the pairing of the monomers contributing to duplex formation between the first and second sequences is different from the stability of the pairing between the first sequence and the target sequence or between the second sequence and the target sequence, wherein the double-stranded RNA comprises a TT overhang sequence.

[0239] In some aspects, the oligomer can be a double-stranded RNA comprising: a sense strand sequence having 4 to 12 asymmetric 2'-O-alkyl modifications, at least 4 of which are present within the 6 terminal nucleotides at the 5' end; and An antisense sequence having at least four asymmetric phosphorothioate modifications.

[0240] In some aspects, the oligomer can be a double-stranded RNA comprising: a sense strand sequence having 4 to 12 asymmetric 2'-O-alkyl modifications, at least 4 of which are present within the 6 terminal nucleotides at the 5' end; and an antisense sequence having at least four asymmetric phosphorothioate modifications; wherein the double-stranded RNA comprises a multivalent galactose or multivalent N-acetyl-galactosamine; and wherein the antisense sequence targets a PCSK9 target sequence.

[0241] In some aspects, the oligomer can be a double-stranded RNA for inhibiting expression of PCSK9 in a cell; wherein the oligomer comprises a sense sequence and an antisense sequence; wherein the sense sequence has one or more asymmetric 2'-O alkyl modifications and the antisense sequence has 4 to 20 phosphorothioate modifications; wherein the double-stranded RNA comprises multivalent galactose or multivalent N-acetyl-galactosamine; and wherein the antisense sequence targets a human PCSK9 sequence.

[0242] In some aspects, the oligomer can be a double-stranded RNA for inhibiting expression of PCSK9 in a cell, wherein the oligomer comprises a sense sequence and an antisense sequence; wherein the sense sequence has one or more asymmetric 2'-O alkyl modifications and the antisense sequence has 4 to 20 phosphorothioate modifications; wherein said antisense sequence comprises a fewer number of asymmetric 2'-O alkyl modifications than said sense sequence; wherein the sense sequence comprises a conjugate group; and wherein the antisense sequence targets a PCSK9 sequence.

[0243] In some aspects, the oligomer may be a double-stranded RNA with reduced off-target RNA inhibitory activity, wherein the oligomer comprises the following (a) and (b): (a) the antisense strand, which is complementary to the target gene; and (b) a sense strand that is complementary to the antisense strand and that contains at least one modified nucleotide in the region corresponding to the target cleavage site; wherein the modified nucleotide is abasic; wherein the reduced off-target RNA inhibitory activity is relative to the corresponding unmodified double-stranded RNA drug.

[0244] In some aspects, the oligomer may be a double-stranded RNA capable of inhibiting PCSk9 expression in vivo; wherein the oligomer comprises (a) and (b): (a) Sense strand; wherein the sense strand comprises the following (i) and (ii): (i) an alternating motif of nucleotides with at least two different chemical modifications; and (ii) one or more carbohydrate ligands; and (b) antisense strand; wherein the antisense strand comprises (i): (i) an alternating motif of at least two different chemically modified nucleotides; wherein said alternating motif is present within said double-stranded region, and said composition optionally further comprises one or more overhanging and / or capping groups.

[0245] In some aspects, the oligomer can be a double-stranded RNA capable of inhibiting PCSK9 expression; wherein the oligomer comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides; wherein the duplex is represented by the following chemical formula: Sense: 5' n p -N a -(XXX) i -N b -YYY -N b -(ZZZ)j -N a -n q 3' Antisense: 3' n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III) wherein i, j, k, and l are each independently 0 or 1 when at least one of i, j, k, and l is 1; p and q each independently represent 0 to 6; Na and Na' each independently represent an oligonucleotide sequence containing 2 to 20 nucleotides that are either modified, unmodified, or a combination thereof, each sequence containing nucleotides with at least two different types of modification; Nb and Nb' each independently represent an oligonucleotide sequence containing 1 to 10 modified nucleotides; n p , n p ',n q and nq' each independently represent an overhanging nucleotide sequence containing 0 to 6 nucleotides; and XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif of three identical modifications on three consecutive nucleotides; and wherein the modification on Nb is different from the modification on Y, and the modification on Nb' is different from the modification on Y'.

[0246] In some aspects, the oligomer can be a double-stranded RNA capable of inhibiting expression of PCSK9, wherein the oligomer comprises a sense strand and an antisense strand, each comprising a strand having 14 to 40 nucleotides, and wherein the dsRNA agent has the following chemical formula: [ka] wherein B1, B2, B3, B1', B2', B3', and B4' each independently represent a nucleotide containing a modification selected from the group consisting of 2'-O alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halo, ENA, and BNA / LNA; C1 is a heat-labile nucleotide selected from the group consisting of i), ii), and iii) below: i) a nucleotide that forms a mismatch pair with the opposing nucleotide in the antisense strand; ii) nucleotides with abasic modifications; and iii) nucleotides with sugar modifications; wherein C1 is positioned opposite the seed region (positions 2 to 8) of the antisense strand; T1, T1', T2', and T3' each independently represent a nucleotide containing a modification that confers on said nucleotide less steric bulk than the steric bulk of a 2'-OMe modification; wherein the modification is at the 2' position of the ribose sugar of the nucleotide, or at a position of a non-ribose nucleotide analogous to the 2' position of the ribose sugar; and where T1' and T3' are 11 nucleotides apart in length; n 1 , n 3 , and q 1 are each independently 4 to 15 nucleotides in length; n 5 , q 3 , and q 7 are each independently 1 to 6 nucleotides in length; n 2 , n 4 , and q 6 are each independently 1 to 3 nucleotides in length; q 5 are independently 0 to 10 nucleotides in length; and n 2 , n 4 , and q 4 are each independently 0 to 3 nucleotides in length; and wherein the dsRNA agent has two blunt ends at either end of the dsRNA duplex.

[0247] In some aspects, the oligomer can be a double-stranded RNA capable of inhibiting the expression of PCSK9; wherein the oligomer comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides; wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference; wherein the antisense strand comprises at least one thermodestabilizing modification in the duplex located within the first 9 nucleotides of the 5' region or precursor thereof; wherein the sense strand comprises an ASGPR ligand.

[0248] In some aspects, the oligomer comprises a modification motif (e.g., a distribution pattern of nucleotide analogs in sense and antisense sequences, internucleoside linkages, conjugate moieties, etc.) disclosed in U.S. Pat. Nos. 8,110,674, 8,420,799, 8,809,516, 9,222,091, 9,708,615, 10,273,477, 9,290,760, 10,233,448, or 9,796,974; U.S. Patent Publication No. 2018-0258427 A1; or International Patent Publication WO2018098328 A1; all of which references are incorporated herein by reference in their entirety.

[0249] In some aspects, the nucleotide analogue is LNA, such as oxy-LNA (such as beta-D-oxy-LNA and alpha-L-oxy-LNA), and / or amino-LNA (such as beta-D-amino-LNA and alpha-L-amino-LNA), and / or thio-LNA (such as beta-D-thio-LNA and alpha-L-thio-LNA), and / or ENA (such as beta-D-ENA and alpha-L-ENA). In some aspects, the LNA is beta-D-oxy-LNA. In some aspects, only one type of nucleotide analogue is present; for example, LNA present in the antisense oligonucleotide of the present disclosure, or in its continuous nucleotide sequence.

[0250] The antisense oligonucleotide of the present disclosure comprises at least one type of locked nucleic acid (LNA) unit, such as 1, 2, 3, 4, 5, 6, 7, or 8 LNA units (such as 3 to 7, or 4 to 8 LNA units).In some aspects, at least one of the nucleotide analogues is locked nucleic acid (LNA); for example, at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8 of the nucleotide analogues can be LNA.In some aspects, all of the nucleotide analogues can be LNA.

[0251] In some aspects, the antisense oligonucleotide of the present disclosure may contain both nucleotide analogs (e.g., LNA) and DNA units. In some aspects, the total number of nucleotide analogs (preferably LNA) and DNA units combined is 5 to 22, for example, 16 to 18, or 16 to 20, or 16 to 22. In some aspects, the total number of nucleotide analogs (e.g., LNA) and DNA units combined is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.

[0252] In some aspects, the nucleotide sequence (such as the consecutive nucleotide sequence) of the antisense oligonucleotide of the present disclosure is composed of at least one kind of nucleotide analogue (LNA), and the remaining nucleotide units are DNA units.In some aspects, the antisense oligonucleotide of the present disclosure only comprises LNA nucleotide analogues and natural nucleotides (such as RNA or DNA, most preferably DNA nucleotides), and optionally has modified internucleoside linkages such as phosphorothioate.

[0253] When reference is made to a particular nucleotide sequence motif or sequence consisting solely of nucleotides, it will be understood that an oligomer of the disclosure defined by that sequence may contain, in place of one or more nucleotides present in the sequence, a corresponding nucleotide analogue (such as an LNA unit or other nucleotide analogue) that enhances duplex stability / increases the Tm of the oligomer / target duplex (i.e., an affinity-enhancing nucleotide analogue).

[0254] Tm assay The oligonucleotide and RNA target (PO) duplexes were diluted to 3 mM in 500 ml of RNase-free water and mixed with 500 ml of 2x Tm-buffer (200 mM NaCl, 0.2 mM EDTA, 20 mM sodium phosphate, pH 7.0). The solution was heated to 95°C for 3 minutes and then annealed at room temperature for 30 minutes. The duplex melting temperature (Tm) was measured using PE Templab software on a Lambda40 UV / VIS spectrophotometer equipped with a Peltier temperature programmable device PTP6 (Perkin Elmer). The temperature was increased from approximately 20°C to approximately 95°C, then decreased to approximately 25°C, and the absorbance at 260 nm was recorded. The first derivative and the local maxima of both melting and annealing were used to estimate the Tm of the duplex.

[0255] In some aspects, any mismatches between the nucleotide sequence of the oligomer and the target sequence are found in regions outside the affinity-enhancing nucleotide analogs, referred to as region Y' in the "Gapmer Design" section, and / or at unmodified positions such as DNA nucleotides in the oligonucleotide, and / or in regions 5' or 3' to the consecutive nucleotide sequence.

[0256] Section 2e:LNA In some aspects, an oligomer (e.g., an ASO) of the present disclosure comprises at least one LNA unit. The terms "locked nucleic acid" and "LNA" refer to any bicyclic nucleoside analog containing a bridge between the ribose ring, i.e., the 2' and 4' positions of a 2' to 4 bicyclic nucleotide analog. LNA is sometimes referred to in the literature as BNA (bridged nucleic acid or bicyclic nucleic acid); the two terms may be used synonymously. The term LNA generally refers to an LNA monomer. The term "LNA oligonucleotide" refers to an oligonucleotide (e.g., an ASO, such as a gapmer) containing one or more such bicyclic nucleotide analogs. In some aspects, the bicyclic nucleoside analog is an LNA nucleotide, and therefore these terms may be used synonymously. In such aspects, both are characterized by the presence of a linker group (e.g., a bridge) between the C2' and C4' positions of the ribose sugar ring.

[0257] In some aspects, the antisense oligonucleotides of the present disclosure may comprise beta-D-oxy-LNA, and may contain one or more of the following LNA units in either the beta-D or alpha-L configuration or a combination thereof: thio-LNA, amino-LNA, oxy-LNA, 5'-methyl-LNA and / or ENA.

[0258] In some aspects, all LNA cytosine units are 5'-methyl-cytosine. In some aspects, at least one nucleoside analog present in the first region (X') is a bicyclic nucleoside analog. In some aspects, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight nucleoside analogs present in the first region (X') are bicyclic nucleoside analogs. Thus, in some aspects, all nucleosides in the oligonucleotides of the present disclosure, except for the DNA and / or RNA nucleosides of region Y', are sugar-modified nucleoside analogs, for example, bicyclic nucleoside analogs such as LNA (e.g., beta-DX-LNA or alpha-LX-LNA, where X is oxy, amino, or thio), other LNAs disclosed herein (including, but not limited to, (R / S)cET, cMOE, or 5'-Me-LNA, or any combination thereof).

[0259] In some aspects, LNAs used in the oligonucleotides of the present disclosure have the structure of the following general formula II: [ka] Here, Y is -O-, -CHO-, -S-, -NH-, N(R e ) and / or -CH2-; Z and Z * is independently selected from an internucleoside linkage, R, a terminal group, or a protecting group; B constitutes a natural or unnatural nucleotide base moiety (nucleobase), and R is selected from hydrogen and C1-4-alkyl; R a , R b , R c , R d , and R e are optionally and independently: Hydrogen, optionally substituted C1-12-alkyl, optionally substituted C2-12-alkenyl, optionally substituted C2-12-alkynyl, hydroxy, C1-12-alkoxy, C2-12-alkoxyalkyl, C2-12-alkenyloxy, carboxy, C1-12-alkoxycarbonyl, C1-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di( selected from the group consisting of mono- and di(C1-6-alkyl)amino, carbamoyl, mono- and di(C1-6-alkyl)amino-carbonyl, amino-C1-6-alkyl-aminocarbonyl, mono- and di(C1-6-alkyl)amino-C1-6-alkyl-aminocarbonyl, C1-6-alkyl-carbonylamino, carbamido, C1-6-alkanoyloxy, sulfono, C1-6-alkylsulfonyloxy, nitro, azido, sulfanyl, C1-6-alkylthio, halogen, DNA intercalator, photochemically active group, thermochemically active group, chelating group, reporter group, and ligand; wherein aryl and heteroaryl are optionally substituted, and wherein two geminal substituents R a and R b both refer to optionally substituted methylene (=CH2); and RH is selected from hydrogen and C1-4-alkyl.

[0260] In some situations, R a , R b , R c , R d , and R e are optionally independently selected from the group consisting of hydrogen and C1-6 alkyl (e.g., methyl). For all chiral centers, the asymmetric groups can be in either the R or S configuration, and for example, two exemplary stereochemical isomers include the beta-D and alpha-L isoforms, which are illustrated as follows: [ka]

[0261] A specific example of an LNA unit is shown below: [ka]

[0262] The term "thio-LNA" includes locked nucleotides in which Y in the general formula above is selected from S or -CH2-S-. Thio-LNA can be in both the beta-D and alpha-L configurations.

[0263] The term "amino-LNA" includes locked nucleotides in which Y in the above general formula is selected from -N(H)-, N(R)-, CH2-N(H)-, and -CH2-N(R)-, where R is selected from hydrogen and C1-4-alkyl. Amino-LNA can be in both the beta-D and alpha-L configurations.

[0264] The term "oxy-LNA" includes locked nucleotides in which Y in the general formula above represents -O. Oxy-LNA can be in both the beta-D and alpha-L configurations.

[0265] The term "ENA" includes locked nucleotides in the general formula above where Y is -CH2-O- (where the oxygen atom of -CH2-O- is linked to the 2' position of the base B). e is hydrogen or methyl.

[0266] In some exemplary aspects, the LNA is selected from beta-D-oxy-LNA, alpha-L-oxy-LNA, beta-D-amino-LNA and beta-D-thio-LNA, in particular beta-D-oxy-LNA.

[0267] As used herein, "bicyclic nucleoside" refers to a modified nucleoside containing a bicyclic sugar moiety. Examples of bicyclic nucleosides include, but are not limited to, nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In some aspects, the compounds provided herein contain one or more bicyclic nucleosides, wherein the bridge comprises a 4' to 2' bicyclic nucleoside. Such 4' to 2' bicyclic nucleosides include, but are not limited to, one of the following formulas: 4'-(CH2)-O-2'(LNA);4'-(CH2)-S-2';4'-(CH2)2-O-2'(ENA);4'-CH(CH3)-O-2' and 4'-CH(CH2OCH3)-O-2 * and analogs thereof (see U.S. Pat. No. 7,399,845, the entire contents of which are incorporated herein by reference); 4'-C(CH3)(CH3)-O-2', and analogs thereof (see International Patent Publication WO2009 / 006478, the entire contents of which are incorporated herein by reference); 4'-CH2-N(OCH3)-2', and analogs thereof (see International Patent Publication WO2008 / 150729, the entire contents of which are incorporated herein by reference); 4'-CH2-ON(CH3)-2' (see U.S. Patent Application No. 2004 / 0171570, the entire contents of which are incorporated herein by reference); 4'-CH2-N(R)-O-2', where R is H, C1-C10 alkyl, or a protecting group (see U.S. Pat. No. 7,427,672, the entire contents of which are incorporated herein by reference); 4'-CH2-C(H), see J. Org. Chem. 2009, 74:118-134, the contents of which are incorporated herein by reference in their entirety; and 4'-CH2-C(=CH2)-2', and analogs thereof (see International Patent Publication WO2008 / 154401, the entire contents of which are incorporated herein by reference). See also, for example: Singh et al., Chem. Commun., 1998, 4:455-456; Koshkin et al., Tetrahedron, 1998, 54:3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97:5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8:2219-2222; Singh et al., J. Org. Chem., 1998, 63:10035-10039; Srivastava et al., J. Am. Chem. Soc., 2007, 129:8362-8379; Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2:558-561; Braasch et al., Chem. Biol. 2001, 8:1-7; Oram et al., Curr. Opinion Mol. Ther., 2001, 3:239-243; U.S. Patent Nos. 6,670,461, 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, and 7,399,845; International Patent Publications WO2004 / 106356, WO94 / 14226, WO2005 / 021570, and WO2007 / 134181; U.S. Patent Application Nos. 2004 / 0171570, 2007 / 0287831, and 2008 / 003961 No. 8; and U.S. Patent Application Nos. 12 / 129,154, 60 / 989,574, 61 / 026,995, 61 / 026,998, 61 / 056,564, 61 / 086,231, 61 / 097,787, and 61 / 099,844; and PCT International Patent Application Nos. PCT / US2008 / 064591, PCT / US2008 / 066154, and PCT / US2008 / 068922; all of which are incorporated herein by reference in their entireties.

[0268] Each of the bicyclic nucleosides described above can be prepared with one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see, e.g., International Patent Application WO 99 / 14226). In some aspects, the bicyclic sugar moiety of an LNA nucleoside includes, but is not limited to, compounds having at least one bridge between the 4' and 2' positions of the pentofuranosyl sugar moiety; wherein such bridges are independently selected from the group consisting of -[CiR a XR b )]-, -C(R a )=C(R b )-, -C(R a )=N-, -C(=NR a )-, -C(=O)-, -C(=S)-, -O-, -Si(R a )2-, -S(=O)x-, and -N(R a )-; where x is 0, 1, or 2; n is 1, 2, 3, or 4; R a and R b each independently represents H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, a heterocyclic radical, a substituted heterocyclic radical, heteroaryl, a substituted heteroaryl, a C5-C7 alicyclic radical, a substituted C5-C7 alicyclic radical, a halogen, or OJ J1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1), where J1 and J2 are each independently H, C1-C6 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or a protecting group.

[0269] In some aspects, the bridge of the bicyclic sugar moiety is —[C(R a )(R b )]n-, -[C(R a )(R b )]nO-, -C(R a R b )-N(R)-O- or -C(R a R b )-ON(R)-. In some aspects, the bridge is 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2', 4 * -(CH2)2-O-2', 4'-CH2-ON(R)-2', and 4'-CH2-N(R)-O-2'-, where each R is independently H, a protecting group, or a C1-C12 alkyl.

[0270] In some aspects, bicyclic nucleosides are further defined by their isomeric configuration. For example, nucleosides containing a 4'-2' methylene-oxy bridge can be in the α-L or β-D configuration. α-L-methyleneoxy (4'-CH2-O-2') BNAs have previously been incorporated into antisense oligonucleotides and demonstrated antisense activity. See Frieden et al. (2003) Nucleic Acids Research 21:6365-6372.

[0271] In some aspects, bicyclic nucleosides include, but are not limited to, the following: (A) α-L-methyleneoxy(4'-CH2-O-2')BNA, (B) β-D-methyleneoxy(4'-CH2-O-2')BNA, (C) Ethyleneoxy(4'-(CH2)2-O-2')BNA, (D) aminooxy(4'-CH2-ON(R)-2')BNA, (E) oxyamino(4'-CH2-N(R)-O-2')BNA, (F) methyl(methyleneoxy)(4'-CH(CH3)-O-2')BNA, (G) methylene-thio(4'-CH2-S-2')BNA, (H) methylene-amino(4'-CH2-N(R)-2')BNA, (I) methyl carbocyclic (4'-CH2-CH(CH3)-2') BNA, and (J) Propylene carbocyclic (4'-(CH2)3-2') BNA as shown below: [ka] where Bx is the base moiety and R is independently H, a protecting group, or C1-C2 alkyl. [ka]

[0272] In some aspects, the bicyclic nucleoside is defined by the following formula: [ka] where: Bx is a heterocyclic base moiety; -Qa-Qb-Qc is -CH2-N(Rc)-CH2-, -C(=O)-N(Rc)-CH2-, -CH2-ON(Rc)-, -CH2-N(Rc)-O-, or -N(Rc)-O-CH2; Rc is a C1-C12 alkyl or amino protecting group; and Ta and Tb are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent linkage to a support medium.

[0273] In some aspects, the bicyclic nucleoside is defined by the following formula: [ka] where: Bx is a heterocyclic base moiety; Ta and Tb are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent linkage to a support medium; and Za is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, substituted amido, thiol, or substituted thio.

[0274] In some aspects, each substituted group is independently mono- or poly-substituted with substituents independently selected from halogen, oxo, hydroxyl, OJc, NJd, SJc, N3, OC(=X)Jc, and NJeC(=X)NJcJd; wherein Jc, Jd, and Je are each independently H, C1-C6 alkyl, or substituted C1-C6 alkyl; and X is O or NJc.

[0275] In some aspects, the bicyclic nucleoside is defined by the following formula: [ka] where: Bx is a heterocyclic base moiety; Ta and Tb are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent linkage to a support medium; and Rd is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, or substituted acyl (C(=O)-).

[0276] In some aspects, the bicyclic nucleoside is defined by the following formula: [ka] where: Bx is a heterocyclic base moiety; Ta and Tb are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent linkage to a support medium; Rd is C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl; qb, qc, and qd are each independently H, halogen, C-C alkyl, substituted C1-C alkyl, C2-Ce alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl, C1-C6 alkoxyl, substituted C1-C6 alkoxyl, acyl, substituted acyl, C1-C6 aminoalkyl, or substituted C1-C6 aminoalkyl.

[0277] In some aspects, the bicyclic nucleoside is defined by the following formula: [ka] where: Bx is a heterocyclic base moiety; Ta and Tb are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent linkage to a support medium; qa, qb, qc, and qf are each independently hydrogen, halogen, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C1-C12 alkoxy, substituted C1-C12 alkoxy, OJj, SJj, SOJj, SO2Jj, NJjJk, N3, CN, C(=O)OJj, C(=O)NJj Jk, C(=O)Jj, OC(=O)NJj Jk, N(H)C(=NH)NJj Jk, N(H)C(=O)NJj Jk, or N(H)C(=S)NJjJk; or, qe and qf are both = C(qg)(qh), where qg and qh are each independently H, halogen, C1-C12 alkyl, or substituted C1-C12 alkyl.

[0278] In some aspects, the bicyclic nucleoside is defined by the formula: [ka] where: Bx is a heterocyclic base moiety; Ta and Tb are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent linkage to a support medium; qj, qj, qk and ql are each independently H, halogen, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C1-C12 alkoxyl, substituted C2-C12 alkoxyl, OJj, SJj, SOJj, SO2Jj, NJjJk, N3, CN, C(=O)OJj, C(=O)NJjJk, C(=O)Jj, OC(=O)NJjJk, N(H)C(=NH)NJjJk, N(H)C(=O)NJjJk, or (H)C(=S)NJjJk; and qi and qj or ql and qk together = C(qg)(qh); where qg and qh are each independently H, halogen, C1-C12 alkyl, or substituted C1-C6 alkyl.

[0279] Carbocyclic bicyclic nucleosides having a 4'-(CH2)3-2' bridge and alkenyl analogs having a 4'-CH=CH-CH2-2' bridge are described in the following references: Freier et al. (1997) Nucleic Acids Research 25:4429-4443; and Albaek et al. (2006) J. Org. Chem. 71:7731-7740.

[0280] Herein, a "4'-2' bicyclic nucleoside" or "4' to 2' bicyclic nucleoside" refers to a bicyclic nucleoside containing a furanosyl ring containing a bridge connecting the 2' and 4' carbon atoms. Herein, a "monocyclic nucleoside" refers to a nucleoside containing a modified sugar moiety that is not a bicyclic sugar moiety. In some aspects, the sugar moiety of a nucleoside, or sugar moiety analog, can be modified or substituted at any position. Herein, a "2'-modified sugar" refers to a furanosyl sugar modified at the 2' position. In some aspects, such modifications include a substituent selected from halides, including, but not limited to, substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted aminoalkyl, substituted and unsubstituted alkyl, substituted and unsubstituted aryl, and substituted and unsubstituted alkynyl. In some aspects, the 2' modifications are selected from the following substituents, including, but not limited to: O[(CH2)nO]mCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, OCH2C(=O)N(H)CH3, and O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10.

[0281] Other 2'-substituents may also be selected from the following: C1-C12 alkyl; substituted alkyl; alkenyl; alkynyl; alkaryl; aralkyl; O-alkaryl or O-aralkyl; SH; SCH3; OCN; Cl; Br; CN; CF3; OCF3; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; R; cleavage groups; reporter groups; intercalators; groups that improve the pharmacokinetic properties; and groups that improve the pharmacokinetic properties of antisense compounds, and other substituents with similar properties.

[0282] In some aspects, modified nucleosides comprise 2'-MOE side chains.It is described that such 2'-MOE substitutions improve binding affinity compared to unmodified nucleosides and compared to other modified nucleosides such as 2'-O-methyl, O-propyl, and O-aminopropyl.Oligonucleotides with 2'-MOE substitutions are potent antisense inhibitors of gene expression in vivo.

[0283] As used herein, "modified tetrahydropyran nucleoside" or "modified THP nucleoside" refers to a nucleoside having a six-membered tetrahydropyran "sugar" (sugar surrogate) replacing the pentofuranosyl residue of a normal nucleoside. Modified THP nucleosides include, but are not limited to, those referred to in the art as hexitol nucleic acid (HNA), altitol nucleic acid (ANA), mannitol nucleic acid (MNA), fluoroHNA (F-HNA), or compounds defined by the following chemical formula: [ka] where: Bx is a heterocyclic base moiety; T3 and T4 are each independently an internucleoside linking group linking a tetrahydropyran nucleoside analog to the antisense compound, or either T3 and T4 is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T3 and T4 is H, a hydroxyl protecting group, a linking conjugate group, or a 5'- or 3'-terminal group; q1, q2, q3, q4, q5, q6, and q7 are each independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl; and one of R1 and R2 is hydrogen and the other is selected from halogen, substituted or unsubstituted alkoxy, NJ, J2, SJ, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, and CN, where X is O, S, or NJ1, and each of J1, J2, and J3 is independently H or C1-C6 alkyl. In some aspects, modified THP nucleosides of the above formula are provided, wherein qm, qn, qp, qr, qs, qt, and qu are each H. In some aspects, at least one of qm, qn, qp, qr, qs, qt, and qu is other than H. In some aspects, at least one of qm, qn, qp, qr, qs, qt, and qu is methyl. In some aspects, THP nucleosides of the above formula are provided, wherein one of R1 and R2 is F. In some aspects, R1 is fluoro and R2 is H; R1 is methoxy and R2 is H; or R1 is methoxyethoxy and R2 is H.

[0284] As used herein, "2'-modified" or "2'-substituted" refers to a nucleoside containing a sugar containing a substituent other than H and OH at the 2' position. 2'-Modified nucleosides include, but are not limited to, nucleosides with non-bridging 2' substituents such as allyl, amino, azido, thio, O-allyl, O-C1-C10 alkyl, -OCF3, O-(CH2)2-O-CH3, 2'-O(CH2)2SCH3, O-(CH2)2-ON(Rm)(Rn), or O-CH2-C(=O)-N(Rm)(Rn); where Rm and R are each independently H or substituted or unsubstituted C1-C10 alkyl. 2'-Modified nucleosides can further contain other modifications, for example, at other positions on the sugar and / or at the nucleobase.

[0285] In some aspects, the LNA unit has a structure selected from the following groups: [ka]

[0286] Inclusion of affinity-enhancing nucleotide analogs in the oligomer (such as LNA or 2'-substituted sugars) allows for the size of the specifically binding oligomer to be reduced, and also lowers the upper size limit for the oligomer before non-specific or aberrant binding occurs.

[0287] Section 2f: RNAse Recruitment Oligomeric compounds of the present disclosure can function through non-RNase-mediated degradation of target mRNA (such as by steric hindrance of translation or other methods). In some aspects, oligomers of the present disclosure are capable of recruiting endoribonucleases (RNases), such as RNase H. In some oligomers, the region A, or contiguous nucleotide sequence, consists of a region of at least 4 contiguous nucleotide units (residues), such as at least 5 contiguous, such as at least 6 contiguous, such as at least 7 contiguous nucleotide units, such as at least 8 or at least 9 contiguous nucleotide units (residues) (including 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleotides), capable of RNase recruitment when forming a duplex with a complementary target RNA (such as a DNA unit). When referring to a gapmer as described herein, the contiguous sequence capable of RNase recruitment may be region Y'. In some aspects, the contiguous sequence capable of RNase recruitment, such as region Y', may be larger in size, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotide units.

[0288] An oligomer is considered to be capable of RNase recruitment if, in the presence of the complementary RNA target with the oligomer, the initial rate, measured in pmol / l / min using, for example, the methodology provided in EP 1222309, is at least 1%, such as at least 5%, such as at least 10% or more than 20%, of the initial rate determined using a DNA-only oligonucleotide having the same base sequence but containing only DNA monomers (no 2' substitutions and containing phosphorothioate linkages between all monomers in the oligonucleotide).

[0289] In some aspects, an oligomer is considered to be essentially incapable of recruiting RNase H if, in the presence of the complementary RNA target and RNase H together with the oligomer, the initial rate of RNase H, measured in pmol / l / min using, for example, the methodology provided in EP 1222309, is less than 1%, such as less than 5%, such as less than 10%, or such as less than 20%, of the initial rate determined using an equivalent DNA of only the oligonucleotide (which contains no 2' substitutions and which contains phosphorothioate linkages between all nucleotides in the oligonucleotide).

[0290] In other aspects, an oligomer is considered capable of RNase H recruitment if, in the presence of the complementary RNA target and RNase H together with the oligomer, the initial rate of RNase H, measured in pmol / l / min using, for example, the methodology provided in EP 1222309, is at least 20%, such as at least 40%, such as at least 60%, such as at least 80%, of the initial rate determined using the equivalent DNA of only the oligonucleotide (which contains no 2' substitutions and which contains phosphorothioate linkages between all nucleotides in the oligonucleotide).

[0291] Typically, the region of the oligomer that forms consecutive nucleotide units capable of RNase recruitment when duplexed with the complementary target RNA consists of nucleotide units that form a DNA / RNA-like duplex with the RNA target. Oligomers of the present disclosure, such as the first region, can include a nucleotide sequence that includes both nucleotides and nucleotide analogs, and are in the form of an LNA gapmer. Section 2g: Gappar Design

[0292] In some aspects, the oligomer of the present disclosure is an LNA gapmer. A gapmer oligomer is an oligomer comprising a contiguous stretch of nucleotides (e.g., as a region of at least six or seven DNA nucleotides) capable of recruiting an RNAse (e.g., RNAse H), referred to herein as region Y' (Y'), where region Y' is flanked on both its 5' and 3' sides by regions of affinity-enhancing nucleotide analogs (e.g., one to six nucleotide analogs 5' and 3' to the contiguous stretch of nucleotides capable of recruiting an RNAse); these regions flanking Y' are referred to as regions X' (X') and Z' (Z'), respectively. The X' and Z' regions may also be referred to as the wings of the gapmer. Examples of gapmers are disclosed, for example, in WO2004 / 046160, WO2008 / 113832, and WO2007 / 146511.

[0293] In some aspects, the monomer capable of recruiting RNAse is selected from the group consisting of a DNA monomer, an alpha-L-LNA monomer, a C4' alkylated DNA monomer, and a UNA (unlinked nucleic acid) nucleotide. A UNA is typically an unlocked nucleic acid in which the C2-C3 C-C bond of the ribose has been removed to form an unlocked "sugar" residue. In some aspects, the LNA gapmer comprises a (poly)nucleotide sequence of the formula (5' to 3' direction) X'-Y'-Z', where region X' (X') (5' region) consists of, or comprises, at least one nucleotide analogue, such as at least one LNA unit, such as 1 to 6 nucleotide analogues, such as LNA units; and region Y' (Y') consists of, or comprises, at least four or at least five consecutive nucleotides (such as DNA nucleotides) that are capable of recruiting RNAse (when duplexed with a complementary RNA molecule (such as an mRNA target)).

[0294] In some aspects, region X' consists of 1, 2, 3, 4, 5 or 6 nucleotide analogues, such as LNA units, such as 2 to 5 nucleotide analogues, such as 2 to 5 LNA units, such as 3 or 4 nucleotide analogues, such as 3 or 4 LNA units; and / or region Z consists of 1, 2, 3, 4, 5 or 6 nucleotide analogues, such as LNA units, such as 2 to 5 nucleotide analogues, such as 2 to 5 LNA units, such as 3 or 4 nucleotide analogues, such as 3 or 4 LNA units.

[0295] In some aspects, Y' consists of or comprises 4, 5, 6, 7, 8, 9, 10, 11, or 12 consecutive nucleotides capable of recruiting RNAse, or 4 to 12, 6 to 10, or 7 to 9 consecutive nucleotides, such as 8 consecutive nucleotides, capable of recruiting RNAse. In some aspects, region Y' consists of or comprises at least one DNA nucleotide unit (such as 1 to 12 DNA units), preferably 4 to 12 DNA units, more preferably 6 to 10 DNA units, such as 7 to 10 DNA units, and most preferably 8, 9, or 10 DNA units. In some aspects, region X' consists of 3 or 4 nucleotide analogues (such as LNA); region X' consists of 7, 8, 9, or 10 DNA units; and region Z' consists of 3 or 4 nucleotide analogues (such as LNA). Such designs include (X'-Y'-Z')3-10-3, 3-10-4, 4-10-3, 3-9-3, 3-9-4, 4-9-3, 3-8-3, 3-8-4, 4-8-3, 3-7-3, 3-7-4, 4-7-3. In some aspects, the gapmer is a 3-9-4 gapmer. In some aspects, the gapmer is a 3-10-3 gapmer.

[0296] In some aspects, the oligomer (e.g., region X') consists of a contiguous nucleotide sequence of a total of 10, 11, 12, 13, or 14 nucleotide units, wherein the contiguous nucleotide sequence comprises or is represented by the formula (5'-3'), X'-Y'-Z'; where X' consists of 1, 2, or 3 nucleotide analog units (e.g., LNA units); Y' consists of 7, 8, or 9 contiguous nucleotide units that are capable of RNAse recruitment when duplexed with a complementary RNA molecule (e.g., an mRNA target); and Z' consists of 1, 2, or 3 nucleotide analog units (e.g., LNA units).

[0297] In some aspects, X' consists of 1 LNA unit. In some aspects, X' consists of 2 LNA units. In some aspects, X' consists of 3 LNA units. In some aspects, Z' consists of 1 LNA unit. In some aspects, Z' consists of 2 LNA units. In some aspects, Z' consists of 3 LNA units. In some aspects, Y' consists of 7 nucleotide units. In some aspects, Y' consists of 8 nucleotide units. In some aspects, Y' consists of 9 nucleotide units. In particular aspects, region Y' consists of 10 nucleoside monomers. In some aspects, region Y' consists of 1 to 10 DNA monomers, or the same inclusive. In some aspects, Y' comprises 1 to 9 DNA units (such as 2, 3, 4, 5, 6, 7, 8 or 9 DNA units). In some aspects, Y' consists of DNA units. In some aspects, Y' comprises at least one LNA unit in the α-L configuration (such as 2, 3, 4, 5, 6, 7, 8 or 9 LNA units in the α-L configuration), hi some aspects, Y' comprises at least one α-L-oxy LNA unit, or in which case all LNA units in the α-L configuration are α-L-oxy LNA units. In some aspects, the number of nucleotides in X'-Y'-Z' is selected from the group consisting of (nucleotide analog unit-region Y'-nucleotide analog unit): 1-8-1, 1-8-2, 2-8-1, 2-8-2, 3-8-3, 2-8-3, 3-8-2, 4-8-1, 4-8-2, 1-8-4, 2-8-4, 1-9-1, 1-9-2, 2-9-1, 2-9-2, 2-9-3, 3-9-2, 1-9-3, 3-9-1, 4-9-1, 1-9-4; or 1-10-1, 1-10-2, 2-10-1, 2-10-2, 1-10-3, 3-10-1, 2-10-3, or 3-10-2. In some aspects, the number of nucleotides in X'-Y'-Z' is selected from the group consisting of: 2-7-1, 1-7-2, 2-7-2, 3-7-3, 2-7-3, 3-7-2, 3-7-4, and 4-7-3.

[0298] In certain aspects, regions X' and Y' each consist of three LNA monomers and region Y' consists of eight, nine or ten nucleoside monomers, preferably DNA monomers. In some aspects, X' and Z' both consist of two LNA units each, or Y' consists of eight or nine nucleotide units, preferably DNA units. In some aspects, other gapmer designs include those in which regions X' and / or Z' are comprised of three, four, five, or six nucleoside analogs (e.g., monomers containing 2'-O-methoxyethyl-ribose sugars (2'-MOE) or monomers containing 2'-fluoro-deoxyribose sugars) and region Y' is comprised of eight, nine, ten, eleven, or twelve nucleosides (e.g., DNA monomers), where the region X'-Y'-Z' has 3-9-3, 3-10-3, 5-10-5, or 4-12-4 monomers. Additional gapmer designs are disclosed, for example, in WO2007 / 146511A2, the entire contents of which are incorporated herein by reference.

[0299] In some aspects, a gapmer oligomer of the present disclosure comprises the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 40. In some aspects, a gapmer oligomer of the present disclosure consists of the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 40.

[0300] Section 2h: Internucleoside linkages In some aspects, the nucleoside monomers of the oligonucleotides of the present disclosure are linked to one another via internucleoside linking groups. Each monomer is linked to its 3'-adjacent monomer via a linking group. Each nucleotide is also linked to its 3'-adjacent nucleotide via a linking group. For purposes of the present disclosure, the 5'-monomer located at the end of the oligomer does not contain a 5'-linking group, but the 5'-terminal group, or a linking group for conjugation, may or may not contain one.

[0301] The term "linking group" or "internucleoside linkage" is intended to mean a group capable of linking two nucleotides by covalent bonding. Specific examples include phosphate and phosphorothioate groups. Internucleoside linkage is used synonymously with internucleoside bond.

[0302] Suitable internucleoside linkages include, for example, those described in WO2007 / 031091; the entire contents of this reference are incorporated herein by reference. In some aspects, other than the phosphodiester linkage in region B (if present), the internucleoside linkage can be modified from normal phosphodiester to a more resistant linkage (such as phosphorothioate or boranophosphate) to improve the linkage to be more resistant to nuclease attack. These two types can be cleaved by RNase H, which also enables an antisense inhibition pathway to reduce the expression of the target gene.

[0303] In some aspects, the oligomers of the present disclosure contain one or more nucleoside linkages selected from the group consisting of phosphorothioate, phosphorodithioate, boranophosphate, methylphosphonate, phosphoramidate, or any combination thereof. Suitable sulfur-containing (S) internucleoside linkages as provided herein can be, for example, phosphorothioate, phosphodithioate, or a combination thereof. Phosphorothioate internucleoside linkages are also preferred, particularly for the first region (such as those contained in gapmers, mixmers, antimir splice-switching oligomers, and totalmers).

[0304] The term "mixmer" refers to an oligomer containing both natural and non-natural nucleotides, but where, unlike gapmers, tailers, and headmers, there are no more than five consecutive sequences, and in some aspects there are no more than four consecutive, three consecutive, etc. natural nucleotides (e.g., DNA units).

[0305] The term "totalmer" refers to a single-stranded oligomer that contains only unnatural nucleosides (such as sugar-modified nucleoside analogs).

[0306] For gapmers, the internucleoside linkages in the oligomer can be, for example, phosphorothioates or boranophosphates, which allow for cleavage of the target RNA by RNase H. Phosphorothioates are preferred for improved nuclease resistance and for other reasons, such as ease of manufacture.

[0307] In some aspects, with the exception of the phosphodiester linkage between the first and second regions and, optionally, the phosphodiester linkage within region B, for the remaining internucleoside linkages of the oligomer of the present disclosure, the nucleotides and / or nucleotide analogs are linked to each other by phosphorothioate groups.

[0308] In some aspects, 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 95%, or about 110% of the total internucleoside linkages of nucleosides in the first region are selected from the group consisting of phosphorothioates, phosphorodithioates, or boranophosphates. In some aspects, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% of the total internucleoside linkages of nucleosides in the first region are selected from the group consisting of phosphorothioates, phosphorodithioates, or boranophosphates.

[0309] In some aspects, 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 95%, or about 110% of the total internucleoside linkages of nucleosides in the first region are phosphorothioate. In some aspects, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% of the total internucleoside linkages of nucleosides in the first region are phosphorothioate.

[0310] In some aspects, the oligonucleotides of the present disclosure contain only one phosphorothioate linkage. In some aspects, the oligonucleotides of the present disclosure contain two phosphorothioate linkages. In some aspects, the oligonucleotides of the present disclosure contain three phosphorothioate linkages. In some aspects, the oligonucleotides of the present disclosure contain four phosphorothioate linkages. In some aspects, the oligonucleotides of the present disclosure contain five phosphorothioate linkages. In some aspects, the oligonucleotides of the present disclosure contain six phosphorothioate linkages. In some aspects, the oligonucleotides of the present disclosure contain seven phosphorothioate linkages. In some aspects, the oligonucleotides of the present disclosure contain eight phosphorothioate linkages. In some aspects, the oligonucleotides of the present disclosure contain nine phosphorothioate linkages. In some aspects, the oligonucleotides of the present disclosure contain ten phosphorothioate linkages. In some aspects, the oligonucleotides of the present disclosure comprise 11 phosphorothioate linkages. In some aspects, the oligonucleotides of the present disclosure comprise 12 phosphorothioate linkages. In some aspects, the oligonucleotides of the present disclosure comprise 13 phosphorothioate linkages. In some aspects, the oligonucleotides of the present disclosure comprise 14 phosphorothioate linkages. In some aspects, the oligonucleotides of the present disclosure comprise 15 phosphorothioate linkages. In some aspects, the oligonucleotides of the present disclosure comprise 16 phosphorothioate linkages. In some aspects, the oligonucleotides of the present disclosure comprise 17 phosphorothioate linkages. In some aspects, the oligonucleotides of the present disclosure comprise 18 phosphorothioate linkages. In some aspects, the oligonucleotides of the present disclosure comprise 19 phosphorothioate linkages. In some aspects, the oligonucleotides of the present disclosure comprise 20 phosphorothioate linkages. In some aspects, the oligonucleotides of the present disclosure comprise 21 phosphorothioate linkages.

[0311] WO09124238 refers to oligomeric compounds having at least one bicyclic nucleoside linked to the 3' or 5' end via a neutral internucleoside linkage. Thus, the oligomers of the present disclosure may have at least one or more bicyclic nucleosides linked to the 3' or 5' end via a neutral internucleoside linkage (such as one or more phosphotriester, methylphosphonate, MMI, amide-3, formacetal, or thioformacetal). The remaining linkages may be phosphorothioates.

[0312] Section 3: Oligonucleotide conjugates In some aspects, the antisense oligonucleotide conjugates of the present disclosure optionally comprise at least one non-nucleotide or non-polynucleotide moiety (C) covalently attached to the oligomer (A) via a linker region disposed between the contiguous sequence of the oligomer and the conjugate moieties (B and / or Y).

[0313] Representative conjugate moieties that have been used with oligonucleotides include lipid-soluble molecules (aromatic and non-aromatic), including steroid molecules; proteins (e.g., antibodies, enzymes, serum proteins); peptides; vitamins (water-soluble or lipid-soluble); polymers (water-soluble or lipid-soluble); small molecules, including drugs, toxins, reporter molecules, and receptor ligands; carbohydrate conjugates; nucleic acid cleavage conjugates; metal chelators (e.g., porphyrins, texaphyrins, crown ethers, etc.); intercalators, including photonuclease / intercalator hybrids; crosslinkers (e.g., photoreactive, redox-active), and combinations and derivatives thereof.

[0314] In some aspects, the disclosure provides an antisense oligonucleotide conjugate comprising: (a) a nucleotide sequence selected from the group consisting of: (a) an antisense oligomer (A) of 16 to 22 nucleotides in length containing a sequence of 16 consecutive nucleotides complementary to the corresponding length of SEQ ID NO: 31; wherein the antisense oligomer is an LNA gapmer; and (b) at least one conjugate moiety (C) covalently attached to said oligomer (A), said conjugate moiety (C) targeting an asialoglycoprotein receptor.

[0315] In some aspects, the oligomers of the present disclosure are targeted to the liver; i.e., after systemic administration, the compounds accumulate in liver cells (e.g., hepatocytes). Liver targeting can be significantly enhanced by adding a conjugate moiety (C). However, to maximize the efficacy of the oligomer, it is often desirable to link the conjugate (or targeting moiety) to the oligomer via a biocleavable linker (B) (e.g., a nucleotide phosphate linker). Therefore, it is desirable to utilize a conjugate moiety that enhances uptake and activity in hepatocytes. Enhanced activity can be due to enhanced uptake or enhanced efficacy of the compound in hepatocytes. Thus, the present disclosure also provides oligomeric compounds in the form of LNA oligomers (such as gapmers) or LNA antisense oligomers (which may also be referred to herein as Region A), for example, comprising an antisense oligomer, optionally a biocleavable linker (such as Region B), and a carbohydrate conjugate (which may also be referred to as Region C). The LNA antisense oligomer may be 7 to 30 nucleosides in length, for example 8 to 26 nucleosides in length, comprising at least one LNA unit (nucleoside).

[0316] In some aspects, the conjugate is or can include a carbohydrate or can include a carbohydrate group. In some aspects, the carbohydrate is selected from the group consisting of galactose, lactose, n-acetylgalactosamine, mannose, and mannose-6-phosphate. In some aspects, the conjugate group can be or can include mannose or mannose-6-phosphate. Carbohydrate conjugates can be used to enhance delivery or activity in various tissues (e.g., liver and / or muscle). In some aspects, the conjugates of the present disclosure include a moiety that targets the asialoglycoprotein receptor, the conjugate moiety being in the form of a carbohydrate moiety (e.g., a GalNAc moiety) (which may also be referred to as Region C). The carbohydrate moieties may be multivalent (e.g., 2, 3, 4, or 4 identical or non-identical carbohydrate moieties); and may be covalently linked to the oligomer, optionally via a single linker or multiple linkers (such as region Y).

[0317] In some aspects, the carbohydrate moiety is not a linear carbohydrate polymer, but may be multivalent, e.g., two, three, four, or four identical or non-identical carbohydrate moieties may be covalently linked by the oligomer, optionally via a single linker or multiple linkers, etc.

[0318] Section 3a: GalNAc conjugate moiety The present disclosure provides a conjugate comprising an oligomer of the present disclosure and an asialoglycoprotein receptor targeting moiety (such as a GalNAc moiety), which can form part of an additional region (termed region C). The present disclosure also provides an antisense oligonucleotide conjugate comprising: (a) and (b) (a) an antisense oligomer (A) of 16 to 22 nucleotides in length containing a sequence of 16 consecutive nucleotides complementary to the corresponding length of SEQ ID NO: 31; wherein the antisense oligomer is an LNA gapmer comprising at least one LNA unit; and (b) at least one conjugate moiety (C) covalently attached to said oligomer (A), said conjugate moiety (C) targeting an asialoglycoprotein receptor.

[0319] In some aspects, the conjugate moiety (e.g., third region or region C) comprises an asialoglycoprotein receptor targeting moiety selected from the group consisting of galactose, galactosamine, N-formyl-galactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, Nn-butanoyl-galactosamine, and N-isobutanoylgalactosamine. In some aspects, the conjugate comprises a galactose cluster (e.g., an N-acetylgalactosamine trimer). In some aspects, the conjugate moiety comprises GalNAc (N-acetylgalactosamine) (e.g., monovalent, divalent, trivalent, or tetravalent GalNAc).

[0320] Trivalent GalNAc conjugates can be used to target the compound to the liver. GalNAc conjugates have been used with methylphosphonate and PNA antisense oligonucleotides and siRNA. WO 2012 / 083046 discloses siRNAs containing GalNAc conjugate moieties with cleavable pharmacokinetic modifiers suitable for use in the present disclosure; preferred pharmacokinetic modifiers are C16 hydrophobic groups such as palmitoyl, hexadec-8-enoyl, oleyl, (9E,12E)-octadeca-9,12-dienoyl, dioctanoyl, and C16-C20 acyl. The cleavable pharmacokinetic modifier disclosed in WO 2012 / 083046 can also be cholesterol.

[0321] The "targeting moiety" (conjugate moiety) may be selected from the group consisting of galactose, galactosamine, N-formyl-galactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, Nn-butanoyl-galactosamine, N-isobutanoylgalactosamine, galactose cluster, and N-acetylgalactosamine trimer; the "targeting moiety" may also have a pharmacokinetic modifier selected from the group consisting of a hydrophobic group having 16 or more carbon atoms, a hydrophobic group having 16 to 20 carbon atoms, palmitoyl, hexadec-8-enoyl, oleyl, (9E,12E)-octadeca-9,12-dienoyl, dioctanoyl, and C16 to C20 acyl, and cholesterol. Specific GalNAc clusters disclosed in WO2012 / 083046 include (E)-hexadec-8-enoyl (C16), oleyl (C18), (9,E,12E)-octadeca-9,12-dienoyl (C18), octanoyl (C8), dodecanoyl (C12), C20 acyl, C24 acyl, and dioctanoyl (2xC8). The targeting moiety of the targeting moiety / pharmacokinetic modifier can be linked to the polynucleotide via a physiologically labile bond or, for example, a disulfide bond or a PEG linker. The present disclosure also relates to the use of a phosphodiester linker between the oligomer and the conjugate group (referred to herein as Region B, and preferably positioned between the LNA oligomer and the carbohydrate conjugate group).

[0322] A preferred targeting ligand for hepatocyte targeting in the liver is a galactose cluster. Galactose clusters include molecules having (e.g., including) two to four terminal galactose derivatives. In some aspects, the galactose derivative is N-acetyl-galactosamine (GalNAc). Other saccharides having affinity for the asialoglycoprotein receptor can be selected from the list including: galactosamine, Nn-butanoylgalactosamine, and N-isobutanoylgalactosamine. The term "galactose derivative" herein includes both galactose and galactose derivatives, where the galactose derivative has affinity for the asialoglycoprotein receptor that is similar to or stronger than that of galactose. The terminal galactose derivative is linked to the molecule through the CI carbon. The asialoglycoprotein receptor (ASGPr) is primarily expressed in hepatocytes and binds to branched galactose-terminal glycoproteins. Galactose clusters have three terminal galactosamines or galactosamine derivatives, each of which has affinity for the asialoglycoprotein receptor. Another galactose cluster has three terminal N-acetylgalactosamines. Other terms commonly used in the art include triheaded galactose, trivalent galactose, and galactose trimer. Triheaded galactose derivative clusters are known to bind to the ASGPr with higher affinity than biantennary or monoantennary galactose derivative structures. Multivalency is necessary to achieve nM affinity.

[0323] A galactose cluster can contain two or preferably three galactose derivatives, each linked to a central branch point. The galactose derivatives are linked to the central branch point via the CI carbon of the sugar. The galactose derivatives are preferably linked to the branch point via a linker or spacer. In some aspects, the spacer is a flexible hydrophilic spacer, such as a PEG spacer (e.g., a PEG3 spacer). The branch point can be any small molecule that allows linkage to three galactose derivatives and further linkage of the branch point to the oligomer. An example of a branch point group is dilysine. A dilysine molecule contains three amine groups and one carboxyl-reactive group, allowing linkage of three galactose derivatives via the amine groups and linkage of the dilysine to the oligomer via the carboxyl group. The branch point can be linked to the oligomer via a linker or spacer. In some aspects, the spacer is a flexible hydrophilic spacer, such as a PEG spacer (e.g., a PEG spacer (e.g., three ethylene units)). The galactose cluster can be linked to the 3' or 5' end of the oligomer using methods known in the art.

[0324] In some aspects of the disclosure, the conjugate portion of the antisense oligonucleotide conjugate comprises or consists of Conj1, 2, 3, 4, and Conj1a, 2a, 3a, and 4a. In some aspects, the conjugate portion comprises or consists of Conj2a. In some aspects, the antisense oligonucleotide conjugate is selected from the group consisting of SEQ ID NOs: 18 and 19.

[0325] Therefore, each carbohydrate moiety (e.g., GalNAc) of a GalNAc cluster can be linked to the oligomer via a spacer such as a (poly)ethylene glycol linker (PEG) (di-, tri-, tetra-, penta-, hexa-ethylene glycol linker, etc.). As shown above, the PEG moiety forms a spacer between the galactose sugar moiety and the peptide (trilysine shown) linker.

[0326] In some aspects, the GalNAc cluster comprises a peptide linker, e.g., a Tyr-Asp (Asp) tripeptide or an Asp (Asp) dipeptide, that is linked to the oligomer (or region Y or region B) via a biradical linker; for example, the GalNAc cluster comprises the following biradical linker: [ka]

[0327] R1 is preferably a biradical selected from: -C2H4-, -C3H6-, -C4H8-, -C5H 10 -, -C6H 12 -, 1,4-cyclohexyl (-CH 10 -), 1,4-phenyl (-C6H4-), -C2H4OC2H4-, -C2H4(OC2H4)2- or -C2H4(OC2H4)3-, -C(O)CH2-, -C(O)C2H4-, -C(O)C3H6-, -C(O)C4H8-, -C(O)C5H 10 -, -C(O)CH 12 -, 1,4-cyclohexyl (-C(O)CH 10 -), 1,4-phenyl (-C(O)C6H4-), -C(O)C2H4OC2H4-, -C(O)C2H4(OC2H4)2- or -C(O)C2H4(OC2H4)3-.

[0328] In some aspects, R1 is preferably a biradical selected from the following: -C2H4-, -C3H6-, -C4H8-, -C5H10 -, -C6H 12 -, 1,4-cyclohexyl (-CH 10 -), 1,4-phenyl (-C6H4-), -C2H4OC2H4-, -C2H4(OC2H4)2- or -C2H4(OC2H4)3-.

[0329] The carbohydrate conjugate (e.g., GalNAc) or carbohydrate / linker moiety (e.g., carbohydrate / PEG moiety) can be covalently attached (linked) to the oligomer via a branch-point group (e.g., amino acid or peptide) containing two or more amino groups (e.g., 3, 4, or 5) (e.g., lysine, di-, tri-, or tetralysine). The trilysine molecule contains four amine groups and one carboxyl-reactive group, allowing attachment of three types of carbohydrate conjugate groups (e.g., galactose and derivatives (e.g., GalNAc)) and additional conjugates (e.g., hydrophobic or lipophilic moieties / groups) via the amine groups, and the trilysine can be linked to the oligomer via the carboxyl-reactive group. Additional conjugates, such as lipophilic / hydrophobic moieties, can be linked to the lysine residues added to the oligomer.

[0330] GalNAc conjugates used with the LNA oligomers of the present disclosure do not require a pharmacokinetics modifier (e.g., a lipid-soluble moiety as disclosed below). Thus, in some aspects, the GalNAc conjugate is not covalently linked to a lipid-soluble or hydrophobic moiety (as described herein); e.g., it does not contain a C8-C36 fatty acid or a sterol. Thus, the present disclosure provides LNA oligomer-GalNAc conjugates that do not include conjugation with a lipid-soluble or hydrophobic pharmacokinetics modifier.

[0331] Section 3b: Lipid-soluble part Oligonucleotides of the present disclosure can further comprise one or more additional conjugate moieties (e.g., instead of or in addition to a GalNAc moiety), with lipophilic or hydrophobic moieties of the additional conjugate moiety being of particular interest, such as when the conjugate group is a carbohydrate moiety. Such lipophilic or hydrophobic moieties can act as pharmacokinetic modifiers and can be covalently linked, optionally via a linker (e.g., a biocleavable linker), to either the carbohydrate conjugate, the linker connecting the carbohydrate conjugate to the oligomer, or the linker connecting multiple carbohydrate conjugates (multivalent conjugates) to the oligomer.

[0332] Thus, the oligomer or conjugate moiety can include a pharmacokinetic modifier (such as a lipophilic or hydrophobic moiety). Such moieties are disclosed in connection with siRNA conjugates in WO2012 / 082046; the entire contents of this reference are incorporated herein by reference. The hydrophobic moiety can include a C8-C36 fatty acid, which can be saturated or unsaturated. In some aspects, C10, C12, C14, C16, C18, C20, C22, C24, C26, C28, C30, C32, and C34 fatty acids can be used. The hydrophobic group can have 16 or more carbon atoms.

[0333] Examples of suitable hydrophobic groups can be selected from the group consisting of sterol, cholesterol, palmitoyl, hexadec-8-enoyl, oleyl, (9E,12E)-octadeca-9,12-dienoyl, dioctanoyl, and C16-C20 acyl. Hydrophobic groups with fewer than 16 carbon atoms are less effective in enhancing polynucleotide targeting, but they can be used in multiple copies (e.g., 2x (2x C8 or C10, C12 or C14, etc.)) for enhanced potency. Pharmacokinetic modifiers useful as polynucleotide targeting moieties can be selected from the group consisting of cholesterol, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups, each of which can be linear, branched, or cyclic. Pharmacokinetic modifiers are preferably hydrocarbons containing only carbon and hydrogen atoms. However, substitutions or heteroatoms (eg, fluorine) that maintain hydrophobicity are acceptable.

[0334] Section 3c: Lipid-soluble conjugates In some aspects, the conjugate moiety can be or include a lipid-soluble moiety such as a sterol (e.g., cholesterol, cholesteryl, cholestanol, stigmasterol, cholanic acid, and ergosterol). In some aspects, the conjugate moiety can be or include a tocopherol (e.g., Conj6 and Conj6a in Figure 2). In some aspects, the conjugate moiety can be or include a cholesterol (e.g., Conj5 and Conj5a in Figure 2).

[0335] In some aspects, the conjugate moiety can be or include a lipid, phospholipid, or fat-soluble alcohol, such as a cationic lipid, a neutral lipid, a sphingolipid, and a fatty acid, such as stearic acid, oleic acid, elaidic acid, linoleic acid, linolenic acid, and myristic acid. In some aspects, the fatty acid comprises a C4-C30 saturated or unsaturated alkyl chain. The alkyl chain can be linear or branched.

[0336] Lipid-soluble conjugate moieties can be used, for example, to counteract the hydrophilic nature of oligomeric compounds and to facilitate intracellular penetration.

[0337] Lipid-soluble moieties include, for example, sterols, stanols, and steroids and related compounds (such as cholesterol, thiocholesterol, lanosterol, coprostanol, stigmasterol, ergosterol, calciferol, cholic acid, deoxycholic acid, estrone, estradiol, estratriol, progesterone, stilbestrol, testosterone, androsterone, deoxycorticosterone, cortisone, 17-hydroxycorticosterone, and derivatives thereof). In some aspects, the conjugate can be selected from the group consisting of cholesterol, thiocholesterol, lanosterol, coprostanol, stigmasterol, ergosterol, calciferol, cholic acid, deoxycholic acid, estrone, estradiol, estratriol, progesterone, stilbestrol, testosterone, androsterone, deoxycorticosterone, cortisone, and 17-hydroxycorticosterone. Other lipid-soluble conjugate moieties include aliphatic groups such as linear, branched, and cyclic alkyls, alkenyls, and alkynyls. The aliphatic groups can have, for example, from 5 to about 50 carbon atoms, from 6 to about 50 carbon atoms, from 8 to about 50 carbon atoms, or from 10 to about 50 carbon atoms. Aliphatic groups include, for example, undecyl, dodecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, terpenes, bornyl, adamantyl, and derivatives thereof. In some aspects, one or more carbon atoms in the aliphatic group can be replaced with a heteroatom (O, S, or N (e.g., geranyloxyhexyl)). Additional suitable lipid-soluble conjugate moieties include aliphatic derivatives of glycerols, such as alkylglycerols, bis(alkyl)glycerols, tris(alkyl)glycerols, monoglycerides, diglycerides, and triglycerides. In some aspects, the lipid-soluble conjugate is di-hexyldecyl-rac-glycerol or 1,2-di-O-hexyldecyl-rac-glycerol or a phosphonate thereof.For example, saturated and unsaturated fatty functional groups, such as fatty acids, fatty alcohols, fatty acid esters, and fatty amines, can also serve as lipid-soluble conjugate moieties. In some aspects, the fatty functional group can contain from about 6 carbons to about 30 carbons or from about 8 carbons to about 22 carbons. Fatty acids include, for example, capric acid, caprylic acid, lauric acid, palmitic acid, myristic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, and eicosenoic acids.

[0338] In a further aspect, the lipid-soluble conjugate group can be a polycyclic aromatic group having 6 to about 50 carbon atoms, 10 to about 50 carbon atoms, or 14 to about 40 carbon atoms. Examples of polycyclic aromatic groups include pyrenes, purines, acridines, xanthenes, fluorenes, phenanthrenes, anthracenes, quinolines, isoquinolines, naphthalenes, and derivatives thereof. Other suitable lipid-soluble conjugate moieties include trityles (e.g., dimethoxytrityl (DMT)), phenoxazines, lipoic acid, phospholipids, ethers, thioethers (e.g., hexyl-S-tritylthiol), and derivatives thereof.

[0339] Oligomeric compounds containing conjugate moieties with affinity for low-density lipoprotein (LDL) can help provide an effective targeted delivery system. When LDL receptors are expressed at high levels on tumor cells, LDL serves as an attractive carrier for selectively delivering drugs to these cells. Moieties with affinity for LDL include many lipid-soluble groups, such as steroids (e.g., cholesterol), fatty acids, their derivatives, and combinations thereof. In some aspects, the conjugate moiety with LDL affinity can be dioleyl esters of cholic acid (e.g., chenodeoxycholic acid and lithocholic acid). In some aspects, the lipid-soluble conjugate can be or include biotin. In some aspects, the lipid-soluble conjugate moiety can be or include a glyceride or glyceride ester.

[0340] Lipid-soluble conjugate moieties, such as sterols, stanols, and stains (such as cholesterol or substances disclosed herein), can be used, for example, to facilitate delivery of the oligonucleotide to the liver (typically to hepatocytes). In some aspects of the present disclosure, the conjugate moiety of the antisense oligonucleotide conjugate comprises or consists of Conj5, 5a, 6, or 6a. In some aspects, the conjugate moiety comprises or consists of Conj5a. In some aspects, the antisense oligonucleotide conjugate is selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 11.

[0341] Section 3d: Linker A bond or linker is a linkage between two atoms that connects one chemical group or segment of interest to another chemical group or segment of interest via one or more covalent bonds. A conjugate moiety (or targeting moiety or blocking moiety) can be linked to an oligomeric compound directly or via a linking moiety (linker or tether), i.e., a linker. A linker is a bifunctional moiety that serves to covalently link a third region, e.g., a conjugate moiety, to an oligomeric compound (e.g., region A). In some aspects, the linker comprises a chain structure or oligomer of repeating units (e.g., ethylene glycol or amino acid units). The linker can have at least two types of functionality, one of which is a bond to the oligomeric compound and the other of which is a bond to the conjugate moiety. Examples of linker functionality can be electrophilic, which reacts with a nucleophilic group on the oligomer or conjugate moiety, or nucleophilic, which reacts with an electrophilic group. In some aspects, the linker functional group includes amino, hydroxyl, carboxylic acid, thiol, phosphoramidate, phosphorothioate, phosphate, phosphite, unsaturation (e.g., double or triple bond), etc.Some examples of linkers include 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-I-carboxylate (SMCC), 6-aminohexanoic acid (AHEX or AHA), 6-aminohexyloxy, 4-aminobutyric acid, 4-aminocyclohexylcarboxylic acid, succinimidyl 4-(N-maleimidomethyl)cyclohexane-I-carboxy-(6-amido-caproate) (LCSMCC), succinimidyl m-maleimido-benzoate (MBS), succinimidyl Ne-maleimido-benzoate (NE-maleimido-benzoate), and succinimidyl methyl ... Examples of such amino acids include beta-aminocyclohexanoic acid (ACHC), beta-cyclopropyl-2-methylpropanol (beta-cyclopropyl-2-methylpropanol), beta-isopropyl-2-methylpropanol (beta-iso ...

[0342] A variety of linker groups that may be useful for linking oligomeric compounds to conjugate moieties are further known in the art. Reviews of many of these useful linker groups can be found, for example, in: Antisense Research and Applications, edited by ST Crooke and B. Lebleu, CRC Press, Boca Raton, Fla., 1993, pp. 303-350.

[0343] Linkers and their use in preparing conjugates of oligomeric compounds are widely available in the art, examples of which include: International Patent Publications WO96 / 11205 and WO98 / 52614; and U.S. Patent Nos. 4,948,882; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,580,731; 5,486,603; 5,608,046; 4,587,044; 4,667,025; 5,254,469; 5,245,022 ; 5,112,963; 5,391,723; 5,510475; 5,512,667; 5,574,142; 5,684,142; 5,770,716; 6,096,875; 6,335,432; and 6,335,437; and International Patent Publication WO2012 / 083046; the contents of these references are incorporated herein by reference in their entireties.

[0344] A "physiologically labile bond" herein is a labile bond that is cleavable under normal conditions in a mammalian body, or similar conditions (also referred to as a cleavable linker, shown as region B in Figures 12 and 13). A physiologically labile linking group is selected such that a chemical transformation (e.g., cleavage) occurs when present under specific physiological conditions. Conditions within a mammalian cell include chemical conditions such as pH, temperature, oxidizing or reducing conditions or oxidizing or reducing agents, and salt concentration within a mammalian cell, or similar conditions. Conditions within a mammalian cell also include the presence of enzymatic activities (such as proteolytic or hydrolytic enzymes) normally present in mammalian cells. In some aspects, the cleavable linker is For example, it is susceptible to nucleases that may be expressed in the target cell; therefore, as described in detail herein, the linker may be a short (e.g., 1-10) nucleoside region (such as DNA nucleosides) linked by phosphodiester bonds.

[0345] Chemical transformation (cleavage of the labile bond) can be initiated by the addition of a pharmaceutically acceptable agent to a cell, or can occur spontaneously when a molecule containing the labile bond reaches the appropriate intracellular and / or extracellular environment. For example, a pH-labile bond can be cleaved when the molecule enters an acidic endosome. Thus, a pH-labile bond can be considered an intraendosomal cleavable bond. An enzymatically cleavable bond can be cleaved when exposed to an enzyme, such as an enzyme present in an endosome or lysosome within the cytoplasm. A disulfide bond can be cleaved when the molecule enters the more reducing environment of the cytoplasm. Thus, a disulfide can be considered an intracytoplasmic cleavable bond. As used herein, a "pH-labile bond" is a labile bond that selectively degrades under acidic conditions (pH<7). Because the pH of cellular endosomes and lysosomes is below 7, such bonds can also be referred to as intraendosomal labile bonds.

[0346] Section 3e: Oligomers linked to biocleavable conjugates The oligomeric compound may optionally include a second region (region B) disposed between the oligomer (referred to as region A) and the conjugate (referred to as region C). For illustrative illustrations, see Figures 12 and 13. Region B may be a linker, such as a cleavable linker (also referred to as a physiologically labile linkage). Nuclease-Sensitive Physiologically Labile Linkage: In some aspects, the oligomers (also referred to as oligomeric compounds) (or conjugates) of the present disclosure include three regions: (i) a first region (region A) (e.g., comprising 16 to 18 consecutive nucleotides); (ii) a second region (region B) (comprising a biocleavable linker); and (iii) a third region (C) (comprising a conjugate moiety, a targeting moiety, an activating moiety, wherein the third region is covalently linked to the second region).

[0347] In some aspects, region B can be a phosphate nucleotide linker. For example, such linkers can be used when the conjugate is a lipid-soluble conjugate, such as a lipid, fatty acid, or sterol (e.g., cholesterol or tocopherol). For other conjugates, such as carbohydrate conjugates (e.g., GalNAc), phosphate nucleotide linkers can also be used.

[0348] Section 3 e.1: Peptide Linkers In some aspects, the biocleavable linker (region B) is a peptide, such as a trilysine peptide linker, which can be used in polyGalNAc conjugates (such as triGalNAc conjugates). See also peptide biradicals described herein.

[0349] Other linkers known in the art that can be used include disulfide linkers.

[0350] Section 3e.2: Phosphate Nucleotide Linkers In some aspects, region B comprises 1 to 6 nucleotides covalently linked, such as via an internucleoside linkage group (such as a phosphodiester bond), to the 5' or 3' nucleotide of the first region (region A); where: (a) the internucleoside linkages between the first and second regions are phosphodiester bonds, and the nucleosides of the second region adjacent (e.g., immediately adjacent) to the first region are either DNA or RNA; and / or (b) at least one nucleoside of the second region is a phosphodiester-linked DNA or RNA nucleoside;

[0351] In some aspects, region A and region B form a single contiguous nucleotide sequence 16 to 22 nucleotides in length.

[0352] In some aspects, the internucleoside linkages between a first region and a second region can be considered part of the second region.

[0353] In some aspects, a phosphorus-containing linking group is present between the second region and the third region. The phosphorus-containing linking group can be, for example, a phosphate (phosphodiester), phosphorothioate, phosphorodithioate, or boranophosphate group. In some aspects, the phosphorus-containing linking group is present between the second region and the linker region (which connects to the third region). In some aspects, the phosphate group is a phosphodiester.

[0354] Thus, in some aspects, the oligomeric compound comprises at least two phosphodiester groups, where at least one is as described above and the other is disposed between the second and third regions, optionally between the linker group and the second region.

[0355] In some aspects, the third region is an activating group, such as an activating group for use in conjugation. In this regard, the present disclosure also provides activated oligomers comprising region A and region B and an activating group (e.g., an intermediate suitable for subsequent linkage to a third region, such as for conjugation).

[0356] In some aspects, the third region is a reactive group (e.g., a reactive group for use in conjugation). In this regard, the present disclosure also provides oligomers comprising region A and region B and a reactive group (e.g., an intermediate suitable for subsequent linkage to a third region, such as a conjugation intermediate). The reactive group, in some aspects, comprises an amine of an alcohol group (e.g., an amine group).

[0357] In some aspects, region A includes at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21) internucleoside linkage other than phosphodiester, wherein the internucleoside linkage is (optionally independently) selected from the group consisting of phosphorothioate, phosphorodithioate, and boranophosphate, and methylphosphonate (e.g., phosphorothioate). In some aspects, region A includes at least one phosphorothioate linkage. In some aspects, at least 50% of the internucleoside linkages (e.g., at least 75%, such as at least 90%, such as all internucleoside linkages in region A) are other than phosphodiester (e.g., are phosphorothioate linkages). In some aspects, all internucleoside linkages in region A are other than phosphodiester linkages.

[0358] In some aspects, the oligomeric compound comprises an antisense oligonucleotide (such as an antisense oligonucleotide conjugate). The antisense oligonucleotide may comprise the first region and, optionally, the second region, or the same. In this regard, in some aspects, region B may form part of a contiguous nucleobase sequence that is complementary to the (nucleic acid) target. In other aspects, region B lacks complementarity to the target.

[0359] In other words, in some aspects, the present disclosure provides non-phosphodiester-linked (e.g., phosphorothioate-linked) oligonucleotides (e.g., antisense oligonucleotides) having at least one terminal (5' and / or 3') DNA or RNA nucleoside linked via a phosphodiester bond to adjacent nucleosides of the oligonucleotide, wherein the terminal DNA or RNA nucleoside is optionally further covalently linked, via a linker moiety, to a conjugate moiety, targeting moiety, or blocking moiety.

[0360] In some aspects, the oligomeric compound comprises an antisense oligonucleotide (such as an antisense oligonucleotide conjugate). The antisense oligonucleotide may comprise the first region and optionally the second region, or may comprise the same regions. In this regard, in some aspects, region B may form part of a contiguous nucleobase sequence that is complementary to the (nucleic acid) target. In other aspects, region B lacks complementarity to the target.

[0361] In some aspects, at least two consecutive nucleosides of the second region are DNA nucleosides (such as at least 3 or 4 or 5 consecutive DNA nucleotides).

[0362] In one such aspect, the oligonucleotides of the disclosure can be described as according to the following formula: 5'-A-PO-B[Y]X-3' or 3'-A-PO-B[Y]X-5'; where A is region A, PO is a phosphodiester bond, B is region B, Y is an optional linking group, and X is a conjugate, targeting group, blocking group, or reactive or activating group.

[0363] In some aspects, region B comprises, in a 3'-5' or 5'-3' orientation: (i) a phosphodiester bond to the 5′ or 3′ nucleoside of region A; (ii) DNA nucleosides or RNA nucleosides (e.g., DNA nucleosides); and (iii) an additional phosphodiester bond, 5'-A-PO-B-PO-3' or 3'-A-PO-B-PO-5'; Includes.

[0364] Additional phosphodiester bonds link the Region B nucleosides to one or more further nucleosides (such as one or more DNA or RNA nucleosides) or, optionally, to X via a linking group (Y), where X is a conjugate, targeting group, or blocking group, or reactive group, or activating group.

[0365] In some aspects, region B comprises, in a 3'-5' or 5'-3' orientation: (i) a phosphodiester bond to the 5′ or 3′ nucleoside of region A; (ii) 2 to 10 nucleosides linked by DNA nucleosides or RNA phosphodiesters (e.g., DNA nucleosides); and optionally, (iii) an additional phosphodiester bond; Contains 5'-A-[PO-B]n - [Y]-X 3' or 3'-A-[PO-B]n -[Y]-X 5' 5'-A-[PO-B]n - PO-[Y]-X 3' or 3'-A-[PO-B]n - PO-[Y]-X 5', where A represents region A, [PO-B]n represents region B, where n is 1 to 10 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), and PO is an optional phosphodiester linking group between region B and X (or Y, if present).

[0366] In some aspects, the disclosure provides compounds according to (or including) one of the following formulas: 5' [Area A] - PO - [Area B] 3' -Y - X 5' [Area A] - PO - [Area B] -PO 3' -Y - X 5' [Area A] - PO - [Area B] 3' - X 5' [Area A] - PO - [Area B] -PO 3' - X 3' [Area A] - PO - [Area B] 5' -Y - X 3' [Area A] - PO - [Area B] -PO 5' -Y - X 3' [Area A] - PO - [Area B] 5' - X 3' [Area A] - PO - [Area B] -PO 5' - X

[0367] Area B could be, for example: 5' DNA 3' 3' DNA 5' 5' DNA-PO-DNA-3' 3' DNA-PO-DNA-5' 5' DNA-PO-DNA-PO-DNA 3' 3' DNA-PO-DNA-PO-DNA 5' 5' DNA-PO-DNA-PO-DNA-PO-DNA 3' 3' DNA-PO-DNA-PO-DNA-PO-DNA 5' 5' DNA-PO-DNA-PO-DNA-PO-DNA-PO-DNA 3' 3' DNA-PO-DNA-PO-DNA-PO-DNA-PO-DNA 5', It may include or consist of the same.

[0368] It is understood that nucleosides other than DNA and RNA can be used for the phosphate-linked biocleavable linker. Biocleavable nucleotide linkers can be identified using the assay of Example 6.

[0369] In some aspects, the compounds of the present disclosure include a biocleavable linker (also called a physiologically labile linker, a nuclease-sensitive physiologically labile bond, or a nuclease-sensitive linker) connecting the oligomer (or contiguous nucleotide sequence or region A) to the conjugate moiety (or region C) (e.g., a phosphate nucleotide linker (such as region B) or a peptide linker).

[0370] Susceptibility to cleavage in the assay shown in Example 6 can be used to determine whether a linker is biocleavable or physiologically labile.

[0371] Bio-cleavable linkers according to the present disclosure include linkers that are susceptible to cleavage (i.e., physiologically unstable) in target tissues (e.g., liver and / or kidney). The cleavage rate in the target tissue is preferably faster than the rate in serum. A suitable method for determining the percent cleavage level in tissues (e.g., liver or kidney) and serum is described in Example 6. In some aspects, a bio-cleavable linker (also referred to as a physiologically labile linker or nuclease-sensitive linker), such as region B in a compound of the present disclosure, is at least about 20% cleaved (e.g., at least about 30% cleaved, such as at least about 40% cleaved, such as at least about 50% cleaved, such as at least about 60% cleaved, such as at least about 70% cleaved, such as at least about 75% cleaved) in the liver or kidney homogenate assay of Example 6. In some aspects, the percent cleavage in serum, as in the assay of Example 6, is less than about 30% (such as less than about 20%, such as less than about 10%, such as less than 5%, such as less than about 1%).

[0372] In some aspects, which may be the same or different, a bio-cleavable linker (also referred to as a physiologically labile linker or a nuclease-sensitive linker) such as region B in a compound of the present disclosure is susceptible to cleavage by S1 nuclease. Susceptibility to cleavage by S1 can be assessed using the S1 nuclease assay shown in Example 6. In some aspects, a bio-cleavable linker (also referred to as a physiologically labile linker or a nuclease-sensitive linker) such as region B in a compound of the present disclosure is at least about 30% cleaved (e.g., at least about 40% cleaved, such as at least about 50% cleaved, such as at least about 60% cleaved, such as at least about 70% cleaved, such as at least about 80% cleaved, such as at least about 90% cleaved, such as at least 95% cleaved) after 120 minutes of incubation with S1 nuclease according to the assay used in Example 6.

[0373] Sequence selection in region B In some aspects, oligomers of the present disclosure include a region (region B) that is not complementary to the target sequence. In other aspects, region B forms a sequence complementary to the target sequence. In some aspects, region A and region B together can form a single contiguous sequence complementary to the target sequence.

[0374] In some aspects, the sequence of bases in region B is selected to provide an optimal endonuclease cleavage site based on the predominant endonuclease cleavage enzyme present in the target tissue or cell or subcellular compartment. In this regard, by isolating cell extracts from target and non-target tissues, the endonuclease cleavage sequence used in region B can be selected based on the preferential cleavage activity in the desired target cells (e.g., liver / hepatocytes) compared to non-target cells (e.g., kidney). In this regard, the efficacy of targeting downregulation by the compound can be optimized for the desired tissue / cell.

[0375] In some aspects, region B comprises a dinucleotide of the sequence AA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG, GA, GT, GC, or GG, where C can be 5-methylcytosine and / or T can be substituted with U. In some aspects, region B comprises the trinucleotide sequence of AAA, AAT, AAC, AAG, ATA, ATT, ATC, ATG, ACA, ACT, ACC, ACG, AGA, AGT, AGC, AGG, TAA, TAT, TAC, TAG, TTA, TTT, TTC, TAG, TCA, TCT, TCC, TCG, TGA, TGT, TGC, TGG, CAA, CAT, CAC, CAG, CTA, CTG, CTC, CTT, CCA, CCT, CCC, CCG, CGA, CGT, CGC, CGG, GAA, GAT, GAC, CAG, GTA, GTT, GTC, GTG, GCA, GCT, GCC, GCG, GGA, GGT, GGC, and GGG, where C can be 5-methylcytosine and / or T can be substituted with U.

[0376] In some aspects, region B includes AAAX, AATX, AACX, AAGX, ATAX, ATTX, ATCX, ATGX, ACAX, ACTX, ACCX, ACGX, AGAX, AGTX, AGCX, AGGX, TAAX, TATX, TACX, TAGX, TTAX, TTTX, TTCX, TAGX, TCAX, TCTX, TCCX, TCGX, TGAX, TGTX, TGCX, TGGX, CAAX, CATX, CACX, CAGX, CTAX, CTGX, CTCX, CTT X, CCAX, CCTX, CCCX, CCGX, CGAX, CGTX, CGCX, CGGX, GAAX, GATX, GACX, CAGX, GTAX, GTTX, GTCX, GTGX, GCAX, GCTX, GCCX, GCGX, GGAX, GGTX, GGCX, and GGGX, where X can be selected from the group consisting of A, T, U, G, C, and analogs thereof, where C can be 5-methylcytosine and / or T can be substituted with U. It will be understood that when referring to (natural) nucleobases A, T, U, G, C, these can be replaced with nucleobase analogs that function equivalently to the natural nucleobases (e.g., base pairs with complementary nucleosides). In some aspects, region B does not contain T or U.

[0377] Section 4: Medical Indications In some aspects, pharmaceutical compositions of the present disclosure (e.g., oral formulations (e.g., oral formulations comprising CIVI008 and an oral delivery agent (such as SNAC, C10, or 5-CNAC, a derivative thereof, a pharmaceutically acceptable hydrate, solvate, or salt thereof; or any combination thereof)) can be used to treat conditions associated with overexpression or expression of a normal form, mutant, allelic variant, or splice variant of the PCSK9 gene. The present disclosure further provides pharmaceutical compositions of the present disclosure (e.g., oral formulations (e.g., oral formulations comprising CIVI008 and an oral delivery agent (such as SNAC, C10, or 5-CNAC, a derivative thereof, a pharmaceutically acceptable hydrate, solvate, or salt thereof; or any combination thereof)) for use in the manufacture of a therapeutic medicament for the treatment of a disease, disorder, or condition as referred to herein.

[0378] The present disclosure provides a method of treating a mammal, e.g., a human, suffering from or susceptible to a condition associated with abnormal levels and / or activity of PCSK9, comprising administering to the mammal a therapeutically effective amount of a pharmaceutical composition of the present disclosure (e.g., an oral formulation (e.g., an oral formulation comprising CIVI008 and an oral delivery agent (such as SNAC, C10, or 5-CNAC, derivatives thereof, pharmaceutically acceptable hydrates, solvates, or salts thereof; or any combination thereof)).

[0379] In some aspects, the diseases or disorders referred to herein may be associated with mutations in the PCSK9 gene or mutations in the gene of a protein product that binds to or interacts with PCSK9. Thus, in some aspects, the target is RNA. In some aspects, the mRNA is a pre-mRNA. In some aspects, the RNA is an intron of an mRNA. In some aspects, the RNA is an exon of an RNA. In some aspects, the RNA is an intron-exon boundary. In some aspects, the RNA is a normal PCSK9 mRNA that is upregulated, for example, by a defective promoter or by other components of a metabolic or signaling pathway that includes PCSK9. In some aspects, the mRNA is derived from an allelic variant of the PCSK9 gene. In some aspects, the mRNA is derived from a splice variant of a normal or mutant PCSK9 gene. In some aspects, the mRNA is derived from a mutated form of the PCSK9 gene (e.g., a gain-of-function mutant).

[0380] PCSK9 plays a major regulatory role in cholesterol homeostasis, primarily by reducing LDLR levels on the cell membrane. Decreased LDLR levels result in decreased metabolism of LDL particles, which can lead to hypercholesterolemia. Binding of LDL to LDLR induces internalization of the LDLR / LDL complex into endosomes. When PCSK9 binds to LDLR (via its EGF-A domain), it inhibits a conformational change in the receptor / ligand complex. This inhibition redirects LDLR to the lysosome instead.

[0381] PCSK9 also plays an important role in the production of triglyceride-rich intestinal apoB lipoproteins in the small intestine and postprandial dyslipidemia. PCSK9 variants can lower or increase circulating cholesterol. Another variant is associated with a rare autosomal dominant familial hypercholesterolemia (HCHOLA3). These mutations increase its protease activity, thereby decreasing LDLR levels and inhibiting intracellular cholesterol uptake. PCSK9 is highly expressed in the arterial wall, including in endothelium, smooth muscle cells, and macrophages, and has local effects that may regulate vascular homeostasis and atherosclerosis. Thus, PCSK9 has proatherosclerotic effects and regulates lipoprotein synthesis.

[0382] PCSK9 is involved in the regulation of glucose metabolism and renal sodium reabsorption, which is associated with obesity and hypertension. Additionally, PCSK9 may be involved in bacterial or viral infections and sepsis.

[0383] In some aspects, the PCSK9 gene is an allelic variant selected from Pro174Ser PCSK0, Ser127Arg PCSK9, Phe216Leu PCSK9, and Asp374Tyr PCSK9.

[0384] The method disclosed herein is preferably used for the treatment and prevention of diseases caused by abnormal levels and / or activity of PCSK9. The present disclosure provides a method for treating abnormal levels and / or activity of PCSK9 in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present disclosure (e.g., an oral formulation (e.g., an oral formulation comprising CIVI008 and an oral delivery agent (e.g., SNAC, C10, or 5-CNAC, its derivatives, their pharmaceutically acceptable hydrates, solvates, or salts; or any combination thereof)).

[0385] The present disclosure also relates to pharmaceutical compositions of the present disclosure for use as therapeutic agents (e.g., oral formulations (e.g., oral formulations comprising CIVI008 and an oral delivery agent (such as SNAC, C10, or 5-CNAC, derivatives thereof, pharmaceutically acceptable hydrates, solvates, or salts thereof; or any combination thereof)).

[0386] The present disclosure further relates to the use of a therapeutically effective amount of a pharmaceutical composition of the present disclosure (e.g., an oral formulation (e.g., an oral formulation comprising CIVI008 and an oral delivery agent (such as SNAC, C10, or 5-CNAC, derivatives thereof, pharmaceutically acceptable hydrates, solvates, or salts thereof; or any combination thereof)) in the manufacture of a therapeutic medicament for the treatment of abnormal levels of PCSK9 and / or abnormal activity of PCSK9 and / or expression of a mutant (e.g., a gain-of-function mutant) of PCSK9, an allelic variant of PCSK9, or a splice variant of PCSK9.

[0387] The present disclosure provides methods for treating a disease or condition caused by an abnormal expression level and / or activity of PCSK9 in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition of the present disclosure (e.g., an oral formulation (e.g., an oral formulation comprising CIVI008 and an oral delivery agent (such as SNAC, C10, or 5-CNAC, derivatives thereof, pharmaceutically acceptable hydrates, solvates, or salts thereof; or any combination thereof)), wherein administration of the pharmaceutical composition reduces serum PCSK9 levels and / or reduces serum LDL cholesterol levels in the subject. In some aspects, In some aspects, the disease or condition is selected from the group consisting of atherosclerosis, hypercholesterolemia, HDL / LDL cholesterol imbalance, dyslipidemia, coronary artery disease (CAD), and coronary heart disease (CHD). In some aspects, the dyslipidemia is familial hyperlipidemia (FCHL) or acquired hyperlipidemia. In some aspects, the hypercholesterolemia is familial hypercholesterolemia or statin-resistant hypercholesterolemia. In some aspects, the subject or patient in need of treatment is a subject or patient suffering from or likely to suffer from a disease or disorder disclosed herein.

[0388] In some instances, the term "treatment" herein refers to both treatment of an existing disease (e.g., a disease or disorder disclosed herein) and prevention (i.e., preventing disease). Thus, in some aspects, the treatment referred to herein can be prophylactic.

[0389] In some aspects, treatment with a pharmaceutical composition of the present disclosure (e.g., an oral formulation (e.g., an oral formulation comprising CIVI008 and an oral delivery agent (such as SNAC, C10, or 5-CNAC, a derivative thereof, a pharmaceutically acceptable hydrate, solvate, or salt thereof; or any combination thereof)) can treat, ameliorate, or prevent symptoms of a disease disclosed herein. In some aspects, treatment with a pharmaceutical composition of the present disclosure (e.g., an oral formulation (e.g., an oral formulation comprising CIVI008 and an oral delivery agent (such as SNAC, C10, or 5-CNAC, a derivative thereof, a pharmaceutically acceptable hydrate, solvate, or salt thereof; or any combination thereof)) can prevent, delay, or reduce sequelae associated with a disease disclosed herein.

[0390] For example, in some aspects, hypercholesterolemia can be treated in a subject by administering a pharmaceutical composition of the present disclosure (e.g., an oral formulation (e.g., an oral formulation comprising CIVI008 and an oral delivery agent (SNAC, C10, or 5-CNAC, a derivative thereof, a pharmaceutically acceptable hydrate, solvate, or salt thereof; or any combination thereof, etc.)). In some aspects, the development of hypercholesterolemia can be prevented in a subject at risk for hypercholesterolemia by administering a pharmaceutical composition of the present disclosure (e.g., an oral formulation (e.g., an oral formulation comprising CIVI008 and an oral delivery agent (SNAC, C10, or 5-CNAC, a derivative thereof, a pharmaceutically acceptable hydrate, solvate, or salt thereof; or any combination thereof, etc.)). In some aspects, the onset of hypercholesterolemia can be delayed in a subject at risk of hypercholesterolemia by administering a pharmaceutical composition of the present disclosure (e.g., an oral formulation (e.g., an oral formulation comprising CIVI008 and an oral delivery agent (SNAC, C10, or 5-CNAC, a derivative thereof, a pharmaceutically acceptable hydrate, solvate, or salt thereof; or any combination thereof, etc.)). In some aspects, the onset of hypercholesterolemia can be prevented in a subject at risk of hypercholesterolemia by administering a pharmaceutical composition of the present disclosure (e.g., an oral formulation (e.g., an oral formulation comprising CIVI008 and an oral delivery agent (SNAC, C10, or 5-CNAC, a derivative thereof, a pharmaceutically acceptable hydrate, solvate, or salt thereof; or any combination thereof, etc.)).In some aspects, administration of a pharmaceutical composition of the present disclosure (e.g., an oral formulation (e.g., an oral formulation comprising CIVI008 and an oral delivery agent (e.g., SNAC, C10, or 5-CNAC, derivatives thereof, pharmaceutically acceptable hydrates, solvates, or salts thereof; or any combination thereof)) can cause or reduce symptoms of hypercholesterolemia in a subject (e.g., chest pain during activity, xanthomas (fatty deposits frequently found in tendons and the elbows, buttocks, and knees), xanthelasma (cholesterol deposits around the eyelids), arcus senilis (gray-white cholesterol deposits around the cornea), etc.). In some aspects, administering a pharmaceutical composition of the present disclosure (e.g., an oral formulation (e.g., an oral formulation comprising CIVI008 and an oral delivery agent (such as SNAC, C10, or 5-CNAC, a derivative thereof, a pharmaceutically acceptable hydrate, solvate, or salt thereof; or any combination thereof)) can prevent or delay the onset or occurrence of sequelae or complications of hypercholesterolemia (e.g., heart disease, circulatory disease, heart attack, long-term atherosclerosis, stroke, etc.) in a subject.

[0391] In some aspects, the present disclosure provides methods for treating a disease or condition (e.g., hypercholesterolemia) caused by an abnormal expression level and / or activity of PCSK9 in a subject in need thereof, the method comprising administering to the subject an effective amount of an oral formulation (e.g., in the form of a pill or capsule) comprising CIVI008 and an oral delivery agent (an oral formulation comprising SNAC, C10, or 5-CNAC, derivatives thereof, pharmaceutically acceptable hydrates, solvates, or salts thereof; or any combination thereof), wherein administration of the pharmaceutical composition reduces serum PCSK9 levels in the subject, thereby reducing serum LDL cholesterol levels. In some aspects, the present disclosure provides methods for treating hypercholesterolemia in a subject in need thereof, comprising administering to the subject an effective amount of an oral formulation (e.g., in the form of a pill or capsule) comprising CIVI008 and an oral delivery agent (such as SNAC, C10, or 5-CNAC), wherein administration of the pharmaceutical composition reduces serum PCSK9 levels in the subject, thereby reducing serum LDL cholesterol levels. In some aspects, the present disclosure provides methods for treating hypercholesterolemia in a subject in need thereof, comprising administering to the subject an effective amount of an oral formulation (e.g., in the form of a pill or capsule) comprising CIVI008 and SNAC, wherein administration of the pharmaceutical composition reduces serum PCSK9 levels in the subject, thereby reducing serum LDL cholesterol levels. In some aspects, the present disclosure provides methods of treating hypercholesterolemia in a subject in need thereof, comprising administering to the subject an effective amount of an oral formulation (e.g., in the form of a pill or capsule) comprising CIVI008 and C10, wherein administration of the pharmaceutical composition reduces serum PCSK9 levels in the subject, thereby reducing serum LDL cholesterol levels.

[0392] In some aspects, the present disclosure provides a method of treating hypercholesterolemia in a subject in need thereof, comprising administering to the subject an effective amount of an oral formulation (e.g., in the form of a pill or capsule) comprising CIV008 and 5-CNACI, wherein administration of the pharmaceutical composition reduces serum PCSK9 levels in the subject, thereby reducing serum LDL cholesterol levels.

[0393] Section 5: Combination Therapy In some aspects, a pharmaceutical composition of the present disclosure (e.g., an oral formulation, (e.g., an oral formulation comprising CIVI008 and an oral delivery agent (SNAC, C10, or 5-CNAC, a derivative thereof, a pharmaceutically acceptable hydrate, solvate, or salt thereof; or any combination thereof, etc.)) can be used in combination therapy with another therapeutic agent. In some aspects, the additional therapeutic agents can be co-administered as part of the same pharmaceutical composition, e.g., as additional components of the same pill or capsule. In some aspects, the additional therapeutic agents can be co-administered separately; i.e., the pharmaceutical composition of the present disclosure can be administered, e.g., as a pill or capsule, and the additional component can be administered, e.g., as a separate pill or capsule.

[0394] Additional therapeutic agents that can be co-administered with the pharmaceutical compositions of the present disclosure include, for example, inhibitors of HMG CoA reductase, such as statins, which are widely used, for example, in the treatment of metabolic diseases (see WO2009 / 043354; the entire contents of this reference are incorporated herein by reference, e.g., for combination therapy).

[0395] Therapeutic agents that can be co-administered with the pharmaceutical compositions of the present disclosure can be other cholesterol-lowering compounds, such as those selected from the group consisting of: (i) bile salt adsorbent resins (e.g., cholestyramine, colestipol, and colesevelam hydrochloride); (ii) HMGCoA-reductase inhibitors (e.g., lovastatin, cerivastatin, pravastatin, atorvastatin, simvastatin, rosuvastatin, and fluvastatin); (iii) nicotinic acid; (iv) fibric acid derivatives (e.g., clofibrate, gemfibrozil, fenofibrate, bezafibrate, and ciprofibrate); (v) Probucol; (vi) neomycin; (vii) dextrothyroxine; (viii) plant stanol esters, (ix) cholesterol absorption inhibitors (e.g., ezetimibe); (x) Implitapide; (xi) inhibitors of bile acid transporters (e.g., apical sodium-dependent bile acid transporters); (xii) regulators of hepatic CYP7a; (xiii) estrogen replacement therapy (e.g., tamoxifen); (xiv) anti-inflammatory agents (e.g., glucocorticoids); and (xv) any combination thereof.

[0396] In one aspect, the combination therapy comprises a pill or capsule for oral delivery containing an oral pharmaceutical composition (e.g., an oral formulation) of the present disclosure and a statin, where the oral pharmaceutical composition comprises, for example, CIVI008 and an oral delivery agent (such as SNAC, C10, or 5-CNAC, a derivative thereof, a pharmaceutically acceptable hydrate, solvate, or salt thereof; or any combination thereof). In one aspect, the combination therapy comprises a pill or capsule for oral delivery containing (i) CIVI008, (ii) an oral delivery agent (such as SNAC, C10, or 5-CNAC, a derivative thereof, a pharmaceutically acceptable hydrate, solvate, or salt thereof; or any combination thereof), and (iii) a statin. In one aspect, the combination therapy comprises a pill or capsule for oral delivery containing (i) CIVI008, (ii) SNAC, and (iii) a statin. In one aspect, the combination therapy comprises a pill or capsule for oral delivery comprising (i) CIVI008, (ii) C10, and (iii) a statin. In one aspect, the combination therapy comprises a pill or capsule for oral delivery comprising (i) CIVI008, (ii) 5-CNAC, and (iii) a statin.

[0397] Section 6: Controlled-Release Formulations In some aspects, oral pharmaceutical compositions of the present disclosure include components that facilitate transit through the stomach and upper intestinal tract (e.g., enteric coatings, pH-sensitive materials, and enzyme inhibitors). In some aspects, oral pharmaceutical compositions of the present disclosure may also include gelatin, for example, as a coating or thickening agent.

[0398] Enteric (slightly gastric soluble) coating materials, e.g., polymers, can dissolve in intestinal fluids (such as in the small intestine) at pH levels higher than that of the stomach (e.g., greater than 4.5), allowing release of the active agent in that region of the small intestine, but substantially preventing release in the upper gastrointestinal tract. In one aspect, the enteric material begins to dissolve in aqueous solution at a pH of about 4.5 to about 5.5. In another aspect, the enteric material rapidly dissolves in aqueous solution at a pH of about 5. In another aspect, the enteric material rapidly dissolves in aqueous solution at a pH of about 5.5.

[0399] Suitable enteric (poorly soluble in the stomach) materials include, but are not limited to: Crosslinked polyvinylpyrrolidone;Non-crosslinked polyvinylpyrrolidone;Hydroxypropyl methylcellulose phthalate, Hydroxypropyl methylcellulose acetate succinate, Cellulose acetate succinate;Cellulose acetate phthalate, Hydroxypropyl methylcellulose acetate succinate, Cellulose acetate trimellitate;Starch acetate phthalate;Polyvinyl acetate phthalate;Carboxymethylcellulose;Methylcellulose phthalate;Methylcellulose succinate;Methylcellulose phthalate succinate;Methylcellulose phthalate succinate;Methylcellulose phthalate half ester;Ethylcellulose succinate;Carboxymethyl Amides; potassium methacrylate / divinylbenzene copolymers; polyvinyl alcohols; polyoxyethylene glycols; polyethylene glycol; sodium alginate; galactomannan; carboxypolymethylene; sodium carboxymethyl starch; copolymers of acrylic acid and / or methacrylic acid with monomers selected from the following: methyl methacrylate, ethyl methacrylate, ethyl acrylate, butyl methacrylate, hexyl methacrylate, decyl methacrylate, lauryl methacrylate, phenyl methacrylate, methyl acrylate, isopropyl acrylate, isobutyl acrylate, or octadecyl acrylate, e.g., Evonik EUDRAGIT™-L series and -S series (including L100-55, L30 D-55, L100, S100, L12.5, and S12.5) available from EUDRAGIT Industries; polyvinyl acetate; fats; oils; waxes; fatty alcohols; shellac; zein; gluten; ethyl acrylic acid / maleic anhydride copolymer; maleic anhydride / vinyl methyl ether copolymer; styrene / maleic acid copolymer; 2-ethyl-hexyl-acrylic acid maleic anhydride; crotonic acid / vinyl acetate copolymer; glutamic acid / glutamic acid ester copolymer; carboxymethylethyl cellulose glycerol monooctanoate; polyarginine; polyethylene; polypropylene; polyethylene oxide; poly(ethylene terephthalic acid); poly(vinyl isobutyl ether); poly(vinyl chloride); and polyurethane.

[0400] Combinations of enteric materials may also be used. In some aspects, the enteric material dissolves rapidly at pH 5.5 or above, providing rapid dissolution in the upper intestinal tract. For example, the enteric material may be selected from copolymers of methacrylic acid and methyl methacrylic acid, and copolymers of methacrylic acid and ethyl acrylate. For example, the enteric polymer is poly(methacrylic acid-co-ethyl acrylic acid) 1:1 (EUDRAGIT® L30D-55 and EUDRAGIT® L100-55).

[0401] Other suitable enteric coatings include beeswax and glyceryl monostearate; beeswax, shellac, and cellulose; cetyl alcohol, mastic resin, and shellac; shellac and stearic acid; polyvinyl acetate and ethyl cellulose; and neutral copolymers of polymethacrylates (EUDRAGIT® L30D); copolymers of methacrylic acid and methyl methacrylate, or neutral copolymers of polymethacrylates containing metal stearates. Such coatings include mixtures of fats and fatty acids, shellac and shellac derivatives, and cellulose phthalates; e.g., those with free carboxyls.

[0402] As is known in the art, one or more plasticizers can be added to enteric polymers to increase their flexibility and reduce their brittleness. Suitable plasticizers include, for example, butyl citrate, triethyl citrate, diethyl phthalate, dibutyl sebacate, polyethylene glycols (PEGs such as PEG6000), acetyltriethyl citrate, and triacetin. In one aspect, the plasticizer is triethyl citrate. Some enteric materials are flexible and do not require plasticizers, but for more brittle polymers (e.g., EUDRAGIT® L / S series, EUDRAGIT® RL / RS, and EUDRAGIT® FS30D), plasticizers are effective; for example, for 1:1 poly(methacrylate-co-ethyl acrylate), triethyl citrate in the range of 5% to 30% by weight, and about 8% to about 12% by weight, based on the dry polymer weight.

[0403] In certain aspects, the enteric coating includes one or more anti-adherents (anti-adherents) to reduce film tack and prevent clumping, as is known in the art. Suitable anti-adherents include, but are not limited to, talc, glyceryl monostearate, fumed silica (e.g., AEROSIL™ 200), precipitated silica (e.g., SIPERNAT™ PQ), and magnesium stearate. The anti-adherent can be used in any suitable amount, for example, from about 10% to 100% by weight, from about 10% to about 50% by weight, from about 10% to about 30% by weight, or from about 15% to about 30% by weight, based on the dry polymer weight. For example, in one aspect, the amount is from 15% to about 30% by weight, based on the dry polymer weight.

[0404] As is known in the art, one or more surfactants may be added to the enteric coating mixture to increase substrate wettability and / or stabilize the suspension, including polysorbate 80, sorbitan monooleate, and sodium dodecyl sulfate, as well as other surfactants described herein.

[0405] The enteric coating can be carried out by any suitable process. Coating processes include, for example, pan coating, fluidized bed coating, and dry coating (e.g., toasted dry coating and electrostatic dry coating). Pan coating and fluidized bed coating using a solvent are established processes. In liquid coating, the enteric material and optional additives (e.g., colorants, plasticizers, anti-adherents) are mixed in an organic solvent or water to form a solution or dispersion. In a pan coater or fluidized bed dryer, the coating solution or coating dispersion is sprayed into a solid dosage form and then dried with hot air. For example, in the Wurster fluidized bed coating process, the coating liquid is sprayed from the bottom of the fluidized bed equipment. Alternatively, the coating liquid is used by top spray. In certain aspects, tangential spraying is used.

[0406] The amount of enteric material used is sufficient to achieve the desired acid resistance and release characteristics. For example, in one aspect, the amount of enteric coating agent meets the requirements of the United States Pharmacopeia Section 711 for delayed-release dosage forms (USP36-NF31) so that 10.0% by weight of the drug is not released after 2 hours in 0.1N HCl. In certain aspects, for example, when using the dissolution method of the United States Pharmacopeia Section 711 (USP36-NF31), the formulation releases at least 80% of the active ingredient in 20 minutes in a pH 6.8 buffer solution.

[0407] In one aspect, the enteric coating is present in an amount ranging from about 10% to 40%, or 25% to about 35%, as measured by weight gain compared to the uncoated particle core; or alternatively, a weight gain ranging from about 25% to about 31%, a weight gain ranging from about 27% to about 31%, or a weight gain ranging from about 28.5% to about 31%, based on the weight of the uncoated particle core.

[0408] The formulation may include a capsule shell. Soft and hard capsule shells are known. In one aspect, the capsule shell is a hard capsule shell, such as a gelatin capsule shell or a vegetable-based hard capsule shell. In certain aspects, the capsule shell includes one or more enteric coating agents described herein. During storage under accelerated conditions, gelatin capsules may disintegrate. Therefore, in certain aspects, the formulation may include a hydroxypropylmethylcellulose capsule shell.

[0409] The solid dosage forms of the present invention may be formulated to prevent or delay disintegration in the stomach. Controlled release formulations suitable for use in the present invention may, for example, include an enteric coating or may ...

Claims

1. A composition for oral administration to a subject in need thereof, in a dosage form including an enteric coating. (i) CIVI008, and (ii) N-(5-chlorosalicyloyl)-8-aminocaprylic acid (5-CNAC) or a salt thereof, such as the monosodium salt, the disodium salt, or a combination thereof. Including, The composition is formulated for release of CIVI008 in the small intestine of a subject following oral administration. composition.

2. The composition of claim 1, further comprising at least one excipient or a combination thereof.

3. The composition of claim 1, wherein the dosage form comprises a hydrogel, a drug delivery system, a liposome, a micelle, a lipid nanoparticle, a microsphere, a nanoparticle, a tablet, or a capsule.

4. The composition of claim 1, wherein the enteric coating comprises a pH-sensitive coating that dissolves in intestinal fluids at pH values ​​higher than the pH value of the stomach, e.g., a pH value greater than 4.5, or a pH value of about 5.5 to about 7.5, or a pH value of about 7.2 to about 7.

3.

5. The composition described in claim 1, wherein the enteric coating does not substantially disintegrate or dissolve in the stomach and dissolves in the intestine.

6. The composition of claim 4, wherein the pH-sensitive coating comprises hydroxypropyl methylcellulose, such as hydroxypropyl methylcellulose acetate succinate, e.g., AQOAT (registered trademark), and / or an anionic copolymer, such as a copolymer of methacrylic acid and methacrylic methacrylic acid, e.g., EUDRAGIT (registered trademark) polymer.

7. The composition of claim 1, further comprising one or more additional therapeutic agents selected from the group consisting of statins (e.g., lovastatin, cerivastatin, pravastatin, atorvastatin, simvastatin, rosuvastatin, and fluvastatin), ezetimibe, bile-sorbing resins, nicotinic acid, fibric acid derivatives, probucol, neomycin, dextrothyroxine, plant stanol esters, cholesterol absorption inhibitors, implitapide, inhibitors of bile acid transporters, regulators of hepatic CYP7a, estrogen replacement therapy agents, and anti-inflammatory agents.

8. The composition of claim 1, further comprising at least one enzyme inhibitor that prevents enzymatic degradation of CIVI008 and / or additional therapeutic agents in the stomach and / or intestine.

9. The dosage form is a capsule or a tablet, (i) an amount of CIVI008 in the range of about 1 mg to about 100 mg, about 5 mg to about 100 mg, about 10 mg to about 100 mg, about 20 mg to about 100 mg, or about 20 mg to about 50 mg, and (ii) about 100 mg to about 200 mg of 5-CNAC The composition of claim 1 , comprising:

10. The capsule or tablet comprises: i. about 5 mg CIVI008 and about 200 mg 5-CNAC; ii. about 10 mg CIVI008 and about 200 mg 5-CNAC; iii. about 15 mg CIVI008 and about 200 mg 5-CNAC; iv. about 20 mg of CIVI008 and about 200 mg of 5-CNAC; v. about 25 mg of CIVI008 and about 200 mg of 5-CNAC, or vi. about 30 mg of CIVI008 and about 200 mg of 5-CNAC; and about 200 mg of 5-CNAC, The composition of claim 9.

11. (a) i. about 5 mg CIVI008 and about 200 mg 5-CNAC; ii. about 10 mg CIVI008 and about 200 mg 5-CNAC; iii. about 15 mg CIVI008 and about 200 mg 5-CNAC; iv. about 20 mg of CIVI008 and about 200 mg of 5-CNAC; v. about 25 mg of CIVI008 and about 200 mg of 5-CNAC, or vi. about 30 mg of CIVI008 and about 200 mg of 5-CNAC; (b) an enteric coating; and (c) one or more additional therapeutic agents selected from the group consisting of statins (e.g., lovastatin, cerivastatin, pravastatin, atorvastatin, simvastatin, rosuvastatin, and fluvastatin), ezetimibe, bile-adsorbing resins, nicotinic acid, fibric acid derivatives, probucol, neomycin, dextrothyroxine, plant stanol esters, cholesterol absorption inhibitors, implitapide, inhibitors of bile acid transporters, regulators of hepatic CYP7a, estrogen replacement therapy agents, and anti-inflammatory agents. A tablet or capsule comprising a composition comprising: the composition is formulated for release of CIVI008 and any one or more additional therapeutic agents in the small intestine of a subject following oral administration; Tablets or capsules.

12. The tablet or capsule of claim 11, wherein the capsule is a gelatin capsule, such as a hard shell gelatin capsule, and comprises CIVI008 and 5-CNAC in a dry mix, and optionally one or more excipients and / or additional therapeutic agents.

13. The tablet or capsule of claim 11 for use in reducing PCSK9 expression levels and / or activity, or cholesterol levels, in a subject in need thereof, such as a subject with atherosclerosis, hyperlipidemia, hypercholesterolemia, HDL / LDL cholesterol imbalance, coronary artery disease (CAD), or coronary heart disease (CHD).

14. A tablet or capsule as described in claim 13 suitable for administration at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes before a meal.

15. A method for producing a dosage form comprising the composition of claim 1 or the tablet or capsule of claim 11, comprising the steps of: (a) (1) a first composition comprising CIVI008; (2) a second composition comprising 5-CNAC or a salt thereof; (3) optionally, one or more excipients, and (4) Optionally, an additional therapeutic agent dry mixing the (b) encapsulating the dry blend obtained in step (a) Including, the capsule comprises an enteric coating formulated for release of CIVI008 and an additional therapeutic agent in the small intestine of a subject following oral administration. method.

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