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WO2025064819A1 - Compounds and methods for inhibiting lpa - Google Patents

Compounds and methods for inhibiting lpa Download PDF

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Publication number
WO2025064819A1
WO2025064819A1 PCT/US2024/047698 US2024047698W WO2025064819A1 WO 2025064819 A1 WO2025064819 A1 WO 2025064819A1 US 2024047698 W US2024047698 W US 2024047698W WO 2025064819 A1 WO2025064819 A1 WO 2025064819A1
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nucleosides
modified
certain embodiments
modified oligonucleotide
oligomeric
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Dieter KUBLI
Adam Mullick
Thazha P. Prakash
Eric E. Swayze
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Ionis Pharmaceuticals Inc
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Ionis Pharmaceuticals Inc
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/11—Antisense
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate

Definitions

  • oligomeric duplexes including methods for modulating the amount and/or activity of ApoA expression, LPA RNA, and/or Lp(a), as well as methods for treating or managing inflammatory, cardiovascular and/or metabolic diseases, disorders or conditions, and in certain embodiments, ameliorating at least one symptom of an inflammatory, cardiovascular and/or metabolic disease, disorder or condition.
  • the lipoprotein(a) [Lp(a)] particle comprises a unique LDL particle in which one apolipoprotein B (apoB) protein is linked via a disulfide bond to a single apolipoprotein(a) [apo(a)] protein.
  • the apo(a) protein shares a high degree of homology with plasminogen, particularly within the kringle IV type 2 repetitive domain.
  • Levels of circulating Lp(a) are inversely proportional to the number of kringle IV type 2 variable repeats present in the molecule and, as both alleles are co-expressed within individuals, can display heterozygous plasma isoform profiles among individuals (Kraft et al., Eur J Hum Genet, 1996; 4(2): 74-87).
  • apo(a) may be responsible for its pro- thrombotic and anti-fibrinolytic properties, potentially enhancing atherosclerotic progression.
  • Apo(a) has been shown to preferentially bind oxidized phospholipids and potentiate vascular inflammation (Bergmark et al., J Lipid Res 2008; 49:2230-2239; Tsimikas et al., Circulation. 2009; 119(13): 1711— 1719).
  • Lp(a) particle may also stimulate endothelial permeability, induce plasminogen activator inhibitor type- 1 expression and activate macrophage interleukin-8 secretion (Koschinsky and Marcovina, Curr Opin Lipidol 2004; 15: 167-174).
  • genetic association studies revealed that Lp(a) was an independent risk factor for myocardial infarction, stroke, peripheral vascular disease and abdominal aortic aneurysm (Rifai et al., Clin Chem 2004; 50: 1364-71; Erqou et al., JAMA 2009;302:412-23; Kamstrup et al., Circulation 2008;l 17: 176-84).
  • Examples of indirect apo(a) inhibitors for treating cardiovascular disease in a clinical setting include aspirin, Niaspan, Mipomersen, Anacetrapib, Epirotirome and Lomitapide which reduce plasma Lp(a) levels by 18%, 39%, 32%, 36%, 43% and 17%, respectively.
  • Lp(a) apheresis has also been used in the clinic to reduce apo(a) containing Lp(a) particles.
  • proposed therapeutic strategies to treat cardiovascular disease by directly targeting apo(a) levels have been described (e.g., U.S. Patent Nos. 5,877,022, 8,138,328, 8,673,632 and 7,259,150; U.S. Patent Publication No.
  • oligomeric duplexes for reducing the amount or activity for reducing the amount or activity of LPA RNA and reducing the expression of Apo(a) protein in a cell or subject and/or Lp(a) in a subject.
  • a subject has a disease or disorder associated with misregulation of lipoproteins or a mutation in lipoprotein regulation pathway.
  • the subject has elevated lipoprotein (a).
  • the subject has or is at risk for a severe a cardiovascular disease, disorder or condition.
  • the subject has or is at risk for a metabolic or inflammatory disease, disorder or condition.
  • agents useful for reducing the amount or activity of LPA RNA are oligomeric duplex, oligomeric compound, or composition as provided herein.
  • agents useful for decreasing expression of Apo(a) or levels of lipoprotein (a) are oligomeric compounds, oligomeric duplexes, antisense agents, and/or RNAi agents.
  • modified oligonucleotides and compounds and compositions comprising them including, but not limited to, antisense agents, oligomeric agents, oligomeric duplexes and pharmaceutical compositions comprising modified oligonucleotides.
  • a modified oligonucleotide provided herein comprises a nucleobase sequence at least 80% complementary to an equal length portion of an LPA nucleic acid.
  • the modified oligonucleotide consists of 12 to 35, 14 to 30, 15 to 28, 16 to 25, or 18 to 23 linked nucleosides targeting LPA nucleic acid.
  • a modified oligonucleotide provided herein comprises a sequence of nucleobase s complementary to an equal length portion of the nucleobase sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2.
  • provided oligomeric duplexes comprise a first oligomeric compound and a second oligomeric compound, wherein a first oligomeric compound comprises a modified oligonucleotide consisting of 18 to 50 linked nucleosides, wherein the nucleobase sequence of the first oligomeric compound comprises at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID Nos: 6- 829 and 3078-3083, wherein each of the nucleosides of the modified oligonucleotide comprises a modified sugar moiety or sugar surrogate and wherein no more than 22%, no more than 20%, no more than 18%, no more than 15%,
  • a modified oligonucleotide provided herein comprises at least one modified sugar moiety and/or at least one modified intemucleoside linkage.
  • an oligomeric compound provided herein comprises a modified oligonucleotide comprising at least one 3’-fluoro-hexitol sugar moiety.
  • an oligomeric compound provided herein comprises a modified oligonucleotide comprising at least one a 2’ -deoxynucleoside.
  • an oligomeric compound provided herein comprises a modified oligonucleotide conjugated to a cell targeting agent.
  • Modified oligonucleotides and compositions comprising them including, but not limited to, oligomeric duplexes, oligomeric compounds, modified oligonucleotides and composition described herein are useful for reducing or inhibiting LPA expression in a cell, organ, tissue, system, organism or subject.
  • oligomeric compounds comprising a modified oligonucleotide comprising a nucleobase sequence selected from any one of SEQ ID NO: 6-829 and 3078-3083, wherein the modified oligonucleotide comprises a modified sugar motif independently selected from one of efyyyfyyyyyyyyfyfyyyyyyyy, yfyyyfyyyyyyfyfyyyyyyyyyyyyyyyyyyyyyyyyyy, efyydyyeyyyyyydydyyyyyyyee, efyydyyyyyyyfyfyyyyyee, e[FHNA]yyfyyyyyyfyfyyyyyyyee, and e[FHNA]yyyfyeyyyfyfyyyyyyyyyyyyyy
  • oligomeric compounds comprising a modified oligonucleotide comprising a sequence comprising at least 18, at least 19, or at least 20 contiguous nucleobases of any one of the sequences of SEQ ID NOs: 1541-2364 and 3090-3095.
  • oligomeric compounds comprising a modified oligonucleotide comprising a nucleobase sequence selected from any one of SEQ ID NO: 830-1540 and 3096-3101, wherein the modified oligonucleotide comprises a modified sugar motif independently selected from one of yyyyyyfyfffyyyyyyyyyyy and eeyyyyyyyyyyffyyyyyyyyee, wherein each ‘e’ represents a 2'-M0E sugar moiety, each ‘y’ represents a 2'-0Me sugar moiety, and each ‘f represents a 2'-F sugar moiety, wherein all except 0, 1, or 2 modifications are identical to the sugar motif.
  • oligomeric compounds comprising a modified oligonucleotide comprising a sequence comprising at least 18, at least 19, or at least 20 contiguous nucleobases of any one of the sequences of SEQ ID NOs: 2365-3075 and 3084-3089.
  • oligomeric compounds comprising a modified oligonucleotide having a sequence selected from any one of SEQ ID NO: 6-1540, 3078-3089, and 3096- 3101.
  • oligomeric duplexes comprising an oligomeric compound having a sequence selected from any one of SEQ ID NO: 6-3075 and 3078-3101.
  • oligomeric agents comprising an oligomeric compound having a sequence selected from any one of SEQ ID NO: 1541-3075 and 3084-3095.
  • oligomeric duplexes comprising a first oligomeric compound having a sequence selected from any one of SEQ ID NO: 1541-2364 and 3090-3095, and comprising a second oligomeric compound having a sequence selected from any one of SEQ ID NO: 2365-3075 and 3084-3089.
  • oligomeric duplexes consisting of a first oligomeric compound having a sequence selected from any one of SEQ ID NO: 1541-2364 and 3090-3095, and a second oligomeric compound having a sequence selected from any one of SEQ ID NO: 2365-3075 and 3084-3089.
  • methods for reducing or inhibiting LPA expression, LPA RNA levels and/or Apo(a) protein levels and/or Lp(a) activity in a cell, tissue, organ or subject include contacting a cell or subject with a composition provided herein, comprising, e.g., an oligomeric duplex, oligomeric compound, modified oligonucleotide as described herein.
  • the subject is a human who has or is at risk of having a cardiovascular, metabolic, or inflammatory disease, disorder, condition or injury associated with increased lipoprotein a levels, misregulation of lipoprotein turnover or a mutation in LPA.
  • the subject is a human who has or is at risk of having hypertriglyceridemia. In certain embodiments, the subject is a human who has or is at risk of having atherosclerotic cardiovascular disease (ASCVD) or coronary artery disease (CAD).
  • ASCVD atherosclerotic cardiovascular disease
  • CAD coronary artery disease
  • a method of treating a disease, disorder, condition or injury associated with lipoprotein metabolism misregulation, regulation of lipoprotein turnover or a mutation in LPA comprises administering to a subject, e.g., a human subject, having, or at risk of having, a disease, disorder or condition associated with elevated Lp(a), a provided oligomeric duplex, oligomeric compound, or composition provided herein, wherein the disease, disorder, condition or injury is selected from a cardiovascular disease, disorder, condition, a metabolic disease, disorder, or condition, or an inflammatory disease disorder or condition.
  • the subject has or is at risk for developing cardiovascular disease (CVD) coronary artery disease (CAD), hypercholesterolemia, myocardial infarction (MI), peripheral arterial disease (PAD), calcific aortic valve disease (CAVD), aortic stenosis, atherosclerotic cardiovascular disease (ASCVD), atherosclerosis, dyslipidemia, thrombosis, or stroke.
  • CVD cardiovascular disease
  • CAD coronary artery disease
  • MI myocardial infarction
  • PDA peripheral arterial disease
  • CAVD calcific aortic valve disease
  • ASCVD aortic stenosis
  • methods of treating provided herein result in ameliorating (whether by reduced frequency, severity) a at least one symptom of a disease, disorder, condition or injury associated with lipoprotein metabolism misregulation.
  • methods of treating provided herein result in preventing, delay or postponing, or slowing the development or progression of at least one symptom of a disease, disorder
  • a symptom of hypertriglyceridemia, hyperlipidemia, dyslipidemia, and/or hyperlipoproteinemia include, but are not limited to, abdominal pain, physical fatigue, difficulty thinking, diarrhea, acute pancreatitis, eruptive xanthomas, lipemia retinalis, or hepatosplenomegaly, or a combination of two or more of the foregoing in the subject.
  • methods provided herein for preventing, treating, ameliorating, delaying the onset of, or reducing frequency of at least one symptom of hypertriglyceridemia, hyperlipidemia, dyslipidemia, and/or hyperlipoproteinemia include administering to a subject, e.g., a human subject, having or at risk of having at least one symptom a composition provided herein, e.g., a modified oligonucleotide, oligomeric duplex, oligomeric compound, or pharmaceutical composition provided herein,
  • a substituent at the “2 '-position” means that the substituent is directly attached to the carbon at the 2'-position of a fiiranosyl sugar moiety.
  • 2'-deoxynucleoside means a nucleoside comprising a 2'-deoxyfiiranosyl sugar moiety. Unless otherwise indicated, a 2'-deoxynucleoside is a 2'-p-D-deoxynucleoside which comprises a 2'-P-D-deoxyribosyl sugar moiety, which is in the P-D configuration in naturally occurring deoxyribonucleic acid (DNA).
  • a 2'-deoxynucleoside or a nucleoside comprising an unmodified 2'- deoxyribosyl sugar moiety may be abasic, comprise a modified nucleobase, or may comprise an RNA nucleobase (uracil).
  • 2'-deoxy sugar moiety means a 2'-H(H) deoxyfuranosyl sugar moiety. Unless otherwise indicated, a 2'-deoxy sugar moiety is a 2'-P-D-deoxyribosyl sugar moiety, which has the P-D stereochemical configuration in naturally occurring deoxyribonucleic acid (DNA).
  • DNA deoxyribonucleic acid
  • 2'-M0E means a 2'-OCH2CH2OCH3 group at the 2'-position of a furanosyl sugar moiety.
  • a “2'-M0E sugar moiety” means a sugar moiety with a 2'-OCH2CH2OCH3 group at the 2'-position of a furanosyl sugar moiety. Unless otherwise indicated, a 2'-M0E sugar moiety is in the P-D-ribosyl stereochemical configuration. “MOE” means O-methoxyethyl.
  • 2'-M0E nucleoside or “2'- OCH2CH2OCH3 nucleoside” means a nucleoside comprising a 2'-M0E sugar moiety (or 2'-OCH2CH2OCH3 furanosyl sugar moiety).
  • 2'-0Me means a 2’-O-methyl or 2'-OCH3 group at the 2'-position of a furanosyl sugar moiety.
  • a “2'-0Me sugar moiety” means a sugar moiety with a 2’-O-methyl or a 2'-OCH3 group at the 2'-position of a furanosyl sugar moiety. Unless otherwise indicated, a 2'-0Me sugar moiety is in the P-D-ribosyl stereochemical configuration.
  • 2'-0Me nucleoside means a nucleoside comprising a 2'-0Me sugar moiety.
  • 2'-F means a 2'-fluoro group at the 2'-position of a furanosyl sugar moiety.
  • a “2'-F sugar moiety” means a sugar moiety with a 2'-F group at the 2'-position of a furanosyl sugar moiety. Unless otherwise indicated, a 2'-F sugar moiety is in the P-D-ribosyl configuration.
  • 2'-F nucleoside means a nucleoside comprising a 2'-F sugar moiety.
  • 2'-NMA nucleoside means a nucleoside comprising a 2'-NMA sugar moiety.
  • 2 '-substituted nucleoside means a modified nucleoside comprising a 2'- substituted furanosyl sugar moiety.
  • 2'-substituted nucleosides include, but are not limited to, a 2'-0Me nucleoside, a 2'-M0E nucleoside, a 2'-F nucleoside, a 2'-NMA nucleoside, a cEt nucleoside, and a LNA nucleoside.
  • 2 ’-substitution or “2 '-substituted sugar moiety” means a modified furanosyl sugar moiety wherein the 2'-position is attached to at least one substituent other than H or OH.
  • a 2'- substituted sugar moiety includes a bicyclic sugar moiety wherein the second ring is joined to the furanosyl ring at the 2'-position.
  • 2'-substituted sugar moieties include, but are not limited to, 2'-0Me sugar moieties, 2'-M0E sugar moieties, 2'-F sugar moieties, 2'-NMA sugar moieties, cEt sugar moieties, and LNA sugar moieties.
  • 5-methylcytosine means a cytosine modified with a methyl group attached at the 5 position.
  • a 5-methylcytosine is a modified nucleobase.
  • abasic nucleoside means a modified nucleoside in which the sugar moiety is not attached to a nucleobase.
  • acyclic sugar surrogate nucleoside means a nucleoside having Formula II, Formula III, or Formula IV:
  • Ri is H, OH, halogen, Ci-Ce alkyl, Ci-Ce alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, or (CH2) q Rs;
  • R2, R3, and R4 are each independently H, OH, halogen, Ci-Ce alkyl, Ci-Ce alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, S-CH3, N(CH3)(CH3), OCH2CH2OCH3, O-alkylamino, or (CH2) q Rs;
  • Ei is H, Ci-Ce alkyl or substituted Ci-Ce alkyl
  • Rs and Re are independently H, OH, Ci-Ce alkyl, or N(R 7 ); wherein if Rs is OH, then Re is not OH;
  • Rs is OH, halogen, methoxy, ethoxy, azido, C2-C6 alkenyl, or C2-C6 alkynyl, and q is 1, 2, or 3;
  • Bx is a nucleobase.
  • acyclic sugar surrogate means the sugar moiety of an acyclic sugar surrogate nucleoside.
  • ameliorate with reference to a symptom of a disease, means improvement in, or lessening, or preclusion of, at least one symptom of a disease. Amelioration may be reduction in severity or frequency of a symptom or the delayed onset, prevention of occurrence of, or slowing of progression in the severity or frequency of, a symptom. Progression, frequency, or severity indicators may be determined by subjective or objective measures known in the art and/or described herein.
  • antisense activity means any detectable and/or measurable change attributable (whether directly and/or indirectly) to hybridization of an antisense oligonucleotide to a target nucleic acid.
  • compounds have antisense activity when they alter the amount or activity of a target nucleic acid by 25% or more in an in vitro assay; or, for example compounds have antisense activity when they alter the amount or activity of a target nucleic acid by 25% or more in an in vivo assay.
  • Antisense activity may be assessed in a standard assay.
  • antisense activity is a reduction or inhibition in the amount or expression of a target nucleic acid, or a protein encoded by such target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the oligonucleotide.
  • antisense agent means an oligomeric agent comprising an antisense oligonucleotide.
  • antisense oligonucleotide means an oligonucleotide having at least one region (a “targeting region”) that is complementary to a target nucleic acid (e.g. , a target region).
  • An antisense oligonucleotide may be paired with a second oligonucleotide (herein, a “sense oligonucleotide”) that is complementary to the antisense oligonucleotide (for example, forming an “oligomeric duplex”), may be an unpaired antisense oligonucleotide (herein, a single-stranded antisense oligonucleotide), or may be a “hairpin oligonucleotide” that has at least one region that is self-complementary.
  • bicyclic sugar or “bicyclic sugar moiety” means a modified sugar moiety comprising a furanosyl sugar moiety and a second ring, wherein the second ring is formed via a bridge connecting two non-geminal atoms in the ring of the furanosyl sugar moiety, thereby forming a bicyclic structure.
  • bicyclic sugar moieties include locked nucleic acid (LNA) sugar moieties and constrained ethyl (cEt) sugar moieties as defined herein.
  • bicyclic nucleoside or “BNA” means a nucleoside comprising a bicyclic sugar moiety.
  • cell-targeting moiety means a conjugate group or portion of a conjugate group that has affinity for a particular cell type or particular cell types.
  • a cell-targeting moiety may have affinity for a cell surface moiety, such as a cell surface receptor on a particular cell type.
  • cleavable moiety means a group of atoms comprising at least one bond that is cleaved under physiological conditions, e.g., in a cell, in a subject.
  • a cleavable moiety cleaved inside a cell or sub-cellular compartment, such as an endosome or lysosome.
  • a cleavable moiety may be cleaved by endogenous enzymes, such as nucleases.
  • a cleavable moiety may be cleaved by endogenous change in conditions such as pH.
  • complementary nucleobase(s) or “complementary” in reference to nucleobase(s) means nucleobases that form hydrogen bonds with another when two regions of linked nucleosides (e.g., an oligonucleotide and a target nucleic acid; two oligonucleotides).
  • Complementary nucleobase pairs include, but are not limited to, adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), 5 -methylcytosine ( m C) and guanine (G).
  • hypoxanthine the nucleobase of the nucleoside inosine (I)
  • hypoxanthine (I) is considered a complementary nucleobase to thymine (T), adenine (A), uracil (U), and cytosine (C).
  • nucleobase sequence means the order of contiguous nucleobases in a strand of linked nucleosides or a region thereof (e.g., an oligonucleotide or region thereof, or a target nucleic acid or region thereof) independent of any sugar or intemucleoside linkage modification.
  • Complementary nucleobase sequences may be nucleobase sequences of two separate strands of linked nucleosides or region thereof (e.g., an oligonucleotide and a region of a target nucleic acid); or complementary nucleobase sequences may be two regions of a single strand of linked nucleosides (e.g., self-complementary regions of a hairpin oligonucleotide).
  • first nucleobase sequence e.g., an oligonucleotide
  • second nucleobase sequence e.g., a target nucleic acid or another oligonucleotide
  • complementarity is expressed as a percent, such percent represents the percent of nucleobases within one nucleobase sequence that are complementary to nucleobases within an equal length second sequence when the sequences are aligned.
  • “complementary” is assumed to be at least 70%.
  • Complementary nucleobase sequences may be 75%, 80%, 85%, 90%, 95%, or 100% complementary. For example, if a nucleobase sequence of an oligonucleotide consisting of 20 nucleosides is 80% complementary to another nucleobase sequence, then 16 of the nucleobase pairs are complementary nucleobases, and there are 4 mismatches when the sequences are aligned.
  • nucleobase sequence of an oligonucleotide consisting of 20 nucleosides is at least 80% complementary to another nucleobase sequence, then 16, 17, 18, 19, or 20 of the nucleobase pairs are complementary nucleobases, and there are 0-4 mismatches when the sequences are aligned.
  • “fully complementary” or “100% complementary” means that each nucleobase pair of two nucleobase sequences is complementary when the equal length sequences are aligned.
  • conjugate group means a group of atoms including a conjugate moiety and a conjugate linker that attaches the conjugate moiety to the oligonucleotide.
  • conjugate linker means a single bond or a group of atoms comprising at least one bond that connects a conjugate moiety to an oligonucleotide.
  • conjugate moiety means a group of atoms that when covalently bound to a molecule (e.g., an oligonucleotide) modifies one or more properties of such molecule compared to the same molecule lacking the conjugate moiety, wherein such properties include, but are not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge, and clearance.
  • constraint ethyl or “cEt” or “cEt sugar moiety” means a P-D ribosyl bicyclic sugar moiety wherein the second ring of the bicyclic sugar is formed via a bridge connecting the 4'-carbon and the 2'-carbon of the -D ribosyl sugar moiety, wherein the bridge has the formula 4'-CH(CH3)-O-2', and wherein the methyl group of the bridge is in the S configuration.
  • cEt nucleoside means a nucleoside comprising a cEt sugar moiety.
  • cyclic sugar surrogate nucleoside is a nucleoside having Formula I:
  • J is H, Ci-Ce alkyl, or C2-C6 alkenyl
  • Rs is H, halogen, Ci-Ce alkyl, substituted Ci-Ce alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or N(E2)(E 3 );
  • Ei, E 2 and E 3 are each, independently, H, Ci-Ce alkyl or substituted Ci-Ce alkyl; n is 0 or 1; m is 0 or 1; p is 0 or 1; q is from 1 to 6; s is 0 or 1; j is 0 or 1;
  • Bx is a nucleobase; and provided that if X is O, Z is C G1G2), and Q is CH, then m is 1.
  • cyclic sugar surrogate means the sugar moiety of a cyclic sugar surrogate nucleoside, and is represented by Formula la herein.
  • double-stranded refers to hybridized or bound complementary regions, including those between two separate strands of linked nucleosides (e.g., an antisense oligonucleotide and a sense oligonucleotide) and those within a single strand of linked nucleosides (e.g., a hairpin oligonucleotide).
  • Paired complementary regions of two separate strands of linked nucleosides form a duplex of the separate strands.
  • Paired complementary regions of a single strand of linked nucleosides i.e., a first region of the strand of linked nucleosides and a second region of the strand of linked nucleosides
  • a “furanosyl sugar moiety” is a group of atoms that comprises a furanose ring and optional substituents, and is numbered according to the structure below, with optional additional substituents at any of the 1', 2', 3', 4', and 5' positions.
  • hybridize or “hybridization” means the process of two complementary complementary regions of linked nucleosides (e.g., oligonucleotides, nucleic acids) annealing to form a doublestranded region. While not limited to a particular mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.
  • intemucleoside linkage means the covalent linkage between adjacent nucleosides in an oligonucleotide.
  • unmodified intemucleoside linkage means a phosphodiester intemucleoside linkage.
  • modified intemucleoside linkage means any intemucleoside linkage other than a phosphodiester intemucleoside linkage.
  • a “phosphorothioate intemucleoside linkage” is a modified intemucleoside linkage in which one of the non-bridging oxygen atoms of a phosphodiester intemucleoside linkage is replaced with a sulfur atom.
  • inverted nucleoside means a nucleoside having a 3' to 3' and/or 5' to 5' intemucleoside linkage.
  • linked nucleosides are nucleosides that are connected in a contiguous sequence (z.e., nucleosides immediately adjacent to one another, no additional nucleosides are presented between those that are linked).
  • a “mismatch” between two aligned strands of linked nucleosides means that two nucleobases at a specified position of the aligned nucleobase sequences are not complementary nucleobases as defined herein.
  • modified nucleoside means a compound or subunit comprising a sugar moiety and optionally a nucleobase, wherein the sugar moiety is modified and/or the nucleobase is modified or absent.
  • modified sugar moiety means a sugar moiety other than a P-D-ribosyl sugar moiety in RNA or a -D-deoxyribosyl sugar moiety in DNA.
  • a modified sugar moiety is selected from a modified fiiranosyl sugar moiety, a cyclic sugar surrogate, an acyclic sugar surrogate, or a sugar mimic.
  • a “modified nucleobase” means a nucleobase other than unmodified A, T, C, U, or G capable of pairing with at least one unmodified nucleobase.
  • a “5 -methylcytosine” is a modified nucleobase.
  • Inosine (I) is a nucleoside comprising the modified nucleobase hypoxanthine.
  • motif means a pattern of independently unmodified and/or independently modified sugar moieties, nucleobases, and/or intemucleoside linkages in an oligonucleotide.
  • non-bicyclic modified sugar moiety means a modified fiiranosyl sugar moiety comprising a modification, such as a substituent, that does not form a bridge between two atoms of the sugar to form a second ring.
  • nucleobase means an unmodified nucleobase or a modified nucleobase.
  • nucleobase sequence of a reference SEQ ID NO, refers only to the order of contiguous nucleobases provided in such SEQ ID NO, independent of any sugar or intemucleoside linkage modifications and therefore, unless otherwise indicated, includes compounds wherein each sugar moiety and each intemucleoside linkage, independently, is modified or unmodified, irrespective of the presence or absence of modifications indicated in the referenced SEQ ID NO.
  • nucleoside means an “unmodified nucleoside” or a “modified nucleoside”.
  • oligomeric agent means a compound or complex comprising or consisting of at least one modified oligonucleotide and optionally one or more additional associated features selected from: (a) one or more conjugate groups, which may be covalently attached directly or indirectly to any oligonucleotide of such oligomeric agent; (b) one or more terminal groups; and (c) one or more additional modified or unmodified oligonucleotides, each of which may be hybridized to or covalently linked to the at least one modified oligonucleotide and/or to each other;.
  • oligomeric compound means a compound comprising a modified oligonucleotide and optionally one or more covalently linked chemical features selected from one or more conjugate group and one or more terminal group.
  • oligonucleotide means a strand of linked nucleosides, wherein, independently each nucleoside and/or independently each intemucleoside linkage of the strand of linked nucleosides may be independently modified or unmodified. Unless otherwise indicated, oligonucleotides consist of 12-50 linked nucleosides. Unless otherwise indicated, no more than 10% of the nucleosides of an oligonucleotide are abasic nucleosides. As used herein, “modified oligonucleotide” means an oligonucleotide, wherein at least one nucleoside and/or intemucleoside linkage is modified.
  • unmodified oligonucleotide means an oligonucleotide consisting of unmodified nucleosides linked by phosphodiester intemucleoside linkages.
  • An oligonucleotide may be paired with a second oligonucleotide that is complementary to the oligonucleotide to form an oligomeric duplex, or it may be unpaired.
  • a pharmaceutical composition means a mixture of substances suitable for administration to a subject.
  • a pharmaceutical composition may comprise an agent (e.g., an oligomeric agent, duplex, or antisense agent) and a sterile aqueous solution.
  • a pharmaceutical composition may show activity in certain cell lines.
  • “pharmaceutically acceptable carrier or diluent” means an ingredient in a pharmaceutical composition suitable for use in administering to a subject.
  • a “carrier” or “diluent” lacks pharmacological activity but is desirable in preparing a pharmaceutical composition.
  • pharmaceutically acceptable salts means physiologically and pharmaceutically acceptable salts of compounds. Pharmaceutically acceptable salts retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
  • RNA nucleoside means a nucleoside comprising an unmodified RNA sugar moiety.
  • An RNA nucleoside may comprise a modified or unmodified nucleobase.
  • An RNA nucleoside may comprise a thymine nucleobase or a modified nucleobase, or may be an abasic nucleoside.
  • RNA sugar moiety means an unmodified RNA sugar moiety
  • RNase H agent means an antisense agent that acts, at least in part, through RNase H to modulate a target nucleic acid and/or protein encoded by a target nucleic acid.
  • RNase H agents may be single-stranded, or RNase H agents may be double -stranded.
  • RNase H compounds may comprise conjugate groups and/or terminal groups.
  • RNase H agents may modulate the amount and/or activity of a target nucleic acid.
  • the term RNase H agent excludes antisense agents that act principally through RISC/Ago2.
  • RNAi agent means an antisense agent that acts, at least in part, through RISC or Ago2 to modulate a target nucleic acid and/or a protein encoded by a target nucleic acid.
  • RNAi agents include, but are not limited to double-stranded siRNA, single-stranded RNAi (ssRNAi), and microRNA, including microRNA mimics.
  • RNAi agents may comprise conjugate groups and/or terminal groups.
  • an RNAi agent modulates the amount and/or activity of a target nucleic acid.
  • the term RNAi agent excludes antisense agents that act through RNase H.
  • single -stranded in reference to a nucleic acid (e.g., an oligonucleotide) means that the strand or region is unpaired; that is, the strand of linked nucleosides is not part of a duplex, not part of a double-stranded region.
  • Single-stranded nucleic acids e.g., single-stranded oligonucleotides
  • stabilized phosphate moiety means a 5 '-phosphate analog that is metabolically more stable than a 5 '-phosphate as naturally occurs on DNA or RNA.
  • stereochemical or “stereorandom chiral center” in the context of a population of molecules of identical molecular formula means a chiral center that is not controlled during synthesis, or enriched following synthesis, for a particular absolute stereochemical configuration.
  • the absolute stereochemical configuration of a chiral center can be controlled by using stereochemically-pure starting materials, e.g., using P-D-ribosyl nucleoside monomers for oligonucleotide synthesis.
  • the stereochemical configuration of a chiral center is random when it is the result of a synthetic method that is not designed to control the stereochemical configuration.
  • the number of molecules having the (.S') configuration of the stereorandom chiral center may be the same as the number of molecules having the (/?) configuration of the stereorandom chiral center (“racemic”).
  • the stereorandom chiral center may not be racemic because one absolute configuration predominates following synthesis, e.g., due to the action of non-chiral reagents near the enriched stereochemistry of an adjacent sugar moiety.
  • the stereorandom chiral center may be at the phosphorous atom of a stereorandom phosphorothioate or stereorandom mesyl phosphoramidate intemucleoside linkage.
  • a “strand” or “strand of linked nucleosides” means contiguous linked nucleosides connected via intemucleoside linkages.
  • a strand of linked nucleosides has a nucleobase sequence.
  • subject means a human or a non-human animal.
  • sugar moiety means an unmodified sugar moiety or a modified sugar moiety.
  • “sugar mimic” means a group of atoms forming the portion of a nucleoside corresponding to the P-D-ribosyl sugar in RNA other than a modified furanosyl sugar moiety, a cyclic sugar surrogate, or an acyclic sugar surrogate.
  • sugar surrogate nucleoside means a cyclic sugar surrogate nucleoside or an acyclic sugar surrogate nucleoside.
  • symptom of a disease means any manifestation, indication, sign, or evidence of a disease. Symptoms include subjective and objective indicia of a disease and may be perceived, experienced, detected, observed, measured, and/or quantified. A symptom may be apparent upon diagnostic testing, and in certain instances only upon invasive diagnostic testing, including, but not limited to, post-mortem tests. A symptom may be an absence of a feature, such as failing to reach expected developmental milestones.
  • Symptoms may include but are not limited to, angina, chest pain, shortness of breath, palpitations, weakness, dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling in the lower extremities, cyanosis, fatigue, fainting, numbness of the face, numbness of the limbs, claudication or cramping of muscles, bloating of the abdomen, and fever.
  • target nucleic acid means a LPA nucleic acid that an antisense oligonucleotide is designed to affect.
  • target RNA means an LPA RNA transcript and includes pre-mRNA and/or mRNA unless otherwise specified.
  • target region refers to a portion of a target nucleic acid that is complementary to the targeting region of an antisense oligonucleotide.
  • terminal group means a chemical group or group of atoms that is covalently linked to a terminus of an oligonucleotide.
  • therapeutic index means the ratio of a measure of toxicity or intolerability divided by a measure of potency or activity. Typically, the therapeutic index is expressed as the ratio between the concentration (or dose) at which a compound becomes toxic or induces unacceptable adverse effects (or the highest concentration or dose at which a compound is not toxic, or is tolerated, before it becomes toxic or induces unacceptable adverse effects) to a subject and the concentration (or dose) at which the compound is pharmacologically effective or produces the desired effect.
  • treating means administering a compound or agent to a subject having or at risk for developing such disease. Treating a disease may result in amelioration of at least one symptom of such disease. Treatment may reduce, improve, and/or prevent one or more symptom(s) such that a symptom of the disease is diminished or is no longer apparent.
  • unmodified nucleobase means unmodified adenine (A), unmodified thymine (T), unmodified cytosine (C), unmodified uracil (U), or unmodified guanine (G).
  • an “unmodified nucleoside” means a compound or subunit comprising an unmodified sugar moiety and an unmodified nucleobase.
  • unmodified sugar moiety means a 2'-0H(H) P-D-ribosyl sugar moiety, as found in RNA, or a 2'-H(H) -D-deoxyribosyl sugar moiety, as found in DNA.
  • Unmodified sugar moieties are fiiranosyl or deoxyfuranosyl sugar moieties in the P-D- stereochemical configuration, and have one hydrogen at each of the T, 3 ', and 4' positions, an oxygen at the 3 ' position, two hydrogens at the 5 ' position, and two hydrogens (DNA) or a hydrogen and an OH (RNA) at the 2' position.
  • An oligomeric duplex comprising a first oligomeric compound and a second oligomeric compound, wherein: the first oligomeric compound comprises a first modified oligonucleotide consisting of 18 to 50 linked nucleosides, wherein the nucleobase sequence comprises a nucleobase sequence that is at least 80% identical to at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NOs:6-829 and 3078- 3083, wherein at least one but no more than 22%, no more than 20%, no more than 18%, no more than 15%, no more than 10%, or no more than 5% of the modified nucleosides in the first modified oligonucleotide comprises a 2'-F sugar moiety and/or an FHNA sugar surrogate, and the second oligomeric compound comprises a second modified oligonucleotide consisting of 16 to 50 linked nucleoside
  • the first modified oligonucleotide comprises at least one and no more than four modified nucleosides comprising a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate
  • the second modified oligonucleotide comprises at least one and no more than four modified nucleosides comprising a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate
  • the oligomeric duplex comprises at least one and no more than eight modified nucleosides comprising a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate
  • nucleosides of the first modified oligonucleotide, each independently, and at least 16 nucleosides of the second modified oligonucleotide, each independently, comprise a modified sugar moiety or sugar surrogate selected from a 2'-F sugar moiety, a 2'-MOE sugar moiety, a 2'-OMe sugar moiety, a DNA sugar moiety, and an FHNA sugar surrogate; and/or
  • nucleobase sequence of the first modified oligonucleotide comprises at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NOs:6-829 and 3078-3083
  • nucleobase sequence of the second modified oligonucleotide comprises at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID Nos: 830-1540 and 3096-3101.
  • nucleoside comprising a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety or FHNA sugar surrogate of the first modified oligonucleotide is independently selected from one of: i. the second nucleoside counting from the 5’ end, ii. the second and fourteenth nucleosides counting from the 5’ end, or iii. the second and sixteenth nucleosides counting from the 5’ end, or iv. the second, fourteenth and sixteenth nucleosides counting from the 5’ end, or v. the second, sixth, fourteenth, and sixteenth nucleosides counting from the 5’ end.
  • nucleoside comprising a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate of the second modified oligonucleotide is independently selected from one of: i.the ninth and tenth nucleosides counting from the 5’ end, or ii.the tenth and eleventh nucleosides counting from the 5’ end, or iii .the ninth, tenth and eleventh nucleosides counting from the 5’ end, or iv.the seventh, ninth, tenth and eleventh nucleosides counting from the 5’ end, or v.the seventh, ninth, and eleventh nucleosides counting from the 5 ’ end.
  • oligomeric duplex of any one of embodiments 1-4, wherein one modified nucleoside of the first modified oligonucleotide comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate comprises a 3’-fluoro-hexitol sugar moiety sugar surrogate.
  • oligomeric duplex of embodiment 9, wherein the overhang nucleosides comprise two modified adenosine (AA), two modified uridine (UU) nucleosides, two modified inosine (II) nucleosides, or two modified nucleosides wherein one is an inosine, and one is an adenosine (Al or IA).
  • nucleoside of the first modified oligonucleotide comprising a 2 ’-MOE sugar moiety is an internal nucleoside in a region of the sequence of the first modified oligonucleotide that is any of the ninth and/or tenth nucleosides counting from the 5’ end of the first modified oligonucleotide.
  • oligomeric duplex of embodiment 17, wherein the stabilized phosphate group comprises a methylene phosphonate, cyclopropyl phosphonate or a vinyl phosphonate.
  • each intemucleoside linkage is independently selected from a phosphodiester intemucleoside linkage and a phosphorothioate intemucleoside linkage.
  • ASGPR asialoglycoprotein receptor
  • the oligomeric duplex of embodiment 30, wherein the GalNAc conjugate moiety is selected from Table A.
  • the oligomeric duplex of embodiment 30, wherein the conjugate group consists of a GalNAc ligand and a conjugate linker.
  • An oligomeric duplex comprising i) a first oligomeric compound comprising a modified oligonucleotide consisting of 16 to 50 linked nucleosides, wherein the nucleobase sequence comprises a nucleobase sequence that is at least 80% identical to at least 16 contiguous nucleobases of any one of SEQ ID NOs:6-829 and 3078-3083, wherein all except 0, 1, or 2 nucleobases are identical to the nucleobase sequence SEQ ID NOs:6-829 or 3078-3083, and wherein each of the first modified oligonucleotide comprises a modified sugar moiety selected from: efyyyfyyyyyyfyfyyyyyyyyyyyyyyyyyyyyy, efyyydyyeyyyydydyyyyyee, efyydyyyyyyyf
  • sequence of the second modified oligonucleotide comprises at least 15 or at least 16 contiguous nucleobases of the sequence of any one of SEQ ID NO: 2365-3075 and 3084-3089; and optionally wherein the sequence of the first modified oligonucleotide comprises at least 18, at least 19, or at least 20 contiguous nucleobases of the sequence of any one of SEQ ID NO: 1541-2364 and 3090-3095.
  • oligomeric duplex of embodiment 46 wherein the sequence of the first modified oligonucleotide comprises the sequence of any one of SEQ ID NOs: 1541-2364 and 3090-3095; and optionally wherein the sequence of the second modified oligonucleotide comprises the sequence of any one of SEQ ID NO: 2365-3075 and 3084-3089.
  • oligomeric duplex of embodiment 46 wherein the agent is a duplex compound comprised of any one the following antisense compound and sense compound pairs:
  • a pharmaceutical composition comprising the oligomeric duplex of any one of embodiments 1- 49, or the population of any one of embodiments 50-51, and a pharmaceutically acceptable diluent or carrier.
  • composition 53 The pharmaceutical composition of embodiment 52, wherein the pharmaceutically acceptable diluent is water or phosphate-buffered saline.
  • pharmaceutical composition 54 The pharmaceutical composition of embodiment 52, wherein the pharmaceutical composition consists essentially of the oligomeric duplex and water or phosphate-buffered saline.
  • a method of decreasing the amount of LPA RNA or Lipoprotein(a) protein in a cell, tissue, organ or subject comprising contacting the cell, tissue, organ or subject with the oligomeric duplex of any one of embodiments 1-49, the population of any one of embodiments 50-51, or the pharmaceutical composition of any one of embodiments 52-54.
  • a method comprising administering to a subject the oligomeric duplex of any one of embodiments 1-49, the population of any one of embodiments 50-51, or the pharmaceutical composition of any one of embodiments 52-54; wherein the subject has or is at risk for developing cardiovascular disease (CVD) coronary artery disease (CAD), hypercholesterolemia, myocardial infarction (MI), peripheral arterial disease (PAD), calcific aortic valve disease (CAVD), aortic stenosis, atherosclerotic cardiovascular disease (ASCVD), atherosclerosis, dyslipidemia, thrombosis, or stroke.
  • CVD cardiovascular disease
  • CAD coronary artery disease
  • MI myocardial infarction
  • PDA peripheral arterial disease
  • CAVD calcific aortic valve disease
  • ASCVD atherosclerotic cardiovascular disease
  • atherosclerosis dyslipidemia
  • dyslipidemia thrombosis
  • stroke or stroke.
  • a method of preventing or treating a cardiovascular disease, disorder, condition, a metabolic disease, disorder, or condition, and/or an inflammatory disease disorder or condition in a subject comprising administering to a subject having, or at risk of having, a cardiovascular, metabolic, and/or inflammatory disease, disorder, or condition, an oligomeric duplex of any one of embodiments 1-49, the population of any one of embodiments 50-51, or the pharmaceutical composition of any one of embodiments 52-54; wherein the disease, disorder, condition or injury is hypertriglyceridemia, lipidemia (e.g., hyperlipidemia), dyslipidemia (e.g., atherogenic dyslipidemia, diabetic dyslipidemia, or mixed dyslipidemia), hyperlipoproteinemia, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic valve stenosis, aortic valve calcification, aortic valve regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric
  • 61 The method of any one of embodiments 55-59, wherein the disease, disorder or condition is hypertriglyceridemia or atherosclerotic cardiovascular disease (ASCVD) or coronary artery disease (CAD).
  • ASCVD atherosclerotic cardiovascular disease
  • CAD coronary artery disease
  • 62 The method of embodiment 61, wherein at least one symptom of a disease, disorder or condition associated with elevated lipoprotein(a) is episodes of abdominal pain, physical fatigue, difficulty thinking, diarrhea, recurrent acute pancreatitis, eruptive cutaneous xanthomata, and hepatosplenomegaly or a combination thereof.
  • oligomeric duplex of any one of embodiments 1-49, the population of any one of embodiments 50-51, or the pharmaceutical composition of any one of embodiments 52-54 for treating or preventing a disease, disorder or condition associated with lipoprotein metabolism misregulation or postponing a symptom of a disease, disorder or condition associated with elevated lipoprotein(a).
  • oligomeric duplexes and oligomeric compounds comprising a modified antisense oligonucleotide (e.g., an antisense oligomeric compound) complementary to UPA RNA and a modified sense oligonucleotide (e.g., a sense oligomeric compound) complementary to an antisense oligomeric compound.
  • Modified antisense and/or sense oligonucleotides comprise at least one modified nucleoside (comprising a modified sugar moiety and/or a modified nucleobase) and/or at least one modified intemucleoside linkage. Examples of certain modified nucleosides and modified intemucleoside linkages suitable for use in modified antisense and/or sense oligonucleotides are described herein.
  • Modified nucleosides comprise a modified sugar moiety, a modified nucleobase, or a combination thereof.
  • modified nucleosides comprising the following modified sugar moieties and/or the following modified nucleobases may be incorporated into modified antisense oligonucleotides and/or sense oligonucleotides described herein.
  • Modified sugar moieties include modified fiiranosyl sugar moieties, cyclic sugar surrogates, acyclic sugar surrogates, and sugar mimics.
  • modified sugar moieties are non- bicyclic modified fiiranosyl sugar moieties.
  • modified sugar moieties are bicyclic or tricyclic fiiranosyl sugar moieties.
  • modified sugar moieties are sugar surrogates.
  • Sugar surrogates may comprise one or more substitutions corresponding to those of other types of modified sugar moieties.
  • modified sugar moieties are non-bicyclic modified fiiranosyl sugar moieties comprising one or more substituent groups including, but not limited to, substituents at the 2', 3', 4', and/or 5' positions, as numbered based on ribose:
  • the modified fiiranosyl sugar moiety is a ribosyl sugar moiety that is not an unmodified sugar moiety (i.e. , an unmodified RNA or unmodified DNA moiety).
  • the modified fiiranosyl sugar moiety is a xylosyl, lyxosyl, or arabinosyl sugar moiety.
  • non-bicyclic modified sugar moieties are 2'-substituted sugar moieties and comprise a substituent group at the 2'-position.
  • one or more non-bridging substituent of non-bicyclic modified sugar moieties is branched.
  • substituent groups suitable for the 2'-position of modified sugar moieties include but are not limited to: 2'-F, 2'-OCH3 (“OMe” or “O-methyl”), and 2'-O(CH2)2OCH3 (“MOE” or “O-methoxyethyl”).
  • these 2'-substituent groups may be further substituted with one or more substituent groups independently selected from: halo, cyano, OR a2 , N0 2 , NH 2 , NHR a2 , N(R a2 ) 2 , Ci-Ce alkyl, Ci-Cehaloalkyl, C 2 -Ce alkenyl, C 2 -Ce alkynyl, C3-C10 cycloalkyl, Ce-Cio aryl, heteroaryl, heterocyclyl, Ci-C6 alkylene-NH 2 , Ci-Cealkylene-NHR a2 , Ci-C6 alkylene-N(R a2 ) 2 , C(O)R a3 , C(O)OR a3 , C(O)NHR a3 , C(O)N(Ci-C 4 alkyl)R a3 , SR a3 , S(O) 2 R
  • a non-bridging 2 '-substituent group selected from: F, OCF 3 , OCH 3 , OCH 2 CH 2 OCH 3 , O(CH 2 ) 2 SCH 3 , O(CH 2 ) 2 ON(CH 3 ) 2 , O(CH 2 ) 2 O(CH
  • a 2'-substituted sugar moiety comprises a 2'-substituent group selected from: F, OCH 3 , and OCH 2 CH 2 OCH 3 .
  • modified furanosyl sugar moieties and nucleosides incorporating such modified furanosyl sugar moieties are further defined by stereochemical configuration.
  • a 2'- deoxyfiiranosyl sugar moiety z.e., 2'-(H)H furanosyl sugar moiety
  • Such modified sugar moieties are described in, e.g., WO 2020/072991, incorporated by reference herein.
  • a 2'-modified sugar moiety has an additional stereocenter at the 2'-position relative to a 2'-deoxyfiiranosyl sugar moiety; therefore, such sugar moieties have a total of sixteen possible stereochemical configurations.
  • Modified furanosyl sugar moieties described herein are in the -D-ribosyl stereochemical configuration unless otherwise specified.
  • non-bicyclic modified sugar moieties comprise a substituent group at the 4'-position.
  • substituent groups suitable for the 4'-position of modified sugar moieties include, but are not limited to, alkoxy (e.g., methoxy), alkyl, and those described in Manoharan et al., WO 2015/106128, which is incorporated herein by reference.
  • non-bicyclic modified sugar moieties comprise a substituent group at the 3 '-position.
  • substituent groups suitable for the 3 '-position of modified sugar moieties include, but are not limited to, alkoxy (e.g., methoxy), alkyl (e.g., methyl, ethyl).
  • non-bicyclic modified sugar moieties comprise a substituent group at the 5 '-position.
  • substituent groups suitable for the 5 '-position of modified sugar moieties include, but are not limited to, vinyl, alkoxy (e.g., methoxy), alkynyl, allyl, and alkyl (e.g., methyl (R or .8). ethyl (R or 5)).
  • non-bicyclic modified sugar moieties comprise more than one nonbridging sugar substituent, for example, 2'-F-5'-methyl sugar moieties, such as described in Migawa et al., US 2010/0190837, which is incorporated herein by reference; or alternative 2'- and 5'-modified sugar moieties as described in Rajeev et al., US 2013/0203836, which is incorporated herein by reference.
  • modified sugar moieties are bicyclic sugar moieties and comprise a substituent that bridges two atoms of the fiiranosyl ring to form a second ring.
  • the bicyclic sugar moiety comprises a bridge between the 4' and the 2' furanose ring atoms.
  • 4' to 2' bridging sugar substituents include, but are not limited to: 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2- O-2' (“LNA”), 4'-CH 2 -S-2', 4'-(CH 2 ) 2 -O-2' (“ENA”), 4'-CH(CH 3 )-O-2' (referred to as “constrained ethyl” or “cEt” when in the S configuration), 4'-CH2-O-CH 2 -2', 4'-CH 2 -N(R)-2', 4'-CH(CH 2 OCH3)-O-2' (“constrained MOE” or “cMOE”) and analogs thereof, 4'-C(CH3)(CH 3 )-O-2' and analogs thereof, 4'- CH 2 -N(OCH 3 )-2' and analogs thereof, 4'-CH 2 -O-N(CH 3 )-2', 4'-CH 2 -C
  • the bicyclic sugar moiety comprises a bridge between the 5' and the 3' furanose ring atoms.
  • bicyclic sugar moieties are known in the art, see, for example: Wan, et al., J. Medicinal Chemistry, 2016, 59, 9645-9667; Wengel et al., U.S. 8,080,644; Ramasamy et al., U.S. 6,525,191; Seth et al., U.S. 7,547,684; and Seth et al., U.S. 7,666,854, which are each incorporated herein by reference.
  • bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by stereochemical configuration.
  • an ENA nucleoside (described herein) may be in the a-L configuration or in the P-D configuration.
  • bicyclic nucleosides include both stereochemical configurations.
  • positions of specific bicyclic nucleosides e.g., LNA or cEt
  • they are in the P-D stereochemical configuration, unless otherwise specified.
  • modified sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5 '-substituted and 4'-2' bridged sugars).
  • modified sugar moieties are sugar surrogates, selected from cyclic sugar surrogates and acyclic sugar surrogates.
  • a cyclic sugar surrogate is represented by Formula la: Wherein:
  • J is H, Ci-Ce alkyl, or C2-C6 alkenyl
  • Rs is H, halogen, Ci-Ce alkyl, substituted Ci-Ce alkyl, C 2 -Ce alkenyl, substituted C 2 -Ce alkenyl, C 2 -Ce alkynyl, substituted C 2 -Ce alkynyl or N(E 2 )(E 3 );
  • Ei, E 2 and E 3 are each, independently, H, Ci-Ce alkyl or substituted Ci-Ce alkyl; m is 0 or 1; p is 0 or 1; q is from 1 to 6; s is 0 or 1; j is 0 or 1; and with the proviso that if X is O, Z is C(GIG 2 ), and Q is CH, then m is 1.
  • the oxygen atom of a sugar moiety is replaced, e.g., with a sulfur, carbon or nitrogen atom (X is S, C(RIR 2 ), or N(R 3 )).
  • modified sugar moieties also comprise bridging and/or non-bridging substituents as described herein.
  • certain sugar surrogates comprise a 4 '-sulfur atom and a substitution at the 2 '-position and/or the 5' position.
  • sugar surrogates comprise rings having other than 5 atoms.
  • a sugar surrogate comprises a six-membered tetrahydropyran (“THP”), where X is O-C(RIR 2 ), p is 1, Z is C(GIG 2 ), and m is 0.
  • THP tetrahydropyran
  • X O-C(RIR 2 )
  • p is 1
  • Z is C(GIG 2 )
  • m is 0.
  • Such tetrahydropyrans may be further modified or substituted.
  • FHNA can also be referred to as a F-THP or 3 '-fluoro tetrahydropyran or 3 '-FHNA), each of which are incorporated herein by reference.
  • sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom.
  • nucleosides comprising morpholino sugar moieties and their use in oligonucleotides have been reported.
  • morpholino means a sugar surrogate having Formula la, above, wherein X is O, Y and Z are each CH2, and Q is N.
  • a morpholino is modified, for example by adding or altering various substituent groups from the above morpholino structure.
  • modified morpholines Such sugar surrogates are referred to herein as “modified morpholines.”
  • sugar surrogates are acyclic sugar surrogates and have Formula Ila or Illa:
  • Ri is H, OH, halogen, Ci-Ce alkyl, Ci-Ce alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, or (CH2) q R?
  • R2 and R3 are each independently H, OH, halogen, Ci-Ce alkyl, Ci-Ce alkoxy, C2-C6 alkenyl, C2- Ce alkynyl, S-CH3, N(CH3)(CH3), OCH2CH2OCH3, O-alkylamino, or (CH2) q R?; Ei is H, Ci-Ce alkyl or substituted Ci-Ce alkyl;
  • R4 and R5 are independently H, OH, Ci-Ce alkyl, or N(Rêt): wherein if R4 is OH, then R 5 is not OH;
  • R7 is OH, halogen, methoxy, ethoxy, azido, and C2-C6 alkenyl, or C2-C6 alkynyl, and q is 1, 2, or 3.
  • acyclic sugar surrogates are the “unlocked” sugar structure of UNA (unlocked nucleic acid) nucleosides.
  • UNA locked nucleic acid
  • Representative U.S. publications that teach the preparation of UNA include, but are not limited to, U.S. Patent Publication No. 2011/0313020.
  • acyclic sugar surrogates are the glycerol as found in GNA (glycol nucleic acid) nucleosides, having Formula Ila wherein n is 1, m and o are 0, s is 1, and J2, R2, and R3 are each H, or the butyl as found in acyclic butyl nucleic acid, having Formula Ila wherein n is 2, m and o are 0, s is 1, and J2, R2, and R3 are each H.
  • acyclic sugar surrogates are also known as “C3 spacers” and have Formula Ila wherein n and o are 1; m and s are 0, and Ji, J2, Ri, and R3 are each H.
  • acyclic sugar surrogates include those described in Manoharan et al., U.S. 10,913,767; US patent publication US 2021/0238595; and PCT publication WO 2023/109940.
  • modified oligonucleotides include one or more sugar mimic, in which a group of atoms other than a “furanosyl sugar moiety” or a “sugar surrogate” form the portion of a nucleoside corresponding to the P-D-ribosyl sugar in RNA.
  • a sugar mimic is a portion of the backbone of a peptide nucleic acid, while the remainder of the backbone of the peptide nucleic acid is an intemucleoside linkage.
  • Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262.
  • modified oligonucleotides comprise one or more nucleoside comprising an unmodified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleoside comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more abasic nucleoside. In certain embodiments, modified oligonucleotides contain only nucleosides comprising nucleobases. In certain embodiments, modified oligonucleotides comprise one or more inosine nucleosides (z.e., nucleosides comprising a hypoxanthine nucleobase). In certain embodiments modified oligonucleotides comprise one or more nucleosides comprising a 5- methylcytosine.
  • modified adenine has structure (I):
  • R 2A is H, Ci-Ce alkyl, substituted Ci-Ce alkyl, Ci-Ce thioalkyl, or substituted Ci-Ce thioalkyl, Ci-Ce alkyloxy, or substituted Ci-Ce alkyloxy;
  • R 6A is H, N(R a )(R b ), oxo, acetyl, formyl, or O- phenyl;
  • Y 7A is N and R 7A is absent or is Ci-Ce alkyl; or Y 7A is C and R 7A is H, Ci-Ce alkyl, or N(R a )(R b );
  • Y 8A is N and R 8A is absent, or Y 8A is C and R 8A is H, a halogen, OH, Ci-Ce alkyl, or substituted Ci-Ce alkyl;
  • R a and R b are each independently H, Ci-Ce alkyl, substitute
  • modified guanine has structure (II): wherein: R 2G is N(R a )(R b ); R 6G is oxo and R 1G is H, or R 6G is O-Ci-C 6 alkyl or S-Ci-C 6 alkyl and R 1G is absent; Y 7G is N and R 7G is absent or is Ci-Ce alkyl; or Y 7G is C and R 7G is H, Ci-Ce alkyl, or N(R a )(R b ); Y 8G is N and R 8G is absent, or Y 8G is C and R 8G is H, a halogen, OH, Ci-C 6 alkyl, or substituted Ci-Ce alkyl; R a and R b are independently H, Ci-Ce alkyl, substituted Ci-Ce alkyl, Ci-Ce alkenyl, substituted Ci-Ce alkenyl, acetyl, or for
  • modified thymine or modified uracil has structure (III):
  • X is O or S and R 5U is H, OH, halogen, 0-Ci-C 2 o alkyl, O-Ci-Ci 2 substituted alkyl, Ci- Ci 2 alkyl, substituted C1-C12 alkyl, C1-C12 alkenyl, substituted C1-C12 alkenyl, C1-C12 alkynyl, or substituted C1-C12 alkynyl; wherein if each X is O, R 5U is not H or CH, (unmodified uracil and unmodified thymine, respectively).
  • modified cytosine has structure (IV):
  • R 4C is N(R a )(R b );
  • R 5C is H, OH, halogen, O-Ci-Ci 2 alkyl, O-Ci-Ci 2 substituted alkyl, C1-C12 alkyl , substituted C1-C12 alkyl, C1-C12 alkenyl, or substituted C1-C12 alkenyl;
  • R a and R b are independently H, Ci-Ce alkyl, substituted Ci-Ce alkyl, Ci-Ce alkenyl, substituted Ci-Ce alkenyl, C1-C12 alkynyl, substituted C1-C12 alkynyl, acetyl, or formyl, or together form a 5-7-membered heterocycle; excluding where X is O, R 4C is NH 2 and R 5C is H (unmodified cytosine).
  • modified nucleobases of a modified oligonucleotide are selected from: 5 -substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6, and 0-6 substituted purines.
  • modified nucleobases are selected from: 5 -methylcytosine, hypoxanthine, 1 -methylpseudouridine, 2-aminopropyladenine, 5- hydroxymethyl cytosine, xanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2- propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (-C°C-CH3) uracil, 5- propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5 -ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo (particularly 5-bromo), 5-trifluoromethyl, 5-halouracil), 5-flu
  • nucleobases include tricyclic pyrimidines, such as l,3-diazaphenoxazine-2-one, l,3-diazaphenothiazine-2-one, and 9-(2- aminoethoxy)-l,3-diazaphenoxazine-2-one (G-clamp).
  • Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example, 7-deaza-adenine, 7- deazaguanosine, 2-aminopyridine, and 2-pyridone.
  • Further nucleobases include those disclosed in Englisch, U. et al., Angew. Chem. Int. Ed.
  • each nucleobase of a modified oligonucleotide is selected from unmodified A, unmodified G, unmodified C, unmodified T, unmodified U, and m C. In certain embodiments, each nucleobase of a modified oligonucleotide is selected from unmodified A, unmodified G, unmodified C, unmodified T, unmodified U, m C, or hypoxanthine.
  • oligomeric compounds comprising a modified antisense oligonucleotide (e.g., an antisense oligomeric compound) and/or a modified sense oligonucleotide (e.g., a sense oligomeric compound) provided herein comprise or consist of a modified oligonucleotide comprising at least one modified intemucleoside linkage.
  • the naturally occurring intemucleoside linkage of RNA and DNA is a 3' to 5' phosphodiester linkage.
  • all intemucleoside linkages between furanosyl sugar moieties are 3' to 5' intemucleoside linkages unless otherwise indicated.
  • nucleosides of modified oligonucleotides are linked together using one or more modified intemucleoside linkages.
  • the two main classes of intemucleoside linkages are defined by the presence or absence of a phosphoms atom.
  • Modified intemucleoside linkages compared to naturally occurring phosphodiester linkages, may be used to alter, typically increase, nuclease resistance of the oligonucleotide.
  • a modified intemucleoside linkage is any of those described in WO 2021/030778, incorporated by reference herein.
  • a modified intemucleoside linkage has the formula: wherein independently for each intemucleoside linkage of the modified oligonucleotide: X is selected from O and S;
  • Ri is selected from H, Ci-Ce alkyl, and substituted Ci-Ce alkyl
  • R3 is selected from an aryl, a substituted aryl, CH3, N(CH3)2, OCH3, and a conjugate group;
  • R4 is selected from OCH3, OH, Ci-Ce alkyl, substituted Ci-Ce alkyl, and a conjugate group; and Rs is selected from OCH3, OH, Ci-Ce alkyl, and substituted Ci-Ce alkyl.
  • a modified oligonucleotide comprises a mesyl phosphoramidate linkage having a formula:
  • Further neutral intemucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research,' Y.S. Sanghvi and P.D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral intemucleoside linkages include nonionic linkages comprising mixed N, O, S and CFF component parts.
  • a modified oligonucleotide comprises an intemucleoside linkage comprising a triazole, alkyne, or cyclic guanidine moiety.
  • a modified oligonucleotide comprises an intemucleoside linkage having a formula: which may be stereorandom, or may be enriched for the Rp or Sp configuration.
  • intemucleoside linkages are not 3'-to-5' intemucleoside linkages.
  • modified oligonucleotides comprise one or more inverted nucleoside, where a sugar moiety is linked 3' to 3' and/or 5' to 5', as shown below: wherein each Bx independently represents any nucleobase.
  • an inverted nucleoside is terminal (i. e. , the last nucleoside on one end of an oligonucleotide) and so only one intemucleoside linkage depicted above will be present.
  • additional features e.g., a conjugate group
  • Such terminal inverted nucleosides may be attached to either or both ends of an oligonucleotide.
  • inverted nucleosides lack a nucleobase (are abasic nucleosides).
  • additional features e.g., a conjugate group
  • a terminal inverted nucleoside may be attached to either or both ends of an oligonucleotide.
  • nucleosides are linked 2' to 5' rather than the 3' to 5' linkage. Such a linkage is illustrated below. wherein each Bx represents any nucleobase.
  • a bicyclic sugar moiety may be linked via an atom on the non-furanosyl ring.
  • a bicyclic sugar moiety is linked 7’ to 5’, as shown below:
  • intemucleoside linkages have at least one chiral center.
  • a chiral atom can be prepared as a racemic mixture, or as separate enantiomers.
  • Representative intemucleoside linkages having a chiral center include but are not limited to alkylphosphonates, mesyl phosphoramidates, and phosphorothioates. The mesyl phosphoramidate intemucleoside linkage comprises a chiral center.
  • modified oligonucleotides comprising (Rp) and/or (.S'p) mesyl phosphoramidates comprise one or more of the following formulas, respectively, wherein “Bx” indicates a nucleobase:
  • the phosphorothioate intemucleoside linkage comprises a chiral center.
  • modified oligonucleotides comprising (Rp) and/or (.S'p) phosphorothioates comprise one or more of the following formulas, respectively, wherein “Bx” indicates a nucleobase:
  • Modified oligonucleotides comprising intemucleoside linkages having a chiral center may be prepared as populations of modified oligonucleotides comprising stereorandom intemucleoside linkages, or as populations of modified oligonucleotides comprising intemucleoside linkages containing chiral centers in particular stereochemical configurations.
  • populations of modified oligonucleotides comprise one or more phosphorothioate intemucleoside linkages wherein all of the phosphorothioate intemucleoside linkages are stereorandom.
  • populations of modified oligonucleotides comprise one or more mesyl phosphoramidate intemucleoside linkages wherein all of the mesyl phosphoramidate intemucleoside linkages are stereorandom.
  • modified oligonucleotides can be generated using synthetic methods that result in random selection of the stereochemical configuration of each phosphorothioate and/or mesyl phosphoramidate linkage. Nonetheless, each individual phosphorothioate and/or mesyl phosphoramidate of each individual oligonucleotide molecule has a defined stereoconfiguration.
  • populations of modified oligonucleotides are enriched for modified oligonucleotides comprising one or more particular phosphorothioate and/or mesyl phosphoramidate intemucleoside linkages in a particular, independently selected stereochemical configuration.
  • the particular configuration of the particular phosphorothioate and/or mesyl phosphoramidate linkage is present in at least 65% of the molecules in the population.
  • the particular configuration of the particular phosphorothioate and/or mesyl phosphoramidate linkage is present in at least 70% of the molecules in the population.
  • the particular configuration of the particular phosphorothioate and/or mesyl phosphoramidate linkage is present in at least 80% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate and/or mesyl phosphoramidate linkage is present in at least 90% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate and/or mesyl phosphoramidate linkage is present in at least 99% of the molecules in the population.
  • Such chirally enriched populations of modified oligonucleotides can be generated using synthetic methods known in the art, e.g., methods described in Oka, N., et al. J. Am. Chem. Soc. 2003, 125, 8307-8317; Wan, W. B., et al. Nucleic Acids Res. 2014, 42, 13456, and WO 2017/015555.
  • chirally enriched in reference to a population means a plurality of molecules of identical molecular formula, wherein the number or percentage of molecules within the population that contain a particular stereochemical configuration at a particular chiral center is greater than the number or percentage of molecules expected to contain the same particular stereochemical configuration at the same particular chiral center within the population if the particular chiral center were stereorandom as defined herein.
  • Populations of molecules having multiple chiral centers within each molecule may contain one or more stereorandom chiral centers.
  • the molecules are modified oligonucleotides.
  • the chiral center is at the phosphorous atom of a phosphorothioate intemucleoside linkage.
  • the chiral center is at the phosphorous atom of a mesyl phosphoramidate intemucleoside linkage.
  • a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one indicated phosphorothioate and/or mesyl phosphoramidate in the (.S'p) configuration.
  • a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one phosphorothioate and/or mesyl phosphoramidate in the (/?p) configuration.
  • chiral intemucleoside linkages of modified oligonucleotides described herein may be stereorandom or in a particular stereochemical configuration.
  • the chiral center is at positions 1', 2', 3', and/or 4' of a furanosyl sugar moiety.
  • each chiral center of each furanosyl sugar moiety is enriched such that the sugar moieties have the P-D ribosyl stereochemical configuration.
  • modified oligonucleotides of a population are enriched for -D ribosyl sugar moieties, and all of the phosphorothioate intemucleoside linkages are stereorandom and all of the mesyl phosphoramidate intemucleoside linkages are stereorandom.
  • modified oligonucleotides of a population are enriched for P-D ribosyl sugar moieties, at least one particular phosphorothioate intemucleoside linkage in a particular stereochemical configuration is enriched, and all of the mesyl phosphoramidate intemucleoside linkages are stereorandom.
  • modified oligonucleotides of a population are enriched for P-D ribosyl sugar moieties, at least one particular mesyl phosphoramidate intemucleoside linkage in a particular stereochemical configuration is enriched, and all of the phosphorothioate intemucleoside linkages are stereorandom.
  • modified oligonucleotides of a population are enriched for both P-D ribosyl sugar moieties and at least one, particular phosphorothioate intemucleoside linkage in a particular stereochemical configuration and at least one particular mesyl phosphoramidate intemucleoside linkage in a particular stereochemical configuration is enriched.
  • modified oligonucleotides comprise one or more modified nucleosides comprising a modified sugar moiety. In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more modified intemucleoside linkage. In certain such embodiments, the modified, unmodified, and differently modified sugar moieties, nucleobases, and/or intemucleoside linkages of a modified oligonucleotide define a pattern or motif. In certain embodiments, the patterns of sugar moieties, nucleobases, and intemucleoside linkages are each independent of one another.
  • a modified oligonucleotide may be described by its sugar motif, nucleobase motif, and/or intemucleoside linkage motif (as used herein, nucleobase motif describes the modifications to the nucleobases independent of the nucleobase sequence).
  • oligonucleotides comprise one or more type of modified sugar and/or unmodified sugar moiety arranged along the oligonucleotide or region thereof in a defined pattern or sugar motif.
  • sugar motifs include but are not limited to any of the sugar modifications discussed herein.
  • the sugar moiety of at least one nucleoside of an antisense oligomeric compound is a modified sugar moiety.
  • the sugar moiety of at least one nucleoside of a sense oligomeric compound is a modified sugar moiety.
  • modified oligonucleotides comprise or consist of a region having a fully modified sugar motif.
  • each nucleoside of the fully modified region of the modified oligonucleotide comprises a modified sugar moiety.
  • each nucleoside of the entire modified oligonucleotide comprises a modified sugar moiety.
  • modified oligonucleotides comprise or consist of a region having a fully modified sugar motif, wherein each nucleoside within the fully modified region comprises the same modified sugar moiety, referred to herein as a uniformly modified sugar motif.
  • a fully modified oligonucleotide is a uniformly modified oligonucleotide.
  • each nucleoside of a uniformly modified oligonucleotide comprises the same 2 ’-modification. In certain embodiments, every other nucleoside of a uniformly modified oligonucleotide comprises the same 2 ’-modification, resulting in an alternating 2’- modifications. In certain embodiments, neighboring nucleosides comprise different 2 ’-modification, and every other nucleoside of a uniformly modified oligonucleotide comprises the same 2 ’-modification, resulting in a uniform, alternating 2 ’-modification motif.
  • At least one nucleoside of a modified oligonucleotide comprises a 2’- OMe sugar moiety.
  • at least 8 nucleosides comprise 2’-0Me sugar moieties.
  • at least 10 nucleosides comprise 2’-OMe sugar moieties.
  • at least 12 nucleosides comprise 2’-OMe sugar moieties.
  • at least 13 nucleosides comprise 2’-OMe sugar moieties.
  • at least 14 nucleosides comprise 2’-OMe sugar moieties.
  • at least 15 nucleosides comprise 2’-OMe sugar moieties.
  • At least 16 nucleosides comprise 2’-OMe sugar moieties. In certain embodiments, at least 17 nucleosides comprise 2’-OMe sugar moieties. In certain such embodiments, at least 18 nucleosides comprise 2’-OMe sugar moieties. In certain such embodiments, at least 20 nucleosides comprise 2’-OMe sugar moieties.
  • At least one nucleoside of a modified oligonucleotide comprises a 2’-F sugar moiety (i.e., a 2’-F modified nucleoside).
  • at least 2 nucleosides comprise 2’-F sugar moieties.
  • at least 3 nucleosides comprise 2’-F sugar moieties.
  • 4 nucleosides comprise a 2’-F sugar moiety.
  • at least one, but not more than four nucleosides comprises a 2’-F sugar moiety.
  • 1 or 2 nucleosides comprise 2’-F sugar moieties.
  • nucleosides comprise 2’-F sugar moieties. In certain embodiments, only one nucleoside comprises a 2’-F sugar moiety. In certain embodiments, an antisense oligomeric compound comprises 2 to 4 non-contiguous 2’-F modified nucleosides. In certain embodiments, 4 nucleosides of an antisense oligomeric compound are 2’-F modified nucleosides and none of those 2’-F modified nucleosides are contiguous. In certain embodiments, 1, 2, 3, or 4 nucleosides of an antisense oligomeric compound are 2’-F modified nucleosides and each of those 2’-F modified nucleosides are non-contiguous.
  • At least fifteen of the remainder of the nucleosides are 2’-0Me modified nucleosides.
  • one nucleoside of an antisense oligomeric compound is a 2’-F modified nucleoside and at least fifteen of the remainder of the nucleosides are 2’-0Me modified nucleosides.
  • At least one nucleoside of a modified oligonucleotide comprises a 2’- deoxyribosyl sugar moiety that has no additional modifications.
  • at least one nucleoside comprises a 2 ’-deoxyribosyl sugar moiety.
  • at least 2 nucleosides comprise a 2 ’-deoxyribosyl sugar moiety.
  • at least 3 nucleosides comprise a 2’- deoxyribosyl sugar moiety.
  • at least 4 nucleosides comprise a 2 ’-deoxyribosyl sugar moiety.
  • one nucleoside comprises a 2 ’-deoxyribosyl sugar moiety. In certain embodiments, 1 or 3 nucleosides comprise a 2 ’-deoxyribosyl sugar moiety. In certain embodiments, 1-3 nucleosides comprise a 2’-deoxyribosyl sugar moiety. In certain embodiments, three nucleosides comprise a 2 ’-deoxyribosyl sugar moiety. In certain embodiments, 1, 2, 3, or 4 nucleosides of an antisense oligomeric compound are a 2 ’-deoxyribosyl sugar modified nucleoside and each 2’- deoxyribosyl modified nucleoside is non-contiguous.
  • nucleosides of an antisense oligomeric compound are a 2 ’-deoxyribosyl sugar modified nucleoside and each 2’- deoxyribosyl modified nucleoside is non-contiguous.
  • no nucleosides of a sense oligomeric compound are a 2’ -deoxyribosyl sugar modified nucleoside.
  • three nucleosides of an antisense oligomeric compound are 2 ’-deoxyribosyl sugar modified nucleosides and no nucleoside of a sense oligomeric compound is a 2 ’-deoxyribosyl modified nucleoside.
  • one nucleosides of an antisense oligomeric compound are 2 ’-deoxyribosyl sugar modified nucleosides and no nucleoside of a sense oligomeric compound is a 2 ’-deoxyribosyl modified nucleoside.
  • At least one nucleoside of a modified oligonucleotide comprises a sugar surrogate moiety. In certain embodiments, at least one nucleoside of a modified oligonucleotide comprises a sugar surrogate moiety comprising FHNA. In certain embodiments, at least one nucleosides comprises a sugar surrogate. In certain embodiments, at least two nucleosides comprise a sugar surrogate. In certain embodiments, only one nucleoside comprises a sugar surrogate. In certain embodiments, two nucleosides of an antisense oligomeric compound are sugar surrogates and each of those surrogates are non-contiguous.
  • At least one nucleosides comprises an FHNA sugar surrogate. In certain embodiments, at least two nucleosides comprise an FHNA sugar surrogate. In certain embodiments, only one nucleoside comprises an FHNA sugar surrogate. In certain embodiments, two nucleosides of an antisense oligomeric compound are FHNA sugar surrogates and each of those surrogates are non-contiguous. In certain embodiments, one nucleoside of an antisense oligomeric compound in an FHNA, located at position 2 from the 5’ end of the antisense oligonucleotide.
  • a sugar moiety of an antisense oligomeric compound is modified, wherein the modified sugar modifications and/or sugar surrogate is selected from 2’-F, a 3'-fluoro- hexitol, 2’-M0E, 2’-0Me, and 2’ -deoxyribosyl.
  • a sugar motif (from 5’ to 3’) of the antisense oligomeric compound is selected from efyyyfyyyyyyyyfyfyyyyyyyyyyyyyy, yfyyyfyyyyyyfyfyyyyyyyyyyyyyyyy, efyyydyyeyyyydydyyyyyyee, efyydyyyyyyyyfyfyyyyyee, efyydyyeyyyyfyfyyyyyee, e[FHNA]yyfyyyyyyyfyfyyyyyyee, and e[FHNA]yyyfyeyyyyfyfyyyyyyee wherein each ‘e’ represents a 2'-M0E sugar moiety, each ‘y’ represents a 2'-0
  • a sugar moiety of a sense oligomeric compound is modified, wherein the modified sugar moiety is selected from 2’-F, 2 ’-MOE, and 2’-0Me.
  • a sugar motif (from 5’ to 3’) of a sense oligomeric compound is selected from among: yyyyyyfyfffyyyyyyyyyyyy and eeyyyyyyyyyffyyyyyyyyee, wherein each ‘e’ represents a 2'-M0E sugar moiety, each ‘y’ represents a 2'-0Me sugar moiety, and each ‘f represents a 2'-F sugar moiety.
  • oligonucleotides comprise modified and/or unmodified nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or motif.
  • at least one nucleobase is modified. In certain embodiments, none of the nucleobases are modified.
  • at least one purine and/or at least pyrimidine is modified.
  • at least one adenine is modified.
  • at least one guanine is modified.
  • at least one thymine is modified.
  • at least one uracil is modified.
  • at least one cytosine is modified.
  • At least one of the cytosine nucleobases in a modified oligonucleotide is 5 -methylcytosine. In certain embodiments, all of the cytosine nucleobases are 5 -methylcytosines and all of the other nucleobases of the modified oligonucleotide are unmodified nucleobases. In certain embodiments, one or two of the cytosine nucleobases are 5- methylcytosines and all of the other nucleobases of the modified oligonucleotide are unmodified nucleobases.
  • each nucleobase is selected from 5-methylcytosine, unmodified cytosine, unmodified thymine, unmodified uracil, unmodified adenine, unmodified guanine, and unmodified hypoxanthine. In certain embodiments, each nucleobase is selected from 5-methylcytosine, unmodified cytosine, unmodified thymine, unmodified uracil, unmodified adenine, unmodified guanine, and unmodified hypoxanthine. In certain embodiments, each nucleobase is selected from 5-methylcytosine, unmodified cytosine, unmodified thymine, unmodified adenine, and unmodified guanine.
  • each nucleobase is selected from 5-methylcytosine, unmodified cytosine, unmodified thymine, unmodified adenine, and unmodified guanine. In certain embodiments, each nucleobase is selected from unmodified cytosine, unmodified thymine, unmodified uracil, unmodified adenine, and unmodified guanine. In certain embodiments, each nucleobase is selected from unmodified cytosine, unmodified thymine, unmodified adenine, and unmodified guanine.
  • oligonucleotides comprise modified and unmodified intemucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or motif.
  • each intemucleoside linkage is a phosphodiester intemucleoside linkage.
  • each intemucleoside linkage of a modified oligonucleotide is a phosphorothioate intemucleoside linkage.
  • each intemucleoside linkage of a modified oligonucleotide is independently selected from a phosphorothioate intemucleoside linkage, a mesyl phosphoramidate intemucleoside linkage, and a phosphodiester intemucleoside linkage.
  • each intemucleoside linkage of a modified oligonucleotide is independently selected from a phosphorothioate intemucleoside linkage and a phosphodiester intemucleoside linkage.
  • each intemucleoside linkage of a modified oligonucleotide is independently selected from a mesyl phosphoramidate intemucleoside linkage and a phosphorothioate intemucleoside linkage.
  • each phosphorothioate intemucleoside linkage is independently selected from a stereorandom phosphorothioate, a (.S'p) phosphorothioate, and a (/?p) phosphorothioate.
  • each mesyl phosphoramidate intemucleoside linkage is independently selected from a stereorandom mesyl phosphoramidate, a (.S'p) mesyl phosphoramidate, and a (/?p) mesyl phosphoramidate .
  • the modified antisense oligonucleotide has an intemucleoside linkage motif independently selected from (5' to 3') of: ssooooooooooooooooooss, ssooosooooooososooooss and ssooosooooooppss, wherein each “s” represents a phosphorothioate intemucleoside linkage and each “o” represents a phosphodiester intemucleoside.
  • the modified sense oligonucleotide has an intemucleoside linkage motif independently selected from (5' to 3') of: ssooooooooooooooooss and ssooooooosooooppss, wherein each “s” represents a phosphorothioate intemucleoside linkage, and each “o” represents a phosphodiester intemucleoside.
  • oligomeric compounds comprise one or more modifications, (e.g., a modified sugar moiety, a modified nucleobase, a modified intemucleoside linkage), and/or combinations thereof, incorporated into a modified oligonucleotide.
  • a modified oligonucleotide is characterized by modification motif(s) and overall length. In certain embodiments, such parameters are each independent of one another.
  • each intemucleoside linkage of a modified oligonucleotide having one or more modified sugar moiety and/or sugar motif independently, is modified or unmodified and may or may not follow the modification pattern of the sugar modifications or sugar motif.
  • intemucleoside linkages within a region of a modified oligonucleotide comprising certain sugar modifications may be the same or different from one another and may be the same or different from the intemucleoside linkages of the region of the modified oligonucleotide comprising different sugar modifications.
  • modified oligonucleotides may comprise one or more modified nucleobase independent of the pattern of the sugar modifications or sugar motif and independent of the intemucleoside linkages or intemucleoside linkage motif. Unless specifically indicated, all modifications are independent of nucleobase sequence.
  • oligonucleotide it is possible to increase or decrease the length of an oligonucleotide without eliminating activity.
  • Woolf et al. Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992
  • a series of oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model.
  • Oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the oligonucleotides were able to direct specific cleavage of the target RNA, albeit to a lesser extent than the oligonucleotides that contained no mismatches.
  • target specific cleavage was achieved using 13 nucleobase oligonucleotides, including those with 1 or 3 mismatches.
  • oligonucleotides can have any of a variety of ranges of lengths.
  • oligonucleotides consist of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number nucleosides in the range.
  • X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X ⁇ Y.
  • oligonucleotides consist of 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to
  • modified oligonucleotides comprise 16 linked nucleosides having no more than 1 to 3 mismatches to a target sequence. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) comprise 17 linked nucleosides having no more than 1 to 3 mismatches to a target sequence. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) comprise 18 linked nucleosides having no more than 1 to 3 mismatches to a target sequence.
  • modified oligonucleotides comprise 19 linked nucleosides having no more than 1 to 3 mismatches to a target sequence. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) comprise 20 linked nucleosides having no more than 1 to 3 mismatches to a target sequence. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) comprise 21 linked nucleosides having no more than 1 to 3 mismatches to a target sequence.
  • modified oligonucleotides comprise 22 linked nucleosides having no more than 1 to 3 mismatches to a target sequence. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) comprise 23 linked nucleosides having no more than 1 to 3 mismatches to a target sequence.
  • modified oligonucleotides consist of 16 linked nucleosides. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) consist of 17 linked nucleosides. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) consist of 18 linked nucleosides. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) consist of 19 linked nucleosides. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) consist of 20 linked nucleosides.
  • modified oligonucleotides consist of 21 linked nucleosides. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) consist of 22 linked nucleosides. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) consist of 23 linked nucleosides.
  • antisense oligomeric compounds consist of 12-30 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 17-25 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 17-23 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 17-21 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 18-30 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 20-30 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 21-30 linked nucleosides.
  • antisense oligomeric compounds consist of 23-30 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 18-25 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 20-22 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 21-23 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 23-24 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 20 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 21 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 22 linked nucleosides.
  • antisense oligomeric compounds consist of 23 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 12-30 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 16-25 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 16-23 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 16-21 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 16-30 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 18-30 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 19-30 linked nucleosides.
  • sense oligomeric compounds consist of 16- 25 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 18-25 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 18-20 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 19-21 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 18 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 19 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 20 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 21 linked nucleosides.
  • modified oligonucleotides are further described by their nucleobase sequence.
  • oligonucleotides of oligomeric compounds have a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid.
  • a region of an oligonucleotide has a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid.
  • the nucleobase sequence of a region or entire length of an oligonucleotide is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the second oligonucleotide or nucleic acid, such as a target nucleic acid.
  • a first oligomeric compound comprises a modified oligonucleotide consisting of 18 to 50 linked nucleosides, wherein the nucleobase sequence of the first oligomeric compound comprises at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NOs: 6-829 and 3078-3083.
  • a second oligomeric compound comprises a modified oligonucleotide consisting of 18 to 50 linked nucleosides, wherein the nucleobase sequence of the second oligomeric compound comprises at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NOs: 830-1540 and 3096-3101.
  • oligomeric compounds comprising one or more modified oligonucleotide and one or more conjugate groups.
  • an oligomeric compound optionally further comprises one or more terminal groups.
  • Conjugate groups comprise or consist of a conjugate moiety and a conjugate linker.
  • a conjugate group may be attached at the 3' end and/or the 5' end of an oligonucleotide and/or at any internal position.
  • conjugate groups are atached through a modified sugar moiety or a modified intemucleoside linkage.
  • oligomeric compounds comprise a modified oligonucleotide, a cell-targeting moiety, and a conjugate linker.
  • a conjugate group comprises a conjugate moiety and a conjugate linker.
  • a conjugate moiety modifies one or more properties of an attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance. In certain embodiments, a conjugate moiety imparts a new property on the atached oligonucleotide.
  • a conjugate moiety comprises or consists of a cell-targeting moiety.
  • a cell-targeting moiety is capable of binding the cell-surface receptor or the cellsurface moiety.
  • an agent comprising a cell-targeting moiety is capable of being internalized when it interacts with or binds the cell-surface receptor or the cell-surface moiety.
  • a cell-targeting moiety comprises a liver cell targeting moiety or a liver cell ligand.
  • a liver cell-targeting moiety consists of a cell-targeting moiety having affinity for the hepatic asialoglycoprotein receptor (ASGP-R).
  • oligomeric compounds comprise an oligonucleotide and a conjugate group, wherein the conjugate group comprises a conjugate moiety and a conjugate linker.
  • the conjugate linker links the conjugate moiety to the oligonucleotide.
  • the conjugate linker is a single chemical bond (i.e., the conjugate moiety is attached directly to an oligonucleotide through a single bond).
  • the conjugate linker comprises one or more atoms.
  • the conjugate linker comprises a chemical group.
  • SPAAC strain promoted azido-alkyne cycloaddition
  • CuAAC copper-catalyzed click reaction
  • active ester conjugation to an amino modified oligonucleotide maleimide -thiol Michael addition, ketol/hydroxylamine ligation, the Staudinger ligation, reductive amination, thio ether formation, disulfide formation, reductive alkylation, catalyst-free N- arylation, sulfur fluoride exchange click reaction (SuFEx), and inverse demand Diels Alder reaction.
  • conjugate linkers include but are not limited to pyrrolidine, 8-amino-3,6- dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane- 1 -carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA).
  • ADO 8-amino-3,6- dioxaoctanoic acid
  • SMCC succinimidyl 4-(N-maleimidomethyl) cyclohexane- 1 -carboxylate
  • AHEX or AHA 6-aminohexanoic acid
  • conjugate linkers include but are not limited to substituted or unsubstituted Ci-Cw alkyl, substituted or unsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10 alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.
  • conjugate linkers comprise 1-5 linker-nucleosides. In certain embodiments, conjugate linkers comprise 2-5 linker-nucleosides. In certain embodiments, conjugate linkers comprise exactly 3 linker-nucleosides. In certain embodiments, conjugate linkers comprise the TCA motif. In certain embodiments, such linker-nucleosides are modified nucleosides. In certain embodiments such linker-nucleosides comprise a modified sugar moiety. In certain embodiments, linker- nucleosides are unmodified. In certain embodiments, linker-nucleosides comprise an optionally protected heterocyclic base selected from a purine, substituted purine, pyrimidine or substituted pyrimidine.
  • a cleavable moiety is a nucleoside selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methyl cytosine, 4-N-benzoyl-5-methyl cytosine, adenine, 6-N- benzoyladenine, guanine and 2-N-isobutyrylguanine. It is typically desirable for linker-nucleosides to be cleaved from the oligomeric compound after it reaches a target tissue. Accordingly, linker-nucleosides are typically linked to one another and to the remainder of the oligomeric compound through cleavable bonds. In certain embodiments, such cleavable bonds are phosphodiester bonds.
  • linker-nucleosides are not considered to be part of the oligonucleotide. Accordingly, in embodiments in which an oligomeric compound comprises an oligonucleotide consisting of a specified number or range of linked nucleosides and/or a specified percent complementarity to a reference nucleic acid and the oligomeric compound also comprises a conjugate linker comprising linker-nucleosides, those linker-nucleosides are not counted toward the length of the oligonucleotide and are not used in determining the percent complementarity of the oligonucleotide for the reference nucleic acid.
  • an oligomeric compound may comprise (1) an oligonucleotide consisting of 18-30 nucleosides and (2) a conjugate linker comprising 1-10 linker-nucleosides that are contiguous with the nucleosides of the oligonucleotide.
  • the total number of contiguous linked nucleosides in such an oligomeric compound is more than 30.
  • an oligomeric compound may comprise an oligonucleotide consisting of 18-30 nucleosides and no conjugate linker. The total number of contiguous linked nucleosides in such an oligomeric compound is no more than 30.
  • conjugate linkers comprise no more than 10 linker-nucleosides.
  • conjugate linkers comprise no more than 5 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 3 linker- nucleosides. In certain embodiments, conjugate linkers comprise no more than 2 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 1 linker-nucleoside.
  • a conjugate moiety it is desirable for a conjugate moiety to be cleaved from the oligonucleotide. For example, in certain circumstances oligomeric compounds comprising a particular conjugate moiety are better taken up by a particular cell type, but once the oligomeric compound has been taken up, it is desirable that the conjugate moiety be cleaved to release the unconjugated or parent oligonucleotide.
  • certain conjugate linkers may comprise one or more cleavable moieties.
  • a cleavable moiety is a cleavable bond.
  • a cleavable moiety is a group of atoms comprising at least one cleavable bond.
  • a cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds.
  • a cleavable moiety is selectively cleaved inside a cell or subcellular compartment, such as a lysosome.
  • a cleavable moiety is selectively cleaved by endogenous enzymes, such as nucleases.
  • a cleavable bond is selected from among: an amide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, or a disulfide. In certain embodiments, a cleavable bond is one or both of the esters of a phosphodiester. In certain embodiments, a cleavable moiety comprises a phosphate or phosphodiester. In certain embodiments, the cleavable moiety is a phosphodiester linkage between an oligonucleotide and a conjugate moiety.
  • a cleavable moiety comprises or consists of one or more linker- nucleosides.
  • the one or more linker-nucleosides are linked to one another and/or to the remainder of the oligomeric compound through cleavable bonds.
  • such cleavable bonds are unmodified phosphodiester bonds.
  • a cleavable moiety is 2'-deoxy nucleoside that is attached to either the 3' or 5 '-terminal nucleoside of an oligonucleotide by a phosphate intemucleoside linkage and covalently attached to the remainder of the conjugate linker or conjugate moiety by a phosphate or phosphorothioate linkage.
  • the cleavable moiety is 2'-deoxyadenosine.
  • oligomeric compounds described herein comprise an oligonucleotide linked to a conjugate moiety by a conjugate linker, wherein the oligomeric compound is prepared using Click chemistry known in the art.
  • Compounds have been prepared using Click chemistry wherein alkynyl phosphonate intemucleoside linkages on an oligomeric compound attached to a solid support are converted into the 1,2,3-triazolylphosphonate intemucleoside linkages and then cleaved from the solid support (Krishna et al., J. Am. Chem. Soc. 2012, 134(28), 11618-11631), which is incorporated by reference herein in its entirety.
  • Additional conjugate linkers suitable for use in several embodiments are prepared by Click chemistry described in “Click Chemistry for Biotechnology and Materials Science” Ed. Joerg Laham, Wiley 2009, which is incorporated by reference herein in its entirety.
  • compounds comprise an oligonucleotide, a cell-targeting moiety, and a conjugate linker.
  • oligomeric compounds comprise an oligonucleotide, a hepatic asialoglycoprotein receptor (ASGP-R) ligand, and a conjugate linker.
  • oligomeric compounds comprise an oligonucleotide, a N-acetyl galactosamine (GalNAc) ligand, and a conjugate linker.
  • oligomeric compounds comprise an oligonucleotide, a GalNAc trimer, a branching group, a conjugate linker, and optionally modifications to the GalNAc ligands. In certain embodiments, oligomeric compounds comprise an oligonucleotide, two or more GalNAc ligands, a branching group, a conjugate linker, and optionally modifications to the GalNAc ligands. In certain embodiments, a conjugate linker connects GalNAc ligand to an oligonucleotide.
  • two or more GalNAc ligands are covalently connected to a conjugate linker, and the conjugate linker is covalently connected to the 3’ end of an oligonucleotide.
  • a three GalNAc cluster is covalently connected to a conjugate linker, and the conjugate linker is covalently connected to the 3’ end of an oligonucleotide.
  • two or more GalNAc ligands are covalently connected to a conjugate linker, and the conjugate linker is covalently connected to the 5’ end of an oligonucleotide.
  • a three GalNAc cluster is covalently connected to a conjugate linker, and the conjugate linker is covalently connected to the 5’ end of an oligonucleotide.
  • two or more GalNAc ligands are covalently connected to a conjugate linker, and the conjugate linker is covalently connected to an internal position of an oligonucleotide.
  • a three GalNAc cluster is covalently connected to a conjugate linker, and the conjugate linker is covalently connected to an internal position of an oligonucleotide.
  • an internal position of an oligonucleotide is a 2 ’-position of a modified sugar moiety of a nucleoside within the internal region of an oligonucleotide that is not the 5 ’ terminal nucleoside or the 3’ terminal nucleoside.
  • an internal position of an oligonucleotide is a modified intemucleoside linkage of the oligonucleotide.
  • a sense oligomeric compound is conjugated to a THA-GalNAc conjugate group attached to the 5'-OH of the oligonucleotide.
  • THA-GalNAc The structure of THA-GalNAc is:
  • a sense oligomeric compound is conjugated to a HPPO-GalNAc conjugate group attached to the 3'-OH of the oligonucleotide.
  • the structure of HPPO-GalNAc is: B. Certain Terminal Groups
  • Examples of a terminal group include, but are not limited to, a conjugate group, a capping group, a phosphate moiety, a protecting group, a modified or unmodified nucleoside, and two or more nucleosides that are independently modified or unmodified, wherein one or more groups is attached to either or both ends of an oligonucleotide. In certain embodiments, one or more terminal groups is attached to either or both ends of an oligonucleotide. In certain embodiments, one or more terminal groups is attached at the 3’ and/or 5 ’-end of the oligonucleotide. In certain embodiments, one or more terminal groups is attached at the 3 ’-end of the oligonucleotide.
  • one or more terminal groups is attached at the 5 ’-end of the oligonucleotide. In certain embodiments, one or more terminal groups is attached at the 3 ’-end of the oligonucleotide and one or more terminal groups is attached at the 5 ’-end of the oligonucleotide. In certain embodiments, a terminal group is attached at the 3’ and/or 5 ’-end of the oligonucleotide. In certain embodiments, a terminal group is attached at the 3 ’-end of the oligonucleotide. In certain embodiments, a terminal group is attached near the 3 ’-end of the oligonucleotide.
  • a terminal group is attached at the 5 ’-end of the oligonucleotide. In certain embodiments, a terminal group is attached near the 5 ’-end of the oligonucleotide. In certain embodiments, a terminal group is attached at the 3 ’-end of the oligonucleotide and a terminal group is attached at the 5 ’-end of the oligonucleotide.
  • an oligomeric compound comprises one or more terminal groups.
  • an oligomeric compound comprises a terminal group comprising a stabilized 5’- phosphate.
  • Stabilized 5’-phosphates include, but are not limited to 5 ’-phosphonates, including, but not limited to 5’-vinylphosphonate, 5’-methylphosphonate.
  • a terminal group comprises one or more abasic sugar moieties.
  • a terminal group comprises one or more inverted sugar moieties and/or inverted nucleosides.
  • a terminal group comprises one or more 2’-linked nucleosides or sugar moieties.
  • the 2’-linked terminal group is an abasic sugar moiety.
  • an antisense oligomeric compound comprises a vinylphosphonate.
  • each antisense oligomeric compound has a vinyl phosphonate moiety on the 5'-end (5 ’-VP).
  • an oligomeric compound comprises or consists of a modified oligonucleotide comprising a targeting region that is complementary to an equal-length target region of a target nucleic acid, wherein the target nucleic acid is LPA.
  • LPA nucleic acid has the nucleobase sequence set forth in SEQ ID NO: 1 (the complement of GENBANK Accession No. NC_000006.12, truncated from nucleoside 160528001 to nucleoside 160669000) or SEQ ID NO: 2 (GENBANK Accession No. NM_005577.2), or to both.
  • contacting a cell with an oligomeric duplex comprising an oligomeric compound comprising a modified oligonucleotide that is complementary to an equal-length target region of SEQ ID NO: 1 and/or SEQ ID NO: 2 inhibits LPA RNA in the cell, and in certain embodiments inhibits apo(a) protein or Lp(a) produced in the cell.
  • the oligomeric compound comprises of a modified oligonucleotide.
  • the oligomeric compound comprises a modified oligonucleotide and a conjugate group.
  • the oligomeric compound comprises a modified oligonucleotide and one or more terminal group(s).
  • the oligomeric compound comprises a modified oligonucleotide and a conjugate group and one or more terminal group(s).
  • an oligomeric compound comprises an antisense oligonucleotide comprising a targeting region that is complementary to a target region of a LPA nucleic acid. In certain embodiments, an oligomeric compound comprises an antisense oligonucleotide comprising a targeting region that is complementary to a region of SEQ ID NO: 2.
  • the target nucleic acid is an endogenous LPA RNA molecule.
  • the LPA nucleic acid encodes apo(a) protein.
  • the LPA nucleic acid is a precursor to a nucleic acid that encodes apo(a) protein or Lp(a).
  • the LPA nucleic acid is selected from: a mature mRNA and a pre-mRNA, including intronic, exonic, and untranslated regions.
  • the LPA RNA is a mature mRNA.
  • the LPA nucleic acid is a pre-mRNA.
  • antisense oligonucleotides provided herein are complementary to a target region of a LPA nucleic acid over the entire length of the modified oligonucleotide. In certain embodiments, antisense oligonucleotides are at least 99%, at least 95%, at least 90%, at least 85%, or at least 80% complementary to an equal length portion of the LPA nucleic acid. In certain embodiments, antisense oligonucleotides are at least 80% complementary to a target region of the LPA nucleic acid over the entire length of the antisense oligonucleotide and comprise a targeting region that is 100% or fully complementary to the target region of the LPA nucleic acid.
  • a targeting region is from 6 to 20, 10 to 18, 14 to 18, 16 to 20, or 18 to 20 nucleobases in length.
  • the targeting region comprises or consists of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 contiguous nucleobases.
  • the targeting region comprises or consists of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or 23 contiguous nucleobases.
  • the targeting region constitutes at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the nucleosides of the antisense oligonucleotide. In certain embodiments, the targeting region constitutes all of the nucleosides of the antisense oligonucleotide. In certain embodiments, the targeting region of the antisense oligonucleotide is at least 99%, at least 95%, at least 90%, at least 85%, or at least 80% complementary to a target region of the LPA nucleic acid. In certain embodiments, the targeting region of the antisense oligonucleotide is 100% complementary to a target region of the LPA nucleic acid.
  • antisense oligonucleotides comprise one or more mismatches relative to the target region of the LPA nucleic acid.
  • antisense activity against the target is reduced by such a mismatch, and activity against a non-target is reduced.
  • activity against the non-target is reduced by a greater amount than activity against the target.
  • selectivity of the antisense oligonucleotides is improved.
  • antisense oligonucleotides are at least 80% complementary to the target region of the LPA nucleic acid over the entire length of the antisense oligonucleotide and comprise no more than one to three mismatches with the LPA nucleic acid.
  • antisense oligonucleotides comprise a targeting region that is at least 80% complementary to a target region of the LPA nucleic acid over the entire length of the targeting region, and the targeting region comprises no more than one to three mismatches with the target region.
  • antisense oligonucleotides comprise a targeting region that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to a target region of the LPA nucleic acid over the entire length of the targeting region.
  • additional mismatches may be present at a terminus or at both termini of the antisense oligonucleotide, outside of the targeting region.
  • a mismatch is specifically positioned within an antisense oligonucleotide. In certain embodiments, a mismatch is at position 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 from the 5'-cnd of the antisense oligonucleotide. In certain embodiments, a mismatch is at position 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 from the 3 '-end of the antisense oligonucleotide. In certain embodiments, a mismatch is at position 1, 2, 3, or 4 from the 5 '-end of the antisense oligonucleotide. In certain embodiments, a mismatch is at position 4, 3, 2, or 1 from the 3 '-end of the antisense oligonucleotide.
  • oligomeric compounds comprise or consist of a modified oligonucleotide comprising a targeting region that is complementary to a target region in a LPA nucleic acid, wherein the LPA nucleic acid is expressed in a pharmacologically relevant tissue. In certain embodiments, the LPA nucleic acid is expressed in the liver.
  • oligomeric compounds comprising modified oligonucleotides complementary to a target region in a LPA nucleic acid, such as, for example, a human LPA nucleic acid, such as SEQ ID NO: 1 (the complement of GENBANK Accession No. NC_000006.12, truncated from nucleoside 160528001 to nucleoside 160669000) or SEQ ID NO: 2 (GENBANK Accession No. NM_005577.2), or to both and compositions comprising such oligomeric compounds.
  • a human LPA nucleic acid such as SEQ ID NO: 1 (the complement of GENBANK Accession No. NC_000006.12, truncated from nucleoside 160528001 to nucleoside 160669000) or SEQ ID NO: 2 (GENBANK Accession No. NM_005577.2), or to both and compositions comprising such oligomeric compounds.
  • a modified oligonucleotide has a nucleobase sequence comprising or consisting of a targeting region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% complementary to a region of SEQ ID NOs: 1 and/or 2.
  • a modified oligonucleotide has a complementary region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% complementary to a targeting region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% complementary to a target region of SEQ ID NOs: 1 and/or 2.
  • a modified oligonucleotide has a targeting region that is 100% complementary to a target region of SEQ ID NOs: 1 and/or 2.
  • a modified oligonucleotide has a nucleobase sequence comprising or consisting of a complementary region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% complementary to a targeting region that is 100% complementary to a target region of SEQ ID NOs: 1 and/or 2.
  • a modified antisense oligonucleotide has a nucleobase sequence comprising or consisting of any one of SEQ ID NO: 6-829 and 3078-3083.
  • a second modified sense oligonucleotide has a nucleobase sequence comprising or consisting of a complementary region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% complementary to the first modified antisense oligonucleotide, or which is 100% complementary to the first modified antisense oligonucleotide.
  • a modified sense oligonucleotide has a nucleobase sequence comprising or consisting of any one of SEQ ID NO: 830-1540 and 3096-3101.
  • an oligomeric compound provided herein comprises a modified oligonucleotide having a nucleobase sequence complementary to a sequence in a LPA target nucleic acid paired with a second oligomeric compound to form an oligomeric duplex.
  • Such oligomeric duplex comprises a first oligomeric compound comprising a modified oligonucleotide having a portion complementary to a sequence in an LPA target nucleic acid and a second oligomeric compound comprising a modified oligonucleotide having a portion complementary to the first oligomeric compound.
  • the first oligomeric compound of an oligomeric duplex comprises or consists of (1) a first modified oligonucleotide and optionally a conjugate group and/or terminal group; and the second oligomeric compound of the oligomeric duplex comprises or consists of (2) a second modified oligonucleotide and optionally a terminal group and/or a conjugate group.
  • Either or both oligomeric compounds of an oligomeric duplex may comprise a conjugate group.
  • Either or both oligomeric compounds of an oligomeric duplex may comprise a terminal group.
  • the oligonucleotides of each oligomeric compound of an oligomeric duplex may include non-complementary or unpaired overhanging nucleosides.
  • the non-complementary or unpaired overhanging nucleosides are adenosine or thymine.
  • the non-complementary or unpaired overhanging nucleosides include inosine.
  • the two oligonucleotides have at least one mismatch relative to one another.
  • the oligomeric duplex is an antisense agent.
  • an oligomeric duplex comprises: a first oligomeric compound comprising a first modified oligonucleotide consisting of 18 to 28 linked nucleosides, wherein the nucleobase sequence of the first modified oligonucleotide comprises at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NOs: 6-829 and 3078-3083; and a second oligomeric compound comprising a second modified oligonucleotide consisting of 15 to 25 linked nucleosides, wherein the nucleobase sequence of the second modified oligonucleotide comprises at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NOs: 830-1540 and 3096- 3101; and wherein each of the nucleosides of the first modified oligonucle
  • the oligomeric duplex is an antisense agent.
  • the first oligomeric compound of the oligomeric duplex is an antisense agent, wherein the first modified oligonucleotide is an antisense oligomeric compound.
  • the second oligomeric compound of the oligomeric duplex is a sense agent, wherein the second modified oligonucleotide is a sense oligomeric compound.
  • the first modified oligonucleotide is an antisense RNAi oligonucleotide wherein no more than 22%, no more than 20%, no more than 18%, no more than 16%, no more than 14 %, no more than 12%, no more than 10%, or no more than 7%, of the modified nucleosides in the oligomeric duplex comprise a modified sugar moiety comprising a 2’-F modification.
  • the second modified oligonucleotide is a sense RNAi oligonucleotide.
  • the nucleobase sequence of the second modified oligonucleotide is at least 90%, 95% or 100% complementary to the nucleobase sequence of an equal length portion of the first modified oligonucleotide.
  • an oligomeric duplex comprises a first oligomeric compound comprising a first modified oligonucleotide consisting of 18 to 28 linked nucleosides, wherein the nucleobase sequence of the first modified oligonucleotide comprises at least 18, at least 19, at least 20, at least 21, at least 22, or 23 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NO: 6-829 and 3078-3083; and a second oligomeric compound comprising a second modified oligonucleotide consisting of 15 to 25 linked nucleosides, wherein the nucleobase sequence of the second modified oligonucleotide comprises at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NOs: 830-1540 and 3096- 3101, and wherein each of the nucleosides of the first modified oligonucleotide comprises
  • the first oligomeric compound is an antisense agent, wherein the first modified oligonucleotide is an antisense oligomeric compound.
  • the second oligomeric compound is a sense agent, wherein the second modified oligonucleotide is a sense oligomeric compound.
  • the first modified oligonucleotide is an antisense RNAi oligonucleotide.
  • the second modified oligonucleotide is a sense RNAi oligonucleotide.
  • the nucleobase sequence of the second modified oligonucleotide comprises a complementary region of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or 21 nucleobases that is 100%complementary to the nucleobase sequence of an equal portion of the first modified oligonucleotide.
  • the nucleobase sequence of the second modified oligonucleotide is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the nucleobase sequence of an equal portion of the first modified oligonucleotide.
  • the oligomeric duplex is an antisense agent.
  • the nucleobase sequence of the second modified oligonucleotide comprises a complementary region of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or 21 nucleobases that is 100% complementary to the nucleobase sequence of an equal portion of the first modified oligonucleotide; and the nucleobase sequence of the second modified oligonucleotide is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the nucleobase sequence of an equal portion of the first modified oligonucleotide.
  • the oligomeric duplex is an antisense agent, wherein no more than three nucleosides, no more than four nucleosides, no more than five nucleosides, no more than six nucleosides, no more than seven nucleosides, or no more than eight nucleosides in the oligomeric duplex comprise a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate.
  • an oligomeric duplex comprises a first oligomeric compound comprising a first modified oligonucleotide consisting of 18 to 28 linked nucleosides, wherein the nucleobase sequence of the first modified oligonucleotide comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NO: 6-829 and 3078-3083; and a second oligomeric compound comprising a second modified oligonucleotide consisting of 15 to 25 linked nucleosides, wherein the nucleobase sequence of the second modified oligonucleotide comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleobases the nucleobase sequence of any one of SEQ ID NO: 830
  • the first oligomeric compound is an antisense agent, wherein the first modified oligonucleotide is an antisense oligomeric compound.
  • the second oligomeric compound is a sense agent, wherein the second modified oligonucleotide is a sense oligomeric compound.
  • the first modified oligonucleotide is an antisense RNAi oligonucleotide
  • the second modified oligonucleotide is a sense RNAi oligonucleotide.
  • the nucleobase sequence of the second modified oligonucleotide is at least 95% or 100% complementary to the nucleobase sequence of an equal length portion of the first modified oligonucleotide.
  • the oligomeric duplex is an antisense agent wherein no more than three nucleosides, no more than four nucleosides, no more than five nucleosides, no more than six nucleosides, no more than seven nucleosides, or no more than eight nucleosides in the oligomeric duplex comprise a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate.
  • an oligomeric duplex comprises a first oligomeric compound comprising a first modified oligonucleotide consisting of 18 to 28 linked nucleosides, wherein the nucleobase sequence of the first modified oligonucleotide comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NO: 6-829 and 3078-3083; and a second oligomeric compound comprising a second modified oligonucleotide consisting of 15 to 25 linked nucleosides, wherein the nucleobase sequence of the second modified oligonucleotide comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleobases the nucleobase sequence of any one of SEQ ID NO: 830
  • the first oligomeric compound is an antisense agent, wherein the first modified oligonucleotide is an antisense oligomeric compound comprising a 5’ terminal group.
  • the second oligomeric compound is a sense agent, wherein the second modified oligonucleotide is a sense oligomeric compound optionally conjugated to a cell targeting moiety.
  • the first modified oligonucleotide is an antisense RNAi oligonucleotide
  • the second modified oligonucleotide is a sense RNAi oligonucleotide.
  • the nucleobase sequence of the second modified oligonucleotide is at least 95% or 100% complementary to the nucleobase sequence of an equal length portion of the first modified oligonucleotide.
  • the oligomeric duplex is an antisense agent wherein no more than three nucleosides, no more than four nucleosides, or no more than five nucleosides, in the oligomeric duplex comprise a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate.
  • an oligomeric duplex comprises a first oligomeric compound comprising a first modified oligonucleotide consisting of 18 to 28 linked nucleosides, wherein the nucleobase sequence of the first modified oligonucleotide comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NO: 6-829 and 3078-3083; and a second oligomeric compound comprising a second modified oligonucleotide consisting of 15 to 25 linked nucleosides, wherein the nucleobase sequence of the second modified oligonucleotide comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleobases the nucleobase sequence of any one of SEQ ID NO: 830
  • the first oligomeric compound is an antisense agent, wherein the first modified oligonucleotide is an antisense oligomeric compound comprising a 5 ’ terminal group.
  • the second oligomeric compound is a sense agent, wherein the second modified oligonucleotide is a sense oligomeric compound optionally conjugated to a cell targeting moiety.
  • the first modified oligonucleotide is an antisense RNAi oligonucleotide
  • the second modified oligonucleotide is a sense RNAi oligonucleotide.
  • the nucleobase sequence of the second modified oligonucleotide is at least 95% or 100% complementary to the nucleobase sequence of an equal length portion of the first modified oligonucleotide.
  • the oligomeric duplex is an antisense agent wherein no more than five nucleosides, no more than six nucleosides, no more than seven nucleosides or no more than eight nucleosides, in the oligomeric duplex comprise a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate.
  • an oligomeric duplex comprises a first oligomeric compound comprising a first modified oligonucleotide, wherein the first modified oligonucleotide consists of 21 - 23 linked nucleosides and has nucleobase sequence comprising at least a 19-bp nucleobase sequence of any one of SEQ ID NOs: 6-829 and 3078-3083 having 0, 1, 2 or 3 nucleobases that are different from the corresponding nucleotide in any of SEQ ID NOs: 6-829 or 3078-3083; and a second oligomeric compound comprising a second modified oligonucleotide wherein the second modified oligonucleotide consists of 19 - 21 linked nucleosides and has nucleobase sequence comprising at least a 17-bp nucleobase sequence of any one of SEQ ID NOs: 830-1540 and 3096-3101, having 0, 1, 2 or 3 nucleobases that are different from the corresponding nucleo
  • the first oligomeric compound is an antisense agent, wherein the first modified oligonucleotide is an antisense oligomeric compound comprising a 5 ’ terminal group.
  • the second oligomeric compound is a sense agent, wherein the second modified oligonucleotide is a sense oligomeric compound optionally conjugated to a cell targeting moiety.
  • the first modified oligonucleotide is an antisense RNAi oligonucleotide
  • the second modified oligonucleotide is a sense RNAi oligonucleotide.
  • the nucleobase sequence of the second modified oligonucleotide is at least 95% or 100% complementary to the nucleobase sequence of an equal length portion of the first modified oligonucleotide.
  • the oligomeric duplex is an antisense agent, wherein at least one modified nucleoside and no more than five nucleosides, no more than six nucleosides, no more than seven nucleosides or no more than eight nucleosides, in the oligomeric duplex comprise a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate.
  • one or two of the modified sugar moiety and/or sugar surrogate comprises a 2’-F modification in the first modified oligonucleotide
  • two modified nucleosides of the second modified oligonucleotide comprises a 2’-F modification
  • no more than three nucleosides in the oligomeric duplex comprise a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate.
  • three or four of the modified sugar moiety and/or sugar surrogate comprises a 2’-F modification in the first modified oligonucleotide, and two or four modified nucleosides of the second modified oligonucleotide comprises a 2’-F modification.
  • the oligomeric duplex comprises one or two unpaired nucleosides at either or both ends, forming one or two overhang ends.
  • an overhang end is one or two nucleosides of the antisense oligomeric compound.
  • an overhang end is one or two 3'- nucleosides of the antisense oligomeric compound.
  • the last two 3 '-nucleosides of the antisense oligomeric compound are overhang nucleosides not paired with the sense oligomeric compound.
  • the last one or two 3 '-unpaired overhang nucleosides comprise an adenine nucleobase.
  • the last one or two 3 '-unpaired overhang nucleosides comprise a thymine nucleobase.
  • the last one or two 3 '-unpaired overhang nucleosides comprise a uridine nucleobase.
  • the last one or two 3 '-unpaired overhang nucleosides comprise an inosine nucleobase.
  • the oligomeric duplex is an antisense agent wherein no more than five nucleosides, no more than six nucleosides, no more than seven nucleosides or no more than eight nucleosides, in the oligomeric duplex comprise a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate.
  • an oligomeric duplex comprises a first oligomeric compound comprising a first modified oligonucleotide, wherein the first modified oligonucleotide consists of 21-23 linked nucleosides and has a nucleobase sequence comprising at least a 19-bp sequence of any one of SEQ ID NOs: 1541-2346 and 3090-,3095 having 0, 1, 2 or 3 mismatches with a sequence in a target LPA nucleic acid sequence; and a second oligomeric compound comprising a second modified oligonucleotide wherein the second modified oligonucleotide consists of 19-21 linked nucleosides, comprising at least a 19-bp sequence of any one of SEQ ID NOs: 2365-3075 and 3084-3089, having 0, 1, 2 or 3 mismatches to the first modified oligonucleotide.
  • the first modified oligonucleotide is an antisense RNAi oligonucleotide
  • the second modified oligonucleotide is a sense RNAi oligonucleotide.
  • the nucleobase sequence of the second modified oligonucleotide is at least 85%, 90%, 95% or 100% complementary to the nucleobase sequence of an equal length portion of the first modified oligonucleotide.
  • the oligomeric duplex is an antisense agent wherein each of the nucleosides of the first modified oligonucleotide independently and the second modified oligonucleotide independently comprises a modified sugar moiety or sugar surrogate independently selected from 2’-F, 2’-M0E, 2’-OMe, 2 ’-deoxyribosyl, and 3'-fluoro-hexitol, and wherein at least one modified nucleoside and no more than four modified nucleosides of the first modified oligonucleotide comprises a 2’-F modification, and at least one modified nucleoside and no more than four modified nucleosides of the second modified oligonucleotide comprises a 2’-F modification.
  • the oligomeric duplex comprises one or two unpaired nucleosides at either or both ends, forming one or two overhang ends.
  • an overhang end is one or two nucleosides of the antisense oligomeric compound.
  • an overhang end is one or two 3 '-nucleosides of the antisense oligomeric compound.
  • the last two 3 '-nucleosides of the antisense oligomeric compound are overhang nucleosides not paired with the sense oligomeric compound.
  • the last one or two 3 '-unpaired overhang nucleosides comprise an adenine nucleobase.
  • the last one or two 3 '-unpaired overhang nucleosides comprise a thymine nucleobase. In certain embodiments the last one or two 3 '-unpaired overhang nucleosides comprise a uridine nucleobase. In certain embodiments the last one or two 3'- unpaired overhang nucleosides comprise inosine nucleobase. In certain embodiments the last two 3'- unpaired overhang nucleosides comprise a thymine nucleobase and an inosine nucleobase. In certain embodiments the last two 3 '-unpaired overhang nucleosides comprise 5’ to 3’ a thymine nucleobase and an inosine nucleobase.
  • the last two 3 '-unpaired overhang nucleosides comprise 5’ to 3’ an inosine nucleobase and a thymine nucleobase. In certain embodiments the last two 3'- unpaired overhang nucleosides comprise 5’ to 3’ an adenine nucleobase and an inosine nucleobase. In certain embodiments the last two 3 '-unpaired overhang nucleosides comprise 5’ to 3’ an inosine nucleobase and an adenine nucleobase.
  • an oligomeric duplex comprises a first oligomeric compound comprising a first modified oligonucleotide consisting of 19 to 25 linked nucleosides and a second oligomeric compound comprising a second modified oligonucleotide consisting of 16 to 24 linked nucleosides, wherein the nucleobase sequence of the first modified oligonucleotide and the nucleobase sequence of the second modified oligonucleotide each comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 contiguous nucleobases of any of the following pairs selected from a first oligomeric compound selected from any one of SEQ ID NOs: 1541-2364 and 3090-3095, and a second oligomeric compound selected from any one of SEQ ID NOS: 2365-3075 and 3084-3089.
  • the first oligomeric compound is an antisense agent. In certain embodiments, the first modified oligonucleotide is an antisense oligomeric compound. In certain embodiments, the second oligomeric compound is a sense agent. In certain embodiments, the second modified oligonucleotide is a sense oligomeric compound. In certain embodiments, the first modified oligonucleotide is an antisense RNAi oligonucleotide. In certain embodiments, the second oligomeric compound is a sense agent. In certain embodiments, the second modified oligonucleotide is a sense RNAi oligonucleotide.
  • an oligomeric duplex comprises a first oligomeric compound comprising a first modified oligonucleotide consisting of 21 or 23 linked nucleosides and a second oligomeric compound comprising a second modified oligonucleotide consisting of 19 or 21 linked nucleosides, wherein the nucleobase sequences of the first modified oligonucleotide and second modified oligonucleotide consist of any of the following pairs of selected from a first oligomeric compound selected from any one of SEQ ID NOs: 1541-2364 and 3090-3095, and a second oligomeric compound selected from any one of SEQ ID NOS: 2365-3075 and 3084-3089.
  • the first oligomeric compound is an antisense agent. In certain embodiments, the first modified oligonucleotide is an antisense oligomeric compound. In certain embodiments, the second oligomeric compound is a sense agent. In certain embodiments, the second modified oligonucleotide is a sense oligomeric compound. In certain embodiments, the first modified oligonucleotide is an antisense RNAi oligonucleotide. In certain embodiments, the second oligomeric compound is a sense agent. In certain embodiments, the second modified oligonucleotide is a sense RNAi oligonucleotide.
  • the oligomeric duplex comprises one or two unpaired nucleosides at either or both ends, forming one or two overhang ends.
  • an overhang end is one or two 3 '-nucleosides of the antisense oligomeric compound.
  • the last two 3 '-nucleosides of the antisense oligomeric compound are overhang nucleosides not paired with the sense oligomeric compound.
  • the last one or two 3 '-unpaired overhang nucleosides comprise an adenine nucleobase.
  • the last one or two 3'-unpaired overhang nucleosides comprise a thymine nucleobase.
  • the last one or two 3 '-unpaired overhang nucleosides comprise a uridine nucleobase. In certain embodiments the last one or two 3 '-unpaired overhang nucleosides comprise an inosine nucleobase.
  • the antisense oligomeric compound comprises a 5 ’-terminal group. In certain embodiments the sense strand comprises a conjugate group attached at the 5’ or 3’ end of the sense oligomeric compound.
  • At least one nucleoside of the first modified oligonucleotide and/or the second modified oligonucleotide comprises a modified sugar moiety.
  • suitable modified sugar moieties include, but are not limited to, a bicyclic sugar moiety, such as a 2’-4’ bridge selected from -O-CH 2 -; and -O-CH(CH 3 )-, and a non-bicyclic sugar moiety, such as a 2 ’-MOE sugar moiety, a 2’-F sugar moiety, a 2’-OMe sugar moiety, or a 2’-NMA sugar moiety.
  • At least one nucleoside of the first modified oligonucleotide and/or the second modified oligonucleotide comprises a modified 2’-deoxyribosyl sugar moiety. In certain embodiments, at least one nucleoside of the first modified oligonucleotide and/or the second modified oligonucleotide comprises a modified 3'-fluoro-hexitol sugar moiety.
  • At least 80%, at least 90%, or 100% of the nucleosides of the first modified oligonucleotide and/or the second modified oligonucleotide comprises a modified sugar moiety independently selected from 2’-F, 2’-MOE, 2’-OMe, 2 ’-deoxyribosyl, and 3'- fluoro-hexitol.
  • at least 80%, at least 90%, or 100% of the nucleosides of the first modified oligonucleotide and the second modified oligonucleotide comprises a modified sugar moiety independently selected from 2’-F, 2’-MOE, 2’-OMe, and 2 ’-deoxyribosyl.
  • At least one nucleoside of the first modified oligonucleotide and/or the second modified oligonucleotide comprises a modified sugar moiety and/or sugar surrogate.
  • a sugar moiety of the first modified oligonucleotide is modified, wherein the modified sugar moiety and/or sugar surrogate is selected from 2’-F, 2’-MOE, 2’-OMe, 2 ’-deoxyribosyl, and 3'-fluoro-hexitol.
  • a sugar motif (from 5’ to 3’) of the first modified oligonucleotide is selected from: efyyyfyyyyyyyfyfyyyyyyyyyyyyyy, yfyyyfyyyyyyfyfyyyyyyyyyyyy, efyyydyyeyyyydydyyyyyyyee, efyydyyyyyyyyfyfyyyyyee, efyydyyeyyyyfyfyyyyyee, e[FHNA]yyfyyyyyyyfyfyyyyyyee, and e[FHNA]yyyfyeyyyyfyfyyyyyyee, wherein each ‘e’ represents a 2'-MOE sugar moiety, each ‘y’ represents a 2
  • a sugar moiety of the second modified oligonucleotide is modified, wherein the modified sugar moiety is selected from 2’-F, 2’-MOE, 2’-OMe, 2 ’-deoxyribosyl and 3'-fluoro-hexitol.
  • a sugar motif (from 5’ to 3’) of the second modified oligonucleotide is selected from among: yyyyyyfyfffyyyyyyyyyyyy and eeyyyyyyyyyyyyyyee, wherein each ‘y’ represents a 2'- OMe sugar moiety, each ‘f represents a 2’-F sugar moiety, and each ‘e’ represents a 2’-MOE sugar moiety.
  • a sugar motif (from 5’ to 3’) of the first modified oligonucleotide and the second modified oligonucleotide is selected from: efyyyfyyyyyyyyfyfyyyyyyyyyyyy and yyyyyyyfyfffyyyyyyyyyyyy; yfyyyfyyyyyyyyfyfyyyyyyyyyy and yyyyyyfyfffyyyyyyyyyyyyyyyyyyy; efyydyyeyyydydydyyyyyee and eeyyyyyyyyyyyyyffyyyyyyyyyyyee; efyydyyyyyyyfyfyyyyyee and eeyyyyyyyyyyyyffyyyyyy
  • At least one intemucleoside linkage of the first modified oligonucleotide and/or the second modified oligonucleotide comprises a modified intemucleoside linkage.
  • the modified intemucleoside linkage is a phosphorothioate intemucleoside linkage.
  • at least one of the first, second, or third intemucleoside linkages from the 5 ’ end and/or the 3 ’ end of the first modified oligonucleotide comprises a phosphorothioate linkage.
  • at least one of the first, second, or third intemucleoside linkages from the 5 ’ end and/or the 3 ’ end of the second modified oligonucleotide comprises a phosphorothioate linkage.
  • each intemucleoside linkage of the first modified oligonucleotide is independently selected from a phosphodiester and a phosphorothioate, intemucleoside linkage
  • each intemucleoside linkage of the second modified oligonucleotide is independently selected from a phosphodiester and a phosphorothioate, intemucleoside linkage.
  • At least one linkage of the antisense oligomeric compound is a modified linkage.
  • an intemucleoside linkage of the first modified oligonucleotide is modified, wherein the 5’-most intemucleoside linkage (i.e., linking the first nucleoside from the 5’-end to the second nucleoside from the 5 ’-end) is modified.
  • the intemucleoside linkage motif (from 5’ to 3’) of the first modified oligonucleotide is selected from 5’- ssooooooooooooooss -3’, 5’- ssooosoooooooososooooss -3’, and 5’- ssooosooooooooooss -3’, each “s” is a phosphorothioate intemucleoside intemucleoside linkage and each “o” is a phosphodiester intemucleoside linkage.
  • an intemucleoside linkage of the second modified oligonucleotide is modified, wherein the 5 ’-most intemucleoside linkage (i.e., linking the first nucleoside from the 5’-end to the second nucleoside from the 5 ’-end) is modified.
  • the intemucleoside linkage motif (from 5’ to 3’) of the second modified oligonucleotide is selected from (from 5’ to 3’) of: 5’- ssooooooooooooooooss -3’ and 5’- ssooooooosooooppss -3’, wherein each ‘o’ represents a phosphodiester intemucleoside linkage and each ‘s’ represents a phosphorothioate intemucleoside linkage.
  • the two 5 ’-most intemucleoside linkages are modified.
  • the first one or 2 intemucleoside linkages from the 3 ’-end are modified.
  • the modified intemucleoside linkage is a phosphorothioate linkage.
  • At least one nucleobase of the first modified oligonucleotide and/or at least one nucleobase of the second modified oligonucleotide is a modified nucleobase.
  • the modified nucleobase is methylcytosine.
  • the modified nucleobase is inosine.
  • the first oligomeric compound comprises a terminal group comprising a stabilized phosphate group attached to the 5’ position of the 5’- most nucleoside.
  • the stabilized phosphate group comprises a cyclopropyl phosphonate or an (E)-vinyl phosphonate.
  • the stabilized phosphate group is an (E)-vinyl phosphonate.
  • the first modified oligonucleotide optionally is attached to a conjugate group.
  • the conjugate group comprises a conjugate linker and a conjugate moiety.
  • the conjugate group is attached to the first modified oligonucleotide at the 5 ’-end of the first modified oligonucleotide.
  • the conjugate group is attached to the first modified oligonucleotide at the 3 ’-end of the modified oligonucleotide.
  • the conjugate group is attached to the first modified oligonucleotide at an internal position.
  • the conjugate group is attached to the first modified oligonucleotide through a 2 ’-modification of a fiiranosyl sugar moiety. In certain embodiments, the conjugate group is attached to the first modified oligonucleotide through a modified intemucleoside linkage. In certain embodiments, the conjugate group comprises N-acetyl galactosamine.
  • the second modified oligonucleotide optionally is attached to a conjugate group.
  • the conjugate group comprises a conjugate linker and a conjugate moiety.
  • the conjugate group is attached to the second modified oligonucleotide at the 5 ’-end of the second modified oligonucleotide.
  • the conjugate group is attached to the second modified oligonucleotide at the 3 ’-end of the modified oligonucleotide.
  • the conjugate group is attached to the second modified oligonucleotide at an internal position.
  • the conjugate group is attached to the second modified oligonucleotide through a 2 ’-modification of a furanosyl sugar moiety. In certain embodiments, the conjugate group is attached to the second modified oligonucleotide through a modified intemucleoside linkage. In certain embodiments, the conjugate group comprises N-acetyl galactosamine.
  • an oligomeric agent comprises at least two oligomeric duplexes linked together. In certain embodiments, an oligomeric agent comprises two oligomeric duplexes wherein at least one oligomeric duplex is targeted to LPA RNA as described herein. In certain embodiments, an oligomeric agent comprises two or more of the same oligomeric duplex, which is any of the oligomeric duplexes described herein. In certain embodiments, the two or more oligomeric duplexes are covalently linked together. In certain embodiments, the second modified oligonucleotides of the two or more oligomeric duplexes are covalently linked together.
  • the second modified oligonucleotides of two or more oligomeric duplexes are covalently linked together at their 3’ ends. In certain embodiments, the second modified oligonucleotides of two or more oligomeric duplexes are covalently linked together at the 3 ’ end of one to the 5 ’ end of the other. In certain embodiments, the two or more oligomeric duplexes are covalently linked together by a glycol linker, such as a tetraethylene glycol linker. A structure of oligomeric duplexes covalently linked by a glycol linker is described in, e.g., Alterman, et al., Nature Biotech., 37:844-894, 2019.
  • a first modified oligonucleotide of a first oligomeric duplex is covalently linked to a second modified oligonucleotide of a second oligomeric duplex and a first modified oligonucleotide of the second oligomeric duplex is covalently linked to a second modified oligonucleotide of the first oligomeric duplex (see, e.g., PCT International Patent Application Publication W02020/065602 for a description of an example of a structure of linked oligomeric duplexes).
  • oligomeric duplexes provided herein comprise an oligomeric compound that is capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity; such oligomeric duplexes and oligomeric compounds are antisense agents.
  • RNAi agents are capable of RISC-mediated modulation of a target nucleic acid in a cell. In certain embodiments, such compounds reduce or inhibit the amount or activity of a target nucleic acid by 25% or more in the standard in vitro assay. In certain embodiments, RNAi agents selectively affect one or more target nucleic acid. Such RNAi agents comprise a nucleobase sequence that hybridizes to one or more target nucleic acid, resulting in one or more desired antisense activity. In certain embodiments, an RNAi agent does not hybridize to one or more non-target nucleic acid or does not hybridize to one or more non-target nucleic acid in such a way that results in significant undesired antisense activity.
  • hybridization of an antisense agent to a target nucleic acid results in inhibition of a binding interaction between the target nucleic acid and a protein or other nucleic acid (e.g., miRNA, IncRNA, sncRNA). In certain embodiments, hybridization of an antisense agent to a target nucleic acid results in modulation of translation of the target nucleic acid. In certain embodiments, hybridization of an oligomeric compound to a target nucleic acid results in an increase in the amount or activity of a target nucleic acid. In certain embodiments, hybridization of an antisense oligonucleotide to a target nucleic acid results in increased translation of the target nucleic acid. In certain embodiments, hybridization of an antisense oligonucleotide to a target nucleic acid results in reduced translation of the target nucleic acid.
  • a protein or other nucleic acid e.g., miRNA, IncRNA, sncRNA
  • Antisense activities may be observed directly or indirectly.
  • observation or detection of an antisense activity involves observation or detection of a change in an amount of a target nucleic acid or protein encoded by such target nucleic acid, a change in the ratio of a nucleic acid or protein and/or a phenotypic change in a cell or animal.
  • compositions and methods for reducing LPA RNA levels Certain embodiments provide compositions and methods for reducing apo(a) and/or Lp(a) levels. In certain embodiments, reducing apo(a) levels in a tissue, organ and/or subject improves the ratio of LDL to HDL or the ratio of TG to HDL. In certain embodiments, provided are methods for using an oligomeric antisense agent targeted to an apo(a) nucleic acid for modulating the expression of apo(a) in a subject. In certain embodiments, expression of apo(a) is reduced.
  • inhibition of apo(a) or Lp(a) expression occurs in a cell, tissue or organ. In certain embodiments, inhibition of apo(a) or Lp(a) occurs in a cell, tissue or organ in a subject. In certain embodiments, inhibition is a reduction in apo(a) mRNA level. In certain embodiments, inhibition is a reduction in apo(a) protein level. In certain embodiments, both apo(a) mRNA and protein levels are reduced. In certain embodiments, inhibition is a reduction in Lp(a) level. Such reduction may occur in a time -dependent or in a dose-dependent manner.
  • a method of modulating expression of LPA or modulating apo(a) protein or Lp(a) in a cell comprises contacting the cell with an oligomeric duplex comprising or consisting of an oligomeric compound comprising a modified oligonucleotide having a targeting region complementary to a target region of a LPA nucleic acid.
  • a method of inhibiting expression of LPA or inhibiting apo(a) protein or Lp(a) protein in a cell comprises contacting the cell with an oligomeric duplex comprising or consisting of an oligomeric compound comprising a modified oligonucleotide having a nucleobase sequence complementary to a target region of a LPA nucleic acid.
  • the cell is a hepatocyte.
  • provided herein are methods of inhibiting or reducing LPA expression, LPA RNA levels and/or apo(a) protein or Lp(a) levels and/or activity, in a subject having, or at risk of having, a disease, disorder, condition or injury associated with LPA and/or apo(a) protein or Lp(a), such as a disease, disorder, condition or injury associated with inflammatory cardiovascular disease, wherein the method includes administering to the subject an oligomeric duplex comprising or consisting of an oligomeric antisense agent comprising a modified oligonucleotide targeting a LPA nucleic acid thereby inhibiting expression of LPA nucleic acid in the subject. In certain embodiments, expression of LPA nucleic acid is inhibited.
  • administering an oligomeric duplex inhibits LPA expression, LPA RNA levels and/or apo(a) protein or Lp(a) levels and/or activity in the plasma/serum blood. In certain embodiments, administering an oligomeric duplex inhibits or reduces LPA expression, LPA RNA levels, and/or apo(a) protein or Lp(a) levels, and/or activity in the liver. In certain embodiments, administering such an oligomeric duplex inhibits or reduces LPA expression, LPA RNA levels and/or apo(a) protein or Lp(a) levels and/or activity in the plasma/serum blood and the liver of the subject.
  • detectable amount of the LPA RNA may be reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.
  • an oligomeric compound comprising or consisting of a modified oligonucleotide comprising a targeting region complementary to a target region of SEQ ID NO: 1 or SEQ ID NO: 2 is capable of decreasing or reducing a detectable amount of a apo(a) protein or Lp(a) in a cell, organ or tissue, e.g., the liver of the subject, when the compound is administered to the cell, a tissue, and/or subject.
  • detectable amount of the apo(a) protein or Lp(a) may be reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.
  • an oligomeric duplex comprising or consisting of an oligomeric antisense agent has LPA RNA and/or apo(a) protein or Lp(a) reduction activity, and, in particular embodiments, liver LPA RNA and/or apo(a) protein or Lp(a) reduction activity, that is comparable to or greater than the LPA RNA and/or apo(a) protein or Lp(a) reduction activity of a comparator agent.
  • the comparator agent is an agent that comprises a comparator modified oligonucleotide having a targeting region complementary to a target region of LPA.
  • the comparator modified oligonucleotide is complementary to the same or a similar target region as the modified oligonucleotide of the oligomeric compound.
  • the comparator modified oligonucleotide is complementary to a different target region from the modified oligonucleotide of the oligomeric compound.
  • the amount of LPA RNA is reduced by at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% in a cell (e.g., hepatocyte), organ (e.g., livery), or subject (e.g., animal) that has been contacted with or administered an oligomeric duplex provided herein (or a composition comprising such an oligomeric duplex) compared to a control (e.g., a cell, organ, tissue, system or subject that has not been contacted with or administered the oligomeric duplex, or was contacted with or administered a control substance (e.g., PBS)).
  • a control e.g., a cell, organ, tissue, system or subject that has not been contacted with or administered the oligomeric duplex, or was contacted
  • the percentage of LPA RNA decrease or reduction in a cell e.g., a hepatocyte
  • organ e.g., a livery
  • tissue e.g., system, or subject (e.g., animal) contacted with or administered an oligomeric duplex or composition provided herein is 0.1% to 30% greater than or less than, 0. 1% to 25% greater than or less than, 0.1% to 20% greater than or less than, 0.1% to 15% greater than or less than, 0.1% to 10% greater than or less than, or 0. 1% to 5% greater than or less than, 0.
  • a cell e.g., a cardiomyocyte
  • organ e.g., a heart
  • tissue e.g., system or subject (e.g., animal) contacted with or administered the same concentration or dose of a comparator agent.
  • LDL-lowering therapies reduce the risk of major cardiac events, residual cardiovascular risk encountered in some patients with low LDL levels implies other mechanisms of cardiovascular pathology.
  • Lp(a) concentration is associated with a higher risk of coronary artery disease and atherosclerosis-related disorders (Clarke et al., N. Engl. J. Med., Vol. 361 :2518-2528, 2009; Kamstrup et al., JAMA, Vol.
  • LPA gene mutations can increase LPA RNA levels, result in abnormal apo(a) cleavage that leads to increased apo(a) levels (and Lp(a) levels), or decreased degradation and clearance, and/or abnormal interactions between Lp(a) and other proteins or other endogenous or exogenous substances (e.g., plasminogen receptor) such that Lp(a) level is increased or degradation is decreased.
  • a condition associated with high Lp(a) levels may be relatively insensitive to lifestyle changes and common statin drugs, and therefore hard to treat.
  • Lp(a) levels are at a significant risk of disease (Lippi et al., Clinica Chimica Acta, 2011, 412:797-801; Solfrizz et al.). For example, subjects will Lp(a) levels greater than > 75 nanomole s/liter (nmol/L) or > 30 mg/dL are considered to have increased risk for various diseases. In many subjects with high Lp(a) levels, current treatments cannot reduce Lp(a) levels to safe levels. Apo(a) plays an important role in the formation of Lp(a), hence reducing apo(a) can reduce Lp(a) and prevent, treat or ameliorate a disease associated with Lp(a).
  • treatment with oligomeric duplexes and methods provided herein is indicated for a subject with elevated apo(a) levels and/or Lp(a) levels.
  • the subject has apo(a) levels > 10 mg/dL, > 20 mg/dL, > 30 mg/dL, > 40 mg/dL, > 50 mg/dL, > 60 mg/dL, > 70 mg/dL, > 80 mg/dL, > 90 mg/dL or > 100 mg/dL.
  • the subject has Lp(a) levels > 10 mg/dL, > 15 mg/dL, > 20 mg/dL, > 25 mg/dL, > 30 mg/dL, > 35 mg/dL, > 40 mg/dL, > 50 mg/dL, > 60 mg/dL, > 70 mg/dL, > 80 mg/dL, > 90 mg/dL, > 100 mg/dL, > 110 mg/dL, > 120 mg/dL, > 130 mg/dL, > 140 mg/dL, > 150 mg/dL, > 160 mg/dL, > 170 mg/dL, > 175 mg/dL, > 180 mg/dL, > 190 mg/dL, > 200 mg/dL.
  • a subject has apo(a) level greater than the upper limit of normal, e.g. wherein the subject has apo(a) levels
  • provided herein are methods for preventing, treating, or delaying or preventing the development or progression of, diseases, disorders, conditions or injuries associated with LPA and/or apo(a) protein or Lp(a) increased levels, wherein the method comprises administering to a subject an oligomeric duplex described herein (e.g., an antisense oligomeric compound comprising or consisting of a modified oligonucleotide comprising a targeting region complementary to a target region of a LPA nucleic acid).
  • an oligomeric duplex described herein e.g., an antisense oligomeric compound comprising or consisting of a modified oligonucleotide comprising a targeting region complementary to a target region of a LPA nucleic acid.
  • a Lp(a)-associated condition may be, e.g., a cardiovascular condition, a metabolic condition, an inflammatory condition.
  • an Lp(a)-associated condition is an inflammatory, cardiovascular or metabolic disease or disorder.
  • an Lp(a) associated condition or lipoprotein metabolism disorder is a cardiometabolic disorder.
  • an oligomeric duplex comprising or consisting of an oligomeric antisense agent described herein is used to treat a subject with a cardiovascular disease (CVD) such as hypertriglyceridemia and/or other condition selected from lipidemia (e.g., hyperlipidemia), dyslipidemia (e.g., atherogenic dyslipidemia, diabetic dyslipidemia, or mixed dyslipidemia), hyperlipoproteinemia, hyperapobetalipoproteinemia, chronic heart disease, including coronary artery disease (CAD) or any symptoms or conditions associated with a CVD, e.g., hypercholesterolemia (e.g., statin-resistant hypercholesterolemia, heterozygous or homozygous familial hypercholesterolemia) myocardial infarction (MI), acute coronary syndrome, mesenteric ischemia, superior mesenteric artery occlusion, restenosis, renal
  • CVD cardiovascular disease
  • a method comprises administering to a subject an oligomeric duplex comprising or consisting of an oligomeric antisense agent comprising a targeting region complementary to a target region of a LPA nucleic acid.
  • the subject has or is at risk for developing a cardiovascular, metabolic and/or inflammatory injury, disease, condition or disorder.
  • administration of an oligomeric duplex comprising or consisting of an oligomeric antisense agent provided herein to the subject reduces or delays the onset or progression of at least one at least one symptom of aortic stenosis.
  • Certain embodiments provide compositions and methods for preventing, delaying, slowing the progression of apo(a) related diseases, disorders, and conditions in a subject in need thereof.
  • Certain embodiments provide compositions and methods for ameliorating apo(a) related diseases, disorders, and conditions in a subject in need thereof.
  • Certain embodiments provide compositions and methods for preventing, delaying, slowing the progression Lp(a) related diseases, disorders, and conditions in a subject in need thereof.
  • Certain embodiments provide compositions and methods for ameliorating Lp(a) related diseases, disorders, and conditions in a subject in need thereof.
  • such diseases, disorders, and conditions include inflammatory, cardiovascular and/or metabolic diseases, disorders, and conditions.
  • the individual has elevated apo(a) levels.
  • the individual has elevated Lp(a) levels.
  • the individual has an inflammatory, cardiovascular and/or metabolic disease, disorder or condition.
  • administration of a therapeutically effective amount of an oligomeric duplex is accompanied by monitoring of apo(a) or Lp(a) levels.
  • administration of an oligomeric duplex is accompanied by monitoring of markers of inflammatory, cardiovascular and/or metabolic disease, or other disease process associated with the expression of apo(a), to determine an individual’s response to an oligomeric duplex.
  • An individual’s response to administration of an oligomeric duplex can be used by a physician to determine the amount and duration of therapeutic intervention with an oligomeric duplex.
  • cardiovascular diseases, disorders or conditions include, but are not limited to, aortic stenosis, aneurysm (e.g., abdominal aortic aneurysm), angina, arrhythmia, atherosclerosis, cerebrovascular disease, coronary artery disease, coronary heart disease, dyslipidemia, hypercholesterolemia, hyperlipidemia, hypertension, hypertriglyceridemia, myocardial infarction, peripheral vascular disease (e.g., peripheral artery disease, peripheral artery occlusive disease), retinal vascular occlusion, stroke, elevated Lp(a) associated CVD risk, recurrent cardiovascular events with elevated Lp(a), calcific aortic valve stenosis associated with high Lp(a), and the like.
  • Certain embodiments provide compositions and methods for preventing, delaying, slowing the progression of aortic stenosis.
  • Certain embodiments provide compositions and methods for ameliorating aortic stenosis.
  • an oligomeric duplex modulates physiological markers or phenotypes of a cardiovascular disease, disorder or condition.
  • administration of an oligomeric duplex to a human can decrease Lp(a), LDL and cholesterol levels compared to untreated subjects.
  • modulation of the physiological markers or phenotypes can be associated with inhibition of apo(a) by an oligomeric duplex.
  • physiological markers of the cardiovascular disease, disorder or condition can be quantifiable.
  • Lp(a), LDL or cholesterol levels can be measured and quantified by, for example, standard lipid tests.
  • the marker can be decreased by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values.
  • treating results in reducing the rate of onset of a symptom associated with the cardiovascular disease, disorder or condition.
  • treating results in reducing the severity of a symptom associated with the cardiovascular or condition.
  • a cardiovascular disease, disorder or condition can be characterized by numerous physical symptoms. Any symptom known to one of skill in the art to be associated with the cardiovascular disease, disorder or condition can be prevented, treated, ameliorated or otherwise modulated with the compounds and methods described herein.
  • the symptom can be any of, but not limited to, angina, chest pain, shortness of breath, palpitations, weakness, dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling in the lower extremities, cyanosis, fatigue, fainting, numbness of the face, numbness of the limbs, claudication or cramping of muscles, bloating of the abdomen or fever.
  • a method of treatment of a subject comprises administering to the subject an oligomeric duplex comprising or consisting of an oligomeric antisense agent having a targeting region complementary to a target region of a LPA nucleic acid, thereby treating the subject.
  • the subject has or is at risk for developing a cardiovascular disease, e.g., an atherosclerotic cardiovascular disease, an atherosclerotic cerebrovascular disease, a hyperlipidaemia, wherein the disease is associated with elevated levels of Lp(a)-containing particles.
  • treatment is to prevent and/or reduce the risk of suffering from and/or treat stroke, atherosclerosis, thrombosis calcific aortic stenosis such as calcific aortic stenosis, ischaemic stroke, coronary artery disease, peripheral arterial disease, abdominal aortic aneurysm, heart failure secondary to ischaemic cardiomyopathy, or familial hypercholesterolaemia, wherein the disease is associated with elevated levels of Lp(a)-containing particles.
  • treatment with an oligomeric duplex is to prevent and/or reduce the risk of suffering from and/or treat or cardiovascular disease such as coronary heart disease and any other disease or pathology associated with elevated levels of Lp(a)-containing particles.
  • treament is to prevent and/or reduce a risk of suffering from and/or treat, a cardiovascular disease, e.g., an atherosclerotic cardiovascular disease, an atherosclerotic cerebrovascular disease, a hyperlipidaemia, wherein the disease is associated with elevated levels of Lp(a)-containing particles.
  • treatment is to prevent and/or reduce the risk of suffering from and/or treat stroke, atherosclerosis, thrombosis calcific aortic stenosis such as calcific aortic stenosis, ischaemic stroke, coronary artery disease, peripheral arterial disease, abdominal aortic aneurysm, heart failure secondary to ischaemic cardiomyopathy, or familial hypercholesterolaemia, wherein the disease is associated with elevated levels of Lp(a)-containing particles.
  • treatment with an oligomeric duplex results in reducing the risk of a cardiovascular event in a patient with atherosclerotic cardiovascular disease.
  • the cardiovascular event is cardiovascular death, myocardial infarction, stroke, and/or coronary revascularization.
  • a subject has a history of coronary revascularization, a history of coronary artery bypass grafting, a diagnosis of coronary artery disease, a diagnosis of atherosclerotic cerebrovascular disease, a diagnosis of peripheral artery disease, and/or a history of myocardial infarction.
  • administration of a pharmaceutical composition comprising an oligomeric duplex comprising or consisting of an oligomeric antisense agent to the subject reduces or delays the onset or progression of at least one of stroke, atherosclerosis, thrombosis calcific aortic stenosis such as calcific aortic stenosis, ischaemic stroke, coronary artery disease, peripheral arterial disease, abdominal aortic aneurysm, heart failure secondary to ischaemic cardiomyopathy, or familial hypercholesterolaemia.
  • administration of a pharmaceutical composition comprising an oligomeric duplex comprising or consisting of an oligomeric antisense agent to the subject reduces or delays the onset or progression of at least one of angina, chest pain, shortness of breath, palpitations, weakness, dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling in the lower extremities, cyanosis, fatigue, fainting, numbness of the face, numbness of the limbs, claudication or cramping of muscles, bloating of the abdomen, and fever, or a combination thereof.
  • treatment with an oligomeric duplex is to ameliorate one or more symptoms of cardiovascular disease such as coronary heart disease and/or other disease or pathology associated with elevated levels of Lp(a) containing particles.
  • treatment is to ameliorate symptoms associated with a cardiovascular disease such as coronary artery disease, carotid artery disease, peripheral artery disease, myocardial infarction, cerebrovascular disease, stroke, aortic valve stenosis, stable or unstable angina, atrial fibrillation, heart failure, hyperlipidemia, heterozygous familial hypercholesterolemia, or homozygous familial hypercholesterolemia.
  • a subject is diagnosed with or at risk of a cardiovascular disease; diagnosed myocardial infarction, acute coronary syndrome.
  • the symptoms include, but are not limited to, angina, chest pain, shortness of breath, palpitations, weakness, dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling in the lower extremities, cyanosis, fatigue, fainting, numbness of the face, numbness of the limbs, claudication or cramping of muscles, bloating of the abdomen, and fever. Certain embodiments provide a method of reducing at least one symptom of aortic stenosis.
  • administration of a pharmaceutical composition comprising an oligomeric duplex comprising or consisting of an oligomeric antisense agent to the subject ameliorates or reduces one or more symptoms of at least one of stroke, atherosclerosis, thrombosis calcific aortic stenosis such as calcific aortic stenosis, ischaemic stroke, coronary artery disease, peripheral arterial disease, abdominal aortic aneurysm, heart failure secondary to ischaemic cardiomyopathy, or familial hypercholesterolaemia.
  • administration of a pharmaceutical composition comprising an oligomeric duplex comprising or consisting of an oligomeric antisense agent to the subject ameliorates one or more symptoms of angina, chest pain, shortness of breath, palpitations, weakness, dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling in the lower extremities, cyanosis, fatigue, fainting, numbness of the face, numbness of the limbs, claudication or cramping of muscles, bloating of the abdomen, and fever, or a combination thereof.
  • metabolic diseases, disorders or conditions include, but are not limited to, hyperglycemia, prediabetes, diabetes (type I and type II), obesity, insulin resistance, metabolic syndrome and diabetic dyslipidemia.
  • an oligomeric duplex modulates physiological markers or phenotypes of a metabolic disease, disorder or condition.
  • administration of an oligomeric duplex to humans can decrease glucose and insulin resistance levels in those subjects compared to untreated subjects.
  • the modulation of the physiological markers or phenotypes can be associated with inhibition of apo(a) by an oligomeric duplex.
  • physiological markers of the metabolic disease, disorder or condition can be quantifiable. For example, glucose levels or insulin resistance can be measured and quantified by standard tests known in the art.
  • the marker can be decreased by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values.
  • insulin sensitivity can be measured and quantified by standard tests known in the art.
  • the marker can be increase by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values.
  • treating results in reducing the rate of onset of a symptom associated with the metabolic disease, disorder or condition.
  • treating results in reducing the severity of a symptom associated with the metabolic disease, disorder or condition.
  • a metabolic disease, disorder or condition can be characterized by numerous physical symptoms. Any symptom known to one of skill in the art to be associated with the metabolic disease, disorder or condition can be prevented, treated, ameliorated or otherwise modulated with the compounds and methods described herein.
  • a symptom can be any of, but not limited to, excessive urine production (polyuria), excessive thirst and increased fluid intake (polydipsia), blurred vision, unexplained weight loss and lethargy.
  • inflammatory diseases, disorders or conditions may overlap with cardiometabolic disorders or conditions, and include, but are not limited to, elevated Lp(a) associated CVD risk, recurrent cardiovascular events with elevated Lp(a), aortic stenosis (e.g., calcific aortic valve stenosis associated with high Lp(a)), coronary artery disease (CAD), Alzheimer’s Disease and thromboembolic diseases, disorder or conditions.
  • Certain thromboembolic diseases, disorders or conditions include, but are not limited to, stroke, thrombosis, myocardial infarction and peripheral vascular disease.
  • an oligomeric duplex modulates physiological markers or phenotypes of an inflammatory disease, disorder or condition.
  • administration of an oligomeric duplex to a human can decrease inflammatory cytokine or other inflammatory markers levels in compared to untreated subjects.
  • the modulation of the physiological markers or phenotypes can be associated with inhibition of apo(a) by an oligomeric duplex.
  • physiological markers of the inflammatory disease, disorder or condition can be quantifiable. For example, cytokine levels can be measured and quantified by standard tests known in the art.
  • the marker can be decreased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, or a range defined by any two of these values.
  • treating results in reducing the rate of onset of a symptom associated with the inflammatory disease, disorder or condition. In certain embodiments, treating results in reducing the severity of a symptom associated with the inflammatory disease, disorder or condition.
  • an oligomeric duplex has greater LPA RNA and/or protein reduction activity (i.e., greater specificity of action) in a target cell/organ/tissue/system (e.g., hepatocytes, liver) LPA than a non-target (e.g., plasminogen).
  • a target cell/organ/tissue/system e.g., hepatocytes, liver
  • a non-target e.g., plasminogen
  • administration of an oligomeric duplex provided herein reduces the amount or activity of LPA RNA and/or protein at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to a control and has no, or a non-significant, effect on (e.g., reduction in) the amount or activity of plasminogen.
  • administration of an oligomeric duplex results in reduction of LPA expression by at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, or a range defined by any two of these values.
  • apo(a) expression is reduced to at least ⁇ 100 mg/dL, ⁇ 90 mg/dL, ⁇ 80 mg/dL, ⁇ 70 mg/dL, ⁇ 60 mg/dL,
  • administration of an oligomeric duplex results in reduction of apo(a) by at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, or a range defined by any two of these values.
  • Lp(a) containing particles in serum is reduced to at least ⁇ 200 mg/dL, ⁇ 190 mg/dL, ⁇ 180 mg/dL, ⁇ 175 mg/dL, ⁇ 170 mg/dL, ⁇ 160 mg/dL,
  • ⁇ 150 mg/dL ⁇ 140 mg/dL, ⁇ 130 mg/dL, ⁇ 120 mg/dL, ⁇ 110 mg/dL, ⁇ 100 mg/dL, ⁇ 90 mg/dL, ⁇ 80 mg/dL, ⁇ 70 mg/dL, ⁇ 60 mg/dL, ⁇ 55 mg/dL, ⁇ 50 mg/dL, ⁇ 45 mg/dL, ⁇ 40 mg/dL, ⁇ 35 mg/dL, ⁇ 30 mg/dL, ⁇ 25 mg/dL, ⁇ 20 mg/dL, ⁇ 15 mg/dL, or ⁇ 10 mg/dL.
  • compositions comprising an oligomeric duplex are used for the preparation of a medicament for treating a patient suffering or susceptible to an inflammatory, cardiovascular and/or a metabolic disease, disorder or condition.
  • the subject has or is at risk for developing cardiovascular disease (CVD) such as hypertriglyceridemia and/or other condition selected from lipidemia (e.g., hyperlipidemia), dyslipidemia (e.g., atherogenic dyslipidemia, diabetic dyslipidemia, or mixed dyslipidemia), hyperlipoproteinemia, hyperapobetalipoproteinemia, chronic heart disease, including coronary artery disease (CAD) or any symptoms or conditions associated with a CVD, e.g., hypercholesterolemia (e.g., statin-resistant hypercholesterolemia, heterozygous or homozygous familial hypercholesterolemia) myocardial infarction (MI), acute coronary syndrome, mesenteric ischemia, superior mesenteric artery occlusion, restenosis, renal artery stenosis, angina peripheral arterial disease (PAD), calcific aortic valve disease (CAVD), aortic stenosis, aortic valve regurgitation, aor
  • CVD
  • At least one symptom of the cardiovascular injury, disease, condition, or disorder is ameliorated.
  • the at least one symptom is selected from include, but are not limited to, angina, chest pain, shortness of breath, palpitations, weakness, dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling in the lower extremities, cyanosis, fatigue, fainting, numbness of the face, numbness of the limbs, claudication or cramping of muscles, bloating of the abdomen, and fever.
  • an oligomeric duplex provided herein e.g., an oligomeric duplex comprising or consisting of an oligomeric antisense agent
  • administration of an oligomeric duplex provided herein reduces or delays the onset or progression of at least one at least one symptom of aortic stenosis.
  • Certain embodiments are drawn to an oligomeric duplex comprising or consisting of an oligomeric antisense agent comprising or consisting of a modified oligonucleotide having a targeting region complementary to a target region of a LPA nucleic acid, for the manufacture or preparation of a medicament for ameliorating, or delaying or preventing development or progression of a disease, disorder, condition or injury and/or for ameliorating, preventing or delaying the onset of one or more symptoms of a disease, disorder, condition or injury, wherein the disease, disorder, condition or injury is associated with cardiovascular disease (CVD) such as hypertriglyceridemia and/or other condition selected from lipidemia (e.g., hyperlipidemia), dyslipidemia (e.g., atherogenic dyslipidemia, diabetic dyslipidemia, or mixed dyslipidemia), hyperlipoproteinemia, hyperapobetalipoproteinemia, chronic heart disease, including coronary artery disease (CAD) or any symptoms or conditions associated with a CVD, e.g., hyperchol
  • an oligomeric duplex is for the manufacture or preparation of a medicament for improving one or more symptoms selected from angina, chest pain, shortness of breath, palpitations, weakness, dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling in the lower extremities, cyanosis, fatigue, fainting, numbness of the face, numbness of the limbs, claudication or cramping of muscles, bloating of the abdomen, and fever.
  • prophylactic administration of an oligomeric duplex or composition provided herein to a subject at risk for cardiovascular, metabolic, and/or inflammatory disease is able to prevent, ameliorate, postpone or delay a symptom and/or development or progression of cardiovascular, metabolic, an/or inflammatory disease progression.
  • an oligomeric duplex is for the manufacture or preparation of a medicament for improving angina, chest pain, shortness of breath, palpitations, weakness, dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling in the lower extremities, cyanosis, fatigue, fainting, numbness of the face, numbness of the limbs, claudication or cramping of muscles, bloating of the abdomen, and fever.
  • the oligomeric duplex is any oligomeric duplex (e.g., an oligomeric duplex comprising or consisting of an oligomeric antisense agent) described herein.
  • an oligomeric duplex is administered parenterally. In certain embodiments, an oligomeric duplex is administered intravenously, subcutaneously, intramuscularly, or intrathecally. In certain embodiments, an oligomeric duplex or composition is co-administered with a second agent or therapy. In certain embodiments, the oligomeric duplex or composition and second agent are administered concomitantly. In certain embodiments, a second agent is a glucose-lowering agent. In certain embodiments, a second agent is a LDL, TG or cholesterol lowering agent. In certain embodiments, a second agent is an anti-inflammatory agent.
  • a second agent can be, but is not limited to, a non-steroidal anti-inflammatory drug (NSAID e.g., aspirin), niacin (e.g., Niaspan), nicotinic acid, ezetimibe, an apoB inhibitor (e.g., Mipomersen), a CETP inhibitor (e.g., Anacetrapib), an apo(a) inhibitor, a thyroid hormone analog (e.g., Eprotirome), a HMG-CoA reductase inhibitor (e.g., a statin), a fibrate (e.g., Gemfibrozil), a microsomal triglyceride transfer protein inhibitor (e.g., Lomitapide), a PCSK9 inhibitor (e.g., inclisiran), as well as therapies in development, e.g., an ANGPTL3 inhibitor, an LPA inhibitor.
  • NSAID non-steroidal anti-inflammatory drug
  • niacin e.
  • a therapy can be, but is not limited to, Lp(a) apheresis.
  • a second agent is an alzheimer disease drug.
  • Agents or therapies can be co-administered or administered concomitantly. Agents or therapies can be sequentially or subsequently administered.
  • compositions comprising one or more oligomeric duplexes or oligomeric compounds, wherein each oligomeric duplex or compound comprises a modified oligonucleotide (e.g., oligomeric compound).
  • the one or more oligomeric duplex or oligomeric compound each comprises an antisense agent.
  • a pharmaceutical composition comprises a pharmaceutically acceptable diluent or carrier.
  • a pharmaceutical composition comprises or consists of a sterile saline solution and one or more compound or duplex.
  • the sterile saline is pharmaceutical grade saline.
  • a pharmaceutical composition comprises or consists of one or more compound or duplex and sterile water.
  • the sterile water is pharmaceutical grade water.
  • a pharmaceutical composition comprises or consists of one or more compound or duplex and phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • sterile PBS is pharmaceutical grade PBS.
  • the pH of a solution is modulated with a suitable pH- adjusting agent, for example, with acids such as hydrochloric acid and alkalis such as sodium hydroxide, to a range of from about 7.1-7.3, or to about 7.2.
  • a pharmaceutical composition comprises an oligomeric duplex comprising a first oligomeric compound and a second oligomeric compound and sterile saline.
  • a pharmaceutical composition consists of such oligomeric duplex and sterile saline.
  • a pharmaceutical composition consists essentially of such oligomeric duplex and sterile saline.
  • the sterile saline is sterile PBS.
  • the sterile saline is pharmaceutical grade.
  • the pH of a solution is modulated with a suitable pH-adjusting agent, for example, with acids such as hydrochloric acid and alkalis such as sodium hydroxide, to a range of from about 7. 1-7.3, or to about 7.2.
  • pharmaceutical compositions comprise one or more oligomeric duplex and one or more excipients.
  • excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.
  • an oligomeric duplex may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations.
  • compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
  • compositions comprising an oligomeric duplex encompass any pharmaceutically acceptable salts of the oligomeric compound(s) or duplex, esters of the compound(s) or duplex, or salts of such compounds or esters.
  • pharmaceutical compositions comprising an oligomeric compound or oligomeric duplex comprising one or more oligomeric compound upon administration to a subject, including a human, are capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of oligomeric compounds or oligomeric duplexes, and other bioequivalents.
  • pharmaceutically acceptable salts comprise inorganic salts, such as monovalent or divalent inorganic salts. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium, potassium, calcium, and magnesium salts.
  • oligomeric compounds or oligomeric duplexes are lyophilized and isolated, e.g., as sodium salts.
  • a sodium salt of an agent or duplex is mixed with a pharmaceutically acceptable diluent.
  • the pharmaceutically acceptable diluent comprises sterile saline, sterile water, PBS.
  • a sodium salt of an oligomeric compound or oligomeric duplex is mixed with PBS.
  • lipid moieties have been used in nucleic acid therapies in a variety of methods.
  • a nucleic acid such as an oligomeric compound or oligomeric duplex comprising oligomeric compound, is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids.
  • nucleic acid complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid.
  • a lipid moiety is selected to increase distribution of a pharmaceutical compound to a particular cell or tissue.
  • a lipid moiety is selected to increase distribution of a pharmaceutical compound to fat tissue.
  • a lipid moiety is selected to increase distribution of a pharmaceutical compound to muscle tissue.
  • compositions comprise a delivery system.
  • delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds.
  • certain organic solvents such as dimethylsulfoxide are used.
  • pharmaceutical compositions comprise one or more tissue-specific delivery molecules designed to deliver the one or more compounds to specific tissues or cell types.
  • pharmaceutical compositions include liposomes coated with a tissuespecific antibody.
  • compositions comprise a co-solvent system.
  • co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.
  • co-solvent systems are used for hydrophobic compounds.
  • a non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80TM and 65% w/v polyethylene glycol 300.
  • the proportions of such cosolvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics.
  • co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80TM; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
  • a pharmaceutical composition is prepared for administration of an oligomeric duplex to a subject.
  • Suitable administration routes include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppository, through inhalation, intrathecal, intracerebroventricular, intraperitoneal, intranasal, intraocular, intratumoral, and parenteral (e.g., intravenous, intramuscular, intrathecal, intramedullary, and subcutaneous).
  • pharmaceutical compositions are prepared for oral administration.
  • pharmaceutical compositions are prepared for buccal administration.
  • a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, intrathecal (IT), intracerebroventricular (ICV), etc.).
  • a pharmaceutical composition comprises a carrier or diluent and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives).
  • injectable suspensions are prepared using appropriate liquid carriers, diluents, suspending agents and the like.
  • compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers.
  • Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.
  • certain compounds disclosed herein act as acids. Although such compounds may be drawn or described in protonated (free acid) form or ionized and in association with a cation (salt) form, aqueous solutions of such compounds exist in equilibrium among such forms. For example, a phosphodiester linkage of an oligonucleotide in aqueous solution exists in equilibrium among free acid, anion and salt forms. Unless otherwise indicated, compounds described herein are intended to include all such forms. Moreover, certain oligonucleotides have several such linkages, each of which is in equilibrium. Thus, oligonucleotides in solution exist in an ensemble of forms at multiple positions all at equilibrium.
  • oligonucleotide herein is intended to include all such forms. Drawn structures necessarily depict a single form. Nevertheless, unless otherwise indicated, such drawings are likewise intended to include corresponding forms.
  • a structure depicting the free acid of a compound followed by the term “or a pharmaceutically acceptable salt thereof’ expressly includes all such forms that may be fully or partially protonated/de-protonated/in association with a cation or a combination of cations. In certain embodiments, one or more specific cation is identified. The cations include, but are not limited to, sodium, potassium, calcium, and magnesium.
  • a structure depicting the free acid of a compound followed by the term “or a pharmaceutically acceptable salt thereof’ expressly includes all such forms that may be fully or partially protonated/de-protonated/in association with one or more cations selected from sodium, potassium, calcium, and magnesium.
  • oligomeric compounds or oligomeric duplexes are in aqueous solution with sodium. In certain embodiments, oligomeric compounds or oligomeric duplexes are in aqueous solution with potassium. In certain embodiments, oligomeric compounds or oligomeric duplexes are in PBS. In certain embodiments, oligomeric compounds or oligomeric duplexes are in water. In certain such embodiments, the pH of a solution is adjusted with NaOH and/or HC1 to achieve a desired pH.
  • a dose may be in the form of a dosage unit.
  • a dose (or dosage unit) of an agent e.g., oligomeric compound, oligomeric duplex, antisense agent
  • an agent e.g., oligomeric compound, oligomeric duplex, antisense agent
  • milligrams indicates the mass of the free acid form of the compound.
  • the free acid is in equilibrium with anionic and salt forms.
  • the compound e.g., oligomeric compound, oligomeric duplex, antisense agent
  • the compound exists as a solvent-free, sodium-acetate free, anhydrous, free acid.
  • an agent e.g., oligomeric compound, oligomeric duplex, antisense agent
  • sodium e.g., saline
  • the compound may be partially or fully de-protonated and in association with sodium ions.
  • the mass of the protons is nevertheless counted toward the weight of the dose, and the mass of the sodium ions is not counted toward the weight of the dose.
  • an agent comprises a conjugate group
  • the mass of the conjugate group is included in calculating the dose of such compound. If the conjugate group also has an acid, the conjugate group is likewise assumed to be fully protonated for the purpose of calculating dose.
  • RNA nucleoside comprising a 2 ’-OH sugar moiety and a thymine base
  • nucleic acid sequences provided herein are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases.
  • an oligonucleotide having the nucleobase sequence “ATCGATCG” encompasses any oligonucleotides having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence “AUCGAUCG” and those having some DNA bases and some RNA bases such as “AUCGATCG” and oligonucleotides having other modified nucleobases, such as “AT ⁇ CGAUCG,” wherein m C indicates a cytosine base comprising a methyl group at the 5-position.
  • Certain compounds described herein e.g., modified oligonucleotides have one or more asymmetric center and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (.S'), as a or p such as for sugar anomers, or as (D) or (L), such as for amino acids, etc.
  • Compounds provided herein that are drawn or described as having certain stereoisomeric configurations include only the indicated compounds.
  • Compounds provided herein that are drawn or described with undefined stereochemistry include all such possible isomers, including their stereorandom and optically pure forms, unless specified otherwise.
  • tautomeric forms of the compounds herein are also included unless otherwise indicated. Unless otherwise indicated, compounds described herein are intended to include corresponding salt forms.
  • the compounds described herein include variations in which one or more atoms are replaced with a non-radioactive isotope or radioactive isotope of the indicated element.
  • compounds herein that comprise hydrogen atoms encompass all possible deuterium substitutions for each of the ’H hydrogen atoms.
  • Isotopic substitutions encompassed by the compounds herein include but are not limited to: 2 H or 3 H in place of 'H, 13 C or 14 C in place of 12 C, 15 N in place of 14 N, 17 O or 18 O in place of 16 O, and 33 S, 34 S, 35 S, or 36 S in place of 32 S.
  • non-radioactive isotopic substitutions may impart new properties on the oligomeric compound that are beneficial for use as a therapeutic or research tool.
  • radioactive isotopic substitutions may make the compound suitable for research or diagnostic purposes such as imaging.
  • Oligomeric compounds comprising antisense oligomeric compound complementary to a human LPA nucleic acid, and sense oligomeric compound complementary to an antisense oligomeric compound were designed as follows.
  • Antisense oligomeric compounds were prepared as described in Table 1. Each antisense oligomeric compound has a sugar motif (from 5' to 3') as indicated, wherein each ‘e’ represents a 2'-M0E sugar moiety, each ‘y’ represents a 2'-0Me sugar moiety, each ‘f represents a 2'-F sugar moiety, each ‘[FHNA]’ represents a 3 '-fluoro-hexitol sugar moiety, and each ‘d’ represents a 2'-deoxy sugar moiety; and each antisense oligomeric compound has an intemucleoside linkage motif (from 5' to 3') as indicated, wherein each ‘o’ represents a phosphodiester intemucleoside linkage, and each ‘s’ represents a phosphorothioate intemucleoside linkage.
  • Each antisense oligomeric compound has a vinyl phosphonate (VP-) moiety on the 5'-end.
  • Each antisense oligomeric compound is complementary to SEQ ID NO: 1 (the complement of GenBank Accession No. NC_000006.12 truncated from nucleosides 160528001 to 160669000), or SEQ ID NO: 2 (GenBank Accession No. NM_005577.2), or to both. Due to the nature of the target LPA sequence, an oligonucleotide may bind multiple sites within SEQ ID NO: 1 and/or SEQ ID NO: 2.
  • CPD SID indicates the SEQ ID NO: for the relevant compound, including annotation, e.g., sugar motif and intemucleoside motif; and nb SID indicates the SEQ ID NO: for the nucleobase sequence of the relevant compound.
  • Each sense oligomeric compound has a sugar motif (from 5' to 3') as indicated, wherein each ‘y’ represents a 2'-0Me sugar moiety, each ‘e’ represents a 2'-M0E sugar moiety, and each ‘f represents a 2'-F sugar moiety; and an intemucleoside motif (from 5' to 3') as indicated, wherein each ‘o’ represents a phosphodiester intemucleoside linkage, and each ‘s’ represents a phosphorothioate intemucleoside linkage.
  • Each sense oligomeric compound is complementary to an antisense oligomeric compound in Table 1.
  • CPD SID indicates the SEQ ID NO: for the relevant compound, including annotation, e.g., sugar motif and intemucleoside motif; and nb SID indicates the SEQ ID NO: for the nucleobase sequence of the relevant compound.
  • Certain sense oligomeric compound as indicated in Conjugate column of Table 2, comprise a sense modified oligonucleotide conjugated to a GalNAc moiety.
  • a HPPO-GalNAc phosphoryl conjugate group is attached at the 3 '-OH of the oligonucleotide (designated 3 ’ GalNAc in Table 2); and in certain compounds a THA-GalNAc phosphoryl conjugate group is attached at the 5'-OH of the oligonucleotide (designated 5’ GalNAc in Table 2).
  • HPPO-GalNAc phosphoryl conjugate group is: HPPO-GalNAc phosphoryl conjugate.
  • THA -GalNAc phosphoryl conjugate group is:
  • Oligomeric duplexes were prepared with antisense oligomeric compound numbers and corresponding sense oligomeric compound numbers as listed in Table 3. Table 3: Oligomeric duplexes targeted to human LPA
  • Oligomeric duplexes were tested for single dose effects on LPA RNA in vitro in a series of experiments that had the same culture conditions.
  • Primary human hepatocytes (sourced from BioIVT, M0095-P, lot ZFW) were treated with oligomeric duplex at a concentration of 100 nM or 5,000 nM by free uptake at a density of 20,000 cells per well.
  • LPA RNA levels were measured by quantitative real-time RTPCR using human primer-probe set hAPO(a)12kB (forward sequence CCACAGTGGCCCCGGT, designated herein as SEQ ID NO: 3; reverse sequence ACAGGGCTTTTCTCAGGTGGT, designated herein as SEQ ID NO: 4; probe sequence CCAAGCACAGAGGCTCCTTCTGAACAAG, designated herein as SEQ ID NO: 5).
  • LPA RNA levels were normalized to total RNA content, as measured by RIBOGREEN®. Reduction of LPA RNA is presented in the table below as percent LPA RNA relative to the amount of LPA RNA in untreated control cells (% UTC).
  • Example 4 Effect of oligomeric duplexes in transgenic primary mouse hepatocytes
  • Oligomeric duplexes were tested for single dose effects on LPA RNA in vitro in a series of experiments that had the same culture conditions. Each separate experiment is presented in a separate column in Table 5 below.
  • Primary hepatocytes were isolated from transgenic mice previously described in Frazer K, et. al., The apolipoprotein (a) gene is regulated by sex hormones and acute-phase inducers in YAC transgenic mice; Nature Genetics, 1995, 9: 424-431.
  • Transgenic primary mouse hepatocytes were treated with oligomeric duplex at a concentration of 500 nM (Tables 5A and 5B) or 5000 nM (Table 5C) by free uptake at a density of 15,000 cells per well.
  • Oligomeric duplexes selected from Example 4 were tested at various doses in primary human hepatocytes. The oligomeric duplexes were tested in a series of experiments that had the same culture conditions, and results are depicted in Table 6, with different experiments in each of sub-Tables 6A-6I.
  • hepatocytes Primary human hepatocytes (sourced from BioIVT, M0095-P, lot ZFW) plated at a density of 20,000 cells per well were treated using free uptake with various concentrations of modified oligonucleotide as specified in Table 6. After a treatment period of 48 or 72 hours, total RNA was isolated from the cells and LPA RNA levels were measured by quantitative real-time RTPCR. Human LPA primerprobe set hAPO(a)12kB (described herein above) was used to measure RNA levels as described above. LPA RNA levels were normalized to total RNA content, as measured by RIBOGREEN®. Reduction of
  • LPA RNA is presented in Table 6 as percent LPA RNA, relative to the amount of LPA RNA in untreated control cells (% UTC). “N.C.” represents IC50 values that could not be reliably calculated. The half maximal inhibitory concentration (IC50) of each modified oligonucleotide was calculated using a linear regression on a log/linear plot of the data and is also presented in Table 6.
  • Example 6 Dose-dependent inhibition of human LPA in transgenic primary mouse hepatocytes
  • Oligomeric duplexes selected from Example 5 were tested at various doses in transgenic primary mouse hepatocytes.
  • the primary hepatocytes were isolated from transgenic mice previously described in Frazer K, et. al., The apolipoprotein (a) gene is regulated by sex hormones and acute-phase inducers in YAC transgenic mice; Nature Genetics, 1995, 9: 424-431.
  • the oligomeric duplexes were tested in a series of experiments (presented in separate sub-tables 7A-7B) that had the same culture conditions.
  • Transgenic primary hepatocytes plated at a density of 20,000 cells per well were treated using free uptake with various concentrations of modified oligonucleotide as specified in the tables below. After a treatment period of 24 hours, total RNA was isolated from the cells and LPA RNA levels were measured by quantitative real-time RTPCR. Human LPA primer-probe set hAPO(a)12kB (described herein above) was used to measure RNA levels as described above. LPA RNA levels were normalized to total RNA content, as measured by RIBOGREEN®. Reduction of LPA RNA is presented in the table below as percent LPA RNA, relative to the amount of LPA RNA in untreated control cells (% UTC). The half maximal inhibitory concentration (IC50) of each modified oligonucleotide was calculated using a linear regression on a log/linear plot of the data and is also presented in Table 7.
  • IC50 half maximal inhibitory concentration
  • Example 7 Activity of oligomeric duplexes targeting human LPA in LPA transgenic mice
  • Oligomeric duplexes selected from studies described above were analyzed along with a comparator compound for their effects on LPA mRNA and Lp(a) protein in human LPA transgenic mice.
  • LPA transgenic mice previously described (described in Frazer K, et. al., The apolipoprotein (a) gene is regulated by sex hormones and acute-phase inducers in YAC transgenic mice; Nature Genetics, 1995, 9 424-431) were derived by Taconic Biosciences and were treated with selected oligomeric duplexes and evaluated for activity.
  • Duplex Compound No. 1787751 prepared as a comparator compound, comprises antisense Compound No. 1758680, and sense Compound No. 1758681.
  • Antisense Compound No. 1758680 (SEQ ID NO: 3076) has a nucleobase sequence (from 5' to 3') of UCGUAUAACAAUAAGGGGCUG; a sugar motif (from 5' to 3') of yfyfyfyyyyyfyfyfyfyfyfyfyfyfyfyfyfy, and an intemucleoside linkage motif (from 5' to 3') of ssooooooooooooooooss; and sense Compound No.
  • 1758681 (SEQ ID NO: 3077) has a nucleobase sequence (from 5' to 3') of CAGCCCCUUAUUGUUAUACGA; a sugar motif (from 5' to 3') of yyyyyyyyfffyyyyyyyyyyy, and an intemucleoside linkage motif (from 5' to 3') of soooooooooooooos; wherein each “y” represents a 2'-OMe sugar moiety, and each “f ’ represents a 2'-F sugar moiety, and wherein each “s” represents a phosphorothioate intemucleoside linkage, and each “o” represents a phosphodiester intemucleoside linkage.
  • Sense oligonucleotide Compound No. 1758681 has a THA- GalNAc moiety conjugated to the 5 '-end of the oligonucleotide via a phosphorothioate bond: Duplex No.
  • 1787751 is similar to compound AD03851, a duplex targeting LPA previously disclosed in International Patent Application WO 2017/059223, and differs from AD03851 in two changes: first, 1787751 comprises a THA-GalNAc conjugate (as described herein) on the 5' end of the sense oligonucleotide, whereas AD03851 comprises an NAG25 conjugate; and second, 1787751 comprises a 2'-OMe sugar moiety at the 3’ end of the sense oligonucleotide, whereas AD03851 comprises an inverted 2'-deoxyribosyl sugar moiety modification at the 3' end of the sense oligonucleotide.
  • NAG25 phosphorothioate moiety conjugated to the 5'-end of the oligonucleotide via a phosphorothioate bond, as previously described is shown below:
  • mice Groups of two female LPA transgenic mice each received a single subcutaneous injection of oligomeric duplex compound at a dose of 2 mg/kg.
  • One group of 3 female LPA transgenic mice received a single subcutaneous injection of PBS.
  • Seven days post injection mice were sacrificed, and RNA was extracted from liver tissue for quantitative real time RTPCR analysis of LPA RNA using human primer probe set hAPO(a)12kBTS (described in Example 3).
  • LPA RNA levels were normalized to total RNA content, as measured by RIBOGREEN®. Reduction of LPA RNA is presented as percent LPA RNA relative to the amount of LPA in tissue from PBS control animals (% control).
  • oligomeric duplex compounds were evaluated on apo(a) protein levels.
  • blood plasma was collected on Day 1 prior to treatment and on Day 7 when the mice were sacrificed and analyzed using an automated clinical chemistry analyzer (Hitachi Olympus AU400c, Melville, NY), apo(a) protein levels were analyzed with an Lp(a) assay kit from Randox (Catalog #LP2757). The results were averaged for each group of mice and are presented in the tables below as percent apo(a), relative to the amount of apo(a) present at baseline (% baseline).

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Abstract

Provided are oligomeric duplexes, oligomeric compounds and antisense agents, methods, and pharmaceutical compositions for reducing the amount or activity of LPA RNA in a cell or animal, and in certain instances reducing the amount of Lp(a) in a subject. Such oligomeric duplexes, oligomeric compounds and agents, methods, and pharmaceutical compositions are useful to treat or manage inflammatory, cardiovascular and/or metabolic diseases, disorders or conditions.

Description

COMPOUNDS AND METHODS FOR INHIBITING LPA
Cross-Reference to Related Applications
This application claims the benefit of priority to US Provisional Application No. 63/584,489, filed September 21, 2023, which is incorporated by reference herein in its entirety for any purpose. Sequence Eisting
The present application is being filed concurrently with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0478WOSEQ.xml, created on August 27, 2024, which is 19,863 KB in size. The contents of the electronic format of the sequence listing are incorporated herein by reference in their entirety.
Field
Provided herein are oligomeric duplexes, oligomeric compounds, compositions, and uses therefor, including methods for modulating the amount and/or activity of ApoA expression, LPA RNA, and/or Lp(a), as well as methods for treating or managing inflammatory, cardiovascular and/or metabolic diseases, disorders or conditions, and in certain embodiments, ameliorating at least one symptom of an inflammatory, cardiovascular and/or metabolic disease, disorder or condition.
Background
The lipoprotein(a) [Lp(a)] particle comprises a unique LDL particle in which one apolipoprotein B (apoB) protein is linked via a disulfide bond to a single apolipoprotein(a) [apo(a)] protein. The apo(a) protein shares a high degree of homology with plasminogen, particularly within the kringle IV type 2 repetitive domain. Levels of circulating Lp(a) are inversely proportional to the number of kringle IV type 2 variable repeats present in the molecule and, as both alleles are co-expressed within individuals, can display heterozygous plasma isoform profiles among individuals (Kraft et al., Eur J Hum Genet, 1996; 4(2): 74-87). It is thought that the kringle repeat domain in apo(a) may be responsible for its pro- thrombotic and anti-fibrinolytic properties, potentially enhancing atherosclerotic progression. Apo(a) has been shown to preferentially bind oxidized phospholipids and potentiate vascular inflammation (Bergmark et al., J Lipid Res 2008; 49:2230-2239; Tsimikas et al., Circulation. 2009; 119(13): 1711— 1719). Further, studies suggest the Lp(a) particle may also stimulate endothelial permeability, induce plasminogen activator inhibitor type- 1 expression and activate macrophage interleukin-8 secretion (Koschinsky and Marcovina, Curr Opin Lipidol 2004; 15: 167-174). Importantly, genetic association studies revealed that Lp(a) was an independent risk factor for myocardial infarction, stroke, peripheral vascular disease and abdominal aortic aneurysm (Rifai et al., Clin Chem 2004; 50: 1364-71; Erqou et al., JAMA 2009;302:412-23; Kamstrup et al., Circulation 2008;l 17: 176-84). Further, the Precocious Coronary Artery Disease (PROCARDIS) study, Clarke et al. (Clarke et al., NEJM (2009)361; 2518- 2528) described robust and independent associations between coronary heart disease and plasma Lp(a) concentrations; and Solfrizzi et al., suggested that increased serum Lp(a) may be linked to an increased risk for Alzheimer’s Disease (AD) (Solfrizzi et al., J Neurol Neurosurg Psychiatry 2002, 72:732-736. Examples of indirect apo(a) inhibitors for treating cardiovascular disease in a clinical setting include aspirin, Niaspan, Mipomersen, Anacetrapib, Epirotirome and Lomitapide which reduce plasma Lp(a) levels by 18%, 39%, 32%, 36%, 43% and 17%, respectively. Lp(a) apheresis has also been used in the clinic to reduce apo(a) containing Lp(a) particles. To date, proposed therapeutic strategies to treat cardiovascular disease by directly targeting apo(a) levels have been described (e.g., U.S. Patent Nos. 5,877,022, 8,138,328, 8,673,632 and 7,259,150; U.S. Patent Publication No. US20040242516; International patent application publication nos. W02005/000201, W02003/014397, WO2013/177468, WO 2014/179625, WO2017/059223, W02020/099476 and WO 2022/032288; and Merki et al., J Am Coll Cardiol 2011; 57: 1611-1621., and Tsimikas et al. (Lancet. 2015 Oct 10; 386: 1472-83)). Although active therapeutics have been developed, none have been approved for commercial use. There remains a need for agents which can potently and selectively lower Lp(a) levels in subjects, including in patients at enhanced risk for cardiovascular events due to chronically elevated plasma Lp(a) levels. In particular, new therapeutic agents that enable safe, low frequency administration for treatment and prevention of cardiovascular disease are desired.
Summary
Provided herein are oligomeric duplexes, pharmaceutical compositions, and methods of use for reducing the amount or activity for reducing the amount or activity of LPA RNA and reducing the expression of Apo(a) protein in a cell or subject and/or Lp(a) in a subject. In certain embodiments, a subject has a disease or disorder associated with misregulation of lipoproteins or a mutation in lipoprotein regulation pathway. In certain embodiments, the subject has elevated lipoprotein (a). In certain embodiments the subject has or is at risk for a severe a cardiovascular disease, disorder or condition. In certain embodiments, the subject has or is at risk for a metabolic or inflammatory disease, disorder or condition. In certain embodiments, agents useful for reducing the amount or activity of LPA RNA are oligomeric duplex, oligomeric compound, or composition as provided herein. In certain embodiments, agents useful for decreasing expression of Apo(a) or levels of lipoprotein (a) are oligomeric compounds, oligomeric duplexes, antisense agents, and/or RNAi agents.
Provided are modified oligonucleotides and compounds and compositions comprising them, including, but not limited to, antisense agents, oligomeric agents, oligomeric duplexes and pharmaceutical compositions comprising modified oligonucleotides. In certain embodiments, a modified oligonucleotide provided herein comprises a nucleobase sequence at least 80% complementary to an equal length portion of an LPA nucleic acid. In certain embodiments, the modified oligonucleotide consists of 12 to 35, 14 to 30, 15 to 28, 16 to 25, or 18 to 23 linked nucleosides targeting LPA nucleic acid. In certain embodiments, a modified oligonucleotide provided herein comprises a sequence of nucleobase s complementary to an equal length portion of the nucleobase sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2. In certain embodiments, provided oligomeric duplexes comprise a first oligomeric compound and a second oligomeric compound, wherein a first oligomeric compound comprises a modified oligonucleotide consisting of 18 to 50 linked nucleosides, wherein the nucleobase sequence of the first oligomeric compound comprises at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID Nos: 6- 829 and 3078-3083, wherein each of the nucleosides of the modified oligonucleotide comprises a modified sugar moiety or sugar surrogate and wherein no more than 22%, no more than 20%, no more than 18%, no more than 15%, no more than 10%, or no more than 5% of the modified nucleosides in the first oligomeric compound comprises a 2’-fluoro sugar moiety or a 3’-fluoro-hexitol sugar surrogate; and wherein a second oligomeric compound comprises a modified oligonucleotide consisting of 16 to 50 contiguous linked nucleosides wherein the nucleobase sequence of the second oligomeric compound comprises at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NOs: 830-1540 and 3096-3101, wherein each of the nucleosides of the second oligomeric compound comprises a modified sugar moiety or sugar surrogate and wherein no more than 25%, no more than 20%, no more than 18%, no more than 16%, no more than 14 %, no more than 12%, or no more than 10%, of the modified nucleosides in the second oligomeric compound comprises a 2’-fluoro sugar moiety or a 3’-fluoro-hexitol sugar surrogate. In certain embodiments, a modified oligonucleotide provided herein comprises at least one modified sugar moiety and/or at least one modified intemucleoside linkage. In certain embodiments, an oligomeric compound provided herein comprises a modified oligonucleotide comprising at least one 3’-fluoro-hexitol sugar moiety. In certain embodiments, an oligomeric compound provided herein comprises a modified oligonucleotide comprising at least one a 2’ -deoxynucleoside. In certain embodiments, an oligomeric compound provided herein comprises a modified oligonucleotide conjugated to a cell targeting agent. Modified oligonucleotides and compositions comprising them, including, but not limited to, oligomeric duplexes, oligomeric compounds, modified oligonucleotides and composition described herein are useful for reducing or inhibiting LPA expression in a cell, organ, tissue, system, organism or subject.
In certain embodiments provided are oligomeric compounds comprising a modified oligonucleotide comprising a nucleobase sequence selected from any one of SEQ ID NO: 6-829 and 3078-3083, wherein the modified oligonucleotide comprises a modified sugar motif independently selected from one of efyyyfyyyyyyyfyfyyyyyyy, yfyyyfyyyyyyyfyfyyyyyyy, efyyydyyeyyyydydyyyyyee, efyyydyyyyyyyfyfyyyyyee, efyyydyyeyyyyfyfyyyyyee, e[FHNA]yyyfyyyyyyyfyfyyyyyee, and e[FHNA]yyyfyyeyyyyfyfyyyyyee, wherein each ‘e’ represents a 2'-MOE sugar moiety, each ‘y’ represents a 2'-OMe sugar moiety, each ‘f represents a 2'-F sugar moiety, each ‘[FHNA]’ represents a 3'-fluoro-hexitol sugar moiety, and each ‘d’ represents a 2'-deoxy sugar moiety, wherein all except 0, 1, or 2 modifications are identical to the sugar motif. In certain embodiments provided are oligomeric compounds comprising a modified oligonucleotide comprising a sequence comprising at least 18, at least 19, or at least 20 contiguous nucleobases of any one of the sequences of SEQ ID NOs: 1541-2364 and 3090-3095.
In certain embodiments provided are oligomeric compounds comprising a modified oligonucleotide comprising a nucleobase sequence selected from any one of SEQ ID NO: 830-1540 and 3096-3101, wherein the modified oligonucleotide comprises a modified sugar motif independently selected from one of yyyyyyfyfffyyyyyyyyyy and eeyyyyyyyffyyyyyyyyee, wherein each ‘e’ represents a 2'-M0E sugar moiety, each ‘y’ represents a 2'-0Me sugar moiety, and each ‘f represents a 2'-F sugar moiety, wherein all except 0, 1, or 2 modifications are identical to the sugar motif. In certain embodiments provided are oligomeric compounds comprising a modified oligonucleotide comprising a sequence comprising at least 18, at least 19, or at least 20 contiguous nucleobases of any one of the sequences of SEQ ID NOs: 2365-3075 and 3084-3089.
In certain embodiments provided are oligomeric compounds comprising a modified oligonucleotide having a sequence selected from any one of SEQ ID NO: 6-1540, 3078-3089, and 3096- 3101. In certain embodiments provided are oligomeric duplexes comprising an oligomeric compound having a sequence selected from any one of SEQ ID NO: 6-3075 and 3078-3101. In certain embodiments provided are oligomeric agents comprising an oligomeric compound having a sequence selected from any one of SEQ ID NO: 1541-3075 and 3084-3095. In certain embodiments provided are oligomeric duplexes comprising a first oligomeric compound having a sequence selected from any one of SEQ ID NO: 1541-2364 and 3090-3095, and comprising a second oligomeric compound having a sequence selected from any one of SEQ ID NO: 2365-3075 and 3084-3089. In certain embodiments provided are oligomeric duplexes consisting of a first oligomeric compound having a sequence selected from any one of SEQ ID NO: 1541-2364 and 3090-3095, and a second oligomeric compound having a sequence selected from any one of SEQ ID NO: 2365-3075 and 3084-3089.
Additionally provided are methods for reducing or inhibiting LPA expression, LPA RNA levels and/or Apo(a) protein levels and/or Lp(a) activity in a cell, tissue, organ or subject. In certain embodiments, methods include contacting a cell or subject with a composition provided herein, comprising, e.g., an oligomeric duplex, oligomeric compound, modified oligonucleotide as described herein. In certain embodiments, the subject is a human who has or is at risk of having a cardiovascular, metabolic, or inflammatory disease, disorder, condition or injury associated with increased lipoprotein a levels, misregulation of lipoprotein turnover or a mutation in LPA. In certain embodiments, the subject is a human who has or is at risk of having hypertriglyceridemia. In certain embodiments, the subject is a human who has or is at risk of having atherosclerotic cardiovascular disease (ASCVD) or coronary artery disease (CAD).
Provided herein are methods of treating a disease, disorder, condition or injury associated with lipoprotein metabolism misregulation, regulation of lipoprotein turnover or a mutation in LPA. In certain embodiments, a method of treating a disease, disorder, condition or injury associated with lipoprotein metabolism misregulation, regulation of Lp(a) levels or a mutation in LPA comprises administering to a subject, e.g., a human subject, having, or at risk of having, a disease, disorder or condition associated with elevated Lp(a), a provided oligomeric duplex, oligomeric compound, or composition provided herein, wherein the disease, disorder, condition or injury is selected from a cardiovascular disease, disorder, condition, a metabolic disease, disorder, or condition, or an inflammatory disease disorder or condition. In certain embodiments, the subject has or is at risk for developing cardiovascular disease (CVD) coronary artery disease (CAD), hypercholesterolemia, myocardial infarction (MI), peripheral arterial disease (PAD), calcific aortic valve disease (CAVD), aortic stenosis, atherosclerotic cardiovascular disease (ASCVD), atherosclerosis, dyslipidemia, thrombosis, or stroke. In certain embodiments, methods of treating provided herein result in ameliorating (whether by reduced frequency, severity) a at least one symptom of a disease, disorder, condition or injury associated with lipoprotein metabolism misregulation. In certain embodiments, methods of treating provided herein result in preventing, delay or postponing, or slowing the development or progression of at least one symptom of a disease, disorder or condition associated with elevated Lp(a).
Also provided are methods useful for ameliorating at least one symptom of a disorder associated with lipoprotein metabolism misregulation. In certain embodiments the disorder is severe hypertriglyceridemia, hyperlipidemia, dyslipidemia, and/or hyperlipoproteinemia. In certain embodiments, a symptom of hypertriglyceridemia, hyperlipidemia, dyslipidemia, and/or hyperlipoproteinemia include, but are not limited to, abdominal pain, physical fatigue, difficulty thinking, diarrhea, acute pancreatitis, eruptive xanthomas, lipemia retinalis, or hepatosplenomegaly, or a combination of two or more of the foregoing in the subject. In certain embodiments, methods provided herein for preventing, treating, ameliorating, delaying the onset of, or reducing frequency of at least one symptom of hypertriglyceridemia, hyperlipidemia, dyslipidemia, and/or hyperlipoproteinemia include administering to a subject, e.g., a human subject, having or at risk of having at least one symptom a composition provided herein, e.g., a modified oligonucleotide, oligomeric duplex, oligomeric compound, or pharmaceutical composition provided herein,
Detailed Description
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive. Herein, the use of the singular includes the plural unless specifically stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.
DEFINITIONS
The following definitions are provided, along with additional definitions throughout the specification, for a complete understanding of the instant invention. Unless specific definitions are provided herein, nomenclature used in connection with, and procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Unless otherwise indicated, certain terms have the following meanings:
As used herein, a substituent at the “2 '-position” means that the substituent is directly attached to the carbon at the 2'-position of a fiiranosyl sugar moiety.
As used herein, “2'-deoxynucleoside” means a nucleoside comprising a 2'-deoxyfiiranosyl sugar moiety. Unless otherwise indicated, a 2'-deoxynucleoside is a 2'-p-D-deoxynucleoside which comprises a 2'-P-D-deoxyribosyl sugar moiety, which is in the P-D configuration in naturally occurring deoxyribonucleic acid (DNA). A 2'-deoxynucleoside or a nucleoside comprising an unmodified 2'- deoxyribosyl sugar moiety may be abasic, comprise a modified nucleobase, or may comprise an RNA nucleobase (uracil).
As used herein, “2'-deoxy sugar moiety” means a 2'-H(H) deoxyfuranosyl sugar moiety. Unless otherwise indicated, a 2'-deoxy sugar moiety is a 2'-P-D-deoxyribosyl sugar moiety, which has the P-D stereochemical configuration in naturally occurring deoxyribonucleic acid (DNA).
As used herein, “2'-M0E” means a 2'-OCH2CH2OCH3 group at the 2'-position of a furanosyl sugar moiety. A “2'-M0E sugar moiety” means a sugar moiety with a 2'-OCH2CH2OCH3 group at the 2'-position of a furanosyl sugar moiety. Unless otherwise indicated, a 2'-M0E sugar moiety is in the P-D-ribosyl stereochemical configuration. “MOE” means O-methoxyethyl.
As used herein, “2'-M0E nucleoside” or “2'- OCH2CH2OCH3 nucleoside” means a nucleoside comprising a 2'-M0E sugar moiety (or 2'-OCH2CH2OCH3 furanosyl sugar moiety).
As used herein, “2'-0Me” means a 2’-O-methyl or 2'-OCH3 group at the 2'-position of a furanosyl sugar moiety. A “2'-0Me sugar moiety” means a sugar moiety with a 2’-O-methyl or a 2'-OCH3 group at the 2'-position of a furanosyl sugar moiety. Unless otherwise indicated, a 2'-0Me sugar moiety is in the P-D-ribosyl stereochemical configuration.
As used herein, “2'-0Me nucleoside” means a nucleoside comprising a 2'-0Me sugar moiety.
As used herein, “2'-F” means a 2'-fluoro group at the 2'-position of a furanosyl sugar moiety. A “2'-F sugar moiety” means a sugar moiety with a 2'-F group at the 2'-position of a furanosyl sugar moiety. Unless otherwise indicated, a 2'-F sugar moiety is in the P-D-ribosyl configuration.
As used herein, “2'-F nucleoside” means a nucleoside comprising a 2'-F sugar moiety.
As used herein “2'-NMA” means a 2'-OCH2C(=O)-N(H)CH3 group at the 2'-position of a furanosyl sugar moiety. A “2'-NMA sugar moiety” means a sugar moiety with a 2'-OCH2C(=O)-N(H)CH3 group at the 2'-position of a furanosyl sugar moiety.
As used herein, “2'-NMA nucleoside” means a nucleoside comprising a 2'-NMA sugar moiety.
As used herein, “2 '-substituted nucleoside” means a modified nucleoside comprising a 2'- substituted furanosyl sugar moiety. 2'-substituted nucleosides include, but are not limited to, a 2'-0Me nucleoside, a 2'-M0E nucleoside, a 2'-F nucleoside, a 2'-NMA nucleoside, a cEt nucleoside, and a LNA nucleoside.
As used herein, “2 ’-substitution” or “2 '-substituted sugar moiety” means a modified furanosyl sugar moiety wherein the 2'-position is attached to at least one substituent other than H or OH. A 2'- substituted sugar moiety includes a bicyclic sugar moiety wherein the second ring is joined to the furanosyl ring at the 2'-position. 2'-substituted sugar moieties include, but are not limited to, 2'-0Me sugar moieties, 2'-M0E sugar moieties, 2'-F sugar moieties, 2'-NMA sugar moieties, cEt sugar moieties, and LNA sugar moieties.
As used herein, “5-methylcytosine” means a cytosine modified with a methyl group attached at the 5 position. A 5-methylcytosine is a modified nucleobase.
As used herein, “abasic nucleoside” means a modified nucleoside in which the sugar moiety is not attached to a nucleobase. As used herein, “acyclic sugar surrogate nucleoside” means a nucleoside having Formula II, Formula III, or Formula IV:
Figure imgf000008_0001
Formula IV wherein
X is O, S, C(R5Re), N(E1), NC(=O)-(E1); each Ji and J2 are independently H or Ci-Ce alkyl; n is 0, 1 or 2; m is 0, 1, or 2; p is 0 or 1; o is 0 or 1; s is 0 or 1;
Ri is H, OH, halogen, Ci-Ce alkyl, Ci-Ce alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, or (CH2)qRs;
R2, R3, and R4 are each independently H, OH, halogen, Ci-Ce alkyl, Ci-Ce alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, S-CH3, N(CH3)(CH3), OCH2CH2OCH3, O-alkylamino, or (CH2)qRs;
Ei is H, Ci-Ce alkyl or substituted Ci-Ce alkyl;
Rs and Re are independently H, OH, Ci-Ce alkyl, or N(R7); wherein if Rs is OH, then Re is not OH;
R7 is H, Ci-Ce alkyl, or C(=O)Rg, wherein Rj is Ci-Ce alkyl;
Rs is OH, halogen, methoxy, ethoxy, azido, C2-C6 alkenyl, or C2-C6 alkynyl, and q is 1, 2, or 3; and
Bx is a nucleobase. As used herein, “acyclic sugar surrogate” means the sugar moiety of an acyclic sugar surrogate nucleoside.
As used herein, “ameliorate” with reference to a symptom of a disease, means improvement in, or lessening, or preclusion of, at least one symptom of a disease. Amelioration may be reduction in severity or frequency of a symptom or the delayed onset, prevention of occurrence of, or slowing of progression in the severity or frequency of, a symptom. Progression, frequency, or severity indicators may be determined by subjective or objective measures known in the art and/or described herein.
As used herein, “antisense activity” means any detectable and/or measurable change attributable (whether directly and/or indirectly) to hybridization of an antisense oligonucleotide to a target nucleic acid. For example, compounds have antisense activity when they alter the amount or activity of a target nucleic acid by 25% or more in an in vitro assay; or, for example compounds have antisense activity when they alter the amount or activity of a target nucleic acid by 25% or more in an in vivo assay. Antisense activity may be assessed in a standard assay. Herein, antisense activity is a reduction or inhibition in the amount or expression of a target nucleic acid, or a protein encoded by such target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the oligonucleotide.
As used herein, “antisense agent” means an oligomeric agent comprising an antisense oligonucleotide.
As used herein, “antisense oligonucleotide” means an oligonucleotide having at least one region (a “targeting region”) that is complementary to a target nucleic acid (e.g. , a target region). An antisense oligonucleotide may be paired with a second oligonucleotide (herein, a “sense oligonucleotide”) that is complementary to the antisense oligonucleotide (for example, forming an “oligomeric duplex”), may be an unpaired antisense oligonucleotide (herein, a single-stranded antisense oligonucleotide), or may be a “hairpin oligonucleotide” that has at least one region that is self-complementary.
As used herein, “bicyclic sugar” or “bicyclic sugar moiety” means a modified sugar moiety comprising a furanosyl sugar moiety and a second ring, wherein the second ring is formed via a bridge connecting two non-geminal atoms in the ring of the furanosyl sugar moiety, thereby forming a bicyclic structure. Examples of bicyclic sugar moieties include locked nucleic acid (LNA) sugar moieties and constrained ethyl (cEt) sugar moieties as defined herein.
As used herein, “bicyclic nucleoside” or “BNA” means a nucleoside comprising a bicyclic sugar moiety.
As used herein, “cell-targeting moiety” means a conjugate group or portion of a conjugate group that has affinity for a particular cell type or particular cell types. For example, a cell-targeting moiety may have affinity for a cell surface moiety, such as a cell surface receptor on a particular cell type.
As used herein, “cleavable moiety” means a group of atoms comprising at least one bond that is cleaved under physiological conditions, e.g., in a cell, in a subject. For example, a cleavable moiety cleaved inside a cell or sub-cellular compartment, such as an endosome or lysosome. A cleavable moiety may be cleaved by endogenous enzymes, such as nucleases. A cleavable moiety may be cleaved by endogenous change in conditions such as pH. As used herein, “complementary nucleobase(s)” or “complementary” in reference to nucleobase(s) means nucleobases that form hydrogen bonds with another when two regions of linked nucleosides (e.g., an oligonucleotide and a target nucleic acid; two oligonucleotides). Complementary nucleobase pairs include, but are not limited to, adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), 5 -methylcytosine (mC) and guanine (G). Certain modified nucleobases that are complementary to unmodified nucleobases or to other modified nucleobases are known in the art. For example, hypoxanthine, the nucleobase of the nucleoside inosine (I), can pair with adenine, cytosine, thymine, or uracil. Herein, hypoxanthine (I) is considered a complementary nucleobase to thymine (T), adenine (A), uracil (U), and cytosine (C).
As used herein, “complementary sequence(s)” or “complementary” in reference to sequence(s) refers to two nucleobase sequences in which some, a majority, or all of the nucleobases in the two sequences are complementary nucleobases when the sequences are aligned. A “nucleobase sequence” means the order of contiguous nucleobases in a strand of linked nucleosides or a region thereof (e.g., an oligonucleotide or region thereof, or a target nucleic acid or region thereof) independent of any sugar or intemucleoside linkage modification. Complementary nucleobase sequences may be nucleobase sequences of two separate strands of linked nucleosides or region thereof (e.g., an oligonucleotide and a region of a target nucleic acid); or complementary nucleobase sequences may be two regions of a single strand of linked nucleosides (e.g., self-complementary regions of a hairpin oligonucleotide). As used herein, when a first nucleobase sequence (e.g., an oligonucleotide) or region thereof is described as being complementary to a second nucleobase sequence (e.g., a target nucleic acid or another oligonucleotide), it means that a majority of nucleobases of the first nucleobase sequence or region thereof base pair with complementary nucleobases of the second nucleobase sequence when aligned. Not every pair of nucleobases in the aligned nucleobase sequences needs to be a base pair match for the two sequences to be “complementary.” Rather, some mismatches are tolerated. Where complementarity is expressed as a percent, such percent represents the percent of nucleobases within one nucleobase sequence that are complementary to nucleobases within an equal length second sequence when the sequences are aligned. Unless otherwise specified, “complementary” is assumed to be at least 70%. Complementary nucleobase sequences may be 75%, 80%, 85%, 90%, 95%, or 100% complementary. For example, if a nucleobase sequence of an oligonucleotide consisting of 20 nucleosides is 80% complementary to another nucleobase sequence, then 16 of the nucleobase pairs are complementary nucleobases, and there are 4 mismatches when the sequences are aligned. If a nucleobase sequence of an oligonucleotide consisting of 20 nucleosides is at least 80% complementary to another nucleobase sequence, then 16, 17, 18, 19, or 20 of the nucleobase pairs are complementary nucleobases, and there are 0-4 mismatches when the sequences are aligned. As used herein, “fully complementary” or “100% complementary” means that each nucleobase pair of two nucleobase sequences is complementary when the equal length sequences are aligned.
As used herein, “conjugate group” means a group of atoms including a conjugate moiety and a conjugate linker that attaches the conjugate moiety to the oligonucleotide. As used herein, “conjugate linker” means a single bond or a group of atoms comprising at least one bond that connects a conjugate moiety to an oligonucleotide.
As used herein, “conjugate moiety” means a group of atoms that when covalently bound to a molecule (e.g., an oligonucleotide) modifies one or more properties of such molecule compared to the same molecule lacking the conjugate moiety, wherein such properties include, but are not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge, and clearance.
As used herein, “constrained ethyl” or “cEt” or “cEt sugar moiety” means a P-D ribosyl bicyclic sugar moiety wherein the second ring of the bicyclic sugar is formed via a bridge connecting the 4'-carbon and the 2'-carbon of the -D ribosyl sugar moiety, wherein the bridge has the formula 4'-CH(CH3)-O-2', and wherein the methyl group of the bridge is in the S configuration.
As used herein, “cEt nucleoside” means a nucleoside comprising a cEt sugar moiety.
As used herein a “cyclic sugar surrogate nucleoside” is a nucleoside having Formula I:
Figure imgf000011_0001
Wherein
J is H, Ci-Ce alkyl, or C2-C6 alkenyl;
X is O, S, C(RIR2), N(R3); or X is X1-X2, wherein X1-X2 is C(RI)=C(R2), C(RIR2)-C(RIR2), O- C(RIR2), C(RIR2)-O, S-C(RIR2), C(RIR2)-S, N(R3)-C(RIR2), or C(RIR2)-N(R3);
Y is C(RIR2); or Y1.Y2, wherein Y1.Y2, is C(RI)=C(R2) or C(RIR2)-C(RIR2);
Z is C(GIG2); or Z1-Z2, wherein Zi-Z2 is C(Gi)=C(Ri), C(Ri)=C(Gi); C(GIG2)-C(RIR2), C(RIR2)- C(GIG2); or Zi-Z2-Z3, wherein Zi-Z2-Z3 is C(GIG2)-C(RIR2)-C(RIR2) or C(RIR2)-C(RIR2)-C(GIG2);
Q is CH orN; each Ri and R2 is independently H, OH, Ci-Ce alkyl, or N(R4); wherein if Ri is OH, then R2 is not OH; each R3 and R4 is independently H, Ci-Ce alkyl, or C(=0)R5, wherein R5 is Ci-Ce alkyl; each Gi and G2 is independently H, OH, halogen or O-[C(R6)(R7)]q-[(C=O)s-XG]j-R8; wherein if Gi is OH, then G2 is not OH; each Re and R7 is, independently, H, halogen, Ci-Ce alkyl or substituted Ci-Ce alkyl; each XG is O, S or N(Ei);
Rs is H, halogen, Ci-Ce alkyl, substituted Ci-Ce alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or N(E2)(E3);
Ei, E2 and E3 are each, independently, H, Ci-Ce alkyl or substituted Ci-Ce alkyl; n is 0 or 1; m is 0 or 1; p is 0 or 1; q is from 1 to 6; s is 0 or 1; j is 0 or 1;
Bx is a nucleobase; and provided that if X is O, Z is C G1G2), and Q is CH, then m is 1.
As used herein, “cyclic sugar surrogate” means the sugar moiety of a cyclic sugar surrogate nucleoside, and is represented by Formula la herein.
As used herein, “double-stranded” refers to hybridized or bound complementary regions, including those between two separate strands of linked nucleosides (e.g., an antisense oligonucleotide and a sense oligonucleotide) and those within a single strand of linked nucleosides (e.g., a hairpin oligonucleotide). Paired complementary regions of two separate strands of linked nucleosides form a duplex of the separate strands. Paired complementary regions of a single strand of linked nucleosides (i.e., a first region of the strand of linked nucleosides and a second region of the strand of linked nucleosides) form a “hairpin”.
As used herein, a “furanosyl sugar moiety" is a group of atoms that comprises a furanose ring and optional substituents, and is numbered according to the structure below, with optional additional substituents at any of the 1', 2', 3', 4', and 5' positions.
Figure imgf000012_0001
As used herein, “hybridize” or “hybridization” means the process of two complementary complementary regions of linked nucleosides (e.g., oligonucleotides, nucleic acids) annealing to form a doublestranded region. While not limited to a particular mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.
As used herein, “intemucleoside linkage” means the covalent linkage between adjacent nucleosides in an oligonucleotide. As used herein, “unmodified intemucleoside linkage” means a phosphodiester intemucleoside linkage. As used herein, “modified intemucleoside linkage” means any intemucleoside linkage other than a phosphodiester intemucleoside linkage. A “phosphorothioate intemucleoside linkage” is a modified intemucleoside linkage in which one of the non-bridging oxygen atoms of a phosphodiester intemucleoside linkage is replaced with a sulfur atom. A “mesyl phosphoramidate intemucleoside linkage” is a modified intemucleoside linkage in which one of the nonbridging oxygen atoms of a phosphodiester intemucleoside linkage is replaced with NS(=O)2CH3. Unless otherwise indicated, and in the context of linked nucleosides each comprising a furanosyl sugar moiety, an intemucleoside linkage joins the 3 '-carbon of one furanosyl sugar moiety to the 5 '-carbon of the other furanosyl sugar moiety. As used herein, “inverted nucleoside” means a nucleoside having a 3' to 3' and/or 5' to 5' intemucleoside linkage.
As used herein, “linked nucleosides” are nucleosides that are connected in a contiguous sequence (z.e., nucleosides immediately adjacent to one another, no additional nucleosides are presented between those that are linked).
As used herein, a “mismatch” between two aligned strands of linked nucleosides means that two nucleobases at a specified position of the aligned nucleobase sequences are not complementary nucleobases as defined herein.
As used herein, “modified nucleoside” means a compound or subunit comprising a sugar moiety and optionally a nucleobase, wherein the sugar moiety is modified and/or the nucleobase is modified or absent.
As used herein, “modified sugar moiety” means a sugar moiety other than a P-D-ribosyl sugar moiety in RNA or a -D-deoxyribosyl sugar moiety in DNA. A modified sugar moiety is selected from a modified fiiranosyl sugar moiety, a cyclic sugar surrogate, an acyclic sugar surrogate, or a sugar mimic.
As used herein, a “modified nucleobase” means a nucleobase other than unmodified A, T, C, U, or G capable of pairing with at least one unmodified nucleobase. A “5 -methylcytosine” is a modified nucleobase. Inosine (I) is a nucleoside comprising the modified nucleobase hypoxanthine.
As used herein, “motif’ means a pattern of independently unmodified and/or independently modified sugar moieties, nucleobases, and/or intemucleoside linkages in an oligonucleotide.
As used herein, “non-bicyclic modified sugar moiety” means a modified fiiranosyl sugar moiety comprising a modification, such as a substituent, that does not form a bridge between two atoms of the sugar to form a second ring. As used herein, "nucleobase" means an unmodified nucleobase or a modified nucleobase.
As used herein, “the nucleobase sequence of’ a reference SEQ ID NO, refers only to the order of contiguous nucleobases provided in such SEQ ID NO, independent of any sugar or intemucleoside linkage modifications and therefore, unless otherwise indicated, includes compounds wherein each sugar moiety and each intemucleoside linkage, independently, is modified or unmodified, irrespective of the presence or absence of modifications indicated in the referenced SEQ ID NO.
As used herein, “nucleoside” means an “unmodified nucleoside” or a “modified nucleoside”.
As used herein, "oligomeric agent" means a compound or complex comprising or consisting of at least one modified oligonucleotide and optionally one or more additional associated features selected from: (a) one or more conjugate groups, which may be covalently attached directly or indirectly to any oligonucleotide of such oligomeric agent; (b) one or more terminal groups; and (c) one or more additional modified or unmodified oligonucleotides, each of which may be hybridized to or covalently linked to the at least one modified oligonucleotide and/or to each other;. Herein, where two oligonucleotides are described as being covalently attached to one another, such attachment is other than through a direct intemucleoside linkage. Thus, a single, unbranched oligonucleotide comprising only direct intemucleoside linkages cannot be described as two separate covalently linked oligonucleotides. As used herein, “oligomeric compound” means a compound comprising a modified oligonucleotide and optionally one or more covalently linked chemical features selected from one or more conjugate group and one or more terminal group.
As used herein, “oligonucleotide” means a strand of linked nucleosides, wherein, independently each nucleoside and/or independently each intemucleoside linkage of the strand of linked nucleosides may be independently modified or unmodified. Unless otherwise indicated, oligonucleotides consist of 12-50 linked nucleosides. Unless otherwise indicated, no more than 10% of the nucleosides of an oligonucleotide are abasic nucleosides. As used herein, “modified oligonucleotide” means an oligonucleotide, wherein at least one nucleoside and/or intemucleoside linkage is modified. As used herein, “unmodified oligonucleotide” means an oligonucleotide consisting of unmodified nucleosides linked by phosphodiester intemucleoside linkages. An oligonucleotide may be paired with a second oligonucleotide that is complementary to the oligonucleotide to form an oligomeric duplex, or it may be unpaired.
As used herein, “pharmaceutical composition” means a mixture of substances suitable for administration to a subject. For example, a pharmaceutical composition may comprise an agent (e.g., an oligomeric agent, duplex, or antisense agent) and a sterile aqueous solution. A pharmaceutical composition may show activity in certain cell lines.
As used herein, “pharmaceutically acceptable carrier or diluent” means an ingredient in a pharmaceutical composition suitable for use in administering to a subject. Typically, a “carrier” or “diluent” lacks pharmacological activity but is desirable in preparing a pharmaceutical composition.
As used herein, “pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of compounds. Pharmaceutically acceptable salts retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
As used herein, “RNA nucleoside” means a nucleoside comprising an unmodified RNA sugar moiety. An RNA nucleoside may comprise a modified or unmodified nucleobase. An RNA nucleoside may comprise a thymine nucleobase or a modified nucleobase, or may be an abasic nucleoside.
As used herein, “RNA sugar moiety” means an unmodified RNA sugar moiety
As used herein, “RNase H agent” means an antisense agent that acts, at least in part, through RNase H to modulate a target nucleic acid and/or protein encoded by a target nucleic acid. RNase H agents may be single-stranded, or RNase H agents may be double -stranded. RNase H compounds may comprise conjugate groups and/or terminal groups. RNase H agents may modulate the amount and/or activity of a target nucleic acid. The term RNase H agent excludes antisense agents that act principally through RISC/Ago2. An “RNAi agent” means an antisense agent that acts, at least in part, through RISC or Ago2 to modulate a target nucleic acid and/or a protein encoded by a target nucleic acid. RNAi agents include, but are not limited to double-stranded siRNA, single-stranded RNAi (ssRNAi), and microRNA, including microRNA mimics. RNAi agents may comprise conjugate groups and/or terminal groups. In certain embodiments, an RNAi agent modulates the amount and/or activity of a target nucleic acid. The term RNAi agent excludes antisense agents that act through RNase H. As used herein, “single -stranded” in reference to a nucleic acid (e.g., an oligonucleotide) means that the strand or region is unpaired; that is, the strand of linked nucleosides is not part of a duplex, not part of a double-stranded region. Single-stranded nucleic acids (e.g., single-stranded oligonucleotides) are capable of hybridizing to complementary nucleic acids to form duplexes, at which point they are no longer single -stranded.
As used herein, “stabilized phosphate moiety” means a 5 '-phosphate analog that is metabolically more stable than a 5 '-phosphate as naturally occurs on DNA or RNA.
As used herein, “stereorandom” or “stereorandom chiral center” in the context of a population of molecules of identical molecular formula means a chiral center that is not controlled during synthesis, or enriched following synthesis, for a particular absolute stereochemical configuration. The absolute stereochemical configuration of a chiral center can be controlled by using stereochemically-pure starting materials, e.g., using P-D-ribosyl nucleoside monomers for oligonucleotide synthesis. In contrast, the stereochemical configuration of a chiral center is random when it is the result of a synthetic method that is not designed to control the stereochemical configuration. For example, in a population of molecules comprising a stereorandom chiral center, the number of molecules having the (.S') configuration of the stereorandom chiral center may be the same as the number of molecules having the (/?) configuration of the stereorandom chiral center (“racemic”). The stereorandom chiral center may not be racemic because one absolute configuration predominates following synthesis, e.g., due to the action of non-chiral reagents near the enriched stereochemistry of an adjacent sugar moiety. The stereorandom chiral center may be at the phosphorous atom of a stereorandom phosphorothioate or stereorandom mesyl phosphoramidate intemucleoside linkage.
As used herein, a “strand” or “strand of linked nucleosides” means contiguous linked nucleosides connected via intemucleoside linkages. A strand of linked nucleosides has a nucleobase sequence.
As used herein, “subject” means a human or a non-human animal.
As used herein, “sugar moiety” means an unmodified sugar moiety or a modified sugar moiety.
As used herein, “sugar mimic” means a group of atoms forming the portion of a nucleoside corresponding to the P-D-ribosyl sugar in RNA other than a modified furanosyl sugar moiety, a cyclic sugar surrogate, or an acyclic sugar surrogate.
As used herein, “sugar surrogate nucleoside” means a cyclic sugar surrogate nucleoside or an acyclic sugar surrogate nucleoside.
As used herein, “symptom” of a disease means any manifestation, indication, sign, or evidence of a disease. Symptoms include subjective and objective indicia of a disease and may be perceived, experienced, detected, observed, measured, and/or quantified. A symptom may be apparent upon diagnostic testing, and in certain instances only upon invasive diagnostic testing, including, but not limited to, post-mortem tests. A symptom may be an absence of a feature, such as failing to reach expected developmental milestones. Symptoms may include but are not limited to, angina, chest pain, shortness of breath, palpitations, weakness, dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling in the lower extremities, cyanosis, fatigue, fainting, numbness of the face, numbness of the limbs, claudication or cramping of muscles, bloating of the abdomen, and fever.
As used herein, “target nucleic acid” means a LPA nucleic acid that an antisense oligonucleotide is designed to affect. As used herein, “target RNA” means an LPA RNA transcript and includes pre-mRNA and/or mRNA unless otherwise specified.
As used herein, “target region” refers to a portion of a target nucleic acid that is complementary to the targeting region of an antisense oligonucleotide.
As used herein, “terminal group” means a chemical group or group of atoms that is covalently linked to a terminus of an oligonucleotide.
As used herein, “therapeutic index” means the ratio of a measure of toxicity or intolerability divided by a measure of potency or activity. Typically, the therapeutic index is expressed as the ratio between the concentration (or dose) at which a compound becomes toxic or induces unacceptable adverse effects (or the highest concentration or dose at which a compound is not toxic, or is tolerated, before it becomes toxic or induces unacceptable adverse effects) to a subject and the concentration (or dose) at which the compound is pharmacologically effective or produces the desired effect.
As used herein, “treating,” or “treatment,” with respect to a disease, means administering a compound or agent to a subject having or at risk for developing such disease. Treating a disease may result in amelioration of at least one symptom of such disease. Treatment may reduce, improve, and/or prevent one or more symptom(s) such that a symptom of the disease is diminished or is no longer apparent.
As used herein, “unmodified nucleobase” means unmodified adenine (A), unmodified thymine (T), unmodified cytosine (C), unmodified uracil (U), or unmodified guanine (G).
As used herein, an “unmodified nucleoside” means a compound or subunit comprising an unmodified sugar moiety and an unmodified nucleobase.
As used herein, “unmodified sugar moiety” means a 2'-0H(H) P-D-ribosyl sugar moiety, as found in RNA, or a 2'-H(H) -D-deoxyribosyl sugar moiety, as found in DNA. Unmodified sugar moieties are fiiranosyl or deoxyfuranosyl sugar moieties in the P-D- stereochemical configuration, and have one hydrogen at each of the T, 3 ', and 4' positions, an oxygen at the 3 ' position, two hydrogens at the 5 ' position, and two hydrogens (DNA) or a hydrogen and an OH (RNA) at the 2' position.
EMBODIMENTS
1. An oligomeric duplex comprising a first oligomeric compound and a second oligomeric compound, wherein: the first oligomeric compound comprises a first modified oligonucleotide consisting of 18 to 50 linked nucleosides, wherein the nucleobase sequence comprises a nucleobase sequence that is at least 80% identical to at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NOs:6-829 and 3078- 3083, wherein at least one but no more than 22%, no more than 20%, no more than 18%, no more than 15%, no more than 10%, or no more than 5% of the modified nucleosides in the first modified oligonucleotide comprises a 2'-F sugar moiety and/or an FHNA sugar surrogate, and the second oligomeric compound comprises a second modified oligonucleotide consisting of 16 to 50 linked nucleosides, wherein the nucleobase sequence comprises a nucleobase sequence that is at least 80% identical to at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID Nos: 830-1540 and 3096-3101, wherein at least one but no more than 25%, no more than 20%, no more than 18%, no more than 16%, no more than 14%, no more than 12%, or no more than 10%, of the modified nucleosides in the second modified oligonucleotide comprises a 2'-F sugar moiety and/or an FHNA sugar surrogate; wherein the first modified oligonucleotide and the second modified oligonucleotide are complementary to one another; and wherein each of the nucleosides of the first modified oligonucleotide and the second modified oligonucleotide independently comprises a modified sugar moiety or sugar surrogate.
2. The oligomeric duplex of embodiment 1,
1) wherein the first modified oligonucleotide comprises at least one and no more than four modified nucleosides comprising a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate, and/or wherein the second modified oligonucleotide comprises at least one and no more than four modified nucleosides comprising a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate; and/or wherein the oligomeric duplex comprises at least one and no more than eight modified nucleosides comprising a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate; and/or
2) wherein at least 18 nucleosides of the first modified oligonucleotide, each independently, and at least 16 nucleosides of the second modified oligonucleotide, each independently, comprise a modified sugar moiety or sugar surrogate selected from a 2'-F sugar moiety, a 2'-MOE sugar moiety, a 2'-OMe sugar moiety, a DNA sugar moiety, and an FHNA sugar surrogate; and/or
3) wherein the nucleobase sequence of the first modified oligonucleotide comprises at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NOs:6-829 and 3078-3083, and wherein the nucleobase sequence of the second modified oligonucleotide comprises at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID Nos: 830-1540 and 3096-3101.
3. The oligomeric duplex of embodiment 1 or embodiment 2, wherein a nucleoside comprising a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety or FHNA sugar surrogate of the first modified oligonucleotide is independently selected from one of: i. the second nucleoside counting from the 5’ end, ii. the second and fourteenth nucleosides counting from the 5’ end, or iii. the second and sixteenth nucleosides counting from the 5’ end, or iv. the second, fourteenth and sixteenth nucleosides counting from the 5’ end, or v. the second, sixth, fourteenth, and sixteenth nucleosides counting from the 5’ end.
4. The oligomeric duplex of any one of embodiments 1-3, wherein a nucleoside comprising a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate of the second modified oligonucleotide is independently selected from one of: i.the ninth and tenth nucleosides counting from the 5’ end, or ii.the tenth and eleventh nucleosides counting from the 5’ end, or iii .the ninth, tenth and eleventh nucleosides counting from the 5’ end, or iv.the seventh, ninth, tenth and eleventh nucleosides counting from the 5’ end, or v.the seventh, ninth, and eleventh nucleosides counting from the 5 ’ end.
5. The oligomeric duplex of any one of embodiments 1-4, wherein one modified nucleoside of the first modified oligonucleotide comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate comprises a 3’-fluoro-hexitol sugar moiety sugar surrogate.
6. The oligomeric duplex of any one of embodiments 1-4, wherein one or more nucleosides of the first modified oligonucleotide is a 2 ’-deoxynucleoside.
7. The oligomeric duplex of embodiment 6, wherein the one or more 2’ -deoxynucleosides is one or more nucleosides in a region of the sequence of the first modified oligonucleotide between and including the fifth nucleoside to the sixteenth nucleoside counting from the 5’ end of the first modified oligonucleotide.
8. The oligomeric duplex of embodiment 7, wherein the one or more 2’ -deoxynucleosides is any of the sixth, fourteenth, and/or sixteenth nucleosides counting from the 5 ’ end of the first modified oligonucleotide.
9. The oligomeric duplex of any one of embodiments 1-8, wherein two of the 3’ terminal nucleosides of the first modified oligonucleotide comprise a two nucleoside overhang.
10. The oligomeric duplex of embodiment 9, wherein the overhang nucleosides comprise two modified adenosine (AA), two modified uridine (UU) nucleosides, two modified inosine (II) nucleosides, or two modified nucleosides wherein one is an inosine, and one is an adenosine (Al or IA).
11. The oligomeric duplex of any one of embodiments 1-10, wherein one or more of the nucleosides of the first modified oligonucleotide comprises a 2’-OMe sugar moiety and wherein one or more of the nucleosides of the second modified oligonucleotide comprises a 2’-OMe sugar moiety.
12. The oligomeric duplex of embodiment 11, wherein at least thirteen nucleosides, at least fourteen nucleosides, at least fifteen nucleosides, at least sixteen nucleosides, at least seventeen nucleosides, at least eighteen nucleosides, or at least nineteen nucleosides of the first modified oligonucleotide comprise a 2’-OMe sugar moiety; and wherein at least thirteen nucleosides, at least fourteen nucleosides, at least fifteen nucleosides, at least sixteen nucleosides, at least seventeen nucleosides, at least eighteen nucleosides, or at least nineteen nucleosides of the first modified oligonucleotide comprise a 2’-OMe sugar moiety. 13. The oligomeric duplex of any one of embodiments 1-12, wherein one or more of the nucleosides of the first modified oligonucleotide comprise a 2 ’-MOE sugar moiety; and optionally, one or more of the nucleosides of the second modified oligonucleotide comprise a 2’-M0E sugar moiety.
14. The oligomeric duplex of embodiment 13, wherein the 5’ - and/or 3 ’-terminal nucleosides of the first modified oligonucleotide comprise a 2 ’-MOE sugar moiety; and optionally, the 5’- and/or 3’- terminal nucleosides of the second modified oligonucleotide comprise a 2’-M0E sugar moiety.
15. The oligomeric duplex of embodiment 14, wherein two of the 5’- terminal nucleosides and/or two of the 3’-terminal nucleosides of the first modified oligonucleotide comprise a 2’-M0E sugar moiety; and optionally, two of the 5’- terminal nucleosides and/or two of the 3 ’-terminal nucleosides of the second modified oligonucleotide comprise a 2’-M0E sugar moiety.
16. The oligomeric duplex of embodiment 13, wherein at least one nucleoside of the first modified oligonucleotide comprising a 2 ’-MOE sugar moiety is an internal nucleoside in a region of the sequence of the first modified oligonucleotide that is any of the ninth and/or tenth nucleosides counting from the 5’ end of the first modified oligonucleotide.
17. The oligomeric duplex of any one of embodiments 1-16, wherein the first modified oligonucleotide comprises a stabilized phosphate group attached to the 5 ’-terminal nucleoside.
18. The oligomeric duplex of embodiment 17, wherein the stabilized phosphate group comprises a methylene phosphonate, cyclopropyl phosphonate or a vinyl phosphonate.
19. The oligomeric duplex of any one of embodiments 1-18, wherein the first modified oligonucleotide and/or the second modified oligonucleotide comprises at least one modified intemucleoside linkage.
20. The oligomeric duplex of embodiment 19, wherein at least one modified intemucleoside linkage is a phosphorothioate intemucleoside linkage.
21. The oligomeric duplex of embodiment 20, wherein each intemucleoside linkage is independently selected from a phosphodiester intemucleoside linkage and a phosphorothioate intemucleoside linkage.
22. The oligomeric duplex of embodiment 21, wherein the intemucleoside linkages between the first and second nucleosides and the second and third nucleosides counting from the 5’ end of the first modified oligonucleotide are modified intemucleoside linkages, and/or wherein the intemucleoside linkages between the first and second nucleosides and the second and third nucleosides counting from the 3 ’ end of the first modified oligonucleotide are modified intemucleoside linkages; and optionally, wherein the intemucleoside linkages between the first and second nucleosides and the second and third nucleosides counting from the 5 ’ end of the second modified oligonucleotide are modified intemucleoside linkages, and/or wherein the intemucleoside linkages between the first and second nucleosides and the second and third nucleosides counting from the 3’ end of the second modified oligonucleotide are modified intemucleoside linkages.
23. The oligomeric duplex of embodiment 1, wherein the first modified oligonucleotide comprises a modified sugar motif independently selected from one of efyyyfyyyyyyyfyfyyyyyyy, yfyyyfyyyyyyyfyfyyyyyyy, efyyydyyeyyyydydyyyyyee, efyyydyyyyyyyfyfyyyyyee, efyyydyyeyyyyfyfyyyyyee, e[FHNA]yyyfyyyyyyyfyfyyyyyee, and e[FHNA]yyyfyyeyyyyfyfyyyyyee, wherein each ‘e’ represents a 2'-M0E sugar moiety, each ‘y’ represents a 2'-0Me sugar moiety, each ‘f represents a 2'-F sugar moiety, each ‘[FHNA]’ represents a 3'-fluoro-hexitol sugar moiety, and each ‘d’ represents a 2'-deoxy sugar moiety, and wherein all except 0, 1, or 2 modifications are identical to the sugar motif; and/or the second modified oligonucleotide comprises a modified sugar motif independently selected from one of yyyyyyfyfffyyyyyyyyyy and eeyyyyyyyffyyyyyyyyee, wherein each ‘e’ represents a 2'-MOE sugar moiety, each ‘y’ represents a 2'-OMe sugar moiety, and each ‘f represents a 2'-F sugar moiety, and wherein all except 0, 1, or 2 modifications are identical to the sugar motif.
24. The oligomeric duplex of embodiment 1, wherein the first modified oligonucleotide comprises a modified sugar motif independently selected from one of efyyyfyyyyyyyfyfyyyyyyy, yfyyyfyyyyyyyfyfyyyyyyy, efyyydyyeyyyydydyyyyyee, efyyydyyyyyyyfyfyyyyyee, efyyydyyeyyyyfyfyyyyyee, e[FHNA]yyyfyyyyyyyfyfyyyyyee, and e[FHNA]yyyfyyeyyyyfyfyyyyyee, wherein each ‘e’ represents a 2'-MOE sugar moiety, each ‘y’ represents a 2'-OMe sugar moiety, each ‘f represents a 2'-F sugar moiety, each ‘[FHNA]’ represents a 3'-fluoro-hexitol sugar moiety, and each ‘d’ represents a 2'-deoxy sugar moiety; and/or the second modified oligonucleotide comprises a modified sugar motif independently selected from one of yyyyyyfyfffyyyyyyyyyy and eeyyyyyyyffyyyyyyyyee, wherein each ‘e’ represents a 2'-MOE sugar moiety, each ‘y’ represents a 2'-OMe sugar moiety, and each ‘f represents a 2'-F sugar moiety.
25. The oligomeric duplex of embodiment 24, wherein the first modified oligonucleotide comprises a modified sugar motif and is paired with the second modified oligonucleotide comprising a sugar motif selected from: efyyyfyyyyyyyfyfyyyyyyy and yyyyyyfyfffyyyyyyyyyy; yfyyyfyyyyyyyfyfyyyyyyy and yyyyyyfyfffyyyyyyyyyy; efyyydyyeyyyydydyyyyyee and eeyyyyyyyffyyyyyyyyee; efyyydyyyyyyyfyfyyyyyee and eeyyyyyyyffyyyyyyyyee; efyyydyyeyyyyfyfyyyyyee and eeyyyyyyyffyyyyyyyyee; e[FHNA]yyyfyyeyyyyfyfyyyyyee and eeyyyyyyyffyyyyyyyyee; and e[FHNA]yyyfyyyyyyyfyfyyyyyee and eeyyyyyyyffyyyyyyyyee.
26. The oligomeric duplex of any one of embodiments 1-25, wherein the agent comprises a conjugate group comprising a conjugate moiety and a conjugate linker.
27. The oligomeric duplex of embodiment 26, wherein the conjugate group comprises a celltargeting moiety.
28. The oligomeric duplex of embodiment 27, wherein the conjugate group comprises a liver cell targeting moiety.
29. The oligomeric duplex of embodiment 28, wherein the agent comprises a conjugate moiety that binds asialoglycoprotein receptor (ASGPR).
30. The oligomeric duplex of embodiment 29, wherein the conjugate moiety is selected from a GalNAc moiety.
31. The oligomeric duplex of embodiment 30, wherein the GalNAc conjugate moiety is selected from Table A. 32. The oligomeric duplex of embodiment 30, wherein the conjugate group consists of a GalNAc ligand and a conjugate linker.
33. The oligomeric duplex of embodiment 30, wherein the GalNAc ligand has the structure:
Figure imgf000021_0001
34. The oligomeric duplex of embodiment 15, wherein the conjugate group has the structure:
Figure imgf000021_0002
or an ion or salt thereof, wherein the conjugate linker is covalently connected to an oligonucleotide.
35. The oligomeric duplex of embodiment 30, wherein the conjugate group has the structure:
Figure imgf000021_0003
or an ion or salt thereof, wherein the conjugate linker is covalently connected to an oligonucleotide.
36. The oligomeric duplex of any one of embodiments 26-35, wherein the conjugate group is conjugated directly to the modified oligonucleotide.
37. The oligomeric duplex of embodiment 36, wherein the conjugate group is conjugated to the 5’ end or 3’ end of the modified oligonucleotide. 38. The oligomeric duplex of embodiment 37, wherein the conjugate group is attached to the 5’- terminal nucleoside of the modified oligonucleotide.
39. The oligomeric duplex of embodiment 37, wherein the conjugate group is attached to the 3’- terminal nucleoside of the modified oligonucleotide.
40. The oligomeric duplex of embodiment 36, wherein the conjugate linker of the conjugate group consists of a single bond.
41. The oligomeric duplex of embodiment 36, wherein the conjugate linker of the conjugate group is cleavable.
42. The oligomeric duplex of embodiment 36, wherein the conjugate linker comprises 1 to 3 linker- nucleosides.
43. The oligomeric duplex of embodiment 38, wherein the conjugate group having the structure:
Figure imgf000022_0001
or an ion or salt thereof, is attached to the 5 ’-terminal nucleoside of the modified oligonucleotide.
44. The oligomeric duplex of embodiment 39, wherein the conjugate group having the structure:
Figure imgf000022_0002
or an ion or salt thereof, is attached to the 3 ’-terminal nucleoside of the modified oligonucleotide.
45. An oligomeric duplex comprising i) a first oligomeric compound comprising a modified oligonucleotide consisting of 16 to 50 linked nucleosides, wherein the nucleobase sequence comprises a nucleobase sequence that is at least 80% identical to at least 16 contiguous nucleobases of any one of SEQ ID NOs:6-829 and 3078-3083, wherein all except 0, 1, or 2 nucleobases are identical to the nucleobase sequence SEQ ID NOs:6-829 or 3078-3083, and wherein each of the first modified oligonucleotide comprises a modified sugar moiety selected from: efyyyfyyyyyyyfyfyyyyyyy, yfyyyfyyyyyyyfyfyyyyyyy, efyyydyyeyyyydydyyyyyee, efyyydyyyyyyyfyfyyyyyee, efyyydyyeyyyyfyfyyyyyee, e[FHNA]yyyfyyyyyyyfyfyyyyyee, and e[FHNA]yyyfyyeyyyyfyfyyyyyee, wherein each ‘e’ represents a 2'-M0E sugar moiety, each ‘y’ represents a 2'-0Me sugar moiety, each ‘f represents a 2'-F sugar moiety, each ‘[FHNA]’ represents a 3'-fluoro-hexitol sugar moiety, and each ‘d’ represents a 2'-deoxy sugar moiety; and ii) a second oligomeric compound comprising a second modified oligonucleotide consisting of 14-50 linked nucleosides wherein the nucleobase sequence comprises a nucleobase sequence that is at least 80% identical to at least 14 contiguous nucleobases of any one of SEQ ID NOs: 830-1540 and 3096-3101, wherein all except 0, 1, or 2 nucleobases are identical to the nucleobase sequence of SEQ ID NOs: 830-1540 or 3096-3101, and wherein each of the second modified oligonucleotide comprises a modified sugar motif selected from one of yyyyyyfyfffyyyyyyyyyy and eeyyyyyyyffyyyyyyyyee, wherein each ‘e’ represents a 2'-MOE sugar moiety, each ‘y’ represents a 2'-OMe sugar moiety, and each ‘f represents a 2'-F sugar moiety.
46. The oligomeric duplex of embodiment 45, wherein the sequence of the second modified oligonucleotide comprises at least 15 or at least 16 contiguous nucleobases of the sequence of any one of SEQ ID NO: 2365-3075 and 3084-3089; and optionally wherein the sequence of the first modified oligonucleotide comprises at least 18, at least 19, or at least 20 contiguous nucleobases of the sequence of any one of SEQ ID NO: 1541-2364 and 3090-3095.
47. The oligomeric duplex of embodiment 46, wherein the sequence of the first modified oligonucleotide comprises the sequence of any one of SEQ ID NOs: 1541-2364 and 3090-3095; and optionally wherein the sequence of the second modified oligonucleotide comprises the sequence of any one of SEQ ID NO: 2365-3075 and 3084-3089.
48. The oligomeric duplex of embodiment 46, wherein the sequence of the first modified oligonucleotide consists of the sequence of any one of SEQ ID NOs: 1541-2364 and 3090-3095; and optionally wherein the sequence of the second modified oligonucleotide consists of the sequence of any one of SEQ ID NO: 2365-3075 and 3084-3089.
49. The oligomeric duplex of embodiment 46, wherein the agent is a duplex compound comprised of any one the following antisense compound and sense compound pairs:
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000024_0002
Figure imgf000024_0003
Figure imgf000025_0001
Figure imgf000025_0002
Figure imgf000025_0003
Figure imgf000026_0001
Figure imgf000026_0002
Figure imgf000026_0003
Figure imgf000027_0001
Figure imgf000027_0002
Figure imgf000027_0003
Figure imgf000028_0001
Figure imgf000028_0002
Figure imgf000028_0003
Figure imgf000029_0001
Figure imgf000029_0002
Figure imgf000029_0003
Figure imgf000030_0001
Figure imgf000030_0002
Figure imgf000030_0003
Figure imgf000031_0001
50. A population of oligomeric duplexes of any one of embodiments 1-49, wherein the population is enriched for and/or modified oligonucleotides comprising at least one particular phosphorothioate intemucleoside linkage having a particular stereochemical configuration. 51. The population of embodiment 50, wherein the population is enriched for and/or modified oligonucleotides comprising at least one particular phosphorothioate intemucleoside linkage having the (Sp) or (Rp) configuration.
52. A pharmaceutical composition comprising the oligomeric duplex of any one of embodiments 1- 49, or the population of any one of embodiments 50-51, and a pharmaceutically acceptable diluent or carrier.
53. The pharmaceutical composition of embodiment 52, wherein the pharmaceutically acceptable diluent is water or phosphate-buffered saline. 54. The pharmaceutical composition of embodiment 52, wherein the pharmaceutical composition consists essentially of the oligomeric duplex and water or phosphate-buffered saline.
55. A method of decreasing the amount of LPA RNA or Lipoprotein(a) protein in a cell, tissue, organ or subject, comprising contacting the cell, tissue, organ or subject with the oligomeric duplex of any one of embodiments 1-49, the population of any one of embodiments 50-51, or the pharmaceutical composition of any one of embodiments 52-54.
56. The method of embodiment 55, wherein the cell is a liver cell.
57. A method comprising administering to a subject the oligomeric duplex of any one of embodiments 1-49, the population of any one of embodiments 50-51, or the pharmaceutical composition of any one of embodiments 52-54; wherein the subject has or is at risk for developing cardiovascular disease (CVD) coronary artery disease (CAD), hypercholesterolemia, myocardial infarction (MI), peripheral arterial disease (PAD), calcific aortic valve disease (CAVD), aortic stenosis, atherosclerotic cardiovascular disease (ASCVD), atherosclerosis, dyslipidemia, thrombosis, or stroke.
58. A method of preventing or treating a cardiovascular disease, disorder, condition, a metabolic disease, disorder, or condition, and/or an inflammatory disease disorder or condition in a subject, comprising administering to a subject having, or at risk of having, a cardiovascular, metabolic, and/or inflammatory disease, disorder, or condition, an oligomeric duplex of any one of embodiments 1-49, the population of any one of embodiments 50-51, or the pharmaceutical composition of any one of embodiments 52-54; wherein the disease, disorder, condition or injury is hypertriglyceridemia, lipidemia (e.g., hyperlipidemia), dyslipidemia (e.g., atherogenic dyslipidemia, diabetic dyslipidemia, or mixed dyslipidemia), hyperlipoproteinemia, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic valve stenosis, aortic valve calcification, aortic valve regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, restenosis, renal artery stenosis, angina, cerebrovascular atherosclerosis, cerebrovascular disease, or venous thrombosis.
59. A method of decreasing the amount of LPA RNA and/or Lipoprotein(a) protein in the liver of a subject having or at risk of developing a disease, disorder or condition associated with elevated Lp(a), comprising administering to a subject having, or at risk of having, a disease, disorder or condition associated with lipoprotein(a) metabolism misregulation, an oligomeric duplex of any one of embodiments 1-49, the population of any one of embodiments 50-51, or the pharmaceutical composition of any one of embodiments 52-54; wherein the disease, disorder, condition or injury is a cardiovascular disease, disorder, condition, a metabolic disease, disorder, or condition, and/or an inflammatory disease disorder or condition.
60. The method of any one of embodiments 55-59, wherein the amount of LPA RNA and/or lipoprotein(a) protein in liver and/or plasma of the subject is decreased.
61. The method of any one of embodiments 55-59, wherein the disease, disorder or condition is hypertriglyceridemia or atherosclerotic cardiovascular disease (ASCVD) or coronary artery disease (CAD). 62. The method of embodiment 61, wherein at least one symptom of a disease, disorder or condition associated with elevated lipoprotein(a) is episodes of abdominal pain, physical fatigue, difficulty thinking, diarrhea, recurrent acute pancreatitis, eruptive cutaneous xanthomata, and hepatosplenomegaly or a combination thereof.
63. The method of any one or embodiments 55-59, wherein the method prevents or protects against progression of atherosclerotic cardiovascular disease (ASCVD) or coronary artery disease (CAD).
64. The method of any one of embodiments 55-59, wherein administering of the oligomeric duplex of any one of embodiments 1-49, the population of any one of embodiments 50-51, or the pharmaceutical composition of any one of embodiments 52-54 improves hypertriglyceridemia, hyperlipidemia, dyslipidemia hyperlipoproteinemia abdominal pain, physical fatigue, difficulty thinking, diarrhea, acute pancreatitis, eruptive xanthomas, lipemia retinalis, or hepatosplenomegaly, or a combination of two or more of the foregoing in the subject.
65. The method of any one of embodiments 55-59, wherein administering of the oligomeric duplex of any one of embodiments 1-49, the population of any one of embodiments 50-51, or the pharmaceutical composition of any one of embodiments 52-54 is parenteral.
66. The method of any one of embodiments 55-59, wherein administering of the oligomeric duplex of any one of embodiments 1-49, the population of any one of embodiments 50-51, or the pharmaceutical composition of any one of embodiments 52-54 is subcutaneous.
67. The method of any one of embodiments 55-59, wherein administering of the oligomeric duplex of any one of embodiments 1-49, the population of any one of embodiments 50-51, or the pharmaceutical composition of any one of embodiments 52-54 is co-administering with a second agent.
68. The method of any one of embodiment 55-59, wherein administering of the oligomeric duplex of any one of embodiments 1-29, the population of any one of embodiments 30-31, or the pharmaceutical composition of any one of embodiments 32-34 and the agent are administered concomitantly.
69. Use of the oligomeric duplex of any one of embodiments 1-49, the population of any one of embodiments 50-51, or the pharmaceutical composition of any one of embodiments 52-54 for treating or preventing a disease, disorder or condition associated with lipoprotein metabolism misregulation or postponing a symptom of a disease, disorder or condition associated with elevated lipoprotein(a).
70. Use of the oligomeric duplex of any one of embodiments 1-49, the population of any one of embodiments 50-51, or the pharmaceutical composition of any one of embodiments 52-54in the manufacture of a medicament for treating or preventing a cardiovascular disease, disorder, condition, a metabolic disease, disorder, or condition, or an inflammatory disease disorder or condition.
I. Oligonucleotides
Provided herein are oligomeric duplexes and oligomeric compounds comprising a modified antisense oligonucleotide (e.g., an antisense oligomeric compound) complementary to UPA RNA and a modified sense oligonucleotide (e.g., a sense oligomeric compound) complementary to an antisense oligomeric compound. Modified antisense and/or sense oligonucleotides comprise at least one modified nucleoside (comprising a modified sugar moiety and/or a modified nucleobase) and/or at least one modified intemucleoside linkage. Examples of certain modified nucleosides and modified intemucleoside linkages suitable for use in modified antisense and/or sense oligonucleotides are described herein.
A. Modified Nucleosides
Modified nucleosides comprise a modified sugar moiety, a modified nucleobase, or a combination thereof. In certain embodiments, modified nucleosides comprising the following modified sugar moieties and/or the following modified nucleobases may be incorporated into modified antisense oligonucleotides and/or sense oligonucleotides described herein.
/. Modified Sugar Moieties
Modified sugar moieties include modified fiiranosyl sugar moieties, cyclic sugar surrogates, acyclic sugar surrogates, and sugar mimics. In certain embodiments, modified sugar moieties are non- bicyclic modified fiiranosyl sugar moieties. In certain embodiments, modified sugar moieties are bicyclic or tricyclic fiiranosyl sugar moieties. In certain embodiments, modified sugar moieties are sugar surrogates. Sugar surrogates may comprise one or more substitutions corresponding to those of other types of modified sugar moieties.
In certain embodiments, modified sugar moieties are non-bicyclic modified fiiranosyl sugar moieties comprising one or more substituent groups including, but not limited to, substituents at the 2', 3', 4', and/or 5' positions, as numbered based on ribose:
Figure imgf000034_0001
In certain embodiments, the modified fiiranosyl sugar moiety is a ribosyl sugar moiety that is not an unmodified sugar moiety (i.e. , an unmodified RNA or unmodified DNA moiety). In certain embodiments, the modified fiiranosyl sugar moiety is a xylosyl, lyxosyl, or arabinosyl sugar moiety.
In certain embodiments, non-bicyclic modified sugar moieties are 2'-substituted sugar moieties and comprise a substituent group at the 2'-position. In certain embodiments one or more non-bridging substituent of non-bicyclic modified sugar moieties is branched. Examples of substituent groups suitable for the 2'-position of modified sugar moieties include but are not limited to: 2'-F, 2'-OCH3 (“OMe” or “O-methyl”), and 2'-O(CH2)2OCH3 (“MOE” or “O-methoxyethyl”). In certain embodiments, 2'- substituent groups are selected from: halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, C1-C10 alkoxy, C1-C10 substituted alkoxy, C1-C10 alkyl, C1-C10 substituted alkyl, S-alkyl, N(Rm)-alkyl, O-alkenyl, S- alkenyl, N(Rm)-alkenyl, O-alkynyl, S-alkynyl, N(Rm)-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, ( CFE^SCFE, O(CH2)2ON(Rm)(Rn) or 0CH2C(=0)-N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group, or substituted or unsubstituted C1-C10 alkyl, O(CH2)2ON(CH3)2 (“DMAOE”), or 2'-O(CH2)2O(CH2)2N(CH3)2 (“DMAEOE”). Synthetic methods for some of these 2'-substituent groups may be found, e.g., in Cook et al., U.S. 6,531,584; Cook et al., U.S. 5,859,221; and Cook et al., U.S. 6,005,087. Certain embodiments of these 2'-substituent groups may be further substituted with one or more substituent groups independently selected from: halo, cyano, ORa2, N02, NH2, NHRa2, N(Ra2)2, Ci-Ce alkyl, Ci-Cehaloalkyl, C2-Ce alkenyl, C2-Ce alkynyl, C3-C10 cycloalkyl, Ce-Cio aryl, heteroaryl, heterocyclyl, Ci-C6 alkylene-NH2, Ci-Cealkylene-NHRa2, Ci-C6 alkylene-N(Ra2)2, C(O)Ra3, C(O)ORa3, C(O)NHRa3, C(O)N(Ci-C4alkyl)Ra3, SRa3, S(O)2Ra3, S(O)Ra3, NHC(O)Ra3, N(CI-C4 alkyl)C(O)Ra3, NHS(O)Ra3, N(Ci-C4alkyl)S(O)Ra3, NHS(O)2Ra3, and N(Ci-C4alkyl)S(O)2Ra3; each Ra2 is independently selected from C2-Ce alkyl, C2-Ce alkenyl, C2-Ce alkynyl, C3-C10 cycloalkyl, Ce-Cio aryl, heteroaryl, and heterocyclyl; each Ra3 is independently hydrogen, OH, Ci-Ce alkyl, Ci-Cehaloalkyl, C3- C10 cycloalkyl, Ce-Cio aryl, heteroaryl, or heterocyclyl. In certain embodiments, a sugar moiety comprises two of the above substituents at the 2'-position. In certain embodiments, a sugar moiety comprises a 2'-fluoro and a second 2'-substituent.
In certain embodiments, a 2'-substituted sugar moiety comprises a non-bridging 2 '-substituent group selected from: F, NH2, N3, OCF3, OCH3, O(CH2)3NH2, CH2CH=CH2, OCH2CH=CH2, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(Rm)(Rn), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamide (OCH2C(=O)-N(Rm)(Rn)), where each Rm and Rn is, independently, H, an amino protecting group, or substituted or unsubstituted C1-C10 alkyl.
In certain embodiments, a 2'-substituted sugar moiety comprises a non-bridging 2 '-substituent group selected from: F, OCF3, OCH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(CH3)2, O(CH2)2O(CH2)2N(CH3)2, O(CH2)2ON(CH3)2 (“DMAOE”), O(CH2)2O(CH2)2N(CH3)2 (“DMAEOE”), and OCH2C(=O)-N(H)CH3 (“NMA”).
In certain embodiments, a 2'-substituted sugar moiety comprises a non-bridging 2 '-substituent group selected from: F, OCF3, OCH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(CH3)2, O(CH2)2O(CH2)2N(CH3)2, and OCH2C(=O)-N(H)CH3 (“NMA”).
In certain embodiments, a 2'-substituted sugar moiety comprises a 2'-substituent group selected from: F, OCH3, and OCH2CH2OCH3.
In certain embodiments, modified furanosyl sugar moieties and nucleosides incorporating such modified furanosyl sugar moieties are further defined by stereochemical configuration. For example, a 2'- deoxyfiiranosyl sugar moiety (z.e., 2'-(H)H furanosyl sugar moiety) may be in seven isomeric configurations other than the naturally occurring P-D-deoxyribosyl configuration. Such modified sugar moieties are described in, e.g., WO 2020/072991, incorporated by reference herein. A 2'-modified sugar moiety has an additional stereocenter at the 2'-position relative to a 2'-deoxyfiiranosyl sugar moiety; therefore, such sugar moieties have a total of sixteen possible stereochemical configurations. Modified furanosyl sugar moieties described herein are in the -D-ribosyl stereochemical configuration unless otherwise specified.
In certain embodiments, non-bicyclic modified sugar moieties comprise a substituent group at the 4'-position. Examples of substituent groups suitable for the 4'-position of modified sugar moieties include, but are not limited to, alkoxy (e.g., methoxy), alkyl, and those described in Manoharan et al., WO 2015/106128, which is incorporated herein by reference. In certain embodiments, non-bicyclic modified sugar moieties comprise a substituent group at the 3 '-position. Examples of substituent groups suitable for the 3 '-position of modified sugar moieties include, but are not limited to, alkoxy (e.g., methoxy), alkyl (e.g., methyl, ethyl).
In certain embodiments, non-bicyclic modified sugar moieties comprise a substituent group at the 5 '-position. Examples of substituent groups suitable for the 5 '-position of modified sugar moieties include, but are not limited to, vinyl, alkoxy (e.g., methoxy), alkynyl, allyl, and alkyl (e.g., methyl (R or .8). ethyl (R or 5)).
In certain embodiments, non-bicyclic modified sugar moieties comprise more than one nonbridging sugar substituent, for example, 2'-F-5'-methyl sugar moieties, such as described in Migawa et al., US 2010/0190837, which is incorporated herein by reference; or alternative 2'- and 5'-modified sugar moieties as described in Rajeev et al., US 2013/0203836, which is incorporated herein by reference.
Certain modified sugar moieties are bicyclic sugar moieties and comprise a substituent that bridges two atoms of the fiiranosyl ring to form a second ring. In certain embodiments, the bicyclic sugar moiety comprises a bridge between the 4' and the 2' furanose ring atoms. Examples of such 4' to 2' bridging sugar substituents include, but are not limited to: 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2- O-2' (“LNA”), 4'-CH2-S-2', 4'-(CH2)2-O-2' (“ENA”), 4'-CH(CH3)-O-2' (referred to as “constrained ethyl” or “cEt” when in the S configuration), 4'-CH2-O-CH2-2', 4'-CH2-N(R)-2', 4'-CH(CH2OCH3)-O-2' (“constrained MOE” or “cMOE”) and analogs thereof, 4'-C(CH3)(CH3)-O-2' and analogs thereof, 4'- CH2-N(OCH3)-2' and analogs thereof, 4'-CH2-O-N(CH3)-2', 4'-CH2-C(H)(CH3)-2', 4'-CH2-C(=CH2)-2' and analogs thereof, 4'-C(RaRb)-N(R)-O-2', 4'-C(RaRb)-O-N(R)-2', 4'-CH2-O-N(R)-2', and 4'-CH2-N(R)- O-2', wherein each R, Ra, and Rb is, independently, H, a protecting group, or C1-C12 alkyl. Representative U.S. patents that teach the preparation of such bicyclic sugar moieties include, but are not limited to: hnanishi et al., U.S. 7,427,672; Swayze et al., U.S. 7,741,457; Swayze et al., U.S. 8,022,193; Seth et al., U.S. 8,278,283; Prakash et al., U.S. 8,278,425; and Seth et al., U.S. 8,278,426, each of which are incorporated herein by reference.
In certain embodiments, such 4' to 2' bridges independently comprise from 1 to 4 linked groups independently selected from: -[C(Ra)(Rb)]n-, -[C(Ra)(Rb)]n-O-, C(Ra)=C(Rb)-, C(Ra)=N-, C(=NRa)-, - C(=O)-, -C(=S)-, -O-, -Si(Ra)2-, -S(=O)x-, and N(Ra)-; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each Ra and Rb is, independently, 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, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJi, NJ1J2, SJi, N3, COOJi, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=0)2-Ji), or sulfoxyl (S(=O)-Ji); and each Ji and J2 is, independently, H, 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, acyl (C(=O)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or a protecting group.
In certain embodiments, the bicyclic sugar moiety comprises a bridge between the 5' and the 3' furanose ring atoms. Examples of such 5' to 3' bridging sugar substituents include, but are not limited to, 5'-(CH2)2-3' (bcDNA), 5'-(CH2)3-3' (bc43DNA), 5'-C(F)=CH-CH2-3', and 5'-CH2-CHQ-3', wherein Q is an attachment to an intemucleoside linkage.
Additional bicyclic sugar moieties are known in the art, see, for example: Wan, et al., J. Medicinal Chemistry, 2016, 59, 9645-9667; Wengel et al., U.S. 8,080,644; Ramasamy et al., U.S. 6,525,191; Seth et al., U.S. 7,547,684; and Seth et al., U.S. 7,666,854, which are each incorporated herein by reference.
In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by stereochemical configuration. For example, an ENA nucleoside (described herein) may be in the a-L configuration or in the P-D configuration.
Figure imgf000037_0001
LNA ( -D-configuration) LNA (a-L-configuration) bridge = 4'-CH2-O-2' bridge = 4'-CH2-O-2' a-L-methyleneoxy (4'-CH2-O-2') or a-L-LNA bicyclic nucleosides have been incorporated into oligonucleotides that showed antisense activity (Frieden et al. Nucleic Acids Res. 2003, 21, 6365-6372). The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al. Nucleic Acids Res. 2005, 33(1), 439-447; Mook, O. R. et al. Mol. Cane. Ther. 2007, 6(3), 833-843; Grunweller, A. et al. Nucleic Acids Res. 2003, 37(12), 3185- 3193). Herein, general descriptions of bicyclic nucleosides include both stereochemical configurations. When the positions of specific bicyclic nucleosides (e.g., LNA or cEt) are identified in exemplified embodiments herein, they are in the P-D stereochemical configuration, unless otherwise specified.
In certain embodiments, modified sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5 '-substituted and 4'-2' bridged sugars).
In certain embodiments, modified sugar moieties are sugar surrogates, selected from cyclic sugar surrogates and acyclic sugar surrogates.
A cyclic sugar surrogate is represented by Formula la:
Figure imgf000037_0002
Wherein:
J is H, Ci-Ce alkyl, or C2-C6 alkenyl;
X is O, S, C(RIR2), N(R3), C(RI)=C(R2), C(RIR2)-C(RIR2), O-C(RIR2), C(RIR2)-O, S-C(RIR2), C(RIR2)-S, N(R3)-C(RIR2), or C(RIR2)-N(R3);
Y is C(RIR2), C(RI)=C(R2), or C(RIR2)-C(RIR2);
Z is C(GIG2), C(GI)=C(RI), C(RI)=C(GI), C(GIG2)-C(RIR2), C(RIR2)-C(GIG2), C(GIG2)- C(RIR2)-C(RIR2), or C(RIR2)-C(RIR2)-C(GIG2);
Q is CH orN; each Ri and R2 is independently H, OH, Ci-Ce alkyl, or N(R ): wherein if Ri is OH, then R2 is not OH; each R3 and R4 is independently H, Ci-Ce alkyl, or C(=O)R5, wherein R5 is Ci-Ce alkyl; each Gi and G2 is independently H, OH, halogen or O-[C(R6)(R7)]q-[(C=O)s-XG]j-R8; wherein if Gi is OH, then G2 is not OH; each ; and R7 is, independently, H, halogen, Ci-Ce alkyl or substituted Ci-Ce alkyl; each XG is O, S or N(Ei);
Rs is H, halogen, Ci-Ce alkyl, substituted Ci-Ce alkyl, C2-Ce alkenyl, substituted C2-Ce alkenyl, C2-Ce alkynyl, substituted C2-Ce alkynyl or N(E2)(E3);
Ei, E2 and E3 are each, independently, H, Ci-Ce alkyl or substituted Ci-Ce alkyl; m is 0 or 1; p is 0 or 1; q is from 1 to 6; s is 0 or 1; j is 0 or 1; and with the proviso that if X is O, Z is C(GIG2), and Q is CH, then m is 1.
In certain embodiments, the oxygen atom of a sugar moiety is replaced, e.g., with a sulfur, carbon or nitrogen atom (X is S, C(RIR2), or N(R3)). In certain such embodiments, such modified sugar moieties also comprise bridging and/or non-bridging substituents as described herein. For example, certain sugar surrogates comprise a 4 '-sulfur atom and a substitution at the 2 '-position and/or the 5' position.
In certain embodiments, sugar surrogates comprise rings having other than 5 atoms. For example, in certain embodiments, a sugar surrogate comprises a six-membered tetrahydropyran (“THP”), where X is O-C(RIR2), p is 1, Z is C(GIG2), and m is 0. Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include but are not limited to hexitol nucleic acid (“HNA”), altritol nucleic acid (Gi=OH; G2=H; “ANA”), and fluoro HNA:
Figure imgf000039_0001
FHNA
(Gi=F; G2=H; “FHNA”, see e.g., Egli, M. et al. J. Am. Chem. Soc. 2011, 733(41), 16642-16649; Swayze et al., U.S. 8,088,904; and Swayze et al., U.S. 8,440,803); FHNA can also be referred to as a F-THP or 3 '-fluoro tetrahydropyran or 3 '-FHNA), each of which are incorporated herein by reference.
In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example, nucleosides comprising morpholino sugar moieties and their use in oligonucleotides have been reported. As used here, the term “morpholino” means a sugar surrogate having Formula la, above, wherein X is O, Y and Z are each CH2, and Q is N. In certain embodiments, a morpholino is modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are referred to herein as “modified morpholines.”
In certain embodiments, sugar surrogates are acyclic sugar surrogates and have Formula Ila or Illa:
Figure imgf000039_0002
Formula Illa
Wherein:
X is 0, S, C R4R5), N(Ei), N(Ei)-C(=O); each Ji and J2 is independently H or Ci-Ce alkyl; n is 0, 1 or 2; m is 0, 1, or 2; o is 0 or 1; s is 0 or 1;
Ri is H, OH, halogen, Ci-Ce alkyl, Ci-Ce alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, or (CH2)qR?
R2 and R3 are each independently H, OH, halogen, Ci-Ce alkyl, Ci-Ce alkoxy, C2-C6 alkenyl, C2- Ce alkynyl, S-CH3, N(CH3)(CH3), OCH2CH2OCH3, O-alkylamino, or (CH2)qR?; Ei is H, Ci-Ce alkyl or substituted Ci-Ce alkyl;
R4 and R5 are independently H, OH, Ci-Ce alkyl, or N(R„): wherein if R4 is OH, then R5 is not OH;
Re is H, Ci-Ce alkyl, or C(=O)R«, wherein R8 is Ci-Ce alkyl;
R7 is OH, halogen, methoxy, ethoxy, azido, and C2-C6 alkenyl, or C2-C6 alkynyl, and q is 1, 2, or 3.
In certain embodiments, acyclic sugar surrogates are the “unlocked” sugar structure of UNA (unlocked nucleic acid) nucleosides. Representative U.S. publications that teach the preparation of UNA include, but are not limited to, U.S. Patent Publication No. 2011/0313020.
In certain embodiments, acyclic sugar surrogates are the glycerol as found in GNA (glycol nucleic acid) nucleosides, having Formula Ila wherein n is 1, m and o are 0, s is 1, and J2, R2, and R3 are each H, or the butyl as found in acyclic butyl nucleic acid, having Formula Ila wherein n is 2, m and o are 0, s is 1, and J2, R2, and R3 are each H. In certain embodiments, acyclic sugar surrogates are also known as “C3 spacers” and have Formula Ila wherein n and o are 1; m and s are 0, and Ji, J2, Ri, and R3 are each H.
Further acyclic sugar surrogates include those described in Manoharan et al., U.S. 10,913,767; US patent publication US 2021/0238595; and PCT publication WO 2023/109940.
In certain embodiments, modified oligonucleotides include one or more sugar mimic, in which a group of atoms other than a “furanosyl sugar moiety” or a “sugar surrogate” form the portion of a nucleoside corresponding to the P-D-ribosyl sugar in RNA. In certain embodiments, a sugar mimic is a portion of the backbone of a peptide nucleic acid, while the remainder of the backbone of the peptide nucleic acid is an intemucleoside linkage. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262.
2. Modified Nucleobases
In certain embodiments, modified oligonucleotides comprise one or more nucleoside comprising an unmodified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleoside comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more abasic nucleoside. In certain embodiments, modified oligonucleotides contain only nucleosides comprising nucleobases. In certain embodiments, modified oligonucleotides comprise one or more inosine nucleosides (z.e., nucleosides comprising a hypoxanthine nucleobase). In certain embodiments modified oligonucleotides comprise one or more nucleosides comprising a 5- methylcytosine.
Unless otherwise indicated, modified adenine has structure (I):
Figure imgf000040_0001
I wherein: R2A is H, Ci-Ce alkyl, substituted Ci-Ce alkyl, Ci-Ce thioalkyl, or substituted Ci-Ce thioalkyl, Ci-Ce alkyloxy, or substituted Ci-Ce alkyloxy; R6A is H, N(Ra)(Rb), oxo, acetyl, formyl, or O- phenyl; Y7A is N and R7A is absent or is Ci-Ce alkyl; or Y7A is C and R7A is H, Ci-Ce alkyl, or N(Ra)(Rb); Y8A is N and R8A is absent, or Y8A is C and R8A is H, a halogen, OH, Ci-Ce alkyl, or substituted Ci-Ce alkyl; Ra and Rb are each independently H, Ci-Ce alkyl, substituted Ci-Ce alkyl, Ci-Ce alkenyl, substituted Ci-Ce alkenyl, acetyl, or formyl, or together form a 5-7-membered heterocycle; excluding where Y7A is N and R7A is absent; Y8A is C, R8A is H, R2A is H, and R6A is NH2 (unmodified adenine).
Unless otherwise indicated, modified guanine has structure (II):
Figure imgf000041_0001
wherein: R2G is N(Ra)(Rb); R6G is oxo and R1G is H, or R6G is O-Ci-C6 alkyl or S-Ci-C6 alkyl and R1G is absent; Y7G is N and R7G is absent or is Ci-Ce alkyl; or Y7G is C and R7G is H, Ci-Ce alkyl, or N(Ra)(Rb); Y8G is N and R8G is absent, or Y8G is C and R8G is H, a halogen, OH, Ci-C6 alkyl, or substituted Ci-Ce alkyl; Ra and Rb are independently H, Ci-Ce alkyl, substituted Ci-Ce alkyl, Ci-Ce alkenyl, substituted Ci-Ce alkenyl, acetyl, or formyl, or together form a 5-7-membered heterocycle; excluding where Y7G is N and R7G is absent; Y8G is C, R8G is H, R2G is NH2, and R6G is =0 (unmodified guanine).
Unless otherwise indicated, modified thymine or modified uracil has structure (III):
Figure imgf000041_0002
III wherein: X is O or S and R5U is H, OH, halogen, 0-Ci-C2o alkyl, O-Ci-Ci2 substituted alkyl, Ci- Ci2 alkyl, substituted C1-C12 alkyl, C1-C12 alkenyl, substituted C1-C12 alkenyl, C1-C12 alkynyl, or substituted C1-C12 alkynyl; wherein if each X is O, R5U is not H or CH, (unmodified uracil and unmodified thymine, respectively).
Unless otherwise indicated, modified cytosine has structure (IV):
Figure imgf000041_0003
IV wherein: X is O or S; R4C is N(Ra)(Rb); R5C is H, OH, halogen, O-Ci-Ci2 alkyl, O-Ci-Ci2 substituted alkyl, C1-C12 alkyl , substituted C1-C12 alkyl, C1-C12 alkenyl, or substituted C1-C12 alkenyl; Ra and Rb are independently H, Ci-Ce alkyl, substituted Ci-Ce alkyl, Ci-Ce alkenyl, substituted Ci-Ce alkenyl, C1-C12 alkynyl, substituted C1-C12 alkynyl, acetyl, or formyl, or together form a 5-7-membered heterocycle; excluding where X is O, R4C is NH2 and R5C is H (unmodified cytosine).
In certain embodiments, modified nucleobases of a modified oligonucleotide are selected from: 5 -substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6, and 0-6 substituted purines. In certain embodiments, modified nucleobases are selected from: 5 -methylcytosine, hypoxanthine, 1 -methylpseudouridine, 2-aminopropyladenine, 5- hydroxymethyl cytosine, xanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2- propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (-C°C-CH3) uracil, 5- propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5 -ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo (particularly 5-bromo), 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7- methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3 -deazaguanine, 3- deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5- methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as l,3-diazaphenoxazine-2-one, l,3-diazaphenothiazine-2-one, and 9-(2- aminoethoxy)-l,3-diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example, 7-deaza-adenine, 7- deazaguanosine, 2-aminopyridine, and 2-pyridone. Further nucleobases include those disclosed in Englisch, U. et al., Angew. Chem. Int. Ed. 1991, 30, 613; Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6 and \5, Antisense Drug Technology, Crooke S.T., Ed., CRC Press, 2008, 163-166 and 442- 443.
Preparation of certain of the above noted modified nucleobases, as well as other modified nucleobases are known in the art and can be readily identified in publications that include without limitation, Rogers et al., U.S. 5,134,066 ; Benner et al., U.S. 5,432,272; Matteucci et al., U.S. 5,502,177 ; Froehler et al. , U.S. 5,594,121 ; and Cook et al., U.S. 5,681,941.
In certain embodiments, each nucleobase of a modified oligonucleotide is selected from unmodified A, unmodified G, unmodified C, unmodified T, unmodified U, and mC. In certain embodiments, each nucleobase of a modified oligonucleotide is selected from unmodified A, unmodified G, unmodified C, unmodified T, unmodified U, mC, or hypoxanthine.
3. Modified Internucleoside Linkages
In certain embodiments, oligomeric compounds comprising a modified antisense oligonucleotide (e.g., an antisense oligomeric compound) and/or a modified sense oligonucleotide (e.g., a sense oligomeric compound) provided herein comprise or consist of a modified oligonucleotide comprising at least one modified intemucleoside linkage. The naturally occurring intemucleoside linkage of RNA and DNA is a 3' to 5' phosphodiester linkage. Herein, all intemucleoside linkages between furanosyl sugar moieties are 3' to 5' intemucleoside linkages unless otherwise indicated. In certain embodiments, nucleosides of modified oligonucleotides are linked together using one or more modified intemucleoside linkages. The two main classes of intemucleoside linkages are defined by the presence or absence of a phosphoms atom. Representative phosphoms-containing intemucleoside linkages include but are not limited to phosphates, which contain a phosphodiester bond (“P=O”) (also referred to as unmodified linkages), phosphotriesters, methylphosphonates, phosphoramidates, phosphorothioates (“P=S”), and phosphorodithioates (“HS-P=S”). Representative non-phosphoms containing intemucleoside linkages include but are not limited to methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester, thionocarbamate (-O-C(=O)(NH)-S-), siloxane (-O-SitU-O-)- and N,N'-dimethylhydrazine (-CH2- N(CH3)-N(CH3)-). Modified intemucleoside linkages, compared to naturally occurring phosphodiester linkages, may be used to alter, typically increase, nuclease resistance of the oligonucleotide.
In certain embodiments, a modified intemucleoside linkage is any of those described in WO 2021/030778, incorporated by reference herein. In certain embodiments, a modified intemucleoside linkage has the formula:
Figure imgf000043_0001
wherein independently for each intemucleoside linkage of the modified oligonucleotide: X is selected from O and S;
Ri is selected from H, Ci-Ce alkyl, and substituted Ci-Ce alkyl; and T is selected from SO2R2, C(=O)Rs, and P(=O)R4Rs, wherein: R2 is selected from an aryl, a substituted aryl, a heterocycle, a substituted heterocycle, an aromatic heterocycle, a substituted aromatic heterocycle, a diazole, a substituted diazole, a Ci-Ce alkoxy, Ci-Ce alkyl, Ci-Ce alkenyl, Ci-Ce alkynyl, substituted Ci-Ce alkyl, substituted Ci-Ce alkenyl, substituted Ci-Ce alkynyl, and a conjugate group;
R3 is selected from an aryl, a substituted aryl, CH3, N(CH3)2, OCH3, and a conjugate group;
R4 is selected from OCH3, OH, Ci-Ce alkyl, substituted Ci-Ce alkyl, and a conjugate group; and Rs is selected from OCH3, OH, Ci-Ce alkyl, and substituted Ci-Ce alkyl.
In certain embodiments, a modified oligonucleotide comprises a mesyl phosphoramidate linkage having a formula:
Figure imgf000043_0002
Certain intemucleoside linkages having reduced charge (referred to as “neutral intemucleoside linkages”) have been described. Such neutral intemucleoside linkages include, without limitation, phosphotriesters, methylphosphonates, MMI (3'-CH2-N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), formacetal (3'-O-CH2-O-5'), methoxypropyl (MOP) (see US 9,926,556), and thioformacetal (3'-S-CH2-O-5'). Further neutral intemucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research,' Y.S. Sanghvi and P.D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral intemucleoside linkages include nonionic linkages comprising mixed N, O, S and CFF component parts.
In certain embodiments, a modified oligonucleotide comprises an intemucleoside linkage comprising a triazole, alkyne, or cyclic guanidine moiety. In certain embodiments, a modified oligonucleotide comprises an intemucleoside linkage having a formula:
Figure imgf000044_0001
which may be stereorandom, or may be enriched for the Rp or Sp configuration.
In certain embodiments, intemucleoside linkages are not 3'-to-5' intemucleoside linkages.
In certain embodiments, modified oligonucleotides comprise one or more inverted nucleoside, where a sugar moiety is linked 3' to 3' and/or 5' to 5', as shown below:
Figure imgf000044_0002
wherein each Bx independently represents any nucleobase.
In certain embodiments, an inverted nucleoside is terminal (i. e. , the last nucleoside on one end of an oligonucleotide) and so only one intemucleoside linkage depicted above will be present. In certain embodiments, additional features (e.g., a conjugate group) are attached to the inverted nucleoside. Such terminal inverted nucleosides may be attached to either or both ends of an oligonucleotide.
In certain embodiments, inverted nucleosides lack a nucleobase (are abasic nucleosides). In certain such embodiments, additional features (e.g., a conjugate group) are attached to the inverted abasic nucleoside. A terminal inverted nucleoside may be attached to either or both ends of an oligonucleotide.
In certain embodiments, nucleosides are linked 2' to 5' rather than the 3' to 5' linkage. Such a linkage is illustrated below.
Figure imgf000045_0001
wherein each Bx represents any nucleobase.
In certain embodiments, a bicyclic sugar moiety may be linked via an atom on the non-furanosyl ring. In certain such embodiments, a bicyclic sugar moiety is linked 7’ to 5’, as shown below:
Figure imgf000045_0002
In certain embodiments, intemucleoside linkages have at least one chiral center. In such embodiments, a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Representative intemucleoside linkages having a chiral center include but are not limited to alkylphosphonates, mesyl phosphoramidates, and phosphorothioates. The mesyl phosphoramidate intemucleoside linkage comprises a chiral center. In certain embodiments, modified oligonucleotides comprising (Rp) and/or (.S'p) mesyl phosphoramidates comprise one or more of the following formulas, respectively, wherein “Bx” indicates a nucleobase:
Figure imgf000045_0003
The phosphorothioate intemucleoside linkage comprises a chiral center. In certain embodiments, modified oligonucleotides comprising (Rp) and/or (.S'p) phosphorothioates comprise one or more of the following formulas, respectively, wherein “Bx” indicates a nucleobase:
Figure imgf000046_0001
Modified oligonucleotides comprising intemucleoside linkages having a chiral center may be prepared as populations of modified oligonucleotides comprising stereorandom intemucleoside linkages, or as populations of modified oligonucleotides comprising intemucleoside linkages containing chiral centers in particular stereochemical configurations. In certain embodiments, populations of modified oligonucleotides comprise one or more phosphorothioate intemucleoside linkages wherein all of the phosphorothioate intemucleoside linkages are stereorandom. In certain embodiments, populations of modified oligonucleotides comprise one or more mesyl phosphoramidate intemucleoside linkages wherein all of the mesyl phosphoramidate intemucleoside linkages are stereorandom. Such modified oligonucleotides can be generated using synthetic methods that result in random selection of the stereochemical configuration of each phosphorothioate and/or mesyl phosphoramidate linkage. Nonetheless, each individual phosphorothioate and/or mesyl phosphoramidate of each individual oligonucleotide molecule has a defined stereoconfiguration. In certain embodiments, populations of modified oligonucleotides are enriched for modified oligonucleotides comprising one or more particular phosphorothioate and/or mesyl phosphoramidate intemucleoside linkages in a particular, independently selected stereochemical configuration. In certain embodiments, the particular configuration of the particular phosphorothioate and/or mesyl phosphoramidate linkage is present in at least 65% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate and/or mesyl phosphoramidate linkage is present in at least 70% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate and/or mesyl phosphoramidate linkage is present in at least 80% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate and/or mesyl phosphoramidate linkage is present in at least 90% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate and/or mesyl phosphoramidate linkage is present in at least 99% of the molecules in the population. Such chirally enriched populations of modified oligonucleotides can be generated using synthetic methods known in the art, e.g., methods described in Oka, N., et al. J. Am. Chem. Soc. 2003, 125, 8307-8317; Wan, W. B., et al. Nucleic Acids Res. 2014, 42, 13456, and WO 2017/015555.
As used herein, “chirally enriched” in reference to a population means a plurality of molecules of identical molecular formula, wherein the number or percentage of molecules within the population that contain a particular stereochemical configuration at a particular chiral center is greater than the number or percentage of molecules expected to contain the same particular stereochemical configuration at the same particular chiral center within the population if the particular chiral center were stereorandom as defined herein. Populations of molecules having multiple chiral centers within each molecule may contain one or more stereorandom chiral centers. In certain embodiments, the molecules are modified oligonucleotides. In certain embodiments, the chiral center is at the phosphorous atom of a phosphorothioate intemucleoside linkage. In certain embodiments, the chiral center is at the phosphorous atom of a mesyl phosphoramidate intemucleoside linkage. In certain embodiments, a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one indicated phosphorothioate and/or mesyl phosphoramidate in the (.S'p) configuration. In certain embodiments, a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one phosphorothioate and/or mesyl phosphoramidate in the (/?p) configuration. Unless otherwise indicated, chiral intemucleoside linkages of modified oligonucleotides described herein may be stereorandom or in a particular stereochemical configuration. In certain embodiments, the chiral center is at positions 1', 2', 3', and/or 4' of a furanosyl sugar moiety. In certain embodiments, each chiral center of each furanosyl sugar moiety is enriched such that the sugar moieties have the P-D ribosyl stereochemical configuration. In certain embodiments, modified oligonucleotides of a population are enriched for -D ribosyl sugar moieties, and all of the phosphorothioate intemucleoside linkages are stereorandom and all of the mesyl phosphoramidate intemucleoside linkages are stereorandom. In certain embodiments, modified oligonucleotides of a population are enriched for P-D ribosyl sugar moieties, at least one particular phosphorothioate intemucleoside linkage in a particular stereochemical configuration is enriched, and all of the mesyl phosphoramidate intemucleoside linkages are stereorandom. In certain embodiments, modified oligonucleotides of a population are enriched for P-D ribosyl sugar moieties, at least one particular mesyl phosphoramidate intemucleoside linkage in a particular stereochemical configuration is enriched, and all of the phosphorothioate intemucleoside linkages are stereorandom. In certain embodiments, modified oligonucleotides of a population are enriched for both P-D ribosyl sugar moieties and at least one, particular phosphorothioate intemucleoside linkage in a particular stereochemical configuration and at least one particular mesyl phosphoramidate intemucleoside linkage in a particular stereochemical configuration is enriched.
B. Motifs
In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising a modified sugar moiety. In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more modified intemucleoside linkage. In certain such embodiments, the modified, unmodified, and differently modified sugar moieties, nucleobases, and/or intemucleoside linkages of a modified oligonucleotide define a pattern or motif. In certain embodiments, the patterns of sugar moieties, nucleobases, and intemucleoside linkages are each independent of one another. Thus, a modified oligonucleotide may be described by its sugar motif, nucleobase motif, and/or intemucleoside linkage motif (as used herein, nucleobase motif describes the modifications to the nucleobases independent of the nucleobase sequence).
1. Sugar Motifs
In certain embodiments, oligonucleotides comprise one or more type of modified sugar and/or unmodified sugar moiety arranged along the oligonucleotide or region thereof in a defined pattern or sugar motif. In certain instances, such sugar motifs include but are not limited to any of the sugar modifications discussed herein. In certain embodiments, the sugar moiety of at least one nucleoside of an antisense oligomeric compound is a modified sugar moiety. In certain embodiments, the sugar moiety of at least one nucleoside of a sense oligomeric compound is a modified sugar moiety.
In certain embodiments, modified oligonucleotides comprise or consist of a region having a fully modified sugar motif. In such embodiments, each nucleoside of the fully modified region of the modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, each nucleoside of the entire modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, modified oligonucleotides comprise or consist of a region having a fully modified sugar motif, wherein each nucleoside within the fully modified region comprises the same modified sugar moiety, referred to herein as a uniformly modified sugar motif. In certain embodiments, a fully modified oligonucleotide is a uniformly modified oligonucleotide. In certain embodiments, each nucleoside of a uniformly modified oligonucleotide comprises the same 2 ’-modification. In certain embodiments, every other nucleoside of a uniformly modified oligonucleotide comprises the same 2 ’-modification, resulting in an alternating 2’- modifications. In certain embodiments, neighboring nucleosides comprise different 2 ’-modification, and every other nucleoside of a uniformly modified oligonucleotide comprises the same 2 ’-modification, resulting in a uniform, alternating 2 ’-modification motif.
In certain embodiments, at least one nucleoside of a modified oligonucleotide comprises a 2’- OMe sugar moiety. In certain embodiments, at least 8 nucleosides comprise 2’-0Me sugar moieties. In certain embodiments, at least 10 nucleosides comprise 2’-OMe sugar moieties. In certain embodiments, at least 12 nucleosides comprise 2’-OMe sugar moieties. In certain embodiments, at least 13 nucleosides comprise 2’-OMe sugar moieties. In certain embodiments, at least 14 nucleosides comprise 2’-OMe sugar moieties. In certain embodiments, at least 15 nucleosides comprise 2’-OMe sugar moieties. In certain embodiments, at least 16 nucleosides comprise 2’-OMe sugar moieties. In certain embodiments, at least 17 nucleosides comprise 2’-OMe sugar moieties. In certain such embodiments, at least 18 nucleosides comprise 2’-OMe sugar moieties. In certain such embodiments, at least 20 nucleosides comprise 2’-OMe sugar moieties.
In certain embodiments, at least one nucleoside of a modified oligonucleotide comprises a 2’-F sugar moiety (i.e., a 2’-F modified nucleoside). In certain embodiments, at least 2 nucleosides comprise 2’-F sugar moieties. In certain embodiments, at least 3 nucleosides comprise 2’-F sugar moieties. In certain embodiments, 4 nucleosides comprise a 2’-F sugar moiety. In certain embodiments, at least one, but not more than four nucleosides comprises a 2’-F sugar moiety. In certain embodiments, 1 or 2 nucleosides comprise 2’-F sugar moieties. In certain embodiments, 1-3 nucleosides comprise 2’-F sugar moieties. In certain embodiments, only one nucleoside comprises a 2’-F sugar moiety. In certain embodiments, an antisense oligomeric compound comprises 2 to 4 non-contiguous 2’-F modified nucleosides. In certain embodiments, 4 nucleosides of an antisense oligomeric compound are 2’-F modified nucleosides and none of those 2’-F modified nucleosides are contiguous. In certain embodiments, 1, 2, 3, or 4 nucleosides of an antisense oligomeric compound are 2’-F modified nucleosides and each of those 2’-F modified nucleosides are non-contiguous. In certain such embodiments at least fifteen of the remainder of the nucleosides are 2’-0Me modified nucleosides. In certain embodiments, one nucleoside of an antisense oligomeric compound is a 2’-F modified nucleoside and at least fifteen of the remainder of the nucleosides are 2’-0Me modified nucleosides.
In certain embodiments, at least one nucleoside of a modified oligonucleotide comprises a 2’- deoxyribosyl sugar moiety that has no additional modifications. In certain embodiments, at least one nucleoside comprises a 2 ’-deoxyribosyl sugar moiety. In certain embodiments, at least 2 nucleosides comprise a 2 ’-deoxyribosyl sugar moiety. In certain embodiments, at least 3 nucleosides comprise a 2’- deoxyribosyl sugar moiety. In certain embodiments, at least 4 nucleosides comprise a 2 ’-deoxyribosyl sugar moiety. In certain embodiments, one nucleoside comprises a 2 ’-deoxyribosyl sugar moiety. In certain embodiments, 1 or 3 nucleosides comprise a 2 ’-deoxyribosyl sugar moiety. In certain embodiments, 1-3 nucleosides comprise a 2’-deoxyribosyl sugar moiety. In certain embodiments, three nucleosides comprise a 2 ’-deoxyribosyl sugar moiety. In certain embodiments, 1, 2, 3, or 4 nucleosides of an antisense oligomeric compound are a 2 ’-deoxyribosyl sugar modified nucleoside and each 2’- deoxyribosyl modified nucleoside is non-contiguous. In certain embodiments, 1, or 3 nucleosides of an antisense oligomeric compound are a 2 ’-deoxyribosyl sugar modified nucleoside and each 2’- deoxyribosyl modified nucleoside is non-contiguous. In certain embodiments, no nucleosides of a sense oligomeric compound are a 2’ -deoxyribosyl sugar modified nucleoside. In certain embodiments, three nucleosides of an antisense oligomeric compound are 2 ’-deoxyribosyl sugar modified nucleosides and no nucleoside of a sense oligomeric compound is a 2 ’-deoxyribosyl modified nucleoside. In certain embodiments, one nucleosides of an antisense oligomeric compound are 2 ’-deoxyribosyl sugar modified nucleosides and no nucleoside of a sense oligomeric compound is a 2 ’-deoxyribosyl modified nucleoside.
In certain embodiments, at least one nucleoside of a modified oligonucleotide comprises a sugar surrogate moiety. In certain embodiments, at least one nucleoside of a modified oligonucleotide comprises a sugar surrogate moiety comprising FHNA. In certain embodiments, at least one nucleosides comprises a sugar surrogate. In certain embodiments, at least two nucleosides comprise a sugar surrogate. In certain embodiments, only one nucleoside comprises a sugar surrogate. In certain embodiments, two nucleosides of an antisense oligomeric compound are sugar surrogates and each of those surrogates are non-contiguous. In certain embodiments, at least one nucleosides comprises an FHNA sugar surrogate. In certain embodiments, at least two nucleosides comprise an FHNA sugar surrogate. In certain embodiments, only one nucleoside comprises an FHNA sugar surrogate. In certain embodiments, two nucleosides of an antisense oligomeric compound are FHNA sugar surrogates and each of those surrogates are non-contiguous. In certain embodiments, one nucleoside of an antisense oligomeric compound in an FHNA, located at position 2 from the 5’ end of the antisense oligonucleotide.
In certain embodiments, a sugar moiety of an antisense oligomeric compound is modified, wherein the modified sugar modifications and/or sugar surrogate is selected from 2’-F, a 3'-fluoro- hexitol, 2’-M0E, 2’-0Me, and 2’ -deoxyribosyl. In certain embodiments, a sugar motif (from 5’ to 3’) of the antisense oligomeric compound is selected from efyyyfyyyyyyyfyfyyyyyyy, yfyyyfyyyyyyyfyfyyyyyyy, efyyydyyeyyyydydyyyyyee, efyyydyyyyyyyfyfyyyyyee, efyyydyyeyyyyfyfyyyyyee, e[FHNA]yyyfyyyyyyyfyfyyyyyee, and e[FHNA]yyyfyyeyyyyfyfyyyyyee wherein each ‘e’ represents a 2'-M0E sugar moiety, each ‘y’ represents a 2'-0Me sugar moiety, each represents a 2'-F sugar moiety, each ‘[FHNA]’ represents a 3'-fluoro-hexitol sugar moiety, and each ‘d’ represents a 2'-deoxy sugar moiety. In certain embodiments, a sugar moiety of a sense oligomeric compound is modified, wherein the modified sugar moiety is selected from 2’-F, 2 ’-MOE, and 2’-0Me. In certain embodiments, a sugar motif (from 5’ to 3’) of a sense oligomeric compound is selected from among: yyyyyyfyfffyyyyyyyyyy and eeyyyyyyyffyyyyyyyyee, wherein each ‘e’ represents a 2'-M0E sugar moiety, each ‘y’ represents a 2'-0Me sugar moiety, and each ‘f represents a 2'-F sugar moiety.
2. Nucleobase Motifs
In certain embodiments, oligonucleotides comprise modified and/or unmodified nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or motif. In certain embodiments, at least one nucleobase is modified. In certain embodiments, none of the nucleobases are modified. In certain embodiments, at least one purine and/or at least pyrimidine is modified. In certain embodiments, at least one adenine is modified. In certain embodiments, at least one guanine is modified. In certain embodiments, at least one thymine is modified. In certain embodiments, at least one uracil is modified. In certain embodiments, at least one cytosine is modified. In certain embodiments, at least one of the cytosine nucleobases in a modified oligonucleotide is 5 -methylcytosine. In certain embodiments, all of the cytosine nucleobases are 5 -methylcytosines and all of the other nucleobases of the modified oligonucleotide are unmodified nucleobases. In certain embodiments, one or two of the cytosine nucleobases are 5- methylcytosines and all of the other nucleobases of the modified oligonucleotide are unmodified nucleobases. In certain embodiments, each nucleobase is selected from 5-methylcytosine, unmodified cytosine, unmodified thymine, unmodified uracil, unmodified adenine, unmodified guanine, and unmodified hypoxanthine. In certain embodiments, each nucleobase is selected from 5-methylcytosine, unmodified cytosine, unmodified thymine, unmodified uracil, unmodified adenine, unmodified guanine, and unmodified hypoxanthine. In certain embodiments, each nucleobase is selected from 5-methylcytosine, unmodified cytosine, unmodified thymine, unmodified adenine, and unmodified guanine. In certain embodiments, each nucleobase is selected from 5-methylcytosine, unmodified cytosine, unmodified thymine, unmodified adenine, and unmodified guanine. In certain embodiments, each nucleobase is selected from unmodified cytosine, unmodified thymine, unmodified uracil, unmodified adenine, and unmodified guanine. In certain embodiments, each nucleobase is selected from unmodified cytosine, unmodified thymine, unmodified adenine, and unmodified guanine.
3. Internucleoside Linkage Motifs
In certain embodiments, oligonucleotides comprise modified and unmodified intemucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or motif. In certain embodiments, each intemucleoside linkage is a phosphodiester intemucleoside linkage. In certain embodiments, each intemucleoside linkage of a modified oligonucleotide is a phosphorothioate intemucleoside linkage. In certain embodiments, each intemucleoside linkage of a modified oligonucleotide is independently selected from a phosphorothioate intemucleoside linkage, a mesyl phosphoramidate intemucleoside linkage, and a phosphodiester intemucleoside linkage. In certain embodiments, each intemucleoside linkage of a modified oligonucleotide is independently selected from a phosphorothioate intemucleoside linkage and a phosphodiester intemucleoside linkage. In certain embodiments, each intemucleoside linkage of a modified oligonucleotide is independently selected from a mesyl phosphoramidate intemucleoside linkage and a phosphorothioate intemucleoside linkage. In certain embodiments, each phosphorothioate intemucleoside linkage is independently selected from a stereorandom phosphorothioate, a (.S'p) phosphorothioate, and a (/?p) phosphorothioate. In certain embodiments, each mesyl phosphoramidate intemucleoside linkage is independently selected from a stereorandom mesyl phosphoramidate, a (.S'p) mesyl phosphoramidate, and a (/?p) mesyl phosphoramidate .
In certain embodiments, the modified antisense oligonucleotide has an intemucleoside linkage motif independently selected from (5' to 3') of: ssooooooooooooooooooss, ssooosooooooososooooss and ssooosooooooooooooooss, wherein each “s” represents a phosphorothioate intemucleoside linkage and each “o” represents a phosphodiester intemucleoside. In certain embodiments, the modified sense oligonucleotide has an intemucleoside linkage motif independently selected from (5' to 3') of: ssooooooooooooooooss and ssooooooosooooooooss, wherein each “s” represents a phosphorothioate intemucleoside linkage, and each “o” represents a phosphodiester intemucleoside.
Provided oligomeric compounds comprise one or more modifications, (e.g., a modified sugar moiety, a modified nucleobase, a modified intemucleoside linkage), and/or combinations thereof, incorporated into a modified oligonucleotide. In certain embodiments, a modified oligonucleotide is characterized by modification motif(s) and overall length. In certain embodiments, such parameters are each independent of one another. Thus, unless otherwise indicated, each intemucleoside linkage of a modified oligonucleotide having one or more modified sugar moiety and/or sugar motif, independently, is modified or unmodified and may or may not follow the modification pattern of the sugar modifications or sugar motif. For example, intemucleoside linkages within a region of a modified oligonucleotide comprising certain sugar modifications may be the same or different from one another and may be the same or different from the intemucleoside linkages of the region of the modified oligonucleotide comprising different sugar modifications. Likewise, such modified oligonucleotides may comprise one or more modified nucleobase independent of the pattern of the sugar modifications or sugar motif and independent of the intemucleoside linkages or intemucleoside linkage motif. Unless specifically indicated, all modifications are independent of nucleobase sequence.
C. Lengths
It is possible to increase or decrease the length of an oligonucleotide without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the oligonucleotides were able to direct specific cleavage of the target RNA, albeit to a lesser extent than the oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase oligonucleotides, including those with 1 or 3 mismatches.
In certain embodiments, oligonucleotides (including modified oligonucleotides) can have any of a variety of ranges of lengths. In certain embodiments, oligonucleotides consist of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number nucleosides in the range. In certain such embodiments, X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X<Y. For example, in certain embodiments, oligonucleotides consist of 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to
19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30,
13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to
20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16,
15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to
25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to
23, 19 to 24, 19 to 25, 19 to 26, 19 to 27, 19 to 28, 19 to 29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to
30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked nucleosides.
In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) comprise 16 linked nucleosides having no more than 1 to 3 mismatches to a target sequence. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) comprise 17 linked nucleosides having no more than 1 to 3 mismatches to a target sequence. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) comprise 18 linked nucleosides having no more than 1 to 3 mismatches to a target sequence. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) comprise 19 linked nucleosides having no more than 1 to 3 mismatches to a target sequence. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) comprise 20 linked nucleosides having no more than 1 to 3 mismatches to a target sequence. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) comprise 21 linked nucleosides having no more than 1 to 3 mismatches to a target sequence. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) comprise 22 linked nucleosides having no more than 1 to 3 mismatches to a target sequence. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) comprise 23 linked nucleosides having no more than 1 to 3 mismatches to a target sequence.
In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) consist of 16 linked nucleosides. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) consist of 17 linked nucleosides. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) consist of 18 linked nucleosides. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) consist of 19 linked nucleosides. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) consist of 20 linked nucleosides. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) consist of 21 linked nucleosides. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) consist of 22 linked nucleosides. In certain embodiments, modified oligonucleotides (including antisense oligomeric compounds) consist of 23 linked nucleosides.
In certain embodiments, antisense oligomeric compounds consist of 12-30 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 17-25 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 17-23 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 17-21 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 18-30 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 20-30 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 21-30 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 23-30 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 18-25 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 20-22 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 21-23 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 23-24 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 20 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 21 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 22 linked nucleosides. In certain embodiments, antisense oligomeric compounds consist of 23 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 12-30 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 16-25 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 16-23 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 16-21 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 16-30 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 18-30 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 19-30 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 16- 25 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 18-25 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 18-20 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 19-21 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 18 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 19 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 20 linked nucleosides. In certain embodiments, sense oligomeric compounds consist of 21 linked nucleosides.
D. Nucleobase Sequence
In certain embodiments, modified oligonucleotides (e.g., oligomeric compounds) are further described by their nucleobase sequence. In certain embodiments oligonucleotides of oligomeric compounds have a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid. In certain such embodiments, a region of an oligonucleotide has a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid. In certain embodiments, the nucleobase sequence of a region or entire length of an oligonucleotide is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the second oligonucleotide or nucleic acid, such as a target nucleic acid. In certain embodiments a first oligomeric compound comprises a modified oligonucleotide consisting of 18 to 50 linked nucleosides, wherein the nucleobase sequence of the first oligomeric compound comprises at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NOs: 6-829 and 3078-3083. In certain embodiments a second oligomeric compound comprises a modified oligonucleotide consisting of 18 to 50 linked nucleosides, wherein the nucleobase sequence of the second oligomeric compound comprises at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NOs: 830-1540 and 3096-3101.
II. Conjugates
In certain embodiments, provided herein are oligomeric compounds comprising one or more modified oligonucleotide and one or more conjugate groups. In certain embodiments, an oligomeric compound optionally further comprises one or more terminal groups. Conjugate groups comprise or consist of a conjugate moiety and a conjugate linker. A conjugate group may be attached at the 3' end and/or the 5' end of an oligonucleotide and/or at any internal position. In certain embodiments, conjugate groups are atached through a modified sugar moiety or a modified intemucleoside linkage. In certain embodiments, oligomeric compounds comprise a modified oligonucleotide, a cell-targeting moiety, and a conjugate linker.
A. Conjugate Groups
In certain embodiments, a conjugate group comprises a conjugate moiety and a conjugate linker. Conjugate Moieties
In certain embodiments, a conjugate moiety modifies one or more properties of an attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance. In certain embodiments, a conjugate moiety imparts a new property on the atached oligonucleotide.
In certain embodiments, a conjugate moiety comprises or consists of a cell-targeting moiety. In certain embodiments, a cell-targeting moiety is capable of binding the cell-surface receptor or the cellsurface moiety. In certain embodiments, an agent comprising a cell-targeting moiety is capable of being internalized when it interacts with or binds the cell-surface receptor or the cell-surface moiety. In certain embodiments, a cell-targeting moiety comprises a liver cell targeting moiety or a liver cell ligand. In certain embodiments, a liver cell-targeting moiety consists of a cell-targeting moiety having affinity for the hepatic asialoglycoprotein receptor (ASGP-R). In certain embodiments, the cell-targeting moiety comprises more than one ligand, and each ligand has affinity for the ASGP-R. In certain embodiments, each ligand is a carbohydrate. In certain embodiments, each ligand is independently selected from galactose, N-acetyl galactosamine (GalNAc), mannose, glucose, glucosamine, and fucose.
In certain embodiments, each ligand of a cell-targeting moiety is a carbohydrate, carbohydrate derivative, modified carbohydrate, polysaccharide, modified polysaccharide, or polysaccharide derivative. In certain such embodiments, the conjugate group comprises a carbohydrate cluster (see, e.g., Maier et al., “Synthesis of Antisense Oligonucleotides Conjugated to a Multivalent Carbohydrate Cluster for Cellular Targeting,” Bioconjugate Chemistry, 2003, 14, 18-29 or Rensen et al., “Design and Synthesis of Novel A- Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asiaglycoprotein Receptor,” Med. Chem. 2004, 47, 5798-5808). In certain such embodiments, each ligand is an amino sugar or a thio sugar. For example, amino sugars may be selected from any number of compounds known in the art, such as sialic acid, a-D-galactosamine, P-muramic acid, 2-deoxy-2- methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy- 2-sulfoamino-D-glucopyranose and A'-sulfo-D-gliicosaminc. and '-glycoloyl-a-ncuraminic acid. For example, thio sugars may be selected from 5-Thio-P-D-glucopyranose, methyl 2,3,4-tri-O-acetyl- 1 -thio- 6-O-trityl-a-D-glucopyranoside, 4-thio-P-D-galactopyranose, and ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy- 1,5-dithio-a-D-g/Mco-heptopyranoside.
In certain embodiments, each ligand is N-acetyl galactosamine (GalNAc). In certain embodiments, the cell-targeting moiety comprises one GalNAc ligand. In certain embodiments, the celltargeting moiety comprises two GalNAc ligands. In certain embodiments, the cell-targeting moiety comprises three GalNAc ligands. In certain embodiments, the cell-targeting moiety comprises a GalNAc ligand cluster. In certain embodiments, the cell-targeting moiety comprises a three GalNAc ligand cluster. In certain embodiments, the cell-targeting moiety is any one of those described in US 9,127,276, the entire contents of which is incorporated herein by reference. In certain embodiments, a conjugate groups comprises a cell-targeting moiety selected from any one of the formula set forth in TABLE A:
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Conjugate Linkers
In certain embodiments, oligomeric compounds comprise an oligonucleotide and a conjugate group, wherein the conjugate group comprises a conjugate moiety and a conjugate linker. In certain embodiments, the conjugate linker links the conjugate moiety to the oligonucleotide. In certain embodiments, the conjugate linker is a single chemical bond (i.e., the conjugate moiety is attached directly to an oligonucleotide through a single bond). In certain embodiments, the conjugate linker comprises one or more atoms. In certain embodiments, the conjugate linker comprises a chemical group. In certain embodiments, the conjugate linker comprises a chain structure, such as a hydrocarbyl chain, or an oligomer of repeating units such as ethylene glycol, nucleosides, or amino acid units. In certain embodiments, the oligonucleotide is a modified oligonucleotide.
In certain embodiments, a conjugate linker comprises one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino. In certain such embodiments, the conjugate linker comprises one or more groups selected from alkyl, amino, oxo, amide and ether groups. In certain embodiments, the conjugate linker comprises one or more groups selected from alkyl and amide groups. In certain embodiments, the conjugate linker comprises one or more groups selected from alkyl and ether groups. In certain embodiments, the conjugate linker comprises at least one phosphorus moiety. In certain embodiments, the conjugate linker comprises at least one phosphate group. In certain embodiments, the conjugate linker includes at least one neutral linking group.
In certain embodiments, conjugate linkers, including the conjugate linkers described herein, are bifunctional linking moieties, e.g., those known in the art to be useful for attaching conjugate moieties to parent compounds, such as the oligonucleotides provided herein. In general, a bifunctional linking moiety comprises at least two functional groups. One of the functional groups is selected to react with a particular site on a parent compound and the other is selected to react with a peptide extender. Examples of functional groups used in a bifunctional linking moiety include but are not limited to electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In certain embodiments, bifunctional linking moieties comprise one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl.
In certain embodiments, conjugate linkers comprise chemical groups that are formed upon a reaction between a first functional group and a second functional group. In certain embodiments, a modified oligonucleotide is attached to the first functional group during synthesis, and a conjugate moiety is attached to a second functional group during synthesis. Then, the two compounds are mixed under specific conditions to yield the final oligomeric compound. Such reactions that are compatible with both oligonucleotide and peptide chemistry have been previously described and are often called “bioconjugation” reactions. These reactions include strain promoted azido-alkyne cycloaddition (SPAAC), copper-catalyzed click reaction (CuAAC), active ester conjugation to an amino modified oligonucleotide, maleimide -thiol Michael addition, ketol/hydroxylamine ligation, the Staudinger ligation, reductive amination, thio ether formation, disulfide formation, reductive alkylation, catalyst-free N- arylation, sulfur fluoride exchange click reaction (SuFEx), and inverse demand Diels Alder reaction. Certain such reactions are described in, e.g., Jbara, et al., “Oligonucleotide Bioconjugation with Bifunctional Palladium Reagents”, Angew. Chem. Int. Ed. 2021, 60(21)12109-12115; Dong, et al., “Sulfiir(VI) Fluoride Exchange (SuFEx): Another Good Reaction for Click Chemistry,” Angew. Chem. Int. Ed. 2014, 53(36):9430-9448.4; Zhang, et al., “Arylation Chemistry for Bioconjugation,” Angew. Chem. Int. Ed. Engl. 2019; 58(15): 4810-4839; Walsh, et al., “Site-selective modification strategies in antibody-drug conjugates” Chem. Soc. Rev., 2021, 50: 1305-1353; Tiefenbrunn, et al., “Chemoselective ligation techniques: modem applications of time-honored chemistry”, Biopolymers, 2010, 94( 1): 95- 106; Drake, et al., Bioconjug. Chem. 2014, 25(7): 1331-1341; Bode, Acc. Chem. Res., 2017, 50, 9, 2104-2115; J. Magano, B. Bock, et al, Org. Proc. Res. Dev. 2014, 18: 142-151; Craig S. McKay and M.G. Finn, “Click Chemistry in Complex Mixtures: Bioorthogonal Bioconjugation”, Chemistry & Biology 2014; Mitchell P. Christy et al., Org. Lett. 2020, 22: 2365; Ren et al., Angew. Chem. Int. Ed. Engl. 2009, 48, 9658-9662; Rohrbacher, F. et al., Helv. Chim. Acta. 2018, 101; Baalmaan, et al, “A Bioorthogonal Click Chemistry Toolbox for Targeted Synthesis of Branched and Well-Defined Protein-Protein Conjugates”, Angew. Chem. Int. Ed.2Q2Q (59): 12885-12893; Lang, et al, “Biorthogonal Reactions for Labeling Proteins”, J. Am. Chem. Soc, 2014, 9(1): 16-20; Nair, et al., “The Thiol-Michael Addition Click Reaction: A Powerful and Widely Used Tool in Materials Chemistry”, Chem. Mater. 2013 26( 1): 724- 744; Kalia and Raines, “Hydrolytic Stability of Hydrazones and Oximes”, Angew. Chem. Int. Ed., 2008, 47:7523-7526.
Examples of conjugate linkers include but are not limited to pyrrolidine, 8-amino-3,6- dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane- 1 -carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA). Other conjugate linkers include but are not limited to substituted or unsubstituted Ci-Cw alkyl, substituted or unsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10 alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.
In certain embodiments, conjugate linkers comprise 1-5 linker-nucleosides. In certain embodiments, conjugate linkers comprise 2-5 linker-nucleosides. In certain embodiments, conjugate linkers comprise exactly 3 linker-nucleosides. In certain embodiments, conjugate linkers comprise the TCA motif. In certain embodiments, such linker-nucleosides are modified nucleosides. In certain embodiments such linker-nucleosides comprise a modified sugar moiety. In certain embodiments, linker- nucleosides are unmodified. In certain embodiments, linker-nucleosides comprise an optionally protected heterocyclic base selected from a purine, substituted purine, pyrimidine or substituted pyrimidine. In certain embodiments, a cleavable moiety is a nucleoside selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methyl cytosine, 4-N-benzoyl-5-methyl cytosine, adenine, 6-N- benzoyladenine, guanine and 2-N-isobutyrylguanine. It is typically desirable for linker-nucleosides to be cleaved from the oligomeric compound after it reaches a target tissue. Accordingly, linker-nucleosides are typically linked to one another and to the remainder of the oligomeric compound through cleavable bonds. In certain embodiments, such cleavable bonds are phosphodiester bonds.
Herein, linker-nucleosides are not considered to be part of the oligonucleotide. Accordingly, in embodiments in which an oligomeric compound comprises an oligonucleotide consisting of a specified number or range of linked nucleosides and/or a specified percent complementarity to a reference nucleic acid and the oligomeric compound also comprises a conjugate linker comprising linker-nucleosides, those linker-nucleosides are not counted toward the length of the oligonucleotide and are not used in determining the percent complementarity of the oligonucleotide for the reference nucleic acid. For example, an oligomeric compound may comprise (1) an oligonucleotide consisting of 18-30 nucleosides and (2) a conjugate linker comprising 1-10 linker-nucleosides that are contiguous with the nucleosides of the oligonucleotide. The total number of contiguous linked nucleosides in such an oligomeric compound is more than 30. Alternatively, an oligomeric compound may comprise an oligonucleotide consisting of 18-30 nucleosides and no conjugate linker. The total number of contiguous linked nucleosides in such an oligomeric compound is no more than 30. Unless otherwise indicated conjugate linkers comprise no more than 10 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 5 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 3 linker- nucleosides. In certain embodiments, conjugate linkers comprise no more than 2 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 1 linker-nucleoside.
In certain embodiments, it is desirable for a conjugate moiety to be cleaved from the oligonucleotide. For example, in certain circumstances oligomeric compounds comprising a particular conjugate moiety are better taken up by a particular cell type, but once the oligomeric compound has been taken up, it is desirable that the conjugate moiety be cleaved to release the unconjugated or parent oligonucleotide. Thus, certain conjugate linkers may comprise one or more cleavable moieties. In certain embodiments, a cleavable moiety is a cleavable bond. In certain embodiments, a cleavable moiety is a group of atoms comprising at least one cleavable bond. In certain embodiments, a cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds. In certain embodiments, a cleavable moiety is selectively cleaved inside a cell or subcellular compartment, such as a lysosome. In certain embodiments, a cleavable moiety is selectively cleaved by endogenous enzymes, such as nucleases.
In certain embodiments, a cleavable bond is selected from among: an amide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, or a disulfide. In certain embodiments, a cleavable bond is one or both of the esters of a phosphodiester. In certain embodiments, a cleavable moiety comprises a phosphate or phosphodiester. In certain embodiments, the cleavable moiety is a phosphodiester linkage between an oligonucleotide and a conjugate moiety.
In certain embodiments, a cleavable moiety comprises or consists of one or more linker- nucleosides. In certain such embodiments, the one or more linker-nucleosides are linked to one another and/or to the remainder of the oligomeric compound through cleavable bonds. In certain embodiments, such cleavable bonds are unmodified phosphodiester bonds. In certain embodiments, a cleavable moiety is 2'-deoxy nucleoside that is attached to either the 3' or 5 '-terminal nucleoside of an oligonucleotide by a phosphate intemucleoside linkage and covalently attached to the remainder of the conjugate linker or conjugate moiety by a phosphate or phosphorothioate linkage. In certain such embodiments, the cleavable moiety is 2'-deoxyadenosine.
In certain embodiments, oligomeric compounds described herein comprise an oligonucleotide linked to a conjugate moiety by a conjugate linker, wherein the oligomeric compound is prepared using Click chemistry known in the art. Compounds have been prepared using Click chemistry wherein alkynyl phosphonate intemucleoside linkages on an oligomeric compound attached to a solid support are converted into the 1,2,3-triazolylphosphonate intemucleoside linkages and then cleaved from the solid support (Krishna et al., J. Am. Chem. Soc. 2012, 134(28), 11618-11631), which is incorporated by reference herein in its entirety. Additional conjugate linkers suitable for use in several embodiments are prepared by Click chemistry described in “Click Chemistry for Biotechnology and Materials Science” Ed. Joerg Laham, Wiley 2009, which is incorporated by reference herein in its entirety.
In certain embodiments, compounds comprise an oligonucleotide, a cell-targeting moiety, and a conjugate linker. In certain embodiments, oligomeric compounds comprise an oligonucleotide, a hepatic asialoglycoprotein receptor (ASGP-R) ligand, and a conjugate linker. In certain embodiments, oligomeric compounds comprise an oligonucleotide, a N-acetyl galactosamine (GalNAc) ligand, and a conjugate linker. In certain embodiments, oligomeric compounds comprise an oligonucleotide, a GalNAc trimer, a branching group, a conjugate linker, and optionally modifications to the GalNAc ligands. In certain embodiments, oligomeric compounds comprise an oligonucleotide, two or more GalNAc ligands, a branching group, a conjugate linker, and optionally modifications to the GalNAc ligands. In certain embodiments, a conjugate linker connects GalNAc ligand to an oligonucleotide.
In certain embodiments, two or more GalNAc ligands are covalently connected to a conjugate linker, and the conjugate linker is covalently connected to the 3’ end of an oligonucleotide. In certain embodiments, a three GalNAc cluster is covalently connected to a conjugate linker, and the conjugate linker is covalently connected to the 3’ end of an oligonucleotide. In certain embodiments, two or more GalNAc ligands are covalently connected to a conjugate linker, and the conjugate linker is covalently connected to the 5’ end of an oligonucleotide. In certain embodiments, a three GalNAc cluster is covalently connected to a conjugate linker, and the conjugate linker is covalently connected to the 5’ end of an oligonucleotide. In certain embodiments, two or more GalNAc ligands are covalently connected to a conjugate linker, and the conjugate linker is covalently connected to an internal position of an oligonucleotide. In certain embodiments, a three GalNAc cluster is covalently connected to a conjugate linker, and the conjugate linker is covalently connected to an internal position of an oligonucleotide. In certain embodiments, an internal position of an oligonucleotide is a 2 ’-position of a modified sugar moiety of a nucleoside within the internal region of an oligonucleotide that is not the 5 ’ terminal nucleoside or the 3’ terminal nucleoside. In certain embodiments, an internal position of an oligonucleotide is a modified intemucleoside linkage of the oligonucleotide.
In certain embodiments, a sense oligomeric compound is conjugated to a THA-GalNAc conjugate group attached to the 5'-OH of the oligonucleotide. The structure of THA-GalNAc is:
Figure imgf000062_0001
In certain embodiments a sense oligomeric compound is conjugated to a HPPO-GalNAc conjugate group attached to the 3'-OH of the oligonucleotide. The structure of HPPO-GalNAc is:
Figure imgf000062_0002
B. Certain Terminal Groups
Examples of a terminal group include, but are not limited to, a conjugate group, a capping group, a phosphate moiety, a protecting group, a modified or unmodified nucleoside, and two or more nucleosides that are independently modified or unmodified, wherein one or more groups is attached to either or both ends of an oligonucleotide. In certain embodiments, one or more terminal groups is attached to either or both ends of an oligonucleotide. In certain embodiments, one or more terminal groups is attached at the 3’ and/or 5 ’-end of the oligonucleotide. In certain embodiments, one or more terminal groups is attached at the 3 ’-end of the oligonucleotide. In certain embodiments, one or more terminal groups is attached at the 5 ’-end of the oligonucleotide. In certain embodiments, one or more terminal groups is attached at the 3 ’-end of the oligonucleotide and one or more terminal groups is attached at the 5 ’-end of the oligonucleotide. In certain embodiments, a terminal group is attached at the 3’ and/or 5 ’-end of the oligonucleotide. In certain embodiments, a terminal group is attached at the 3 ’-end of the oligonucleotide. In certain embodiments, a terminal group is attached near the 3 ’-end of the oligonucleotide. In certain embodiments, a terminal group is attached at the 5 ’-end of the oligonucleotide. In certain embodiments, a terminal group is attached near the 5 ’-end of the oligonucleotide. In certain embodiments, a terminal group is attached at the 3 ’-end of the oligonucleotide and a terminal group is attached at the 5 ’-end of the oligonucleotide.
In certain embodiments, an oligomeric compound comprises one or more terminal groups. In certain embodiments, an oligomeric compound comprises a terminal group comprising a stabilized 5’- phosphate. Stabilized 5’-phosphates include, but are not limited to 5 ’-phosphonates, including, but not limited to 5’-vinylphosphonate, 5’-methylphosphonate. In certain embodiments, a terminal group comprises one or more abasic sugar moieties. In certain embodiments, a terminal group comprises one or more inverted sugar moieties and/or inverted nucleosides. In certain embodiments, a terminal group comprises one or more 2’-linked nucleosides or sugar moieties. In certain embodiments, the 2’-linked terminal group is an abasic sugar moiety. In certain embodiments, an antisense oligomeric compound comprises a vinylphosphonate. In certain embodiments, each antisense oligomeric compound has a vinyl phosphonate moiety on the 5'-end (5 ’-VP).
III. Target Nucleic Acids
A. LPA
In certain embodiments, an oligomeric compound comprises or consists of a modified oligonucleotide comprising a targeting region that is complementary to an equal-length target region of a target nucleic acid, wherein the target nucleic acid is LPA. In certain embodiments, LPA nucleic acid has the nucleobase sequence set forth in SEQ ID NO: 1 (the complement of GENBANK Accession No. NC_000006.12, truncated from nucleoside 160528001 to nucleoside 160669000) or SEQ ID NO: 2 (GENBANK Accession No. NM_005577.2), or to both. In certain embodiments, contacting a cell with an oligomeric duplex comprising an oligomeric compound comprising a modified oligonucleotide that is complementary to an equal-length target region of SEQ ID NO: 1 and/or SEQ ID NO: 2 inhibits LPA RNA in the cell, and in certain embodiments inhibits apo(a) protein or Lp(a) produced in the cell. In certain embodiments, the oligomeric compound comprises of a modified oligonucleotide. In certain embodiments, the oligomeric compound comprises a modified oligonucleotide and a conjugate group. In certain embodiments, the oligomeric compound comprises a modified oligonucleotide and one or more terminal group(s). In certain embodiments, the oligomeric compound comprises a modified oligonucleotide and a conjugate group and one or more terminal group(s).
In certain embodiments, an oligomeric compound comprises an antisense oligonucleotide comprising a targeting region that is complementary to a target region of a LPA nucleic acid. In certain embodiments, an oligomeric compound comprises an antisense oligonucleotide comprising a targeting region that is complementary to a region of SEQ ID NO: 2.
In certain embodiments, the target nucleic acid is an endogenous LPA RNA molecule. In certain embodiments, the LPA nucleic acid encodes apo(a) protein. In certain embodiments, the LPA nucleic acid is a precursor to a nucleic acid that encodes apo(a) protein or Lp(a). In certain such embodiments, the LPA nucleic acid is selected from: a mature mRNA and a pre-mRNA, including intronic, exonic, and untranslated regions. In certain embodiments, the LPA RNA is a mature mRNA. In certain embodiments, the LPA nucleic acid is a pre-mRNA.
In certain embodiments, antisense oligonucleotides provided herein are complementary to a target region of a LPA nucleic acid over the entire length of the modified oligonucleotide. In certain embodiments, antisense oligonucleotides are at least 99%, at least 95%, at least 90%, at least 85%, or at least 80% complementary to an equal length portion of the LPA nucleic acid. In certain embodiments, antisense oligonucleotides are at least 80% complementary to a target region of the LPA nucleic acid over the entire length of the antisense oligonucleotide and comprise a targeting region that is 100% or fully complementary to the target region of the LPA nucleic acid.
In certain embodiments, a targeting region is from 6 to 20, 10 to 18, 14 to 18, 16 to 20, or 18 to 20 nucleobases in length. In certain embodiments, the targeting region comprises or consists of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 contiguous nucleobases. In certain embodiments, the targeting region comprises or consists of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or 23 contiguous nucleobases. In certain embodiments, the targeting region constitutes at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the nucleosides of the antisense oligonucleotide. In certain embodiments, the targeting region constitutes all of the nucleosides of the antisense oligonucleotide. In certain embodiments, the targeting region of the antisense oligonucleotide is at least 99%, at least 95%, at least 90%, at least 85%, or at least 80% complementary to a target region of the LPA nucleic acid. In certain embodiments, the targeting region of the antisense oligonucleotide is 100% complementary to a target region of the LPA nucleic acid.
In certain embodiments, antisense oligonucleotides comprise one or more mismatches relative to the target region of the LPA nucleic acid. In certain embodiments, antisense activity against the target is reduced by such a mismatch, and activity against a non-target is reduced. In certain embodiments, activity against the non-target is reduced by a greater amount than activity against the target. Thus, in certain embodiments selectivity of the antisense oligonucleotides is improved. In certain embodiments, antisense oligonucleotides are at least 80% complementary to the target region of the LPA nucleic acid over the entire length of the antisense oligonucleotide and comprise no more than one to three mismatches with the LPA nucleic acid. In certain embodiments, antisense oligonucleotides comprise a targeting region that is at least 80% complementary to a target region of the LPA nucleic acid over the entire length of the targeting region, and the targeting region comprises no more than one to three mismatches with the target region. In certain embodiments, antisense oligonucleotides comprise a targeting region that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to a target region of the LPA nucleic acid over the entire length of the targeting region. In certain embodiments, additional mismatches may be present at a terminus or at both termini of the antisense oligonucleotide, outside of the targeting region. In certain embodiments, a mismatch is specifically positioned within an antisense oligonucleotide. In certain embodiments, a mismatch is at position 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 from the 5'-cnd of the antisense oligonucleotide. In certain embodiments, a mismatch is at position 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 from the 3 '-end of the antisense oligonucleotide. In certain embodiments, a mismatch is at position 1, 2, 3, or 4 from the 5 '-end of the antisense oligonucleotide. In certain embodiments, a mismatch is at position 4, 3, 2, or 1 from the 3 '-end of the antisense oligonucleotide.
B. Target Nucleic Acids in Certain Tissues
In certain embodiments, oligomeric compounds comprise or consist of a modified oligonucleotide comprising a targeting region that is complementary to a target region in a LPA nucleic acid, wherein the LPA nucleic acid is expressed in a pharmacologically relevant tissue. In certain embodiments, the LPA nucleic acid is expressed in the liver.
C. Oligonucleotide sequences
Provided herein are oligomeric compounds comprising modified oligonucleotides complementary to a target region in a LPA nucleic acid, such as, for example, a human LPA nucleic acid, such as SEQ ID NO: 1 (the complement of GENBANK Accession No. NC_000006.12, truncated from nucleoside 160528001 to nucleoside 160669000) or SEQ ID NO: 2 (GENBANK Accession No. NM_005577.2), or to both and compositions comprising such oligomeric compounds. In certain embodiments, a modified oligonucleotide has a nucleobase sequence comprising or consisting of a targeting region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% complementary to a region of SEQ ID NOs: 1 and/or 2. In certain embodiments, a modified oligonucleotide has a complementary region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% complementary to a targeting region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% complementary to a target region of SEQ ID NOs: 1 and/or 2. In certain embodiments, a modified oligonucleotide has a targeting region that is 100% complementary to a target region of SEQ ID NOs: 1 and/or 2. In certain embodiments, a modified oligonucleotide has a nucleobase sequence comprising or consisting of a complementary region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% complementary to a targeting region that is 100% complementary to a target region of SEQ ID NOs: 1 and/or 2. In certain embodiments, a modified antisense oligonucleotide has a nucleobase sequence comprising or consisting of any one of SEQ ID NO: 6-829 and 3078-3083. In certain embodiments, a second modified sense oligonucleotide has a nucleobase sequence comprising or consisting of a complementary region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% complementary to the first modified antisense oligonucleotide, or which is 100% complementary to the first modified antisense oligonucleotide. In certain embodiments, a modified sense oligonucleotide has a nucleobase sequence comprising or consisting of any one of SEQ ID NO: 830-1540 and 3096-3101.
IV. Oligomeric Duplexes
In certain embodiments, an oligomeric compound provided herein comprises a modified oligonucleotide having a nucleobase sequence complementary to a sequence in a LPA target nucleic acid paired with a second oligomeric compound to form an oligomeric duplex. Such oligomeric duplex comprises a first oligomeric compound comprising a modified oligonucleotide having a portion complementary to a sequence in an LPA target nucleic acid and a second oligomeric compound comprising a modified oligonucleotide having a portion complementary to the first oligomeric compound. In certain embodiments, the first oligomeric compound of an oligomeric duplex comprises or consists of (1) a first modified oligonucleotide and optionally a conjugate group and/or terminal group; and the second oligomeric compound of the oligomeric duplex comprises or consists of (2) a second modified oligonucleotide and optionally a terminal group and/or a conjugate group. Either or both oligomeric compounds of an oligomeric duplex may comprise a conjugate group. Either or both oligomeric compounds of an oligomeric duplex may comprise a terminal group. The oligonucleotides of each oligomeric compound of an oligomeric duplex may include non-complementary or unpaired overhanging nucleosides. In certain embodiments the non-complementary or unpaired overhanging nucleosides are adenosine or thymine. In certain embodiments the non-complementary or unpaired overhanging nucleosides include inosine. In certain embodiments, the two oligonucleotides have at least one mismatch relative to one another. In certain embodiments, the oligomeric duplex is an antisense agent.
In certain embodiments, an oligomeric duplex comprises: a first oligomeric compound comprising a first modified oligonucleotide consisting of 18 to 28 linked nucleosides, wherein the nucleobase sequence of the first modified oligonucleotide comprises at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NOs: 6-829 and 3078-3083; and a second oligomeric compound comprising a second modified oligonucleotide consisting of 15 to 25 linked nucleosides, wherein the nucleobase sequence of the second modified oligonucleotide comprises at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NOs: 830-1540 and 3096- 3101; and wherein each of the nucleosides of the first modified oligonucleotide comprises a modified sugar moiety or sugar surrogate and wherein no more than 22%, no more than 20%, no more than 18%, no more than 15%, no more than 10%, or no more than 5% of the modified nucleosides in the first modified oligonucleotide comprises a 2’-F modification and each of the nucleosides of the second modified oligonucleotide comprises a modified sugar moiety or sugar surrogate and wherein no more than 25%, no more than 20%, no more than 18%, no more than 16%, no more than 14 %, no more than 12%, or no more than 10%, of the modified nucleosides in the second modified oligonucleotide comprises a 2’-F modification. In certain embodiments, the oligomeric duplex is an antisense agent. In certain embodiments, the first oligomeric compound of the oligomeric duplex is an antisense agent, wherein the first modified oligonucleotide is an antisense oligomeric compound. In certain embodiments, the second oligomeric compound of the oligomeric duplex is a sense agent, wherein the second modified oligonucleotide is a sense oligomeric compound. In certain embodiments, the first modified oligonucleotide is an antisense RNAi oligonucleotide wherein no more than 22%, no more than 20%, no more than 18%, no more than 16%, no more than 14 %, no more than 12%, no more than 10%, or no more than 7%, of the modified nucleosides in the oligomeric duplex comprise a modified sugar moiety comprising a 2’-F modification. In certain embodiments, the second modified oligonucleotide is a sense RNAi oligonucleotide. In certain embodiments, the nucleobase sequence of the second modified oligonucleotide is at least 90%, 95% or 100% complementary to the nucleobase sequence of an equal length portion of the first modified oligonucleotide.
In certain embodiments, an oligomeric duplex comprises a first oligomeric compound comprising a first modified oligonucleotide consisting of 18 to 28 linked nucleosides, wherein the nucleobase sequence of the first modified oligonucleotide comprises at least 18, at least 19, at least 20, at least 21, at least 22, or 23 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NO: 6-829 and 3078-3083; and a second oligomeric compound comprising a second modified oligonucleotide consisting of 15 to 25 linked nucleosides, wherein the nucleobase sequence of the second modified oligonucleotide comprises at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NOs: 830-1540 and 3096- 3101, and wherein each of the nucleosides of the first modified oligonucleotide comprises a modified sugar moiety or sugar surrogate and the first modified oligonucleotide comprises at least one modified nucleoside and no more than four modified nucleosides of the first modified oligonucleotide comprises a 2’-F modification, and each of the nucleosides of the second modified oligonucleotide comprises a modified sugar moiety or sugar surrogate and at least one modified nucleoside and no more than four modified nucleosides of the second modified oligonucleotide comprises a 2’-F modification. In certain embodiments, the first oligomeric compound is an antisense agent, wherein the first modified oligonucleotide is an antisense oligomeric compound. In certain embodiments, the second oligomeric compound is a sense agent, wherein the second modified oligonucleotide is a sense oligomeric compound. In certain embodiments, the first modified oligonucleotide is an antisense RNAi oligonucleotide. In certain embodiments, the second modified oligonucleotide is a sense RNAi oligonucleotide. In certain embodiments, the nucleobase sequence of the second modified oligonucleotide comprises a complementary region of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or 21 nucleobases that is 100%complementary to the nucleobase sequence of an equal portion of the first modified oligonucleotide. In certain embodiments, the nucleobase sequence of the second modified oligonucleotide is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the nucleobase sequence of an equal portion of the first modified oligonucleotide. In certain embodiments, the oligomeric duplex is an antisense agent. In certain embodiments, the nucleobase sequence of the second modified oligonucleotide comprises a complementary region of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or 21 nucleobases that is 100% complementary to the nucleobase sequence of an equal portion of the first modified oligonucleotide; and the nucleobase sequence of the second modified oligonucleotide is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the nucleobase sequence of an equal portion of the first modified oligonucleotide. In certain embodiments, the oligomeric duplex is an antisense agent, wherein no more than three nucleosides, no more than four nucleosides, no more than five nucleosides, no more than six nucleosides, no more than seven nucleosides, or no more than eight nucleosides in the oligomeric duplex comprise a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate.
In certain embodiments, an oligomeric duplex comprises a first oligomeric compound comprising a first modified oligonucleotide consisting of 18 to 28 linked nucleosides, wherein the nucleobase sequence of the first modified oligonucleotide comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NO: 6-829 and 3078-3083; and a second oligomeric compound comprising a second modified oligonucleotide consisting of 15 to 25 linked nucleosides, wherein the nucleobase sequence of the second modified oligonucleotide comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleobases the nucleobase sequence of any one of SEQ ID NO: 830- 1540 and 3096-3101; wherein each of the nucleosides of the first modified oligonucleotide independently and the second modified oligonucleotide independently comprises a modified sugar moiety or sugar surrogate independently selected from 2’-F, 2’-MOE, 2’-OMe, 2 ’-deoxyribosyl, and 3'-fluoro-hexitol, wherein at least one modified nucleoside and no more than four modified nucleosides of the first modified oligonucleotide comprises a 2’-F modification, and at least one modified nucleoside and no more than four modified nucleosides of the second modified oligonucleotide comprises a 2’-F modification. In certain embodiments, the first oligomeric compound is an antisense agent, wherein the first modified oligonucleotide is an antisense oligomeric compound. In certain embodiments, the second oligomeric compound is a sense agent, wherein the second modified oligonucleotide is a sense oligomeric compound. In certain embodiments, the first modified oligonucleotide is an antisense RNAi oligonucleotide, and the second modified oligonucleotide is a sense RNAi oligonucleotide. In certain embodiments, the nucleobase sequence of the second modified oligonucleotide is at least 95% or 100% complementary to the nucleobase sequence of an equal length portion of the first modified oligonucleotide. In certain embodiments, the oligomeric duplex is an antisense agent wherein no more than three nucleosides, no more than four nucleosides, no more than five nucleosides, no more than six nucleosides, no more than seven nucleosides, or no more than eight nucleosides in the oligomeric duplex comprise a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate.
In certain embodiments, an oligomeric duplex comprises a first oligomeric compound comprising a first modified oligonucleotide consisting of 18 to 28 linked nucleosides, wherein the nucleobase sequence of the first modified oligonucleotide comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NO: 6-829 and 3078-3083; and a second oligomeric compound comprising a second modified oligonucleotide consisting of 15 to 25 linked nucleosides, wherein the nucleobase sequence of the second modified oligonucleotide comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleobases the nucleobase sequence of any one of SEQ ID NO: 830- 1540 and 3096-3101; wherein each of the nucleosides of the first modified oligonucleotide independently and the second modified oligonucleotide independently comprises a modified sugar moiety or sugar surrogate independently selected from 2’-F, 2’-MOE, 2’-OMe, 2 ’-deoxyribosyl, and 3'-fluoro-hexitol wherein only one nucleoside or only three nucleosides of the first modified oligonucleotide are 2’- deoxynucleoside and no nucleosides of the second modified oligonucleotide are 2 ’-deoxynucleoside; wherein one or three of the modified sugar moiety and/or sugar surrogate comprises a 2’-F modification in the first modified oligonucleotide, and two modified nucleosides of the second modified oligonucleotide comprises a 2’-F modification. In certain embodiments, the first oligomeric compound is an antisense agent, wherein the first modified oligonucleotide is an antisense oligomeric compound comprising a 5’ terminal group. In certain embodiments, the second oligomeric compound is a sense agent, wherein the second modified oligonucleotide is a sense oligomeric compound optionally conjugated to a cell targeting moiety. In certain embodiments, the first modified oligonucleotide is an antisense RNAi oligonucleotide, and the second modified oligonucleotide is a sense RNAi oligonucleotide. In certain embodiments, the nucleobase sequence of the second modified oligonucleotide is at least 95% or 100% complementary to the nucleobase sequence of an equal length portion of the first modified oligonucleotide. In certain embodiments, the oligomeric duplex is an antisense agent wherein no more than three nucleosides, no more than four nucleosides, or no more than five nucleosides, in the oligomeric duplex comprise a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate.
In certain embodiments, an oligomeric duplex comprises a first oligomeric compound comprising a first modified oligonucleotide consisting of 18 to 28 linked nucleosides, wherein the nucleobase sequence of the first modified oligonucleotide comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NO: 6-829 and 3078-3083; and a second oligomeric compound comprising a second modified oligonucleotide consisting of 15 to 25 linked nucleosides, wherein the nucleobase sequence of the second modified oligonucleotide comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleobases the nucleobase sequence of any one of SEQ ID NO: 830- 1540 and 3096-3101; wherein each of the nucleosides of the first modified oligonucleotide independently and the second modified oligonucleotide independently comprises a modified sugar moiety or sugar surrogate independently selected from 2’-F, 2’-M0E, 2’-OMe, 2 ’-deoxyribosyl, and 3'-fluoro-hexitol, wherein only one nucleoside of the first modified oligonucleotides comprises a 3'-fluoro-hexitol and no nucleosides of the second modified oligonucleotide comprise 3'-fluoro-hexitol; wherein three of the modified sugar moiety and/or sugar surrogate comprises a 2’-F modification in the first modified oligonucleotide, and two, or four modified nucleosides of the second modified oligonucleotide comprises a 2’-F modification. In certain embodiments, the first oligomeric compound is an antisense agent, wherein the first modified oligonucleotide is an antisense oligomeric compound comprising a 5 ’ terminal group. In certain embodiments, the second oligomeric compound is a sense agent, wherein the second modified oligonucleotide is a sense oligomeric compound optionally conjugated to a cell targeting moiety. In certain embodiments, the first modified oligonucleotide is an antisense RNAi oligonucleotide, and the second modified oligonucleotide is a sense RNAi oligonucleotide. In certain embodiments, the nucleobase sequence of the second modified oligonucleotide is at least 95% or 100% complementary to the nucleobase sequence of an equal length portion of the first modified oligonucleotide. In certain embodiments, the oligomeric duplex is an antisense agent wherein no more than five nucleosides, no more than six nucleosides, no more than seven nucleosides or no more than eight nucleosides, in the oligomeric duplex comprise a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate.
In certain embodiments, an oligomeric duplex comprises a first oligomeric compound comprising a first modified oligonucleotide, wherein the first modified oligonucleotide consists of 21 - 23 linked nucleosides and has nucleobase sequence comprising at least a 19-bp nucleobase sequence of any one of SEQ ID NOs: 6-829 and 3078-3083 having 0, 1, 2 or 3 nucleobases that are different from the corresponding nucleotide in any of SEQ ID NOs: 6-829 or 3078-3083; and a second oligomeric compound comprising a second modified oligonucleotide wherein the second modified oligonucleotide consists of 19 - 21 linked nucleosides and has nucleobase sequence comprising at least a 17-bp nucleobase sequence of any one of SEQ ID NOs: 830-1540 and 3096-3101, having 0, 1, 2 or 3 nucleobases that are different from the corresponding nucleotide in any of SEQ ID NOs: 830-1540 or 3096-3101, wherein each of the nucleosides of the first modified oligonucleotide independently and the second modified oligonucleotide independently comprises a modified sugar moiety or sugar surrogate independently selected from 2’-F, 2’-MOE, 2’-OMe, 2 ’-deoxyribosyl, and 3'-fluoro-hexitol, wherein three nucleosides or no nucleosides of the first modified oligonucleotide is 2 ’-deoxynucleoside and no nucleosides of the second modified oligonucleotide are 2 ’-deoxynucleoside; wherein one or two or three or four of the modified sugar moiety and/or sugar surrogate comprises a 2’-F modification in the first modified oligonucleotide, and two, or four modified nucleosides of the second modified oligonucleotide comprises a 2’-F modification. In certain embodiments, the first oligomeric compound is an antisense agent, wherein the first modified oligonucleotide is an antisense oligomeric compound comprising a 5 ’ terminal group. In certain embodiments, the second oligomeric compound is a sense agent, wherein the second modified oligonucleotide is a sense oligomeric compound optionally conjugated to a cell targeting moiety. In certain embodiments, the first modified oligonucleotide is an antisense RNAi oligonucleotide, and the second modified oligonucleotide is a sense RNAi oligonucleotide. In certain embodiments, the nucleobase sequence of the second modified oligonucleotide is at least 95% or 100% complementary to the nucleobase sequence of an equal length portion of the first modified oligonucleotide. In certain embodiments, the oligomeric duplex is an antisense agent, wherein at least one modified nucleoside and no more than five nucleosides, no more than six nucleosides, no more than seven nucleosides or no more than eight nucleosides, in the oligomeric duplex comprise a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate. In certain embodiments, one or two of the modified sugar moiety and/or sugar surrogate comprises a 2’-F modification in the first modified oligonucleotide, and two modified nucleosides of the second modified oligonucleotide comprises a 2’-F modification, and no more than three nucleosides in the oligomeric duplex comprise a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate. In certain embodiments, three or four of the modified sugar moiety and/or sugar surrogate comprises a 2’-F modification in the first modified oligonucleotide, and two or four modified nucleosides of the second modified oligonucleotide comprises a 2’-F modification. In certain embodiments, the oligomeric duplex comprises one or two unpaired nucleosides at either or both ends, forming one or two overhang ends. In certain embodiments an overhang end is one or two nucleosides of the antisense oligomeric compound. In certain embodiments an overhang end is one or two 3'- nucleosides of the antisense oligomeric compound. In certain embodiments the last two 3 '-nucleosides of the antisense oligomeric compound are overhang nucleosides not paired with the sense oligomeric compound. In certain embodiments the last one or two 3 '-unpaired overhang nucleosides comprise an adenine nucleobase. In certain embodiments the last one or two 3 '-unpaired overhang nucleosides comprise a thymine nucleobase. In certain embodiments the last one or two 3 '-unpaired overhang nucleosides comprise a uridine nucleobase. In certain embodiments the last one or two 3 '-unpaired overhang nucleosides comprise an inosine nucleobase. In certain embodiments, the oligomeric duplex is an antisense agent wherein no more than five nucleosides, no more than six nucleosides, no more than seven nucleosides or no more than eight nucleosides, in the oligomeric duplex comprise a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate.
In certain embodiments, an oligomeric duplex comprises a first oligomeric compound comprising a first modified oligonucleotide, wherein the first modified oligonucleotide consists of 21-23 linked nucleosides and has a nucleobase sequence comprising at least a 19-bp sequence of any one of SEQ ID NOs: 1541-2346 and 3090-,3095 having 0, 1, 2 or 3 mismatches with a sequence in a target LPA nucleic acid sequence; and a second oligomeric compound comprising a second modified oligonucleotide wherein the second modified oligonucleotide consists of 19-21 linked nucleosides, comprising at least a 19-bp sequence of any one of SEQ ID NOs: 2365-3075 and 3084-3089, having 0, 1, 2 or 3 mismatches to the first modified oligonucleotide. In certain embodiments, the first modified oligonucleotide is an antisense RNAi oligonucleotide, and the second modified oligonucleotide is a sense RNAi oligonucleotide. In certain embodiments, the nucleobase sequence of the second modified oligonucleotide is at least 85%, 90%, 95% or 100% complementary to the nucleobase sequence of an equal length portion of the first modified oligonucleotide. In certain embodiments, the oligomeric duplex is an antisense agent wherein each of the nucleosides of the first modified oligonucleotide independently and the second modified oligonucleotide independently comprises a modified sugar moiety or sugar surrogate independently selected from 2’-F, 2’-M0E, 2’-OMe, 2 ’-deoxyribosyl, and 3'-fluoro-hexitol, and wherein at least one modified nucleoside and no more than four modified nucleosides of the first modified oligonucleotide comprises a 2’-F modification, and at least one modified nucleoside and no more than four modified nucleosides of the second modified oligonucleotide comprises a 2’-F modification. In certain embodiments, the oligomeric duplex comprises one or two unpaired nucleosides at either or both ends, forming one or two overhang ends. In certain embodiments an overhang end is one or two nucleosides of the antisense oligomeric compound. In certain embodiments an overhang end is one or two 3 '-nucleosides of the antisense oligomeric compound. In certain embodiments the last two 3 '-nucleosides of the antisense oligomeric compound are overhang nucleosides not paired with the sense oligomeric compound. In certain embodiments the last one or two 3 '-unpaired overhang nucleosides comprise an adenine nucleobase. In certain embodiments the last one or two 3 '-unpaired overhang nucleosides comprise a thymine nucleobase. In certain embodiments the last one or two 3 '-unpaired overhang nucleosides comprise a uridine nucleobase. In certain embodiments the last one or two 3'- unpaired overhang nucleosides comprise inosine nucleobase. In certain embodiments the last two 3'- unpaired overhang nucleosides comprise a thymine nucleobase and an inosine nucleobase. In certain embodiments the last two 3 '-unpaired overhang nucleosides comprise 5’ to 3’ a thymine nucleobase and an inosine nucleobase. In certain embodiments the last two 3 '-unpaired overhang nucleosides comprise 5’ to 3’ an inosine nucleobase and a thymine nucleobase. In certain embodiments the last two 3'- unpaired overhang nucleosides comprise 5’ to 3’ an adenine nucleobase and an inosine nucleobase. In certain embodiments the last two 3 '-unpaired overhang nucleosides comprise 5’ to 3’ an inosine nucleobase and an adenine nucleobase.
In certain embodiments, an oligomeric duplex comprises a first oligomeric compound comprising a first modified oligonucleotide consisting of 19 to 25 linked nucleosides and a second oligomeric compound comprising a second modified oligonucleotide consisting of 16 to 24 linked nucleosides, wherein the nucleobase sequence of the first modified oligonucleotide and the nucleobase sequence of the second modified oligonucleotide each comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 contiguous nucleobases of any of the following pairs selected from a first oligomeric compound selected from any one of SEQ ID NOs: 1541-2364 and 3090-3095, and a second oligomeric compound selected from any one of SEQ ID NOS: 2365-3075 and 3084-3089. In certain embodiments, the first oligomeric compound is an antisense agent. In certain embodiments, the first modified oligonucleotide is an antisense oligomeric compound. In certain embodiments, the second oligomeric compound is a sense agent. In certain embodiments, the second modified oligonucleotide is a sense oligomeric compound. In certain embodiments, the first modified oligonucleotide is an antisense RNAi oligonucleotide. In certain embodiments, the second oligomeric compound is a sense agent. In certain embodiments, the second modified oligonucleotide is a sense RNAi oligonucleotide.
In certain embodiments, an oligomeric duplex comprises a first oligomeric compound comprising a first modified oligonucleotide consisting of 21 or 23 linked nucleosides and a second oligomeric compound comprising a second modified oligonucleotide consisting of 19 or 21 linked nucleosides, wherein the nucleobase sequences of the first modified oligonucleotide and second modified oligonucleotide consist of any of the following pairs of selected from a first oligomeric compound selected from any one of SEQ ID NOs: 1541-2364 and 3090-3095, and a second oligomeric compound selected from any one of SEQ ID NOS: 2365-3075 and 3084-3089. In certain embodiments, the first oligomeric compound is an antisense agent. In certain embodiments, the first modified oligonucleotide is an antisense oligomeric compound. In certain embodiments, the second oligomeric compound is a sense agent. In certain embodiments, the second modified oligonucleotide is a sense oligomeric compound. In certain embodiments, the first modified oligonucleotide is an antisense RNAi oligonucleotide. In certain embodiments, the second oligomeric compound is a sense agent. In certain embodiments, the second modified oligonucleotide is a sense RNAi oligonucleotide. In certain embodiments, the oligomeric duplex comprises one or two unpaired nucleosides at either or both ends, forming one or two overhang ends. In certain embodiments an overhang end is one or two 3 '-nucleosides of the antisense oligomeric compound. In certain embodiments the last two 3 '-nucleosides of the antisense oligomeric compound are overhang nucleosides not paired with the sense oligomeric compound. In certain embodiments the last one or two 3 '-unpaired overhang nucleosides comprise an adenine nucleobase. In certain embodiments the last one or two 3'-unpaired overhang nucleosides comprise a thymine nucleobase. In certain embodiments the last one or two 3 '-unpaired overhang nucleosides comprise a uridine nucleobase. In certain embodiments the last one or two 3 '-unpaired overhang nucleosides comprise an inosine nucleobase. In certain embodiments the antisense oligomeric compound comprises a 5 ’-terminal group. In certain embodiments the sense strand comprises a conjugate group attached at the 5’ or 3’ end of the sense oligomeric compound.
In any of the oligomeric duplexes described herein, at least one nucleoside of the first modified oligonucleotide and/or the second modified oligonucleotide comprises a modified sugar moiety. Examples of suitable modified sugar moieties include, but are not limited to, a bicyclic sugar moiety, such as a 2’-4’ bridge selected from -O-CH2-; and -O-CH(CH3)-, and a non-bicyclic sugar moiety, such as a 2 ’-MOE sugar moiety, a 2’-F sugar moiety, a 2’-OMe sugar moiety, or a 2’-NMA sugar moiety. In certain embodiments, at least one nucleoside of the first modified oligonucleotide and/or the second modified oligonucleotide comprises a modified 2’-deoxyribosyl sugar moiety. In certain embodiments, at least one nucleoside of the first modified oligonucleotide and/or the second modified oligonucleotide comprises a modified 3'-fluoro-hexitol sugar moiety. In certain embodiments, at least 80%, at least 90%, or 100% of the nucleosides of the first modified oligonucleotide and/or the second modified oligonucleotide comprises a modified sugar moiety independently selected from 2’-F, 2’-MOE, 2’-OMe, 2 ’-deoxyribosyl, and 3'- fluoro-hexitol. In certain embodiments, at least 80%, at least 90%, or 100% of the nucleosides of the first modified oligonucleotide and the second modified oligonucleotide comprises a modified sugar moiety independently selected from 2’-F, 2’-MOE, 2’-OMe, and 2 ’-deoxyribosyl.
In certain embodiments, in an oligomeric duplex provided herein, at least one nucleoside of the first modified oligonucleotide and/or the second modified oligonucleotide comprises a modified sugar moiety and/or sugar surrogate. In certain embodiments, in an oligomeric duplex provided herein, a sugar moiety of the first modified oligonucleotide is modified, wherein the modified sugar moiety and/or sugar surrogate is selected from 2’-F, 2’-MOE, 2’-OMe, 2 ’-deoxyribosyl, and 3'-fluoro-hexitol. In certain embodiments, in an oligomeric duplex provided herein, a sugar motif (from 5’ to 3’) of the first modified oligonucleotide is selected from: efyyyfyyyyyyyfyfyyyyyyy, yfyyyfyyyyyyyfyfyyyyyyy, efyyydyyeyyyydydyyyyyee, efyyydyyyyyyyfyfyyyyyee, efyyydyyeyyyyfyfyyyyyee, e[FHNA]yyyfyyyyyyyfyfyyyyyee, and e[FHNA]yyyfyyeyyyyfyfyyyyyee, wherein each ‘e’ represents a 2'-MOE sugar moiety, each ‘y’ represents a 2'-OMe sugar moiety, each ‘f represents a 2'-F sugar moiety, each ‘[FHNA]’ represents a 3'-fluoro-hexitol sugar moiety, and each ‘d’ represents a 2'-deoxy sugar moiety. In certain embodiments, in an oligomeric duplex provided herein, a sugar moiety of the second modified oligonucleotide is modified, wherein the modified sugar moiety is selected from 2’-F, 2’-MOE, 2’-OMe, 2 ’-deoxyribosyl and 3'-fluoro-hexitol. In certain embodiments, in an oligomeric duplex provided herein, a sugar motif (from 5’ to 3’) of the second modified oligonucleotide is selected from among: yyyyyyfyfffyyyyyyyyyy and eeyyyyyyyffyyyyyyyyee, wherein each ‘y’ represents a 2'- OMe sugar moiety, each ‘f represents a 2’-F sugar moiety, and each ‘e’ represents a 2’-MOE sugar moiety.
In certain embodiments, in an oligomeric duplex provided herein, a sugar motif (from 5’ to 3’) of the first modified oligonucleotide and the second modified oligonucleotide is selected from: efyyyfyyyyyyyfyfyyyyyyy and yyyyyyfyfffyyyyyyyyyy; yfyyyfyyyyyyyfyfyyyyyyy and yyyyyyfyfffyyyyyyyyyy; efyyydyyeyyyydydyyyyyee and eeyyyyyyyffyyyyyyyyee; efyyydyyyyyyyfyfyyyyyee and eeyyyyyyyffyyyyyyyyee; efyyydyyeyyyyfyfyyyyyee and eeyyyyyyyffyyyyyyyyee; e[FHNA]yyyfyyeyyyyfyfyyyyyee and eeyyyyyyyffyyyyyyyyee; and e[FHNA]yyyfyyyyyyyfyfyyyyyee and eeyyyyyyyffyyyyyyyyee, wherein each ‘e’ represents a 2'-MOE sugar moiety, each ‘y’ represents a 2'-OMe sugar moiety, each represents a 2'-F sugar moiety, each ‘[FHNA]’ represents a 3'-fluoro-hexitol sugar moiety, and each ‘d’ represents a 2'-deoxy sugar moiety.
In certain embodiments, in an oligomeric duplex provided herein, at least one intemucleoside linkage of the first modified oligonucleotide and/or the second modified oligonucleotide comprises a modified intemucleoside linkage. In certain embodiments, the modified intemucleoside linkage is a phosphorothioate intemucleoside linkage. In certain embodiments, at least one of the first, second, or third intemucleoside linkages from the 5 ’ end and/or the 3 ’ end of the first modified oligonucleotide comprises a phosphorothioate linkage. In certain embodiments, at least one of the first, second, or third intemucleoside linkages from the 5 ’ end and/or the 3 ’ end of the second modified oligonucleotide comprises a phosphorothioate linkage.
In certain embodiments, in an oligomeric duplex provided herein, each intemucleoside linkage of the first modified oligonucleotide is independently selected from a phosphodiester and a phosphorothioate, intemucleoside linkage, and each intemucleoside linkage of the second modified oligonucleotide is independently selected from a phosphodiester and a phosphorothioate, intemucleoside linkage.
In certain embodiments, in an oligomeric duplex provided herein, at least one linkage of the antisense oligomeric compound is a modified linkage. In certain embodiments, in an oligomeric duplex provided herein, an intemucleoside linkage of the first modified oligonucleotide is modified, wherein the 5’-most intemucleoside linkage (i.e., linking the first nucleoside from the 5’-end to the second nucleoside from the 5 ’-end) is modified. In certain embodiments, in an oligomeric duplex provided herein, the intemucleoside linkage motif (from 5’ to 3’) of the first modified oligonucleotide is selected from 5’- ssooooooooooooooooooss -3’, 5’- ssooosooooooososooooss -3’, and 5’- ssooosooooooooooooooss -3’, each “s” is a phosphorothioate intemucleoside intemucleoside linkage and each “o” is a phosphodiester intemucleoside linkage. In certain embodiments, in an oligomeric duplex provided herein, an intemucleoside linkage of the second modified oligonucleotide is modified, wherein the 5 ’-most intemucleoside linkage (i.e., linking the first nucleoside from the 5’-end to the second nucleoside from the 5 ’-end) is modified. In certain embodiments, in an oligomeric duplex provided herein, the intemucleoside linkage motif (from 5’ to 3’) of the second modified oligonucleotide is selected from (from 5’ to 3’) of: 5’- ssooooooooooooooooss -3’ and 5’- ssooooooosooooooooss -3’, wherein each ‘o’ represents a phosphodiester intemucleoside linkage and each ‘s’ represents a phosphorothioate intemucleoside linkage. In certain embodiments, the two 5 ’-most intemucleoside linkages are modified. In certain embodiments, the first one or 2 intemucleoside linkages from the 3 ’-end are modified. In certain embodiments, the modified intemucleoside linkage is a phosphorothioate linkage.
In certain embodiments, in an oligomeric duplex provided herein, at least one nucleobase of the first modified oligonucleotide and/or at least one nucleobase of the second modified oligonucleotide is a modified nucleobase. In certain embodiments, the modified nucleobase is methylcytosine. In certain embodiments, the modified nucleobase is inosine.
In certain embodiments, in an oligomeric duplex provided herein, the first oligomeric compound comprises a terminal group comprising a stabilized phosphate group attached to the 5’ position of the 5’- most nucleoside. In certain embodiments, the stabilized phosphate group comprises a cyclopropyl phosphonate or an (E)-vinyl phosphonate. In certain embodiments, the stabilized phosphate group is an (E)-vinyl phosphonate.
In certain embodiments, in an oligomeric duplex provided herein, the first modified oligonucleotide optionally is attached to a conjugate group. In certain embodiments, the conjugate group comprises a conjugate linker and a conjugate moiety. In certain embodiments, the conjugate group is attached to the first modified oligonucleotide at the 5 ’-end of the first modified oligonucleotide. In certain embodiments, the conjugate group is attached to the first modified oligonucleotide at the 3 ’-end of the modified oligonucleotide. In certain embodiments, the conjugate group is attached to the first modified oligonucleotide at an internal position. In certain embodiments, the conjugate group is attached to the first modified oligonucleotide through a 2 ’-modification of a fiiranosyl sugar moiety. In certain embodiments, the conjugate group is attached to the first modified oligonucleotide through a modified intemucleoside linkage. In certain embodiments, the conjugate group comprises N-acetyl galactosamine.
In any of the oligomeric duplexes described herein, the second modified oligonucleotide optionally is attached to a conjugate group. In certain embodiments, the conjugate group comprises a conjugate linker and a conjugate moiety. In certain embodiments, the conjugate group is attached to the second modified oligonucleotide at the 5 ’-end of the second modified oligonucleotide. In certain embodiments, the conjugate group is attached to the second modified oligonucleotide at the 3 ’-end of the modified oligonucleotide. In certain embodiments, the conjugate group is attached to the second modified oligonucleotide at an internal position. In certain embodiments, the conjugate group is attached to the second modified oligonucleotide through a 2 ’-modification of a furanosyl sugar moiety. In certain embodiments, the conjugate group is attached to the second modified oligonucleotide through a modified intemucleoside linkage. In certain embodiments, the conjugate group comprises N-acetyl galactosamine.
In certain embodiments, an oligomeric duplex comprises an oligomeric compound, which is an antisense agent described herein. In certain embodiments, an antisense agent, which is an oligomeric duplex described herein, is an RNAi agent capable of reducing the amount of LPA RNA through the activation of RISC/Ago2.
In certain embodiments, an oligomeric agent comprises at least two oligomeric duplexes linked together. In certain embodiments, an oligomeric agent comprises two oligomeric duplexes wherein at least one oligomeric duplex is targeted to LPA RNA as described herein. In certain embodiments, an oligomeric agent comprises two or more of the same oligomeric duplex, which is any of the oligomeric duplexes described herein. In certain embodiments, the two or more oligomeric duplexes are covalently linked together. In certain embodiments, the second modified oligonucleotides of the two or more oligomeric duplexes are covalently linked together. In certain embodiments, the second modified oligonucleotides of two or more oligomeric duplexes are covalently linked together at their 3’ ends. In certain embodiments, the second modified oligonucleotides of two or more oligomeric duplexes are covalently linked together at the 3 ’ end of one to the 5 ’ end of the other. In certain embodiments, the two or more oligomeric duplexes are covalently linked together by a glycol linker, such as a tetraethylene glycol linker. A structure of oligomeric duplexes covalently linked by a glycol linker is described in, e.g., Alterman, et al., Nature Biotech., 37:844-894, 2019. In certain embodiments, a first modified oligonucleotide of a first oligomeric duplex is covalently linked to a second modified oligonucleotide of a second oligomeric duplex and a first modified oligonucleotide of the second oligomeric duplex is covalently linked to a second modified oligonucleotide of the first oligomeric duplex (see, e.g., PCT International Patent Application Publication W02020/065602 for a description of an example of a structure of linked oligomeric duplexes).
V. Methods and Uses
A. Antisense Activity
In certain embodiments, oligomeric duplexes provided herein comprise an oligomeric compound that is capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity; such oligomeric duplexes and oligomeric compounds are antisense agents.
In certain antisense activities, hybridization of an antisense oligomeric compound to a target nucleic acid results in recruitment of a protein that cleaves the target nucleic acid. For example, in certain antisense activities, an antisense agent or a portion of an antisense agent is loaded into an RNA-induced silencing complex (RISC), ultimately resulting in cleavage of the target nucleic acid. For example, certain antisense agents result in cleavage of the target nucleic acid by Argonaute. Antisense agents that are loaded into RISC are RNAi agents. RNAi agents may be double-stranded (siRNA or dsRNAi) or single-stranded (ssRNA). In certain embodiments, RNAi agents are capable of RISC-mediated modulation of a target nucleic acid in a cell. In certain embodiments, such compounds reduce or inhibit the amount or activity of a target nucleic acid by 25% or more in the standard in vitro assay. In certain embodiments, RNAi agents selectively affect one or more target nucleic acid. Such RNAi agents comprise a nucleobase sequence that hybridizes to one or more target nucleic acid, resulting in one or more desired antisense activity. In certain embodiments, an RNAi agent does not hybridize to one or more non-target nucleic acid or does not hybridize to one or more non-target nucleic acid in such a way that results in significant undesired antisense activity.
In certain embodiments, hybridization of an antisense agent to a target nucleic acid results in inhibition of a binding interaction between the target nucleic acid and a protein or other nucleic acid (e.g., miRNA, IncRNA, sncRNA). In certain embodiments, hybridization of an antisense agent to a target nucleic acid results in modulation of translation of the target nucleic acid. In certain embodiments, hybridization of an oligomeric compound to a target nucleic acid results in an increase in the amount or activity of a target nucleic acid. In certain embodiments, hybridization of an antisense oligonucleotide to a target nucleic acid results in increased translation of the target nucleic acid. In certain embodiments, hybridization of an antisense oligonucleotide to a target nucleic acid results in reduced translation of the target nucleic acid.
Antisense activities may be observed directly or indirectly. In certain embodiments, observation or detection of an antisense activity involves observation or detection of a change in an amount of a target nucleic acid or protein encoded by such target nucleic acid, a change in the ratio of a nucleic acid or protein and/or a phenotypic change in a cell or animal.
Certain embodiments provide compositions and methods for reducing LPA RNA levels. Certain embodiments provide compositions and methods for reducing apo(a) and/or Lp(a) levels. In certain embodiments, reducing apo(a) levels in a tissue, organ and/or subject improves the ratio of LDL to HDL or the ratio of TG to HDL. In certain embodiments, provided are methods for using an oligomeric antisense agent targeted to an apo(a) nucleic acid for modulating the expression of apo(a) in a subject. In certain embodiments, expression of apo(a) is reduced.
In certain embodiments, inhibition of apo(a) or Lp(a) expression occurs in a cell, tissue or organ. In certain embodiments, inhibition of apo(a) or Lp(a) occurs in a cell, tissue or organ in a subject. In certain embodiments, inhibition is a reduction in apo(a) mRNA level. In certain embodiments, inhibition is a reduction in apo(a) protein level. In certain embodiments, both apo(a) mRNA and protein levels are reduced. In certain embodiments, inhibition is a reduction in Lp(a) level. Such reduction may occur in a time -dependent or in a dose-dependent manner.
In certain embodiments, a method of modulating expression of LPA or modulating apo(a) protein or Lp(a) in a cell comprises contacting the cell with an oligomeric duplex comprising or consisting of an oligomeric compound comprising a modified oligonucleotide having a targeting region complementary to a target region of a LPA nucleic acid. In certain embodiments, a method of inhibiting expression of LPA or inhibiting apo(a) protein or Lp(a) protein in a cell comprises contacting the cell with an oligomeric duplex comprising or consisting of an oligomeric compound comprising a modified oligonucleotide having a nucleobase sequence complementary to a target region of a LPA nucleic acid. In certain embodiments, the cell is a hepatocyte.
In certain embodiments, provided herein are methods of inhibiting or reducing LPA expression, LPA RNA levels and/or apo(a) protein or Lp(a) levels and/or activity, in a subject having, or at risk of having, a disease, disorder, condition or injury associated with LPA and/or apo(a) protein or Lp(a), such as a disease, disorder, condition or injury associated with inflammatory cardiovascular disease, wherein the method includes administering to the subject an oligomeric duplex comprising or consisting of an oligomeric antisense agent comprising a modified oligonucleotide targeting a LPA nucleic acid thereby inhibiting expression of LPA nucleic acid in the subject. In certain embodiments, expression of LPA nucleic acid is inhibited. In certain embodiments, administering an oligomeric duplex inhibits LPA expression, LPA RNA levels and/or apo(a) protein or Lp(a) levels and/or activity in the plasma/serum blood. In certain embodiments, administering an oligomeric duplex inhibits or reduces LPA expression, LPA RNA levels, and/or apo(a) protein or Lp(a) levels, and/or activity in the liver. In certain embodiments, administering such an oligomeric duplex inhibits or reduces LPA expression, LPA RNA levels and/or apo(a) protein or Lp(a) levels and/or activity in the plasma/serum blood and the liver of the subject. In certain embodiments, detectable amount of the LPA RNA may be reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In certain embodiments, an oligomeric compound comprising or consisting of a modified oligonucleotide comprising a targeting region complementary to a target region of SEQ ID NO: 1 or SEQ ID NO: 2 is capable of decreasing or reducing a detectable amount of a apo(a) protein or Lp(a) in a cell, organ or tissue, e.g., the liver of the subject, when the compound is administered to the cell, a tissue, and/or subject. In certain embodiments, detectable amount of the apo(a) protein or Lp(a) may be reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some embodiments an oligomeric duplex comprising or consisting of an oligomeric antisense agent has LPA RNA and/or apo(a) protein or Lp(a) reduction activity, and, in particular embodiments, liver LPA RNA and/or apo(a) protein or Lp(a) reduction activity, that is comparable to or greater than the LPA RNA and/or apo(a) protein or Lp(a) reduction activity of a comparator agent. In certain embodiments, the comparator agent is an agent that comprises a comparator modified oligonucleotide having a targeting region complementary to a target region of LPA. In certain embodiments, the comparator modified oligonucleotide is complementary to the same or a similar target region as the modified oligonucleotide of the oligomeric compound. In certain embodiments, the comparator modified oligonucleotide is complementary to a different target region from the modified oligonucleotide of the oligomeric compound. In some embodiments, the amount of LPA RNA is reduced by at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% in a cell (e.g., hepatocyte), organ (e.g., livery), or subject (e.g., animal) that has been contacted with or administered an oligomeric duplex provided herein (or a composition comprising such an oligomeric duplex) compared to a control (e.g., a cell, organ, tissue, system or subject that has not been contacted with or administered the oligomeric duplex, or was contacted with or administered a control substance (e.g., PBS)). In some embodiments, the percentage of LPA RNA decrease or reduction in a cell (e.g., a hepatocyte), organ (e.g., a livery), tissue, system, or subject (e.g., animal) contacted with or administered an oligomeric duplex or composition provided herein is 0.1% to 30% greater than or less than, 0. 1% to 25% greater than or less than, 0.1% to 20% greater than or less than, 0.1% to 15% greater than or less than, 0.1% to 10% greater than or less than, or 0. 1% to 5% greater than or less than, 0. 1% to 1% greater than or less than, 5% to 40% greater than, 5% to 35% greater than, 10% to 40% greater than, at least 5% greater than, at least 10% greater than, at least 15% greater than, at least 20% greater than, at least 25% greater than, or at least 30% greater than the percentage of LPA RNA decrease or reduction in a cell (e.g., a cardiomyocyte), organ (e.g., a heart), tissue, system or subject (e.g., animal) contacted with or administered the same concentration or dose of a comparator agent.
B. Therapeutic indications, methods
Atherosclerotic cardiovascular disease is highly prevalent and continues to be the highest cause of mortality worldwide despite the widespread use of low-density lipoprotein (LDL)- lowering therapies. Though LDL-lowering therapies reduce the risk of major cardiac events, residual cardiovascular risk encountered in some patients with low LDL levels implies other mechanisms of cardiovascular pathology. Over the last decade, compelling evidence from epidemiological studies and meta-analyses, Mendelian randomization studies, and genome wide association studies have shown that elevated serum Lp(a) concentration is associated with a higher risk of coronary artery disease and atherosclerosis-related disorders (Clarke et al., N. Engl. J. Med., Vol. 361 :2518-2528, 2009; Kamstrup et al., JAMA, Vol. 301 :2331-2339, 2009; Nordestgaard et al., European Heart Journal, Vol. 31 :2844-2853, 2010; Helgadottir et al., J. Am. Coll. Cardiol, Vol. 60:722-729, 2012; Thanassoulis et al., J. Am. Coll. Cardiol., Vol. 55:2491- 2498, 2010; Kamstrup et al., J. Am. Coll. Cardiol., Vol. 63:470-477, 2014; Kral et al., Journal of Cardiology, Vol. 118:656-661, 2016; Thanassoulis et al., J. Lipid Res., Vol. 57: 917-924, 2016; Tsimikas et al., J. Am. Coll. Cardiol., Vol. 69:692-711, 2017). It has been noted that this risk relationship is continuous and becomes proportionally more impactful with higher Lp(a) levels, and the association persists after correction for other lipid parameters (Emerging Risk Factors Collaboration, JAMA, Vol. 302:412-423, 2009). LPA gene mutations can increase LPA RNA levels, result in abnormal apo(a) cleavage that leads to increased apo(a) levels (and Lp(a) levels), or decreased degradation and clearance, and/or abnormal interactions between Lp(a) and other proteins or other endogenous or exogenous substances (e.g., plasminogen receptor) such that Lp(a) level is increased or degradation is decreased. A condition associated with high Lp(a) levels may be relatively insensitive to lifestyle changes and common statin drugs, and therefore hard to treat.
Subjects with high Lp(a) levels are at a significant risk of disease (Lippi et al., Clinica Chimica Acta, 2011, 412:797-801; Solfrizz et al.). For example, subjects will Lp(a) levels greater than > 75 nanomole s/liter (nmol/L) or > 30 mg/dL are considered to have increased risk for various diseases. In many subjects with high Lp(a) levels, current treatments cannot reduce Lp(a) levels to safe levels. Apo(a) plays an important role in the formation of Lp(a), hence reducing apo(a) can reduce Lp(a) and prevent, treat or ameliorate a disease associated with Lp(a). In certain embodiments, treatment with oligomeric duplexes and methods provided herein is indicated for a subject with elevated apo(a) levels and/or Lp(a) levels. In certain embodiments, the subject has apo(a) levels > 10 mg/dL, > 20 mg/dL, > 30 mg/dL, > 40 mg/dL, > 50 mg/dL, > 60 mg/dL, > 70 mg/dL, > 80 mg/dL, > 90 mg/dL or > 100 mg/dL. In certain embodiments, the subject has Lp(a) levels > 10 mg/dL, > 15 mg/dL, > 20 mg/dL, > 25 mg/dL, > 30 mg/dL, > 35 mg/dL, > 40 mg/dL, > 50 mg/dL, > 60 mg/dL, > 70 mg/dL, > 80 mg/dL, > 90 mg/dL, > 100 mg/dL, > 110 mg/dL, > 120 mg/dL, > 130 mg/dL, > 140 mg/dL, > 150 mg/dL, > 160 mg/dL, > 170 mg/dL, > 175 mg/dL, > 180 mg/dL, > 190 mg/dL, > 200 mg/dL. In certain embodiments, a subject has apo(a) level greater than the upper limit of normal, e.g. wherein the subject has apo(a) levels
> 30 mg/dL, > 35 mg/dL, > 40 mg/dL, > 50 mg/dL, > 60 mg/dL, > 70 mg/dL, > 80 mg/dL, > 90 mg/dL,
> 100 mg/dL, > 110 mg/dL, > 120 mg/dL, > 130 mg/dL, > 140 mg/dL, > 150 mg/dL, > 160 mg/dL, > 170 mg/dL, > 175 mg/dL, > 180 mg/dL, > 190 mg/dL, > 200 mg/dL.
In certain embodiments, provided herein are methods for preventing, treating, or delaying or preventing the development or progression of, diseases, disorders, conditions or injuries associated with LPA and/or apo(a) protein or Lp(a) increased levels, wherein the method comprises administering to a subject an oligomeric duplex described herein (e.g., an antisense oligomeric compound comprising or consisting of a modified oligonucleotide comprising a targeting region complementary to a target region of a LPA nucleic acid). Also provided are methods of ameliorating, preventing, or delaying the onset of, one or more symptoms associated with diseases, disorders, conditions or injuries associated with LPA or apo(a) protein and/or Lp(a), wherein the method comprises administering to a subject an oligomeric compound comprising or consisting of a modified oligonucleotide having a nucleobase sequence complementary to a nucleobase sequence in a LPA nucleic acid. A Lp(a)-associated condition may be, e.g., a cardiovascular condition, a metabolic condition, an inflammatory condition. In certain embodiments an Lp(a)-associated condition is an inflammatory, cardiovascular or metabolic disease or disorder. In certain embodiments, an Lp(a) associated condition or lipoprotein metabolism disorder is a cardiometabolic disorder. In some embodiments, an oligomeric duplex comprising or consisting of an oligomeric antisense agent described herein is used to treat a subject with a cardiovascular disease (CVD) such as hypertriglyceridemia and/or other condition selected from lipidemia (e.g., hyperlipidemia), dyslipidemia (e.g., atherogenic dyslipidemia, diabetic dyslipidemia, or mixed dyslipidemia), hyperlipoproteinemia, hyperapobetalipoproteinemia, chronic heart disease, including coronary artery disease (CAD) or any symptoms or conditions associated with a CVD, e.g., hypercholesterolemia (e.g., statin-resistant hypercholesterolemia, heterozygous or homozygous familial hypercholesterolemia) myocardial infarction (MI), acute coronary syndrome, mesenteric ischemia, superior mesenteric artery occlusion, restenosis, renal artery stenosis, angina peripheral arterial disease (PAD), calcific aortic valve disease (CAVD), aortic stenosis, aortic valve regurgitation, aortic dissection, retinal artery occlusion, atherosclerotic cardiovascular disease (ASCVD), atherosclerosis, dyslipidemia, thrombosis, cerebrovascular atherosclerosis, cerebrovascular disease, or stroke.
Thus, in certain embodiments, a method comprises administering to a subject an oligomeric duplex comprising or consisting of an oligomeric antisense agent comprising a targeting region complementary to a target region of a LPA nucleic acid. In certain embodiments, the subject has or is at risk for developing a cardiovascular, metabolic and/or inflammatory injury, disease, condition or disorder. In certain embodiments, the subject has or is at risk for developing cardiovascular disease (CVD) such as hypertriglyceridemia and/or other condition selected from lipidemia (e.g., hyperlipidemia), dyslipidemia (e.g., atherogenic dyslipidemia, diabetic dyslipidemia, or mixed dyslipidemia), hyperlipoproteinemia, hyperapobetalipoproteinemia, chronic heart disease, including coronary artery disease (CAD) or any symptoms or conditions associated with a CVD, e.g., hypercholesterolemia (e.g., statin-resistant hypercholesterolemia, heterozygous or homozygous familial hypercholesterolemia) myocardial infarction (MI), acute coronary syndrome, mesenteric ischemia, superior mesenteric artery occlusion, restenosis, renal artery stenosis, angina peripheral arterial disease (PAD), calcific aortic valve disease (CAVD), aortic stenosis, aortic valve regurgitation, aortic dissection, retinal artery occlusion, atherosclerotic cardiovascular disease (ASCVD), atherosclerosis, dyslipidemia, thrombosis, cerebrovascular atherosclerosis, cerebrovascular disease, or stroke. In certain embodiments, at least one symptom of the cardiovascular injury, disease, condition, or disorder is ameliorated. In certain embodiments, the at least one symptom is selected from include, but are not limited to, angina, chest pain, shortness of breath, palpitations, weakness, dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling in the lower extremities, cyanosis, fatigue, fainting, numbness of the face, numbness of the limbs, claudication or cramping of muscles, bloating of the abdomen, and fever. In certain embodiments, administration of an oligomeric duplex comprising or consisting of an oligomeric antisense agent provided herein to the subject reduces or delays the onset or progression of at least one at least one symptom of aortic stenosis. Certain embodiments provide compositions and methods for preventing, delaying, slowing the progression of apo(a) related diseases, disorders, and conditions in a subject in need thereof. Certain embodiments provide compositions and methods for ameliorating apo(a) related diseases, disorders, and conditions in a subject in need thereof. Certain embodiments provide compositions and methods for preventing, delaying, slowing the progression Lp(a) related diseases, disorders, and conditions in a subject in need thereof. Certain embodiments provide compositions and methods for ameliorating Lp(a) related diseases, disorders, and conditions in a subject in need thereof. In certain embodiments, such diseases, disorders, and conditions include inflammatory, cardiovascular and/or metabolic diseases, disorders, and conditions.
In certain embodiments, provided are methods of treating an individual with an apo(a) related disease, disorder or condition comprising administering a therapeutically effective amount of one or more pharmaceutical compositions as described herein. In certain embodiments, the individual has elevated apo(a) levels. In certain embodiments, provided are methods of treating an individual with an Lp(a) related disease, disorder or condition comprising administering a therapeutically effective amount of one or more pharmaceutical compositions as described herein. In certain embodiments, the individual has elevated Lp(a) levels. In certain embodiments, the individual has an inflammatory, cardiovascular and/or metabolic disease, disorder or condition. In certain embodiments, administration of a therapeutically effective amount of an oligomeric duplex is accompanied by monitoring of apo(a) or Lp(a) levels. In certain embodiments, administration of an oligomeric duplex is accompanied by monitoring of markers of inflammatory, cardiovascular and/or metabolic disease, or other disease process associated with the expression of apo(a), to determine an individual’s response to an oligomeric duplex. An individual’s response to administration of an oligomeric duplex can be used by a physician to determine the amount and duration of therapeutic intervention with an oligomeric duplex.
In certain embodiments, provided herein are methods of treating a subject comprising administering one or more pharmaceutical compositions as described herein. In certain embodiments, a method of treating comprises administering an oligomeric duplex for inhibiting or reducing expression of LPA nucleic acid, such as RNA, in a subject having or at risk of a disease, injury, condition or disorder associated with LPA, comprising administering to the subject an oligomeric duplex comprising or consisting of a modified oligonucleotide having a targeting region complementary to a target region of a LPA nucleic acid, thereby inhibiting or reducing expression of LPA nucleic acid in the subject. In certain embodiments, the individual has an apo(a) related disease. In certain embodiments, the individual has an Lp(a) related disease. In certain embodiments, the individual has an inflammatory, cardiovascular and/or a metabolic disease, disorder or condition.
In certain embodiments, a method of treating a subject comprises administering to a subject an oligomeric duplex comprising or consisting of an oligomeric antisense agent comprising or consisting of a modified oligonucleotide having a targeting region complementary to a target region of a LPA nucleic acid, thereby treating the subject. In certain embodiments, the subject has or is at risk for developing cardiovascular, metabolic, and/or inflammatory disease or disorder. In certain embodiments, administering the therapeutically effective amount of the oligomeric duplex improves one or more symptoms of the cardiovascular, metabolic, and/or inflammatory disease or disorder in the subject. In certain embodiments, at least one symptom of the cardiovascular, metabolic, and/or inflammatory disease or disorder is ameliorated. In certain embodiments, administration of a pharmaceutical composition comprising an oligomeric duplex to the subject reduces or delays the onset or progression of at least one or more symptoms.
In certain embodiments cardiovascular diseases, disorders or conditions include, but are not limited to, aortic stenosis, aneurysm (e.g., abdominal aortic aneurysm), angina, arrhythmia, atherosclerosis, cerebrovascular disease, coronary artery disease, coronary heart disease, dyslipidemia, hypercholesterolemia, hyperlipidemia, hypertension, hypertriglyceridemia, myocardial infarction, peripheral vascular disease (e.g., peripheral artery disease, peripheral artery occlusive disease), retinal vascular occlusion, stroke, elevated Lp(a) associated CVD risk, recurrent cardiovascular events with elevated Lp(a), calcific aortic valve stenosis associated with high Lp(a), and the like. Certain embodiments provide compositions and methods for preventing, delaying, slowing the progression of aortic stenosis. Certain embodiments provide compositions and methods for ameliorating aortic stenosis.
In certain embodiments, an oligomeric duplex modulates physiological markers or phenotypes of a cardiovascular disease, disorder or condition. For example, administration of an oligomeric duplex to a human can decrease Lp(a), LDL and cholesterol levels compared to untreated subjects. In certain embodiments, modulation of the physiological markers or phenotypes can be associated with inhibition of apo(a) by an oligomeric duplex. In certain embodiments, physiological markers of the cardiovascular disease, disorder or condition can be quantifiable. For example, Lp(a), LDL or cholesterol levels can be measured and quantified by, for example, standard lipid tests. For such markers, in certain embodiments, the marker can be decreased by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values.
Provided herein are methods for treating a symptom associated with a cardiovascular disease, disorder or condition in a subject in need thereof, disease, disorder. In certain embodiments, treating results in reducing the rate of onset of a symptom associated with the cardiovascular disease, disorder or condition. In certain embodiments, treating results in reducing the severity of a symptom associated with the cardiovascular or condition. A cardiovascular disease, disorder or condition can be characterized by numerous physical symptoms. Any symptom known to one of skill in the art to be associated with the cardiovascular disease, disorder or condition can be prevented, treated, ameliorated or otherwise modulated with the compounds and methods described herein. In certain embodiments, the symptom can be any of, but not limited to, angina, chest pain, shortness of breath, palpitations, weakness, dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling in the lower extremities, cyanosis, fatigue, fainting, numbness of the face, numbness of the limbs, claudication or cramping of muscles, bloating of the abdomen or fever.
In certain embodiments, a method of treatment of a subject comprises administering to the subject an oligomeric duplex comprising or consisting of an oligomeric antisense agent having a targeting region complementary to a target region of a LPA nucleic acid, thereby treating the subject. In certain embodiments, the subject has or is at risk for developing a cardiovascular disease, e.g., an atherosclerotic cardiovascular disease, an atherosclerotic cerebrovascular disease, a hyperlipidaemia, wherein the disease is associated with elevated levels of Lp(a)-containing particles. In certain embodiments, treatment is to prevent and/or reduce the risk of suffering from and/or treat stroke, atherosclerosis, thrombosis calcific aortic stenosis such as calcific aortic stenosis, ischaemic stroke, coronary artery disease, peripheral arterial disease, abdominal aortic aneurysm, heart failure secondary to ischaemic cardiomyopathy, or familial hypercholesterolaemia, wherein the disease is associated with elevated levels of Lp(a)-containing particles.
In certain embodiments treatment with an oligomeric duplex is to prevent and/or reduce the risk of suffering from and/or treat or cardiovascular disease such as coronary heart disease and any other disease or pathology associated with elevated levels of Lp(a)-containing particles. In certain embodiments treament is to prevent and/or reduce a risk of suffering from and/or treat, a cardiovascular disease, e.g., an atherosclerotic cardiovascular disease, an atherosclerotic cerebrovascular disease, a hyperlipidaemia, wherein the disease is associated with elevated levels of Lp(a)-containing particles. In certain embodiments, treatment is to prevent and/or reduce the risk of suffering from and/or treat stroke, atherosclerosis, thrombosis calcific aortic stenosis such as calcific aortic stenosis, ischaemic stroke, coronary artery disease, peripheral arterial disease, abdominal aortic aneurysm, heart failure secondary to ischaemic cardiomyopathy, or familial hypercholesterolaemia, wherein the disease is associated with elevated levels of Lp(a)-containing particles.
In certain embodiments treatment with an oligomeric duplex results in reducing the risk of a cardiovascular event in a patient with atherosclerotic cardiovascular disease. In certain embodiments the cardiovascular event is cardiovascular death, myocardial infarction, stroke, and/or coronary revascularization. In certain embodiments, a subject has a history of coronary revascularization, a history of coronary artery bypass grafting, a diagnosis of coronary artery disease, a diagnosis of atherosclerotic cerebrovascular disease, a diagnosis of peripheral artery disease, and/or a history of myocardial infarction. In certain embodiments, administration of a pharmaceutical composition comprising an oligomeric duplex comprising or consisting of an oligomeric antisense agent to the subject reduces or delays the onset or progression of at least one of stroke, atherosclerosis, thrombosis calcific aortic stenosis such as calcific aortic stenosis, ischaemic stroke, coronary artery disease, peripheral arterial disease, abdominal aortic aneurysm, heart failure secondary to ischaemic cardiomyopathy, or familial hypercholesterolaemia. In certain embodiments, administration of a pharmaceutical composition comprising an oligomeric duplex comprising or consisting of an oligomeric antisense agent to the subject reduces or delays the onset or progression of at least one of angina, chest pain, shortness of breath, palpitations, weakness, dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling in the lower extremities, cyanosis, fatigue, fainting, numbness of the face, numbness of the limbs, claudication or cramping of muscles, bloating of the abdomen, and fever, or a combination thereof. In certain embodiments treatment with an oligomeric duplex is to ameliorate one or more symptoms of cardiovascular disease such as coronary heart disease and/or other disease or pathology associated with elevated levels of Lp(a) containing particles. In certain embodiments treatment is to ameliorate symptoms associated with a cardiovascular disease such as coronary artery disease, carotid artery disease, peripheral artery disease, myocardial infarction, cerebrovascular disease, stroke, aortic valve stenosis, stable or unstable angina, atrial fibrillation, heart failure, hyperlipidemia, heterozygous familial hypercholesterolemia, or homozygous familial hypercholesterolemia. In certain embodiments a subject is diagnosed with or at risk of a cardiovascular disease; diagnosed myocardial infarction, acute coronary syndrome. In certain embodiments, the symptoms include, but are not limited to, angina, chest pain, shortness of breath, palpitations, weakness, dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling in the lower extremities, cyanosis, fatigue, fainting, numbness of the face, numbness of the limbs, claudication or cramping of muscles, bloating of the abdomen, and fever. Certain embodiments provide a method of reducing at least one symptom of aortic stenosis.
In certain embodiments, administration of a pharmaceutical composition comprising an oligomeric duplex comprising or consisting of an oligomeric antisense agent to the subject ameliorates or reduces one or more symptoms of at least one of stroke, atherosclerosis, thrombosis calcific aortic stenosis such as calcific aortic stenosis, ischaemic stroke, coronary artery disease, peripheral arterial disease, abdominal aortic aneurysm, heart failure secondary to ischaemic cardiomyopathy, or familial hypercholesterolaemia. In certain embodiments, administration of a pharmaceutical composition comprising an oligomeric duplex comprising or consisting of an oligomeric antisense agent to the subject ameliorates one or more symptoms of angina, chest pain, shortness of breath, palpitations, weakness, dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling in the lower extremities, cyanosis, fatigue, fainting, numbness of the face, numbness of the limbs, claudication or cramping of muscles, bloating of the abdomen, and fever, or a combination thereof.
In certain embodiments, metabolic diseases, disorders or conditions include, but are not limited to, hyperglycemia, prediabetes, diabetes (type I and type II), obesity, insulin resistance, metabolic syndrome and diabetic dyslipidemia.
In certain embodiments, an oligomeric duplex modulates physiological markers or phenotypes of a metabolic disease, disorder or condition. For example, administration of an oligomeric duplex to humans can decrease glucose and insulin resistance levels in those subjects compared to untreated subjects. In certain embodiments, the modulation of the physiological markers or phenotypes can be associated with inhibition of apo(a) by an oligomeric duplex. In certain embodiments, physiological markers of the metabolic disease, disorder or condition can be quantifiable. For example, glucose levels or insulin resistance can be measured and quantified by standard tests known in the art. For such markers, in certain embodiments, the marker can be decreased by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values. In another example, insulin sensitivity can be measured and quantified by standard tests known in the art. For such markers, in certain embodiments, the marker can be increase by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values.
Provided herein are methods for treating a symptom associated with the metabolic disease, disorder or condition in a subject in need thereof. In certain embodiments, treating results in reducing the rate of onset of a symptom associated with the metabolic disease, disorder or condition. In certain embodiments, treating results in reducing the severity of a symptom associated with the metabolic disease, disorder or condition. A metabolic disease, disorder or condition can be characterized by numerous physical symptoms. Any symptom known to one of skill in the art to be associated with the metabolic disease, disorder or condition can be prevented, treated, ameliorated or otherwise modulated with the compounds and methods described herein. In certain embodiments, a symptom can be any of, but not limited to, excessive urine production (polyuria), excessive thirst and increased fluid intake (polydipsia), blurred vision, unexplained weight loss and lethargy.
In certain embodiments, inflammatory diseases, disorders or conditions may overlap with cardiometabolic disorders or conditions, and include, but are not limited to, elevated Lp(a) associated CVD risk, recurrent cardiovascular events with elevated Lp(a), aortic stenosis (e.g., calcific aortic valve stenosis associated with high Lp(a)), coronary artery disease (CAD), Alzheimer’s Disease and thromboembolic diseases, disorder or conditions. Certain thromboembolic diseases, disorders or conditions include, but are not limited to, stroke, thrombosis, myocardial infarction and peripheral vascular disease.
In certain embodiments, an oligomeric duplex modulates physiological markers or phenotypes of an inflammatory disease, disorder or condition. For example, administration of an oligomeric duplex to a human can decrease inflammatory cytokine or other inflammatory markers levels in compared to untreated subjects. In certain embodiments, the modulation of the physiological markers or phenotypes can be associated with inhibition of apo(a) by an oligomeric duplex. In certain embodiments, physiological markers of the inflammatory disease, disorder or condition can be quantifiable. For example, cytokine levels can be measured and quantified by standard tests known in the art. For such markers, in certain embodiments, the marker can be decreased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, or a range defined by any two of these values.
Provided herein are methods for treating a symptom associated with an inflammatory disease, disorder or condition in a subject in need thereof. In certain embodiments, treating results in reducing the rate of onset of a symptom associated with the inflammatory disease, disorder or condition. In certain embodiments, treating results in reducing the severity of a symptom associated with the inflammatory disease, disorder or condition.
In certain embodiments, an oligomeric duplex has greater LPA RNA and/or protein reduction activity (i.e., greater specificity of action) in a target cell/organ/tissue/system (e.g., hepatocytes, liver) LPA than a non-target (e.g., plasminogen). For example, in some embodiments, administration of an oligomeric duplex provided herein reduces the amount or activity of LPA RNA and/or protein at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to a control and has no, or a non-significant, effect on (e.g., reduction in) the amount or activity of plasminogen.
In certain embodiments, administration of an oligomeric duplex results in reduction of LPA expression by at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, or a range defined by any two of these values. In certain embodiments, apo(a) expression is reduced to at least < 100 mg/dL, < 90 mg/dL, < 80 mg/dL, < 70 mg/dL, < 60 mg/dL,
< 50 mg/dL, < 40 mg/dL, < 30 mg/dL, <20 mg/dL or < 10 mg/dL. In certain embodiments, administration of an oligomeric duplex results in reduction of apo(a) by at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, or a range defined by any two of these values. In certain embodiments, Lp(a) containing particles in serum is reduced to at least < 200 mg/dL, < 190 mg/dL, < 180 mg/dL, < 175 mg/dL, < 170 mg/dL, < 160 mg/dL,
< 150 mg/dL, < 140 mg/dL, < 130 mg/dL, < 120 mg/dL, < 110 mg/dL, < 100 mg/dL, < 90 mg/dL, < 80 mg/dL, < 70 mg/dL, < 60 mg/dL, < 55 mg/dL, < 50 mg/dL, < 45 mg/dL, < 40 mg/dL, < 35 mg/dL, < 30 mg/dL, < 25 mg/dL, < 20 mg/dL, < 15 mg/dL, or < 10 mg/dL.
In certain embodiments, provided are methods for using an oligomeric duplex in the preparation of a medicament. In certain embodiments, pharmaceutical compositions comprising an oligomeric duplex are used for the preparation of a medicament for treating a patient suffering or susceptible to an inflammatory, cardiovascular and/or a metabolic disease, disorder or condition. In certain embodiments, the subject has or is at risk for developing cardiovascular disease (CVD) such as hypertriglyceridemia and/or other condition selected from lipidemia (e.g., hyperlipidemia), dyslipidemia (e.g., atherogenic dyslipidemia, diabetic dyslipidemia, or mixed dyslipidemia), hyperlipoproteinemia, hyperapobetalipoproteinemia, chronic heart disease, including coronary artery disease (CAD) or any symptoms or conditions associated with a CVD, e.g., hypercholesterolemia (e.g., statin-resistant hypercholesterolemia, heterozygous or homozygous familial hypercholesterolemia) myocardial infarction (MI), acute coronary syndrome, mesenteric ischemia, superior mesenteric artery occlusion, restenosis, renal artery stenosis, angina peripheral arterial disease (PAD), calcific aortic valve disease (CAVD), aortic stenosis, aortic valve regurgitation, aortic dissection, retinal artery occlusion, atherosclerotic cardiovascular disease (ASCVD), atherosclerosis, dyslipidemia, thrombosis, cerebrovascular atherosclerosis, cerebrovascular disease, or stroke. In certain embodiments, at least one symptom of the cardiovascular injury, disease, condition, or disorder is ameliorated. In certain embodiments, the at least one symptom is selected from include, but are not limited to, angina, chest pain, shortness of breath, palpitations, weakness, dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling in the lower extremities, cyanosis, fatigue, fainting, numbness of the face, numbness of the limbs, claudication or cramping of muscles, bloating of the abdomen, and fever. In certain embodiments, administration of an oligomeric duplex provided herein (e.g., an oligomeric duplex comprising or consisting of an oligomeric antisense agent) to the subject reduces or delays the onset or progression of at least one at least one symptom of aortic stenosis.
Certain embodiments are drawn to an oligomeric duplex comprising or consisting of an oligomeric antisense agent comprising or consisting of a modified oligonucleotide having a targeting region complementary to a target region of a LPA nucleic acid, for the manufacture or preparation of a medicament for ameliorating, or delaying or preventing development or progression of a disease, disorder, condition or injury and/or for ameliorating, preventing or delaying the onset of one or more symptoms of a disease, disorder, condition or injury, wherein the disease, disorder, condition or injury is associated with cardiovascular disease (CVD) such as hypertriglyceridemia and/or other condition selected from lipidemia (e.g., hyperlipidemia), dyslipidemia (e.g., atherogenic dyslipidemia, diabetic dyslipidemia, or mixed dyslipidemia), hyperlipoproteinemia, hyperapobetalipoproteinemia, chronic heart disease, including coronary artery disease (CAD) or any symptoms or conditions associated with a CVD, e.g., hypercholesterolemia (e.g., statin-resistant hypercholesterolemia, heterozygous or homozygous familial hypercholesterolemia) myocardial infarction (MI), acute coronary syndrome, mesenteric ischemia, superior mesenteric artery occlusion, restenosis, renal artery stenosis, angina peripheral arterial disease (PAD), calcific aortic valve disease (CAVD), aortic stenosis, aortic valve regurgitation, aortic dissection, retinal artery occlusion, atherosclerotic cardiovascular disease (ASCVD), atherosclerosis, dyslipidemia, thrombosis, cerebrovascular atherosclerosis, cerebrovascular disease, or stroke. In certain embodiments, the disease is aortic stenosis In certain embodiments, an oligomeric duplex is for the manufacture or preparation of a medicament for improving one or more symptoms selected from angina, chest pain, shortness of breath, palpitations, weakness, dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling in the lower extremities, cyanosis, fatigue, fainting, numbness of the face, numbness of the limbs, claudication or cramping of muscles, bloating of the abdomen, and fever.
In certain embodiments, prophylactic administration of an oligomeric duplex or composition provided herein to a subject at risk for cardiovascular, metabolic, and/or inflammatory disease, is able to prevent, ameliorate, postpone or delay a symptom and/or development or progression of cardiovascular, metabolic, an/or inflammatory disease progression. In certain embodiments, an oligomeric duplex is for the manufacture or preparation of a medicament for improving angina, chest pain, shortness of breath, palpitations, weakness, dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling in the lower extremities, cyanosis, fatigue, fainting, numbness of the face, numbness of the limbs, claudication or cramping of muscles, bloating of the abdomen, and fever.
In any of the methods or uses described herein, the oligomeric duplex is any oligomeric duplex (e.g., an oligomeric duplex comprising or consisting of an oligomeric antisense agent) described herein.
In certain embodiments an oligomeric duplex is administered parenterally. In certain embodiments, an oligomeric duplex is administered intravenously, subcutaneously, intramuscularly, or intrathecally. In certain embodiments, an oligomeric duplex or composition is co-administered with a second agent or therapy. In certain embodiments, the oligomeric duplex or composition and second agent are administered concomitantly. In certain embodiments, a second agent is a glucose-lowering agent. In certain embodiments, a second agent is a LDL, TG or cholesterol lowering agent. In certain embodiments, a second agent is an anti-inflammatory agent. In certain embodiments, a second agent can be, but is not limited to, a non-steroidal anti-inflammatory drug (NSAID e.g., aspirin), niacin (e.g., Niaspan), nicotinic acid, ezetimibe, an apoB inhibitor (e.g., Mipomersen), a CETP inhibitor (e.g., Anacetrapib), an apo(a) inhibitor, a thyroid hormone analog (e.g., Eprotirome), a HMG-CoA reductase inhibitor (e.g., a statin), a fibrate (e.g., Gemfibrozil), a microsomal triglyceride transfer protein inhibitor (e.g., Lomitapide), a PCSK9 inhibitor (e.g., inclisiran), as well as therapies in development, e.g., an ANGPTL3 inhibitor, an LPA inhibitor. In certain embodiments a therapy can be, but is not limited to, Lp(a) apheresis. In certain embodiments, a second agent is an alzheimer disease drug. Agents or therapies can be co-administered or administered concomitantly. Agents or therapies can be sequentially or subsequently administered.
VI. Pharmaceutical Compositions
In certain embodiments, described herein are pharmaceutical compositions comprising one or more oligomeric duplexes or oligomeric compounds, wherein each oligomeric duplex or compound comprises a modified oligonucleotide (e.g., oligomeric compound). In certain embodiments, the one or more oligomeric duplex or oligomeric compound each comprises an antisense agent. In certain embodiments, a pharmaceutical composition comprises a pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutical composition comprises or consists of a sterile saline solution and one or more compound or duplex. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition comprises or consists of one or more compound or duplex and sterile water. In certain embodiments, the sterile water is pharmaceutical grade water. In certain embodiments, a pharmaceutical composition comprises or consists of one or more compound or duplex and phosphate-buffered saline (PBS). In certain embodiments, sterile PBS is pharmaceutical grade PBS. In certain embodiments, the pH of a solution is modulated with a suitable pH- adjusting agent, for example, with acids such as hydrochloric acid and alkalis such as sodium hydroxide, to a range of from about 7.1-7.3, or to about 7.2.
In certain embodiments, a pharmaceutical composition comprises an oligomeric duplex comprising a first oligomeric compound and a second oligomeric compound and sterile saline. In certain such embodiments, a pharmaceutical composition consists of such oligomeric duplex and sterile saline. In certain embodiments, a pharmaceutical composition consists essentially of such oligomeric duplex and sterile saline. In certain embodiments, the sterile saline is sterile PBS. In certain embodiments, the sterile saline is pharmaceutical grade. In certain embodiments, the pH of a solution is modulated with a suitable pH-adjusting agent, for example, with acids such as hydrochloric acid and alkalis such as sodium hydroxide, to a range of from about 7. 1-7.3, or to about 7.2. In certain embodiments, pharmaceutical compositions comprise one or more oligomeric duplex and one or more excipients. In certain embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.
In certain embodiments, an oligomeric duplex may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
In certain embodiments, pharmaceutical compositions comprising an oligomeric duplex encompass any pharmaceutically acceptable salts of the oligomeric compound(s) or duplex, esters of the compound(s) or duplex, or salts of such compounds or esters. In certain embodiments, pharmaceutical compositions comprising an oligomeric compound or oligomeric duplex comprising one or more oligomeric compound, upon administration to a subject, including a human, are capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of oligomeric compounds or oligomeric duplexes, and other bioequivalents. In certain embodiments, pharmaceutically acceptable salts comprise inorganic salts, such as monovalent or divalent inorganic salts. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium, potassium, calcium, and magnesium salts.
In certain embodiments, oligomeric compounds or oligomeric duplexes are lyophilized and isolated, e.g., as sodium salts. In certain embodiments, a sodium salt of an agent or duplex is mixed with a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent comprises sterile saline, sterile water, PBS. In certain embodiments, a sodium salt of an oligomeric compound or oligomeric duplex is mixed with PBS.
Lipid moieties have been used in nucleic acid therapies in a variety of methods. In certain methods, a nucleic acid, such as an oligomeric compound or oligomeric duplex comprising oligomeric compound, is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, nucleic acid complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical compound to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical compound to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical compound to muscle tissue.
In certain embodiments, pharmaceutical compositions comprise a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used. In certain embodiments, pharmaceutical compositions comprise one or more tissue-specific delivery molecules designed to deliver the one or more compounds to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissuespecific antibody.
In certain embodiments, pharmaceutical compositions comprise a co-solvent system. Certain of such co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol 300. The proportions of such cosolvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
In certain embodiments, a pharmaceutical composition is prepared for administration of an oligomeric duplex to a subject. Suitable administration routes include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppository, through inhalation, intrathecal, intracerebroventricular, intraperitoneal, intranasal, intraocular, intratumoral, and parenteral (e.g., intravenous, intramuscular, intrathecal, intramedullary, and subcutaneous). In certain embodiments, pharmaceutical compositions are prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration. In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, intrathecal (IT), intracerebroventricular (ICV), etc.). In certain of such embodiments, a pharmaceutical composition comprises a carrier or diluent and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, diluents, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.
Under certain conditions, certain compounds disclosed herein act as acids. Although such compounds may be drawn or described in protonated (free acid) form or ionized and in association with a cation (salt) form, aqueous solutions of such compounds exist in equilibrium among such forms. For example, a phosphodiester linkage of an oligonucleotide in aqueous solution exists in equilibrium among free acid, anion and salt forms. Unless otherwise indicated, compounds described herein are intended to include all such forms. Moreover, certain oligonucleotides have several such linkages, each of which is in equilibrium. Thus, oligonucleotides in solution exist in an ensemble of forms at multiple positions all at equilibrium. The term “oligonucleotide” herein is intended to include all such forms. Drawn structures necessarily depict a single form. Nevertheless, unless otherwise indicated, such drawings are likewise intended to include corresponding forms. Herein, a structure depicting the free acid of a compound followed by the term “or a pharmaceutically acceptable salt thereof’ expressly includes all such forms that may be fully or partially protonated/de-protonated/in association with a cation or a combination of cations. In certain embodiments, one or more specific cation is identified. The cations include, but are not limited to, sodium, potassium, calcium, and magnesium. In certain embodiments, a structure depicting the free acid of a compound followed by the term “or a pharmaceutically acceptable salt thereof’ expressly includes all such forms that may be fully or partially protonated/de-protonated/in association with one or more cations selected from sodium, potassium, calcium, and magnesium.
In certain embodiments, oligomeric compounds or oligomeric duplexes are in aqueous solution with sodium. In certain embodiments, oligomeric compounds or oligomeric duplexes are in aqueous solution with potassium. In certain embodiments, oligomeric compounds or oligomeric duplexes are in PBS. In certain embodiments, oligomeric compounds or oligomeric duplexes are in water. In certain such embodiments, the pH of a solution is adjusted with NaOH and/or HC1 to achieve a desired pH.
Herein, a dose may be in the form of a dosage unit. For clarity, a dose (or dosage unit) of an agent (e.g., oligomeric compound, oligomeric duplex, antisense agent) in milligrams indicates the mass of the free acid form of the compound. As described herein, in aqueous solution, the free acid is in equilibrium with anionic and salt forms. However, for the purpose of calculating dose, it is assumed that the compound (e.g., oligomeric compound, oligomeric duplex, antisense agent) exists as a solvent-free, sodium-acetate free, anhydrous, free acid. In certain embodiments, where an agent (e.g., oligomeric compound, oligomeric duplex, antisense agent) is in solution comprising sodium (e.g., saline), the compound may be partially or fully de-protonated and in association with sodium ions. However, the mass of the protons is nevertheless counted toward the weight of the dose, and the mass of the sodium ions is not counted toward the weight of the dose. When an agent comprises a conjugate group, the mass of the conjugate group is included in calculating the dose of such compound. If the conjugate group also has an acid, the conjugate group is likewise assumed to be fully protonated for the purpose of calculating dose.
Nonlimiting disclosure and incorporation by reference
Section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
All documents, websites, URLs, or portions thereof, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated- by-reference for the portions of the document discussed herein and in their entirety. While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references, GenBank accession numbers, ENSEMBL identifiers, and the like recited in the present application is incorporated herein by reference in its entirety.
Although the sequence listing accompanying this filing identifies each sequence as either “RNA” or “DNA” as required, in reality, those sequences may be modified with any combination of chemical modifications. One of skill in the art will readily appreciate that such designation as “RNA” or “DNA” to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2 ’-OH sugar moiety and a thymine base could be described as a DNA having a modified sugar (2 ’-OH in place of one 2’-H of DNA) or as an RNA having a modified base (thymine (methylated uracil) in place of an uracil of RNA). Accordingly, nucleic acid sequences provided herein, including, but not limited to those in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases. By way of further example and without limitation, an oligonucleotide having the nucleobase sequence “ATCGATCG” encompasses any oligonucleotides having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence “AUCGAUCG” and those having some DNA bases and some RNA bases such as “AUCGATCG” and oligonucleotides having other modified nucleobases, such as “AT^CGAUCG,” wherein mC indicates a cytosine base comprising a methyl group at the 5-position.
Certain compounds described herein (e.g., modified oligonucleotides) have one or more asymmetric center and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (.S'), as a or p such as for sugar anomers, or as (D) or (L), such as for amino acids, etc. Compounds provided herein that are drawn or described as having certain stereoisomeric configurations include only the indicated compounds. Compounds provided herein that are drawn or described with undefined stereochemistry include all such possible isomers, including their stereorandom and optically pure forms, unless specified otherwise. Likewise, tautomeric forms of the compounds herein are also included unless otherwise indicated. Unless otherwise indicated, compounds described herein are intended to include corresponding salt forms.
The compounds described herein include variations in which one or more atoms are replaced with a non-radioactive isotope or radioactive isotope of the indicated element. For example, compounds herein that comprise hydrogen atoms encompass all possible deuterium substitutions for each of the ’H hydrogen atoms. Isotopic substitutions encompassed by the compounds herein include but are not limited to: 2H or 3H in place of 'H, 13C or 14C in place of 12C, 15N in place of 14N, 17O or 18O in place of 16O, and 33S, 34S, 35S, or 36S in place of 32S. In certain embodiments, non-radioactive isotopic substitutions may impart new properties on the oligomeric compound that are beneficial for use as a therapeutic or research tool. In certain embodiments, radioactive isotopic substitutions may make the compound suitable for research or diagnostic purposes such as imaging.
EXEMPLIFICATION
The following examples illustrate certain embodiments of the present disclosure and are not limiting. Moreover, where specific embodiments are provided, the inventors have contemplated generic application of those specific embodiments. For example, disclosure of an oligonucleotide having a particular motif provides reasonable support for additional oligonucleotides having the same or similar motif, and, for example, where a particular high-affinity modification appears at a particular position, other high-affinity modifications at the same position are considered suitable, unless otherwise indicated. Example 1: Design of oligomeric compounds
Oligomeric compounds comprising antisense oligomeric compound complementary to a human LPA nucleic acid, and sense oligomeric compound complementary to an antisense oligomeric compound were designed as follows.
DESIGN OF ANTISENSE OLIGOMERIC COMPOUNDS
Antisense oligomeric compounds were prepared as described in Table 1. Each antisense oligomeric compound has a sugar motif (from 5' to 3') as indicated, wherein each ‘e’ represents a 2'-M0E sugar moiety, each ‘y’ represents a 2'-0Me sugar moiety, each ‘f represents a 2'-F sugar moiety, each ‘[FHNA]’ represents a 3 '-fluoro-hexitol sugar moiety, and each ‘d’ represents a 2'-deoxy sugar moiety; and each antisense oligomeric compound has an intemucleoside linkage motif (from 5' to 3') as indicated, wherein each ‘o’ represents a phosphodiester intemucleoside linkage, and each ‘s’ represents a phosphorothioate intemucleoside linkage. Each antisense oligomeric compound has a vinyl phosphonate (VP-) moiety on the 5'-end. Each antisense oligomeric compound is complementary to SEQ ID NO: 1 (the complement of GenBank Accession No. NC_000006.12 truncated from nucleosides 160528001 to 160669000), or SEQ ID NO: 2 (GenBank Accession No. NM_005577.2), or to both. Due to the nature of the target LPA sequence, an oligonucleotide may bind multiple sites within SEQ ID NO: 1 and/or SEQ ID NO: 2. CPD SID indicates the SEQ ID NO: for the relevant compound, including annotation, e.g., sugar motif and intemucleoside motif; and nb SID indicates the SEQ ID NO: for the nucleobase sequence of the relevant compound.
Table 1: Antisense oligomeric compound targeted to human LPA
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
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Figure imgf000112_0001
Ill
Figure imgf000113_0001
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Figure imgf000124_0001
Figure imgf000125_0001
Figure imgf000126_0001
DESIGN OF SENSE OLIGOMERIC COMPOUNDS
Modified sense oligomeric compounds were prepared as described in Table 2. Each sense oligomeric compound has a sugar motif (from 5' to 3') as indicated, wherein each ‘y’ represents a 2'-0Me sugar moiety, each ‘e’ represents a 2'-M0E sugar moiety, and each ‘f represents a 2'-F sugar moiety; and an intemucleoside motif (from 5' to 3') as indicated, wherein each ‘o’ represents a phosphodiester intemucleoside linkage, and each ‘s’ represents a phosphorothioate intemucleoside linkage. Each sense oligomeric compound is complementary to an antisense oligomeric compound in Table 1. CPD SID indicates the SEQ ID NO: for the relevant compound, including annotation, e.g., sugar motif and intemucleoside motif; and nb SID indicates the SEQ ID NO: for the nucleobase sequence of the relevant compound.
Certain sense oligomeric compound, as indicated in Conjugate column of Table 2, comprise a sense modified oligonucleotide conjugated to a GalNAc moiety. In certain compounds a HPPO-GalNAc phosphoryl conjugate group is attached at the 3 '-OH of the oligonucleotide (designated 3 ’ GalNAc in Table 2); and in certain compounds a THA-GalNAc phosphoryl conjugate group is attached at the 5'-OH of the oligonucleotide (designated 5’ GalNAc in Table 2).
The structure of HPPO-GalNAc phosphoryl conjugate group is:
Figure imgf000127_0001
HPPO-GalNAc phosphoryl conjugate.
The structure of THA -GalNAc phosphoryl conjugate group is:
Figure imgf000127_0002
THA-GalNAc phosphoryl conjugate.
Table 2: Sense oligomeric compounds
Figure imgf000127_0003
Figure imgf000128_0001
Figure imgf000129_0001
Figure imgf000130_0001
Figure imgf000131_0001
Figure imgf000132_0001
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Figure imgf000148_0001
Figure imgf000149_0001
Figure imgf000150_0001
Figure imgf000151_0001
Example 2: Design of oligomeric duplexes that target human LPA
Oligomeric duplexes were prepared with antisense oligomeric compound numbers and corresponding sense oligomeric compound numbers as listed in Table 3. Table 3: Oligomeric duplexes targeted to human LPA
Figure imgf000151_0002
Figure imgf000152_0001
Figure imgf000152_0002
Figure imgf000152_0003
Figure imgf000153_0001
Figure imgf000153_0002
Figure imgf000153_0003
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Figure imgf000154_0002
Figure imgf000154_0003
Figure imgf000155_0001
Figure imgf000155_0002
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Figure imgf000156_0001
Figure imgf000156_0002
Figure imgf000156_0003
Figure imgf000157_0001
Figure imgf000157_0002
Figure imgf000157_0003
Figure imgf000158_0001
Example 3: Effect of oligomeric duplexes targeted to human LPA in primary human hepatocytes
Oligomeric duplexes were tested for single dose effects on LPA RNA in vitro in a series of experiments that had the same culture conditions. Primary human hepatocytes (sourced from BioIVT, M0095-P, lot ZFW) were treated with oligomeric duplex at a concentration of 100 nM or 5,000 nM by free uptake at a density of 20,000 cells per well. After a treatment period of 72 hours, total RNA was isolated from the cells and LPA RNA levels were measured by quantitative real-time RTPCR using human primer-probe set hAPO(a)12kB (forward sequence CCACAGTGGCCCCGGT, designated herein as SEQ ID NO: 3; reverse sequence ACAGGGCTTTTCTCAGGTGGT, designated herein as SEQ ID NO: 4; probe sequence CCAAGCACAGAGGCTCCTTCTGAACAAG, designated herein as SEQ ID NO: 5). LPA RNA levels were normalized to total RNA content, as measured by RIBOGREEN®. Reduction of LPA RNA is presented in the table below as percent LPA RNA relative to the amount of LPA RNA in untreated control cells (% UTC). The values marked with a “f” indicate that the oligomeric duplex is targeted to the amplicon region of the primer probe set. Additional assays may be used to measure the potency and efficacy of the modified oligonucleotides complementary to the amplicon region. Each separate experiment described in this example is presented in separate sub-Tables 4A-4K. Table 4: Effect of oligomeric duplexes on human LPA RNA in primary human hepatocytes
Figure imgf000158_0002
Figure imgf000159_0001
Figure imgf000159_0002
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Figure imgf000160_0001
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Figure imgf000160_0006
Figure imgf000161_0001
Figure imgf000161_0002
Figure imgf000161_0003
1749912 69 1749906 87 1749882 99
Figure imgf000162_0001
Figure imgf000162_0002
Figure imgf000162_0003
Figure imgf000162_0004
Figure imgf000162_0005
Figure imgf000163_0001
Example 4: Effect of oligomeric duplexes in transgenic primary mouse hepatocytes
Oligomeric duplexes were tested for single dose effects on LPA RNA in vitro in a series of experiments that had the same culture conditions. Each separate experiment is presented in a separate column in Table 5 below. Primary hepatocytes were isolated from transgenic mice previously described in Frazer K, et. al., The apolipoprotein (a) gene is regulated by sex hormones and acute-phase inducers in YAC transgenic mice; Nature Genetics, 1995, 9: 424-431. Transgenic primary mouse hepatocytes were treated with oligomeric duplex at a concentration of 500 nM (Tables 5A and 5B) or 5000 nM (Table 5C) by free uptake at a density of 15,000 cells per well. After a treatment period of 24 hours, total RNA was isolated from the cells and LPA RNA levels were measured by quantitative real-time RTPCR using human primer-probe set hAPO(a) 12kB (described herein above). LPA RNA levels were normalized to total RNA content, as measured by RIBOGREEN®. Reduction of LPA RNA is presented in the table below as percent LPA RNA relative to the amount of LPA RNA in untreated control cells (% UTC). The values marked with a “f” indicate that the oligomeric duplex is targeted to the amplicon region of the primer probe set. Additional assays may be used to measure the potency and efficacy of the modified oligonucleotides complementary to the amplicon region. Each separate experiment described in this example is presented in a separate column in Table 5.
Figure imgf000164_0001
Figure imgf000165_0001
Figure imgf000165_0002
Figure imgf000165_0003
Figure imgf000166_0001
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Figure imgf000167_0003
C. 5000 nM
Figure imgf000168_0001
Figure imgf000168_0002
Figure imgf000168_0003
Figure imgf000168_0004
Figure imgf000169_0001
Example 5: Dose-dependent inhibition of human LPA in primary human hepatocytes
Oligomeric duplexes selected from Example 4 were tested at various doses in primary human hepatocytes. The oligomeric duplexes were tested in a series of experiments that had the same culture conditions, and results are depicted in Table 6, with different experiments in each of sub-Tables 6A-6I.
Primary human hepatocytes (sourced from BioIVT, M0095-P, lot ZFW) plated at a density of 20,000 cells per well were treated using free uptake with various concentrations of modified oligonucleotide as specified in Table 6. After a treatment period of 48 or 72 hours, total RNA was isolated from the cells and LPA RNA levels were measured by quantitative real-time RTPCR. Human LPA primerprobe set hAPO(a)12kB (described herein above) was used to measure RNA levels as described above. LPA RNA levels were normalized to total RNA content, as measured by RIBOGREEN®. Reduction of
LPA RNA is presented in Table 6 as percent LPA RNA, relative to the amount of LPA RNA in untreated control cells (% UTC). “N.C.” represents IC50 values that could not be reliably calculated. The half maximal inhibitory concentration (IC50) of each modified oligonucleotide was calculated using a linear regression on a log/linear plot of the data and is also presented in Table 6.
Table 6: Dose-dependent reduction of human LPA RNA in primary human hepatocytes
6A. 72 hours
Figure imgf000170_0001
6B. 72 hours
Figure imgf000170_0002
Figure imgf000170_0003
6D. 72 hours
Figure imgf000171_0001
6E. 48 hours
Figure imgf000171_0002
6F. 48 hours
Figure imgf000171_0003
6G. 48 hours
Figure imgf000171_0004
6H. 48 hours
Figure imgf000171_0005
Figure imgf000172_0001
61. 48 hours
Figure imgf000172_0002
Example 6: Dose-dependent inhibition of human LPA in transgenic primary mouse hepatocytes
Oligomeric duplexes selected from Example 5 were tested at various doses in transgenic primary mouse hepatocytes. The primary hepatocytes were isolated from transgenic mice previously described in Frazer K, et. al., The apolipoprotein (a) gene is regulated by sex hormones and acute-phase inducers in YAC transgenic mice; Nature Genetics, 1995, 9: 424-431. The oligomeric duplexes were tested in a series of experiments (presented in separate sub-tables 7A-7B) that had the same culture conditions.
Transgenic primary hepatocytes plated at a density of 20,000 cells per well were treated using free uptake with various concentrations of modified oligonucleotide as specified in the tables below. After a treatment period of 24 hours, total RNA was isolated from the cells and LPA RNA levels were measured by quantitative real-time RTPCR. Human LPA primer-probe set hAPO(a)12kB (described herein above) was used to measure RNA levels as described above. LPA RNA levels were normalized to total RNA content, as measured by RIBOGREEN®. Reduction of LPA RNA is presented in the table below as percent LPA RNA, relative to the amount of LPA RNA in untreated control cells (% UTC). The half maximal inhibitory concentration (IC50) of each modified oligonucleotide was calculated using a linear regression on a log/linear plot of the data and is also presented in Table 7.
Table 7: Dose-dependent reduction of human LPA RNA in transgenic primary mouse hepatocytes
7A.
Figure imgf000173_0001
7B.
Figure imgf000173_0002
Figure imgf000174_0002
Example 7: Activity of oligomeric duplexes targeting human LPA in LPA transgenic mice
Oligomeric duplexes selected from studies described above were analyzed along with a comparator compound for their effects on LPA mRNA and Lp(a) protein in human LPA transgenic mice.
LPA transgenic mice previously described (described in Frazer K, et. al., The apolipoprotein (a) gene is regulated by sex hormones and acute-phase inducers in YAC transgenic mice; Nature Genetics, 1995, 9 424-431) were derived by Taconic Biosciences and were treated with selected oligomeric duplexes and evaluated for activity.
Duplex Compound No. 1787751, prepared as a comparator compound, comprises antisense Compound No. 1758680, and sense Compound No. 1758681. Antisense Compound No. 1758680 (SEQ ID NO: 3076) has a nucleobase sequence (from 5' to 3') of UCGUAUAACAAUAAGGGGCUG; a sugar motif (from 5' to 3') of yfyfyfyyyyyfyfyfyfyfy, and an intemucleoside linkage motif (from 5' to 3') of ssooooooooooooooooss; and sense Compound No. 1758681 (SEQ ID NO: 3077) has a nucleobase sequence (from 5' to 3') of CAGCCCCUUAUUGUUAUACGA; a sugar motif (from 5' to 3') of yyyyyyyyfffyyyyyyyyyy, and an intemucleoside linkage motif (from 5' to 3') of soooooooooooooooooos; wherein each “y” represents a 2'-OMe sugar moiety, and each “f ’ represents a 2'-F sugar moiety, and wherein each “s” represents a phosphorothioate intemucleoside linkage, and each “o” represents a phosphodiester intemucleoside linkage. Sense oligonucleotide Compound No. 1758681 has a THA- GalNAc moiety conjugated to the 5 '-end of the oligonucleotide via a phosphorothioate bond:
Figure imgf000174_0001
Duplex No. 1787751 is similar to compound AD03851, a duplex targeting LPA previously disclosed in International Patent Application WO 2017/059223, and differs from AD03851 in two changes: first, 1787751 comprises a THA-GalNAc conjugate (as described herein) on the 5' end of the sense oligonucleotide, whereas AD03851 comprises an NAG25 conjugate; and second, 1787751 comprises a 2'-OMe sugar moiety at the 3’ end of the sense oligonucleotide, whereas AD03851 comprises an inverted 2'-deoxyribosyl sugar moiety modification at the 3' end of the sense oligonucleotide. NAG25 phosphorothioate moiety conjugated to the 5'-end of the oligonucleotide via a phosphorothioate bond, as previously described is shown below:
Figure imgf000175_0001
Groups of two female LPA transgenic mice each received a single subcutaneous injection of oligomeric duplex compound at a dose of 2 mg/kg. One group of 3 female LPA transgenic mice received a single subcutaneous injection of PBS. Seven days post injection mice were sacrificed, and RNA was extracted from liver tissue for quantitative real time RTPCR analysis of LPA RNA using human primer probe set hAPO(a)12kBTS (described in Example 3). LPA RNA levels were normalized to total RNA content, as measured by RIBOGREEN®. Reduction of LPA RNA is presented as percent LPA RNA relative to the amount of LPA in tissue from PBS control animals (% control).
To evaluate effects of oligomeric duplex compounds on apo(a) protein levels, blood plasma was collected on Day 1 prior to treatment and on Day 7 when the mice were sacrificed and analyzed using an automated clinical chemistry analyzer (Hitachi Olympus AU400c, Melville, NY), apo(a) protein levels were analyzed with an Lp(a) assay kit from Randox (Catalog #LP2757). The results were averaged for each group of mice and are presented in the tables below as percent apo(a), relative to the amount of apo(a) present at baseline (% baseline).
Table 8: Reduction of LPA in LPA transgenic mice
Figure imgf000175_0002
Figure imgf000176_0001
Table 9: Reduction of LPA in LPA transgenic mice
Figure imgf000176_0002
Table 10: Reduction of LPA in LPA transgenic mice
Figure imgf000176_0003
Table 11: Reduction of LPA in LPA transgenic mice
Figure imgf000177_0001

Claims

1. An oligomeric duplex comprising a first oligomeric compound and a second oligomeric compound, wherein: the first oligomeric compound comprises a first modified oligonucleotide consisting of 18 to 50 linked nucleosides, wherein the nucleobase sequence comprises a nucleobase sequence that is at least 80% identical to at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NOs:6-829 and 3078-3083, wherein at least one but no more than 22%, no more than 20%, no more than 18%, no more than 15%, no more than 10%, or no more than 5% of the modified nucleosides in the first modified oligonucleotide comprises a 2'-F sugar moiety and/or an FHNA sugar surrogate, and the second oligomeric compound comprises a second modified oligonucleotide consisting of 16 to 50 linked nucleosides, wherein the nucleobase sequence comprises a nucleobase sequence that is at least 80% identical to at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID Nos:830-1540 and 3096-3101, wherein at least one but no more than 25%, no more than 20%, no more than 18%, no more than 16%, no more than 14%, no more than 12%, or no more than 10%, of the modified nucleosides in the second modified oligonucleotide comprises a 2'-F sugar moiety and/or an FHNA sugar surrogate; wherein the first modified oligonucleotide and the second modified oligonucleotide are complementary to one another; and wherein each of the nucleosides of the first modified oligonucleotide and the second modified oligonucleotide independently comprises a modified sugar moiety or sugar surrogate.
2. The oligomeric duplex of claim 1,
1) wherein the first modified oligonucleotide comprises at least one and no more than four modified nucleosides comprising a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate, and/or wherein the second modified oligonucleotide comprises at least one and no more than four modified nucleosides comprising a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate; and/or wherein the oligomeric duplex comprises at least one and no more than eight modified nucleosides comprising a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate; and/or
2) wherein at least 18 nucleosides of the first modified oligonucleotide, each independently, and at least 16 nucleosides of the second modified oligonucleotide, each independently, comprise a modified sugar moiety or sugar surrogate selected from a 2'-F sugar moiety, a 2'-MOE sugar moiety, a 2'-OMe sugar moiety, a DNA sugar moiety, and an FHNA sugar surrogate; and/or
3) wherein the nucleobase sequence of the first modified oligonucleotide comprises at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID NOs:6-829 and 3078-3083, and wherein the nucleobase sequence of the second modified oligonucleotide comprises at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases of the nucleobase sequence of any one of SEQ ID Nos:830-1540 and 3096-3101.
3. The oligomeric duplex of claim 1 or claim 2, wherein a nucleoside comprising a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety or FHNA sugar surrogate of the first modified oligonucleotide is independently selected from one of: i. the second nucleoside counting from the 5’ end, ii. the second and fourteenth nucleosides counting from the 5’ end, or iii. the second and sixteenth nucleosides counting from the 5’ end, or iv. the second, fourteenth and sixteenth nucleosides counting from the 5’ end, or v. the second, sixth, fourteenth, and sixteenth nucleosides counting from the 5’ end.
4. The oligomeric duplex of any one of claims 1-3, wherein a nucleoside comprising a modified sugar moiety or sugar surrogate comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate of the second modified oligonucleotide is independently selected from one of: i. the ninth and tenth nucleosides counting from the 5’ end, or ii. the tenth and eleventh nucleosides counting from the 5’ end, or iii. the ninth, tenth and eleventh nucleosides counting from the 5’ end, or iv. the seventh, ninth, tenth and eleventh nucleosides counting from the 5’ end, or v. the seventh, ninth, and eleventh nucleosides counting from the 5’ end.
5. The oligomeric duplex of any one of claims 1-4, wherein one modified nucleoside of the first modified oligonucleotide comprising a 2'-F sugar moiety and/or an FHNA sugar surrogate comprises a 3’-fluoro-hexitol sugar moiety sugar surrogate.
6. The oligomeric duplex of any one of claims 1-4, wherein one or more nucleosides of the first modified oligonucleotide is a 2 ’-deoxynucleoside.
7. The oligomeric duplex of claim 6, wherein the one or more 2 ’-deoxynucleosides is one or more nucleosides in a region of the sequence of the first modified oligonucleotide between and including the fifth nucleoside to the sixteenth nucleoside counting from the 5 ’ end of the first modified oligonucleotide.
8. The oligomeric duplex of claim 7, wherein the one or more 2’-deoxynucleosides is any of the sixth, fourteenth, and/or sixteenth nucleosides counting from the 5 ’ end of the first modified oligonucleotide.
9. The oligomeric duplex of any one of claims 1-8, wherein two of the 3’ terminal nucleosides of the first modified oligonucleotide comprise a two nucleoside overhang.
10. The oligomeric duplex of claim 9, wherein the overhang nucleosides comprise two modified adenosine (AA), two modified uridine (UU) nucleosides, two modified inosine (II) nucleosides, or two modified nucleosides wherein one is an inosine and one is an adenosine (Al or IA).
11. The oligomeric duplex of any one of claims 1-10, wherein one or more of the nucleosides of the first modified oligonucleotide comprises a 2’-OMe sugar moiety and wherein one or more of the nucleosides of the second modified oligonucleotide comprises a 2’-OMe sugar moiety.
12. The oligomeric duplex of claim 11, wherein at least thirteen nucleosides, at least fourteen nucleosides, at least fifteen nucleosides, at least sixteen nucleosides, at least seventeen nucleosides, at least eighteen nucleosides, or at least nineteen nucleosides of the first modified oligonucleotide comprise a 2’-0Me sugar moiety; and wherein at least thirteen nucleosides, at least fourteen nucleosides, at least fifteen nucleosides, at least sixteen nucleosides, at least seventeen nucleosides, at least eighteen nucleosides, or at least nineteen nucleosides of the first modified oligonucleotide comprise a 2’-0Me sugar moiety.
13. The oligomeric duplex of any one of claims 1-12, wherein one or more of the nucleosides of the first modified oligonucleotide comprise a 2 ’-MOE sugar moiety; and optionally, one or more of the nucleosides of the second modified oligonucleotide comprise a 2’- MOE sugar moiety.
14. The oligomeric duplex of claim 13, wherein the 5’- and/or 3 ’-terminal nucleosides of the first modified oligonucleotide comprise a 2 ’-MOE sugar moiety; and optionally, the 5’- and/or 3’- terminal nucleosides of the second modified oligonucleotide comprise a 2’-M0E sugar moiety.
15. The oligomeric duplex of claim 14, wherein two of the 5’- terminal nucleosides and/or two of the 3’-terminal nucleosides of the first modified oligonucleotide comprise a 2’-M0E sugar moiety; and optionally, two of the 5’- terminal nucleosides and/or two of the 3 ’-terminal nucleosides of the second modified oligonucleotide comprise a 2’-M0E sugar moiety.
16. The oligomeric duplex of claim 13, wherein at least one nucleoside of the first modified oligonucleotide comprising a 2 ’-MOE sugar moiety is an internal nucleoside in a region of the sequence of the first modified oligonucleotide that is any of the ninth and/or tenth nucleosides counting from the 5’ end of the first modified oligonucleotide.
17. The oligomeric duplex of any one of claims 1-16, wherein the first oligomeric compound comprises a stabilized phosphate group attached to the 5 ’-terminal nucleoside.
18. The oligomeric duplex of claim 17, wherein the stabilized phosphate group comprises a methylene phosphonate, cyclopropyl phosphonate or a vinyl phosphonate.
19. The oligomeric duplex of any one of claims 1-18, wherein the first modified oligonucleotide and/or the second modified oligonucleotide comprises at least one modified intemucleoside linkage.
20. The oligomeric duplex of claim 19, wherein at least one modified intemucleoside linkage is a phosphorothioate intemucleoside linkage.
21. The oligomeric duplex of claim 20, wherein each intemucleoside linkage is independently selected from a phosphodiester intemucleoside linkage and a phosphorothioate intemucleoside linkage.
22. The oligomeric duplex of claim 21, wherein the intemucleoside linkages between the first and second nucleosides and the second and third nucleosides counting from the 5’ end of the first modified oligonucleotide are modified intemucleoside linkages, and/or wherein the intemucleoside linkages between the first and second nucleosides and the second and third nucleosides counting from the 3 ’ end of the first modified oligonucleotide are modified intemucleoside linkages; and optionally, wherein the intemucleoside linkages between the first and second nucleosides and the second and third nucleosides counting from the 5 ’ end of the second modified oligonucleotide are modified intemucleoside linkages, and/or wherein the intemucleoside linkages between the first and second nucleosides and the second and third nucleosides counting from the 3’ end of the second modified oligonucleotide are modified intemucleoside linkages.
23. The oligomeric duplex of claim 1, wherein the first modified oligonucleotide comprises a modified sugar motif independently selected from one of efyyyfyyyyyyyfyfyyyyyyy, yfyyyfyyyyyyyfyfyyyyyyy, efyyydyyeyyyydydyyyyyee, efyyydyyyyyyyfyfyyyyyee, efyyydyyeyyyyfyfyyyyyee, e[FHNA]yyyfyyyyyyyfyfyyyyyee, and e[FHNA]yyyfyyeyyyyfyfyyyyyee, wherein each ‘e’ represents a 2'-M0E sugar moiety, each ‘y’ represents a 2'-0Me sugar moiety, each represents a 2'-F sugar moiety, each ‘[FHNA]’ represents a 3'-fluoro-hexitol sugar moiety, and each ‘d’ represents a 2'-deoxy sugar moiety, and wherein all except 0, 1, or 2 modifications are identical to the sugar motif; and/or the second modified oligonucleotide comprises a modified sugar motif independently selected from one of yyyyyyfyfffyyyyyyyyyy and eeyyyyyyyffyyyyyyyyee, wherein each ‘e’ represents a 2'-M0E sugar moiety, each ‘y’ represents a 2'-OMe sugar moiety, and each ‘f represents a 2'-F sugar moiety, and wherein all except 0, 1, or 2 modifications are identical to the sugar motif.
24. The oligomeric duplex of claim 1, wherein the first modified oligonucleotide comprises a modified sugar motif independently selected from one of efyyyfyyyyyyyfyfyyyyyyy, yfyyyfyyyyyyyfyfyyyyyyy, efyyydyyeyyyydydyyyyyee, efyyydyyyyyyyfyfyyyyyee, efyyydyyeyyyyfyfyyyyyee, e[FHNA]yyyfyyyyyyyfyfyyyyyee, and e[FHNA]yyyfyyeyyyyfyfyyyyyee, wherein each ‘e’ represents a 2'-M0E sugar moiety, each ‘y’ represents a 2'-OMe sugar moiety, each represents a 2'-F sugar moiety, each ‘[FHNA]’ represents a 3'-fluoro-hexitol sugar moiety, and each ‘d’ represents a 2'-deoxy sugar moiety; and/or the second modified oligonucleotide comprises a modified sugar motif independently selected from one of yyyyyyfyfffyyyyyyyyyy and eeyyyyyyyffyyyyyyyyee, wherein each ‘e’ represents a 2'-M0E sugar moiety, each ‘y’ represents a 2'-OMe sugar moiety, and each ‘f represents a 2'-F sugar moiety.
25. The oligomeric duplex of claim 24, wherein the first modified oligonucleotide comprises a modified sugar motif and is paired with the second modified oligonucleotide comprising a sugar motif selected from: efyyyfyyyyyyyfyfyyyyyyy and yyyyyyfyfffyyyyyyyyyy; yfyyyfyyyyyyyfyfyyyyyyy and yyyyyyfyfffyyyyyyyyyy; efyyydyyeyyyydydyyyyyee and eeyyyyyyyffyyyyyyyyee; efyyydyyyyyyyfyfyyyyyee and eeyyyyyyyffyyyyyyyyee; efyyydyyeyyyyfyfyyyyyee and eeyyyyyyyffyyyyyyyyee; e[FHNA]yyyfyyeyyyyfyfyyyyyee and eeyyyyyyyffyyyyyyyyee; and e[FHNA]yyyfyyyyyyyfyfyyyyyee and eeyyyyyyyffyyyyyyyyee.
26. The oligomeric duplex of any one of claims 1-25, wherein the agent comprises a conjugate group comprising a conjugate moiety and a conjugate linker.
27. The oligomeric duplex of claim 26, wherein the conjugate group comprises a celltargeting moiety.
28. The oligomeric duplex of claim 27, wherein the conjugate group comprises a liver cell targeting moiety.
29. The oligomeric duplex of claim 28, wherein the agent comprises a conjugate moiety that binds asialoglycoprotein receptor (ASGPR).
30. The oligomeric duplex of claim 29, wherein the conjugate moiety is selected from a GalNAc moiety.
31. The oligomeric duplex of claim 30, wherein the GalNAc conjugate moiety is selected from Table A.
32. The oligomeric duplex of claim 30, wherein the conjugate group consists of a GalNAc ligand and a conjugate linker.
33. The oligomeric duplex of claim 30, wherein the GalNAc ligand has the structure:
Figure imgf000182_0001
34. The oligomeric duplex of claim 15, wherein the conjugate group has the structure:
Figure imgf000182_0002
or an ion or salt thereof, wherein the conjugate linker is covalently connected to an oligonucleotide.
35. The oligomeric duplex of claim 30, wherein the conjugate group has the structure:
Figure imgf000183_0001
or an ion or salt thereof, wherein the conjugate linker is covalently connected to an oligonucleotide.
36. The oligomeric duplex of any one of claims 26-35, wherein the conjugate group is conjugated directly to the modified oligonucleotide.
37. The oligomeric duplex of claim 36, wherein the conjugate group is conjugated to the 5’ end or 3’ end of the modified oligonucleotide.
38. The oligomeric duplex of claim 37, wherein the conjugate group is attached to the 5’- terminal nucleoside of the modified oligonucleotide.
39. The oligomeric duplex of claim 37, wherein the conjugate group is attached to the 3’- terminal nucleoside of the modified oligonucleotide.
40. The oligomeric duplex of claim 36, wherein the conjugate linker of the conjugate group consists of a single bond.
41. The oligomeric duplex of claim 36, wherein the conjugate linker of the conjugate group is cleavable.
42. The oligomeric duplex of claim 36, wherein the conjugate linker comprises 1 to 3 linker- nucleosides.
43. The oligomeric duplex of claim 38, wherein the conjugate group having the structure:
Figure imgf000183_0002
or an ion or salt thereof, is attached to the 5 ’-terminal nucleoside of the modified oligonucleotide.
44. The oligomeric duplex of claim 39, wherein the conjugate group having the structure:
Figure imgf000184_0001
or an ion or salt thereof, is attached to the 3 ’-terminal nucleoside of the modified oligonucleotide.
45. An oligomeric duplex comprising i) a first oligomeric compound comprising a modified oligonucleotide consisting of 16 to 50 linked nucleosides, wherein the nucleobase sequence comprises a nucleobase sequence that is at least 80% identical to at least 16 contiguous nucleobases of any one of SEQ ID NOs:6-829 and 3078-3083, wherein all except 0, 1, or 2 nucleobases are identical to the nucleobase sequence SEQ ID NOs:6-829 or 3078-3083, and wherein each of the first modified oligonucleotide comprises a modified sugar moiety selected from: efyyyfyyyyyyyfyfyyyyyyy, yfyyyfyyyyyyyfyfyyyyyyy, efyyydyyeyyyydydyyyyyee, efyyydyyyyyyyfyfyyyyyee, efyyydyyeyyyyfyfyyyyyee, e[FHNA]yyyfyyyyyyyfyfyyyyyee, and e[FHNA]yyyfyyeyyyyfyfyyyyyee, wherein each ‘e’ represents a 2'-M0E sugar moiety, each ‘y’ represents a 2'-OMe sugar moiety, each represents a 2'-F sugar moiety, each ‘[FHNA]’ represents a 3'-fluoro-hexitol sugar moiety, and each ‘d’ represents a 2'-deoxy sugar moiety; and ii) a second oligomeric compound comprising a second modified oligonucleotide consisting of 14-50 linked nucleosides wherein the nucleobase sequence comprises a nucleobase sequence that is at least 80% identical to at least 14 contiguous nucleobases of any one of SEQ ID N0s:830-1540 and 3096-3101, wherein all except 0, 1, or 2 nucleobases are identical to the nucleobase sequence of SEQ ID NOs: 830- 1540 or 3096-3101, and wherein each of the second modified oligonucleotide comprises a modified sugar motif selected from one of yyyyyyfyfffyyyyyyyyyy and eeyyyyyyyffyyyyyyyyee, wherein each ‘e’ represents a 2'-M0E sugar moiety, each ‘y’ represents a 2'- OMe sugar moiety, and each ‘f represents a 2'-F sugar moiety.
46. The oligomeric duplex of claim 45, wherein the sequence of the second modified oligonucleotide comprises at least 15 or at least 16 contiguous nucleobases of the sequence of any one of SEQ ID NO: 2365-3075 and 3084-3089; and optionally wherein the sequence of the first modified oligonucleotide comprises at least 18, at least 19, or at least 20 contiguous nucleobases of the sequence of any one of SEQ ID NO: 1541-2364 and 3090-3095.
47. The oligomeric duplex of claim 46, wherein the sequence of the first modified oligonucleotide comprises the sequence of any one of SEQ ID NOs: 1541-2364 and 3090-3095; and optionally wherein the sequence of the second modified oligonucleotide comprises the sequence of any one of SEQ ID NO: 2365-3075 and 3084-3089.
48. The oligomeric duplex of claim 46, wherein the sequence of the first modified oligonucleotide consists of the sequence of any one of SEQ ID NOs: 1541-2364 and 3090-3095; and optionally wherein the sequence of the second modified oligonucleotide consists of the sequence of any one of SEQ ID NO: 2365-3075 and 3084-3089.
49. The oligomeric duplex of claim 46, wherein the agent is a duplex compound comprised of any one the following antisense compound and sense compound pairs:
Figure imgf000185_0001
Figure imgf000186_0001
Figure imgf000186_0002
Figure imgf000186_0003
Figure imgf000187_0001
Figure imgf000187_0002
Figure imgf000187_0003
Figure imgf000188_0001
Figure imgf000188_0002
Figure imgf000188_0003
Figure imgf000189_0001
Figure imgf000189_0002
Figure imgf000189_0003
Figure imgf000190_0001
Figure imgf000190_0002
Figure imgf000190_0003
Figure imgf000191_0001
Figure imgf000191_0002
Figure imgf000191_0003
Figure imgf000192_0001
50. A population of oligomeric duplex of any one of claims 1-49, wherein the population is enriched for and/or modified oligonucleotides comprising at least one particular phosphorothioate intemucleoside linkage having a particular stereochemical configuration.
51. The population of claim 50, wherein the population is enriched for and/or modified oligonucleotides comprising at least one particular phosphorothioate intemucleoside linkage having the (Sp) or (Rp) configuration.
52. A pharmaceutical composition comprising the oligomeric duplex of any one of claims 1- 49, or the population of any one of claims 50-51, and a pharmaceutically acceptable diluent or carrier.
53. The pharmaceutical composition of claim 52, wherein the pharmaceutically acceptable diluent is water or phosphate-buffered saline.
54. The pharmaceutical composition of claim 52, wherein the pharmaceutical composition consists essentially of the oligomeric duplex and water or phosphate-buffered saline.
55. A method of decreasing the amount of LPA RNA or Lipoprotein(a) protein in a cell, tissue, organ or subject, comprising contacting the cell, tissue, organ or subject with the oligomeric duplex of any one of claims 1-49, the population of any one of claims 50-51, or the pharmaceutical composition of any one of claims 52-54.
56. The method of claim 55, wherein the cell is a liver cell.
57. A method comprising administering to a subject the oligomeric duplex of any one of claims 1-49, the population of any one of claims 50-51, or the pharmaceutical composition of any one of claims 52-54; wherein the subject has or is at risk for developing cardiovascular disease (CVD) coronary artery disease (CAD), hypercholesterolemia, myocardial infarction (MI), peripheral arterial disease (PAD), calcific aortic valve disease (CAVD), aortic stenosis, atherosclerotic cardiovascular disease (ASCVD), atherosclerosis, dyslipidemia, thrombosis, or stroke.
58. A method of preventing or treating a cardiovascular disease, disorder, condition, a metabolic disease, disorder, or condition, and/or an inflammatory disease disorder or condition in a subject, comprising administering to a subject having, or at risk of having, a cardiovascular, metabolic, and/or inflammatory disease, disorder, or condition, an oligomeric duplex of any one of claims 1-49, the population of any one of claims 50-51, or the pharmaceutical composition of any one of claims 52-54; wherein the disease, disorder, condition or injury is hypertriglyceridemia, lipidemia (e.g., hyperlipidemia), dyslipidemia (e.g., atherogenic dyslipidemia, diabetic dyslipidemia, or mixed dyslipidemia), hyperlipoproteinemia, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic valve stenosis, aortic valve calcification, aortic valve regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, restenosis, renal artery stenosis, angina, cerebrovascular atherosclerosis, cerebrovascular disease, or venous thrombosis.
59. A method of decreasing the amount of LPA RNA and/or Lipoprotein(a) protein in the liver of a subject having or at risk of developing a disease, disorder or condition associated with elevated Lp(a), comprising administering to a subject having, or at risk of having, a disease, disorder or condition associated with lipoprotein(a) metabolism misregulation, an oligomeric duplex of any one of claims 1-49, the population of any one of claims 50-51, or the pharmaceutical composition of any one of claims 52- 54; wherein the disease, disorder, condition or injury is a cardiovascular disease, disorder, condition, a metabolic disease, disorder, or condition, and/or an inflammatory disease disorder or condition.
60. The method of any one of claims 55-59, wherein the amount of LPA RNA and/or lipoprotein(a) protein in liver and/or plasma of the subject is decreased.
61. The method of any one of claims 55-59, wherein the disease, disorder or condition is hypertriglyceridemia or atherosclerotic cardiovascular disease (ASCVD) or coronary artery disease (CAD).
62. The method of claim 61, wherein at least one symptom of a disease, disorder or condition associated with elevated lipoprotein(a) is episodes of abdominal pain, physical fatigue, difficulty thinking, diarrhea, recurrent acute pancreatitis, eruptive cutaneous xanthomata, and hepatosplenomegaly or a combination thereof.
63. The method of any one or claims 55-59, wherein the method prevents or protects against progression of atherosclerotic cardiovascular disease (ASCVD) or coronary artery disease (CAD).
64. The method of any one of claims 55-59, wherein administering of the oligomeric duplex of any one of claims 1-49, the population of any one of claims 50-51, or the pharmaceutical composition of any one of claims 52-54 improves hypertriglyceridemia, hyperlipidemia, dyslipidemia hyperlipoproteinemia abdominal pain, physical fatigue, difficulty thinking, diarrhea, acute pancreatitis, eruptive xanthomas, lipemia retinalis, or hepatosplenomegaly, or a combination of two or more of the foregoing in the subject.
65. The method of any one of claims 55-59, wherein administering of the oligomeric duplex of any one of claims 1-49, the population of any one of claims 50-51, or the pharmaceutical composition of any one of claims 52-54 is parenteral.
66. The method of any one of claims 55-59, wherein administering of the oligomeric duplex of any one of claims 1-49, the population of any one of claims 50-51, or the pharmaceutical composition of any one of claims 52-54 is subcutaneous.
67. The method of any one of claims 55-59, wherein administering of the oligomeric duplex of any one of claims 1-49, the population of any one of claims 50-51, or the pharmaceutical composition of any one of claims 52-54 is co-administering with a second agent.
68. The method of any one of claim 55-59, wherein administering of the oligomeric duplex of any one of claims 1-29, the population of any one of claims 30-31, or the pharmaceutical composition of any one of claims 32-34 and the agent are administered concomitantly.
69. Use of the oligomeric duplex of any one of claims 1-49, the population of any one of claims 50-51, or the pharmaceutical composition of any one of claims 52-54 for treating or preventing a disease, disorder or condition associated with lipoprotein metabolism misregulation or postponing a symptom of a disease, disorder or condition associated with elevated lipoprotein(a).
70. Use of the oligomeric duplex of any one of claims 1-49, the population of any one of claims 50-51, or the pharmaceutical composition of any one of claims 52-54 in the manufacture of a medicament for treating or preventing a cardiovascular disease, disorder, condition, a metabolic disease, disorder, or condition, or an inflammatory disease disorder or condition.
71. The oligomeric duplex of any one of claims 1-49, the population of any one of claims 50-51, or the pharmaceutical composition of any one of claims 52-54 for use in treating or preventing a disease, disorder or condition associated with lipoprotein metabolism misregulation or postponing a symptom of a disease, disorder or condition associated with elevated lipoprotein(a).
72. The oligomeric duplex of any one of claims 1-49, the population of any one of claims 50-51, or the pharmaceutical composition of any one of claims 52-54 for use in treating or preventing a cardiovascular disease, disorder, condition, a metabolic disease, disorder, or condition, or an inflammatory disease disorder or condition.
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