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Definitions

• Nucleic acid products that modulate, interfere with, or inhibit angiotensinogen (AGT) gene expression are provided.
• methods, compounds, and compositions are provided for reducing expression of AGT mRNA and protein in an animal. Such methods, compounds, and compositions are useful to treat, prevent, or ameliorate AGT-associated diseases or disorders, such as hypertension.
• renin-angiotensin-aldosterone system The renin-angiotensin-aldosterone system (RAAS) is known to play an important role in the regulation of blood pressure.
• the RAAS cascade is initiated by the release due to widely known mechanisms of renin into the circulation, in particular into the plasma.
• Active renin in the plasma cleaves angiotensinogen (AGT), which is produced by the liver, to yield angiotensin I.
• AGT angiotensinogen
• Angiotensin I is converted to angiotensin II by the circulating and locally expressed angiotensin-converting enzyme (ACE).
• ACE angiotensin-converting enzyme
• Angiontensin II is a peptide hormone, which causes vasoconstriction, which in turn, can increase blood pressure and may lead to hypertension and associated diseases.
• Dysregulation of angiotensin II causes hypertension and can lead to increased oxidative stress, promotion of inflammation, hypertrophy, and fibrosis in the heart, kidneys, and arteries, and may finally result in left ventricular fibrosis, arterial remodelling and glomerulosclerosis.
• Hypertension can also occur along with PCSK9- and/or APOC3-associated disorders such as dyslipidaemia, more specifically hypercholesterinaemia, hypertriglyceridemia, hyperchylomicronaemia, and atherosclerotic cardiovascular disease (ASCVD).
• dyslipidaemia more specifically hypercholesterinaemia, hypertriglyceridemia, hyperchylomicronaemia, and atherosclerotic cardiovascular disease (ASCVD).
• ASCVD atherosclerotic cardiovascular disease
• anti-hypertensive drugs may reduce hypertension and reduce the diseases, disorders and/or conditions associated with hypertension (Paulis et al., Nat Rev Cardiol, 2012, 9:276-285).
• therapies currently approved for treating hypertension as a significant subset of all hypertensive patients do not achieve adequate blood pressure control.
• drugs such as ACE inhibitors and angiotensin receptor blockers (ARBs) that target parts of the renin-angiotensin system (RAS) pathway are limited in their ability to inhibit the RAAS pathway (Nobakht et al., Nat Rev Nephrol, 2011 , 7:356-359).
• certain anti-hypertensive drugs such as ACE inhibitors are contra-indicated in hypertensive patients with renal disease due to their potential to compromise renal function in patients.
• Double-stranded RNAs also are provided.
• dsRNAs Double-stranded RNAs
• dsRNAs lack a loop connecting antisense and sense portions and therefore contains two strands. The two strands are not covalently connected to each other but form a duplex region where base pairing occurs.
• nucleic acid construct may contain at least:
• a second nucleic acid portion that is at least partially complementary to at least a second portion of an RNA, which is transcribed from an AGT gene, the second portion being different from the first portion;
• nucleobase means a group of atoms that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into an oligonucleotide, and where the group of atoms is capable of bonding, more specifically hydrogen bonding, with a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. Nucleobases may be naturally occurring or may be modified.
• unmodified nucleobase or “naturally occurring nucleobase” means the naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C), and uracil (U).
• modified nucleobase means any nucleobase that is not a naturally occurring nucleobase.
• bicyclic nucleoside or "BNA” means a nucleoside having a bicyclic sugar moiety.
• locked nucleic acid nucleoside or "LNA” means a nucleoside having a bicyclic sugar moiety having a 4'-CH2-O-2'bridge.
• 2 '-substituted nucleoside means a nucleoside having a substituent at the 2'- position of the sugar moiety other than H or OH. Unless otherwise indicated, a 2 '-substituted nucleoside is not a bicyclic nucleoside.
• deoxynucleoside means a nucleoside having 2'-H furanosyl sugar moiety, as found in naturally occurring deoxyribonucleosides (DNA).
• a 2'-deoxynucleoside may contain a modified nucleobase or may contain an RNA nucleobase (e.g., uracil).
• oligonucleotide means a compound having a plurality of linked nucleosides.
• an oligonucleotide contains one or more unmodified ribonucleosides (RNA) and / or unmodified deoxyribonucleosides (DNA) and / or one or more modified nucleosides.
• modified oligonucleotide means an oligonucleotide having at least one modified nucleoside and / or at least one modified internucleoside linkage.
• Advantageous positions for such modified internucleoside linkages include the termini and the hairpin loop of single-stranded oligomeric compounds.
• the internucleoside linkages connecting first and second nucleoside and second and third nucleoside counting from the 5' terminus, and/or the internucleoside linkages connecting first and second nucleoside and second and third nucleoside counting from the 3' terminus are modified.
• a linkage connecting the terminal nucleoside of the 3' terminus with a ligand, such as GalNAc may be modified.
• linkages in the hairpin loop designates the linkages between nucleosides, which are not engaged in base pairing.
• linkages in the hairpin loop also extends to the linkages connecting the stem to the loop, i.e., those linkages which connect a base-paired nucleoside to a non-based paired nucleoside.
• linkages connecting the stem to the loop i.e., those linkages which connect a base-paired nucleoside to a non-based paired nucleoside.
• modified internucleoside linkages are at both termini and in the hairpin loop.
• linkage means a group of atoms that link together two or more other groups of atoms.
• nucleoside linkage means a covalent linkage between adjacent nucleosides in an oligonucleotide.
• naturally occurring internucleoside linkage means a 3' to 5' phosphodiester linkage.
• modified internucleoside linkage means any internucleoside linkage other than a naturally occurring internucleoside linkage.
• a “modified internucleoside linkage” as referred to herein can include a modified phosphorous linking group such as a phosphorothioate or phosphorodithioate internucleoside linkage.
• terminal internucleoside linkage means the linkage between the last two nucleosides of an oligonucleotide or defined region thereof.
• phosphorus linking group means a linking group having a phosphorus atom and can include naturally occurring phosphorous linking groups as present in naturally occurring RNA or DNA, such as phosphodiester linking groups, or modified phosphorous linking groups that are not generally present in naturally occurring RNA or DNA, such as phosphorothioate or phosphorodithioate linking groups.
• Phosphorus linking groups can therefore include without limitation, phosphodiester, phosphorothioate, phosphorodithioate, phosphonate, methylphosphonate, phosphoramidate, phosphorothioamidate, thionoalkylphosphonate, phosphotriesters, thionoalkylphosphotriester and boranophosphate.
• nucleoside phosphorus linking group means a phosphorus linking group that directly links two nucleosides.
• oligomeric compound means a polymeric structure having two or more substructures.
• an oligomeric compound contains an oligonucleotide, such as a modified oligonucleotide.
• an oligomeric compound further contains one or more conjugate groups and I or terminal groups and I or ligands.
• an oligomeric compound consists of an oligonucleotide.
• an oligomeric compound contains a backbone of one or more linked monomeric sugar moieties, where each linked monomeric sugar moiety is directly or indirectly attached to a heterocyclic base moiety.
• oligomeric compounds may also include monomeric sugar moieties that are not linked to a heterocyclic base moiety, thereby providing abasic sites.
• Oligomeric compounds may be defined in terms of a nucleobase sequence only, i.e., by specifying the sequence of A, G, C, U (or T). In such a case, the structure of the sugar-phosphate backbone is not particularly limited and may or may not contain modified sugars and/or modified phosphates.
• oligomeric compounds may be more comprehensively defined, i.e., by specifying not only the nucleobase sequence, but also the structure of the backbone, in particular the modification status of the sugars (unmodified, 2'-OMe modified, 2'-F modified etc.) and/or of the phosphates.
• An mxRNA is one non-limiting example for an oligomeric compound.
• nucleic acid construct refers to an assembly of two or more, such as four oligomeric compounds.
• the oligomeric compounds may be connected to each other by covalent bonds such phosphodiester bonds as they occur in naturally occurring nucleic acids or modified versions thereof as disclosed herein, or by non-covalent bonds such as hydrogen bonds, advantageously hydrogen bonds between nucleobases such as Watson-Crick base pairing.
• a construct contains four oligomeric compounds, two of which are connected covalently, thereby giving rise to two nucleic acid strands which nucleic acid strands are bound to each other by hydrogen bonds. Complementarity between the strand may be throughout, but is not necessarily so.
• exemplary embodiments provide for an antisense strand targeting a first region of AGT mRNA to be connected covalently with a sense strand of another AGT- targeting double stranded RNA molecule, and of the antisense strand of the AGT mRNA-targeting double stranded RNA molecule to be connected covalently to a sense strand of the other AGT mRNA-targeting double stranded RNA molecule.
• one construct contains a central region where the 3' regions of the antisense portions of the parent single-target-directed RNA molecules face each other. In that region generally no or only partial base pairing will occur, while full complementarity is not excluded. Otherwise, where antisense and sense portions of the respective parent RNA molecules face each other; there is complementarity, advantageously full complementarity or 1 or 2 mismatches.
• An muRNA is non-limiting example for a nucleic acid construct.
• strand has its art-established meaning and refers to a plurality of linked nucleosides, the linker not being particularly limited, but including phosphodiesters and variants thereof as disclosed herein.
• a strand may also be viewed as a plurality of linked nucleotides in which case the linker would be a covalent bond.
• terminal group means one or more atom attached to either, or both, the 3 ' end or the 5' end, also called “terminus” of an oligonucleotide.
• a terminal group contains one or more terminal group nucleosides, whereas a “terminal nucleoside” is only one nucleotide at the respective end (5' end or 3' end).
• conjugate means an atom or group of atoms bound to an oligonucleotide or oligomeric compound.
• a conjugate group links a ligand to a modified oligonucleotide or oligomeric compound.
• conjugate groups can modify one or more properties of the compound to which they are attached, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and I or clearance properties.
• the carbohydrate cluster portion contains 2 GalNAc groups.
• the carbohydrate cluster portion contains 3 GalNAc groups and this is particularly advantageous.
• the carbohydrate cluster portion contains 4 GalNAc groups.
• Such ligand portions are attached to an oligomeric compound via a cleavable moiety, such as a cleavable bond or cleavable nucleoside.
• the ligands can be arranged in a linear or branched configuration, such as a biantennary or triantennary configurations.
• An advantageous carbohydrate cluster has the following formula:
• carbohydrate cluster means a compound having one or more carbohydrate residues attached to a linker group.
• modified carbohydrate means any carbohydrate having one or more chemical modifications relative to naturally occurring carbohydrates.
• carbohydrate derivative means any compound which may be synthesized using a carbohydrate as a starting material or intermediate.
• Carbohydrate means a naturally occurring carbohydrate, a modified carbohydrate, or a carbohydrate derivative.
• a carbohydrate is a biomolecule including carbon (C), hydrogen (H) and oxygen (O) atoms.
• Carbohydrates can include monosaccharide, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides or polysaccharides, such as one or more galactose moieties, one or more lactose moieties, one or more N-Acetyl-Galactosamine moieties, and I or one or more mannose moieties.
• a particularly advantageous carbohydrate is N-Acetyl-Galactosamine.
• short hairpin RNA also denoted as shRNA
• shRNA contains a duplex region and a loop connecting the regions forming the duplex.
• the end of the duplex region, which does not carry the loop, may be blunt-ended or carry (a) 3' and/or (a) 5' overhang(s). Blunt-ended constructs are particularly advantageous.
• shRNA is more generic than "mxRNA", as defined below, and may include compounds in which the loop is not or not exclusively formed out of an antisense strand.
• shRNA includes an antisense strand, also called guide strand, being complementary to a region of a target RNA, and a sense strand, i.e. a passenger strand, being substantially complementary to the antisense strand.
• the antisense strand and the sense strand within the shRNA are directly linked, e.g. by a phosphate or a phosphorothioate, or linked by a third portion of linked nucleosides forming the loop, which means that the 3' end of the antisense strand is linked to the 5' end of the sense strand via covalent bonding over several other groups.
• Such direct linkage does not include a gap or nick.
• nucleobase at a certain position of an oligomeric compound is capable of hydrogen bonding with a nucleobase at a certain position of a target sequence
• the position of hydrogen bonding between the oligomeric compound and the target sequence is considered complementary at that nucleobase pair.
• non-complementary in reference to nucleobases means a pair of nucleobases that do not form hydrogen bonds with one another.
• oligomeric compound or region thereof is capable of pairing with a nucleobase of a complementary nucleic acid target sequence or a self-complementary region of the oligomeric compound.
• a fully complementary oligomeric compound or region thereof contains no mismatches or unhybridized nucleobases with respect to its target sequence or a self- complementary region of the oligomeric compound.
• percent identity means the number of nucleobases in a first nucleic acid that are the same type (independent of chemical modification) as nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid.
• modulation means a change of amount or quality of a molecule, function, or activity when compared to the amount or quality of a molecule, function, or activity prior to modulation.
• modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression.
• nucleoside having a modification of a first type may be an unmodified nucleoside.
• RNA nucleosides that are the same but for the presence of different nucleobases are not differently modified.
• MOE nucleoside and an unmodified naturally occurring RNA nucleoside are “differently modified,” even though the naturally occurring nucleoside is unmodified.
• DNA and RNA oligonucleotides are “differently modified,” even though both are naturally occurring unmodified nucleosides. Nucleosides that are the same but for the presence of different nucleobases are not differently modified.
• alkylene means a saturated straight or branched divalent hydrocarbon radical of the general formula -CnHzn- where n is 1 -6. Methylene or ethylene are typical alkylenes.
• alkynyl means a straight or branched unsaturated C2-6 hydrocarbon radical, with ethynyl being a typical alkynyl as a substituent at the 2'-position of the sugar moiety.
• degree of unsaturation that is present in an alkynyl radical is the presence of at least one carbon to carbon triple bond.
• the alkynyl group typically attaches to an oxygen linking atom at the 2'-position of the sugar, therefore, overall providing a -Oalkynyl substituent on a sugar moiety of an oligomeric compound as described herein.
• alkoxy means a radical formed between an alkyl group, such as a C1-6 alkyl group, and an oxygen atom where the oxygen atom is used to attach the alkoxy group either to a parent molecule (such as at the 2'-position of a sugar moiety), or to another group such as an alkylene group as defined herein.
• alkoxy groups include without limitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy and tert-butoxy.
• Alkoxy groups as used herein may optionally include further substituent groups.
• alkoxyalkylene means an alkoxy group as defined herein that is attached to an alkylene group also as defined herein, and where the oxygen atom of the alkoxy group attaches to the alkylene group and the alkylene attaches to a parent molecule.
• the alkylene group typically attaches to an oxygen linking atom at the 2'-position of the sugar, therefore, overall providing a - Oalkylenealkoxy substituent, such as an -OCH2CH2OCH3 substituent, on a sugar moiety of an oligomeric compound as described herein. This is generally referred to as an MOE substituent as defined herein and as known in the art.
• amino includes primary, secondary and tertiary amino groups.
• an mxRNA is in particular understood as defined in WO 2020/044186 A2, which is incorporated by reference herein in its entirety.
• an mxRNA is a hairpin-shaped RNA molecule consisting of an antisense portion (also referred to as the guide strand) and a sense portion (also referred to the passenger strand).
• the mxRNA contains duplex region and a hairpin loop, where the mxRNA has approximate length of about 34 nucleotides.
• the duplex region contains a region in which parts of the antisense portion and substantially the entire sense portion, typically 14 or 15 nucleotides of each strand, are base-paired.
• the hairpin loop connects both regions, i.e.
• oligomeric compounds are provided that are capable of inhibiting expression of angiotensinogen (AGT), where the compound contains at least a first region of linked nucleosides having at least a first nucleobase sequence that is at least partially complementary to at least a portion of RNA transcribed from an AGT gene, where the first nucleobase sequence is selected from the following sequences, or a portion thereof: sequences of Table 1 a (SEQ ID NOs: 1 to 100), where the portion advantageously has a length of at least 18 nucleosides.
• the 5' terminal nucleoside of the first nucleobase sequence can contain U instead of A; or U instead of G; or U instead of C, respectively.
• the oligomeric compound including the first and second regions of linked nucleosides may contain at least one complementary duplex region that contains at least a portion of the first region of linked nucleosides directly or indirectly linked to at least a portion of the second region of linked nucleosides, where advantageously the duplex region has a length of 10 to 19, 12 to 19, 12 to 15, or 14 or 15, base pairs, where optionally there is one mismatch within the duplex region.
• each of the first and second regions of linked nucleosides has a 5’ to 3’ directionality thereby defining 5’ and 3’ regions respectively thereof.
• the 5’ region of the first region of linked nucleosides may be directly or indirectly linked to the 3’ region of the second region of linked nucleosides, for example by complementary base pairing, where advantageously the 5' terminal nucleoside of the first nucleoside region base pairs with the 3' terminal nucleoside of the second nucleoside region.
• the oligomeric compound may consist of the first region of linked nucleosides and the second region of linked nucleosides.
• Each ofthe regions may constitute a separate strand, thereby giving rise to a double-stranded RNA (dsRNA).
• dsRNAs particularly advantageous dsRNAs are those with a length of the first strand of 19 nucleosides and a length of the second region of 14 or 15, advantageously 14 nucleosides.
• the terms "nucleoside” and “nucleotide” are used equivalently.
• the oligomeric compound contains a single strand having the first and second nucleoside regions, where at least a portion of the first nucleoside region is directly or indirectly linked to at least a portion of the second nucleoside region so as to form the at least partially complementary duplex region.
• the third region is optional.
• the oligomeric compound may contain or may consist of a single strand having or consisting of the first and second regions of linked nucleosides, where at least a portion of the first region of linked nucleosides is directly or indirectly linked to at least a portion of the second region of linked nucleosides so as to form the at least partially complementary duplex region.
• the additional number of linked nucleosides of the first nucleoside region form a hairpin loop linking the first and second regions of linked nucleosides, where advantageously a part of the first nucleobase sequence of the first nucleobase sequence being complementary RNA transcribed from an AGT gene forms the hairpin loop, where the loop contains 2 to 5, advantageously 4 or 5, nucleosides.
• Such compounds are also referred to as hairpins or mxRNAs herein.
• the compound is optimized in terms of size (or miniaturized) as compared to a conventional siRNA which has two regions of comparable length.
• the loop has 4 or 5 linked nucleosides. Particularly advantageous is a length of the first region of 19 nucleosides, of the second region of 14 nucleosides, and of the hairpin loop of 5 nucleosides, where the 5 nucleosides in the hairpin are the 5 3'-terminal nucleosides of the first region.
• Such molecular architecture of a hairpin or mxRNA is also designated "14-5-14" herein.
• an oligomeric single strand as disclosed earlier herein can be selected from Table 2, in particular selected from the group consisting of SEQ IDs NO: 227, 252, 256, 262, 275, 293 and 3602-3603, where advantageously the 5' terminal nucleoside of the first region of linked nucleosides is substituted by an U as the nucleobase, and the 5' terminal nucleoside of the second region of linked nucleosides is substituted by an A as the nucleobase.
• the single strand is selected from Table 3c, in particular from Construct ID NOs: 527, 552, 556, 562, 575, 593 and 3604-3605, where advantageously the 5' terminal nucleoside of the first region of linked nucleosides is substituted by an U as the nucleobase, and the 5' terminal nucleoside of the second region of linked nucleosides is substituted by an A as the nucleobase.
• the first "13" refers to the region of the guide sequence involved in the duplex
• 5 is the length of the loop which is also formed by the guide sequence
• the second 13 refers to the second region of the duplex and is formed by one nucleobase of the guide sequence and 12 nucleobases of the passenger region in 5' to 3' direction.
• a length of the guide sequence of 19 nucleosides is maintained, but the passenger sequence is shortened to 12 nucleosides.
• oligomeric compounds according to the first aspect disclosed herein may be blunt ended.
• the second region may be selected from the sequences of Table 3b, or a portion thereof, especially a portion having a length of 14 nucleosides, in particular from Construct ID NOs: 427, 452, 456, 462, 475 and 493.
• the one or more ligands in particular two or more or three ligands, may be conjugated to the second region of linked nucleosides and/or the first region of linked nucleosides.
• the one or more ligands may be conjugated at the 3' region, advantageously at the 3' terminal nucleoside of the second region of linked nucleosides and/or of the first region of linked nucleosides, and/or to the 5' terminal nucleoside of the second region of linked nucleosides.
• the ligands may be conjugated to the 3' terminal nucleoside.
• the one or more ligands are any cell directing moiety, such as lipids, carbohydrates, aptamers, vitamins and / or peptides that bind cellular membrane or a specific target on cellular surface.
• the one or more ligands may contain one or more, in particular three, carbohydrates.
• the one or more, in particular three, carbohydrates can be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide or polysaccharide.
• the one or more carbohydrates may contain one or more, in particular three, N-Acetyl-Galactosamine moieties.
• the target tissue i.e. the liver where AGT is produced
• the target tissue i.e. the liver where AGT is produced
• the oligomeric compounds can exhibit their inhibition of AGT gene more efficiently.
• the one or more, in particular three, ligands may be attached to the oligomeric compound as a biantennary or triantennary configuration.
• the oligomeric compound according to the first aspect disclosed herein may contain internucleoside linkages and where at least one internucleoside linkage is a modified internucleoside linkage.
• the modified internucleoside linkage may be a phosphorothioate or phosphorodithioate internucleoside linkage.
• the oligomeric compound according to the first aspect disclosed herein may contain 1 to 16 phosphorothioate or phosphorodithioate internucleoside linkages.
• modified internucleoside linkages are the subject of some of the embodiments which follow. Certain modified internucleoside linkages are known in the art and described in, for example, Hu et al., Signal Transduction and Targeted Therapy (2020)5:101 .
• the oligomeric compound may contain 7, 8, 9 or 10 phosphorothioate or phosphorodithioate internucleoside linkages.
• the one or more phosphorothioate or phosphorodithioate internucleoside linkages may present at the 5’ region of the first region of linked nucleosides, where advantageously, the oligomeric compound contains three phosphorothioate internucleoside linkages at three adjacent nucleosides at the 5' region.
• the oligomeric compound may contain phosphorothioate or phosphorodithioate internucleoside linkages between at least two, at least three, at least four, or at least five, adjacent nucleosides of the hairpin loop, dependent on the number of nucleosides present in the hairpin loop.
• the oligomeric compound may contain a phosphorothioate or phosphorodithioate internucleoside linkage between each adjacent nucleoside that is present in the hairpin loop.
• At least one nucleoside may contain a modified sugar.
• modified sugars are the subject of some exemplary embodiments, which follow. Certain modified sugars are known in the art and described in, for example, Hu et al., Signal Transduction and Targeted Therapy (2020)5:101 .
• the 2' modified sugar may be selected from 2'-O-alkyl modified sugar, 2'-O-methyl modified sugar, 2'- O-methoxyethyl modified sugar, 2'-O-allyl modified sugar, 2'-C-allyl modified sugar, 2'-deoxy modified sugar such as 2'-deoxy ribose, 2'-F modified sugar, 2'-arabino-fluoro modified sugar, 2'-O-benzyl modified sugar, and 2'-O-methyl-4-pyridine modified sugar.
• At least one modified sugar may be a 2'- O-methyl modified sugar.
• At least one modified sugar may be a 2'-F modified sugar and, advantageously, at most 16 or 17 sugars are 2'-F modified sugars.
• the sugar is ribose.
• sugars of the nucleosides at any of positions 2 and 14 downstream from the first nucleoside of the 5’ region ofthe first region of linked nucleosides do not contain 2'-O-methyl modifications.
• the 3' terminal position ofthe second region of linked nucleosides does not contain a 2'-O-methyl modification.
• sugars of the nucleosides at any of positions 2 and 14 downstream from the first nucleoside of the 5’ region of the first region of linked nucleosides contain 2'-F modifications.
• sugars of the nucleosides of the second region of linked nucleosides that correspond in position to any of the nucleosides of the first region of linked nucleosides at any of positions 11 to 13 downstream from the first nucleoside of the 5’ region of the first region of linked nucleosides contain 2'-F modifications.
• the 3' terminal nucleoside of the second region of linked nucleosides contains a 2'-F modification.
• one or more of the odd numbered nucleosides starting from the 5’ region of the first region of linked nucleosides may be modified, and I or where one or more of the even numbered nucleosides starting from the 5’ region of the first region of linked nucleosides may be modified, where typically the modification of the even numbered nucleosides is a second modification that is different from the modification of odd numbered nucleosides.
• one or more of the odd numbered nucleosides starting from the 3’ region of the second region of linked nucleosides may be modified by a modification that is different from the modification of odd numbered nucleosides of the first region of linked nucleosides.
• one or more of the even numbered nucleosides starting from the 3’ region of the second region of linked nucleosides are modified by a modification that is different from the modification of even numbered nucleosides of the first region of linked nucleoside.
• At least one or more of the modified even numbered nucleosides ofthe first region of linked nucleosides is adjacent to at least one or more of the differently modified odd numbered nucleosides of the first nucleoside region.
• At least one or more of the modified even numbered nucleosides ofthe second nucleoside region is adjacent to at least one or more of the differently modified odd numbered nucleosides of the second region of linked nucleosides.
• sugars of one or more of the odd numbered nucleosides starting from the 5’ region of the first region of nucleosides may be 2'-O-methyl modified sugars.
• one or more of the even numbered nucleosides starting from the 3’ region of the first region of linked nucleosides may be 2'-F modified sugars.
• one or more of the even numbered nucleosides starting from the 5’ region of the second region of linked nucleosides may be 2'-F modified sugars.
• sugars of a plurality of adjacent nucleosides of the second nucleoside region may be modified by a common or different modification.
• the common modification may be a 2'-O-methyl modified sugar.
• the plurality of adjacent 2'-O-methyl modified sugars may be present in at least eight adjacent nucleosides of the first and / or second nucleoside regions.
• the plurality of adjacent 2'-O-methyl modified sugars may be present in three or four adjacent nucleosides of the hairpin loop.
• hairpin loop may contain at least one nucleoside having a modified sugar.
• the at least one nucleoside is adjacent to a nucleoside with a differently modified sugar, where advantageously all adjacent nucleosides in the hairpin loop have a differently modified sugar.
• the modified sugar is a 2'-O-methyl modified sugar
• the differently modified sugar is a 2'-F modified sugar
• one or more nucleosides of the first region of linked nucleosides and / or the second region of linked nucleosides may be an inverted nucleoside and is attached to an adjacent nucleoside via the 3' carbon of its sugar and the 3' carbon of the sugar of the adjacent nucleoside, and / or one or more nucleosides of the first region of linked nucleosides and / or the second region of linked nucleosides is an inverted nucleoside and is attached to an adjacent nucleoside via the 5' carbon of its sugar and the 5' carbon of the sugar of the adjacent nucleoside.
• nucleic acid construct having at least:
• the construct may be designed such that subsequent to in vivo administration the construct disassembles to yield at least first and second discrete nucleic acid targeting molecules that respectively target the RNA portions transcribed from the target genes of (a) and (b); whereby (i) the first nucleic acid targeting molecule is capable of modulating expression of the target gene of (a), and contains, or is derived from, at least the first nucleic acid portion of (a), and (ii) the second nucleic acid targeting molecule is capable of modulating expression of the target gene of (b), and contains, or is derived from, the second nucleic acid portion of (b).
• the construct according to the second aspect and its aforementioned embodiments may at least contain one labile functionality such that subsequent to in vivo administration the construct is cleaved so as to yield the at least first and second discrete nucleic acid targeting molecules.
• the labile functionality may contain one or more unmodified nucleotides.
• the one or more unmodified nucleotides of the labile functionality represent one or more cleavage positions within the construct whereby subsequent to in vivo administration the construct is cleaved at the one or more cleavage positions so as to yield the at least first and second discrete nucleic acid targeting molecules.
• the cleavage positions may be respectively located within the construct so that subsequent to cleavage the first discrete nucleic acid targeting molecule contains, or is derived from, the first nucleic acid duplex region, and the second discrete nucleic acid targeting molecule contains, or is derived from, the second nucleic acid duplex region.
• the first discrete nucleic acid targeting molecule contains or consists of the first nucleic acid portion of (a) and the third nucleic acid portion of (c), and/or the second discrete nucleic acid targeting molecule contains or consists of the second nucleic acid portion of (b) and the fourth nucleic acid portion of (d).
• the first nucleic acid portion has a nucleobase sequence selected from SEQ ID NOs: 1 to 100 in Table 1a;
• the second nucleic acid portion has a nucleobase sequence selected from Table 1a (SEQ ID NOs: 1 to 100);
• the third nucleic acid portion has a nucleobase sequence selected from Table 1 b SEQ ID NOs: 101 to 200; and/or
• the fourth nucleic acid portion has a nucleobase sequence selected from Table 1 b (SEQ ID NOs: 101 to 200). where the third and fourth nucleobase sequences, to the extent they have a length of 14 nucleobases, may be shorter by one, two or three nucleobases, where advantageously the 5'-terminal nucleobase(s) is/are absent.
• the first nucleic acid portion of (a) may be directly or indirectly linked to the fourth nucleic acid portion of (d) as a primary structure.
• the first and the fourth nucleic acid portions may have the nucleobase sequences of SEQ ID NOs: 27 and 127, 44 and 144, 41 and 141 , 97 and 197, 90 and 190, 62 and 162, 52 and 152, 93 and 193, 49 and 149, 73 and 173, 18 and 118, 37 and 137, 56 and 156, 100 and
• the second and third nucleic acid portions may have the nucleobase sequences of SEQ ID NOs: 27 and 127, 44 and 144, 41 and 141 , 97 and 197, 90 and 190, 62 and 162, 52 and 152, 93 and 193, 49 and 149, 73 and 173, 18 and 118, 37 and 137, 56 and 156, 100 and
• sequences of SEQ ID NOs: 27, 52, 56, 62, 75, and 93 may be shorter by one, two, three or four nucleobases, where advantageously the 5'-terminal nucleobase(s) is/are absent.
• the direct or indirect linking may represent either (i) an internucleotide bond, (ii) an internucleotide nick, or (iii) a nucleic acid linker portion of 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, the nucleic acid linker advantageously being single stranded.
• the linking may be direct, thereby giving rise to (a) contiguous strand(s).
• (i) may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, advantageously 2, 3, 4 or 5 base pairs; and/or
• the unmodified nucleotide is at position 19.
• one, more of all of the duplex regions independently may have a length of 10 to 19, 13 to 19, 13, 14 or 15 base pairs, where optionally there is one mismatch within the duplex region.
• the first nucleic acid portion of (a), and / or the second nucleic acid portion of (b), and / orthe third nucleic acid portion of (c), and / or the fourth nucleic acid portion of (d), and / or, to the extent present, the 1 to 8 additional nucleic acid portions as defined previously herein, and / or the passenger nucleic acid portions as defined previously herein, respectively may have a 5’ to 3’ directionality thereby defining 5’ and 3’ regions thereof.
• one or more ligands are conjugated at the 3 ' region, advantageously the 3' end, of any of (I) the third nucleic acid portion of (c), and / or (ii) the fourth nucleic acid portion of (d), and / or, to the extent present, the (ill) passenger nucleic acid portions as defined previously herein.
• one or more ligands may be conjugated at one or more regions intermediate of the 5’ and 3’ regions of any of the nucleic acid portions, advantageously of the third nucleic acid portion of (c), and I or the fourth nucleic acid portion of (d), and / orthe passenger nucleic acid portions as defined previously herein.
• the hexose moiety may contain two or three N-Acetyl-Galactosamine moieties.
• the hexose moiety may contain three N-Acetyl-Galactosamine moieties.
• the one or more ligands may be attached in a linear configuration, or in a branched configuration.
• the one or more ligands may be attached as a biantennary or triantennary configuration, or as a configuration based on single ligands at different positions.
• the ligand may have the following structure:
• the nucleic acid construct may contain 1 to 15 phosphorothioate or phosphorodithioate internucleotide linkages.
• the nucleic acid construct may contain phosphorothioate or phosphorodithioate internucleotide linkages between at least two adjacent nucleotides of the nucleic acid linker portion as defined in previously herein.
• nucleic acid construct according to the second aspect as described above and its aforementioned embodiments, at least one nucleotide of at least one of the following may be modified: the first nucleic acid portion of (a); and / or the second nucleic acid portion of (b); and / or the third nucleic acid portion of (c); and I or the fourth nucleic acid portion of (d); and I or to the extent present, the 1 to 8 additional nucleic acid portions as defined previously herein; and I or to the extent present, the passenger nucleic acid portions as defined previously herein; and / or to the extent present, the nucleic acid linker portion as further defined previously herein.
• one or more of the odd numbered nucleotides starting from the 5’ region of one of the following may be modified, and / or where one or more of the even numbered nucleotides starting from the 5' region of one of the following are modified, where typically the modification of the even numbered nucleotides is a second modification that is different from the modification of odd numbered nucleotides: the first nucleic acid portion of (a); and / or the second nucleic acid portion of (b); and / or the third nucleic acid portion of (c); and I or the fourth nucleic acid portion of (d); and I or to the extent present, the 1 to 8 additional nucleic acid portions as defined previously herein; and / or to the extent present, the passenger nucleic acid portions as defined previously herein.
• one or more of the odd numbered nucleotides starting from the 3’ region of the third nucleic acid portion of (c) may be modified by a modification that is different from the modification of odd numbered nucleotides starting from the 5’ region of the first nucleic acid portion of (a); and I or one or more of the odd numbered nucleotides starting from the 3’ region of the fourth nucleic acid portion of (d) may be modified by a modification that is different from the modification of odd numbered nucleotides starting from the 5’ region of the second nucleic acid portion of (b); and / or one or more of the odd numbered nucleotides starting from the 3’ region of the passenger nucleic acid portions as defined previously herein, to the extent present, may be modified by a modification that is different from the modification of odd numbered nucleotides starting from the 5’ region of the 1 to 8 additional nucleic acid portions as defined previously herein; and I or where one or more of the nucleotides of a nucleotides
• one or more of the even numbered nucleotides starting from the 3’ region of: (i) the third nucleic acid portion of (c), and I or (ii) the fourth nucleic acid portion of (d), and I or (iii) the passenger nucleic acid portions as defined previously herein, to the extent present, may be modified by a modification that is different from the modification of odd numbered nucleotides starting from the 3’ region of these respective portions.
• At least one or more of the modified even numbered nucleotides of (I) the first nucleic acid portion of (a), and I or (ii) the second nucleic acid portion of (b), and I or (iii), to the extent present, the 1 to 8 additional nucleic acid portions as defined previously herein, may be adjacent to at least one or more differently modified odd numbered nucleotides of these respective portions.
• At least one or more of the modified even numbered nucleotides of (I) the third nucleic acid portion of (c), and I or (ii) the fourth nucleic acid portion of (d), and I or (iii), to the extent present, the passenger nucleic acid portions as defined previously herein, may be adjacent to at least one or more differently modified odd numbered nucleotides of these respective portions.
• a plurality of adjacent nucleotides of (i) the third nucleic acid portion of (c), and I or (ii) the fourth nucleic acid portion of (d), and I or (iii), to the extent present, the passenger nucleic acid portions as defined previously herein, may be modified by a common modification.
• the plurality of adjacent commonly modified nucleotides may be 2 to 4 adjacent nucleotides, advantageously 3 or 4 adjacent nucleotides.
• the plurality of adjacent commonly modified nucleotides may be located in the 5’ region of (i) the third nucleic acid portion of (c), and I or (ii) the fourth nucleic acid portion of (d), and I or (iii), to the extent present, the passenger nucleic acid portions previously herein.
• the one or more of the modified nucleotides of first nucleic acid portion of (a) may not have a common modification present in the corresponding nucleotide of the third nucleic acid portion of (c) of the first duplex region; and I or one or more of the modified nucleotides of second nucleic acid portion of (b) may not have a common modification present in the corresponding nucleotide of the fourth nucleic acid portion of (d) of the second duplex region; and I or one or more of the modified nucleotides of the 1 to 8 additional nucleic acid portions, to the extent present, as defined previously herein, may not have a common modification present in the corresponding nucleotide of the corresponding passenger nucleic acid portions of the respective duplex regions.
• the one or more of the modified nucleotides ofthe first nucleic acid portion of (a) may be shifted by at least one nucleotide relative to a commonly modified nucleotide of the third nucleic acid portion of (c); and / or one or more of the modified nucleotides of the second nucleic acid portion of (b) may be shifted by at least one nucleotide relative to a commonly modified nucleotide of the fourth nucleic acid portion of (d); and / or one or more of the modified nucleotides of the 1 to 8 additional nucleic acid portions, to the extent present, as defined previously herein may be shifted by at least one nucleotide relative to a commonly modified nucleotide of the passenger nucleic acid portions, to the extent present, as defined previously herein.
• the modification and I or modifications may be each and individually sugar, phosphate, or base modifications.
• the modification may be selected from nucleotides with 2' modified sugars; conformationally restricted nucleotides (CRN) sugar such as locked nucleic acid (LNA), (S)- constrained ethyl bicyclic nucleic acid, and constrained ethyl (cEt), tricyclo-DNA; morpholino, unlocked nucleic acid (UNA), glycol nucleic acid (GNA), D-hexitol nucleic acid (HNA), and cyclohexene nucleic acid (CeNA).
• CRN conformationally restricted nucleotides
• At least one modification may be a 2'-O-methyl modification in a ribose moiety.
• At least one modification may be a 2'-F modification in a ribose moiety.
• one, two or all three nucleotides of (i) the third nucleic acid portion of (c); and or (ii) the fourth nucleic acid portion of (d); and / or (iii), to the extent present, the passenger nucleic acid portions as defined previously herein; that respectively correspond in position to any of the nucleotides at any of positions 11 to 13 downstream from the first nucleotide of the 5’ region of (i) the first nucleic acid portion of (a); and I or (ii) the second nucleic acid portion of (b); and I or (ill), to the extent present, the 1 to 8 additional nucleic acid portions as defined previously herein; may contain 2'- F modifications in ribose moieties.
• the first nucleic acid portion may be selected from Table 3a;
• the second nucleic acid portion is selected from Table 5b;
• the first nucleic acid portion is selected from Table 1 a, in particular Table 3a;
• the second nucleic acid portion is selected from Table 10b, in particular Table 12b;
• the fourth nucleic acid portion is selected from Table 11 b, in particular Table 13b.
• the (a) first nucleic acid portion no matter if selected from Table 1a in its unmodified form or from Table 3a in its modified form, is selected from group consisting of the constructs denoted AGT_27, AGT_62, AGT_52, AGT_56, AGT_93 and AGT_75.
• the corresponding (c) third nucleic acid portion has the corresponding denotation in Table 1 b or 3b.
• the second nucleic acid portion is selected from the group consisting of A28(14-4)mF and A277(12-5) in Table 6a, in particular in Table 6b, where the second portion consists of the first 19 nucleotides of these entries.
• the corresponding (d) fourth portion contains the remaining nucleotides from the corresponding entries in Table 6a, in particular Table 6b.
• the nucleic acid portions targeting the AGT gene can be arbitrarily combined with the nucleic acid portions targeting the APOC3 gene as mentioned above, such as AGT_27+A28(14-4)mF, AGT_27+A277(12-5), AGT_62+A28(14-4)mF etc. in orderto arrive at a muRNA molecule targeting both, AGT and APOC3. Such combination is also plausible due to the activity of the respective compounds as set forth in the Examples.
• the target gene different from the AGT gene may be a PCSK9 gene.
• the (b) second nucleic acid portion is e.g. an PCSK9 antisense strand and (d) the fourth nucleic portion a strand at least partially complementary thereto.
• the first nucleic acid portion is selected from Table 3a;
• the second nucleic acid portion is selected from Table 7a
• the third nucleic acid portion is selected from Table 3b
• the fourth nucleic acid portion is selected from 7b.
• the first nucleic acid portion is selected from Table 1 a, in particular Table 3a;
• the second nucleic acid portion is selected from Table 8a, in particular Table 8b;
• the third nucleic acid portion is selected from Table 1 b, in particular Table 3b;
• the first nucleic acid portion is selected from Table 1 a, in particular Table 3a;
• the second nucleic acid portion is selected from Table 9a, in particular Table 9b;
• the third nucleic acid portion is selected from Table 1 b, in particular Table 3b;
• the fourth nucleic acid portion is selected from Table 9c, in particular Table 9d.
• Table 9c the fourth nucleic acid portion is selected from Table 9c, in particular Table 9d.
• the first nucleic acid portion is selected from Table 1 a, in particular Table 3a;
• the second nucleic acid portion is selected from Table 10a, in particular Table 11 a;
• the third nucleic acid portion is selected from Table 1 b, in particular Table 3b;
• the fourth nucleic acid portion is selected from Table 12a, in particular Table 13a.
• the (a) first nucleic acid portion is selected from group consisting of the constructs denoted AGT_27, AGT_62, AGT_52, AGT_56, AGT_93 and AGT_75.
• the corresponding (c) third nucleic acid portion has the corresponding denotation in Table 1 b or 3b.
• the second nucleic acid portion is selected from the group consisting of PCS29, PCS44 and PCS53 in Table 22, where the second portion consists of the first 19 nucleotides of these entries.
• the corresponding (d) fourth portion contains the remaining nucleotides from the corresponding entries in Table 22.
• the nucleic acid portions targeting the AGT gene can be arbitrarily combined with the nucleic acid portions targeting the APOC3 gene as mentioned above, such as AGT_27+PCS29, AGT_27+PCS44, AGT_27+PCS53, AGT_62+PCS29 etc. in order to arrive at a muRNA molecule targeting both, AGT and PCSK9. Such combination is also plausible due to the activity of the respective compounds as set forth in the Examples.
• the construct further contains 1 to 8 additional nucleic acid portions that are respectively at least partially complementary to an additional 1 to 8 portions of RNA transcribed from one or more target genes, which target genes different to each other, and / or the same or different to the target genes defined in (a) and (b), and where each of the 1 to 8 additional nucleic acid portions respectively form additional duplex regions with respective passenger nucleic acid portions that are respectively at least partially complementary therewith.
• the construct contains 1 additional nucleic acid portion.
• the construct targets the target genes selected from the group consisting of:
• AGT gene (b) APOC3 gene and (e) Lp(a) gene
• (a) is the first nucleic acid portion that is at least partially complementary to at least a first portion of an RNA, which is transcribed from an AGT gene
• (b) is the second nucleic acid portion that is at least partially complementary to a first portion of an RNA which is transcribed from the second gene
• (e) is a fifth nucleic acid portion that is at least partially complementary to a third portion of RNA which is transcribed from the third gene
• (f) is a sixth nucleic acid portion that is at least partially complementary to (e)
• the target genes are (a) AGT gene, (b) APOC3 gene and (e) PCSK9 gene.
• the target genes are (a) an AGT gene, (b) an APOC3 gene and (e) a PCSK9 gene.
• the first nucleic acid portion is selected from Table 1 a, in particular Table 3a;
• the third nucleic acid portion is selected from Table 1b, in particular Table 3b;
• the fifth nucleic acid portion is selected from Table 7a;
• the first nucleic acid portion is selected from Table 1 a, in particular Table 3a;
• the second nucleic acid portion is selected from Table 4a, in particular Table 4b;
• the third nucleic acid portion is selected from Table 1 b, in particular Table 3b;
• the fourth nucleic acid portion is selected from Table 4c, in particular Table 4d;
• the fifth nucleic acid portion is selected from Table 8a, in particular Table 8b; and (d) the sixth nucleic acid portion is selected from Table 8c, in particular Table 8d.
• the first nucleic acid portion is selected from Table 1 a, in particular Table 3a;
• the second nucleic acid portion is selected from Table 4a, in particular Table 4b;
• the third nucleic acid portion is selected from Table 1b, in particular Table 3b;
• the fourth nucleic acid portion is selected from Table 4c, in particular Table 4d;
• the fifth nucleic acid portion is selected from Table 8a, in particular Table 8b;
• the first nucleic acid portion is selected from Table 1 a;
• the second nucleic acid portion is selected from Table 5a;
• the third nucleic acid portion is selected from Table 1b;
• the fifth nucleic acid portion is selected from Table 9a, in particular Table 9b;
• (f) the sixth nucleic acid portion is selected from Table 9c, in particular Table 9d.
• the first nucleic acid portion is selected from Table 3a;
• the second nucleic acid portion is selected from Table 5b;
• the fifth nucleic acid portion is selected from Table 9a, in particular Table 9b;
• the first nucleic acid portion is selected from Table 1 a, in particular Table 3a;
• the second nucleic acid portion is selected from Table 10b, in particular Table 12b;
• the third nucleic acid portion is selected from Table 1 b, in particular Table 3b;
• the fourth nucleic acid portion is selected from Table 11 b, in particular Table 13b;
• the fifth nucleic acid portion is selected from Table 10b, in particular Table 11 b;
• (f) the sixth nucleic acid portion is selected from Table 12b, in particular Table 12b.
• nucleic acid portions to be used are set forth under the headings "AGT and APOC3 muRNA” and “AGT and PCSK9 muRNA” above.
• all combinations are possible, such as AGT_27+A28(14-4)mF+PCS44, AGT_62+277(12-5)+PCS29, as long as it contains or consists of one AGT-targeting construct, one APCO3-targeting construct and one PCSK9-targeting construct among these specific constructs.
• Such combinations are also possible due to the activity of the respective compounds as set forth in the Examples.
• AGT-associated diseases or disorders simultaneously with APOC3- associated diseases or disorders and PCSK9-associated diseases or disorders.
• compositions and pharmaceutical compositions including shRNA, mxRNA and/or muRNA oligomeric constructs
• the composition contains an oligomeric compound according to the first aspect and/or a nucleic acid construct according the second aspect as described above, and a physiologically acceptable excipient.
• a pharmaceutical composition containing an oligomeric compound according to the first aspect and/or a nucleic acid construct according to the second aspect as described above.
• the pharmaceutical composition may further contain a pharmaceutically acceptable excipient, diluent, antioxidant, and/or preservative.
• the oligomeric compound according to the first aspect and/or the construct according to the second aspect may be the only pharmaceutically active agent(s).
• the pharmaceutical composition furthermore contains one or more further pharmaceutically active agents.
• the further pharmaceutically active agent(s) may be (an) agent(s) which decrease hypertension, where the further pharmaceutically active agent(s) is/are optionally selected from the group consisting of a diuretic, an angiotensin converting enzyme (ACE) inhibitor, an angiotensin II receptor antagonist, a beta-blocker, a vasodilator, a calcium channel blocker, an aldosterone antagonist, an alpha2-agonist, a renin inhibitor, an alpha-blocker, a peripheral acting adrenergic agent, a selective D1 receptor partial agonist, a nonselective alpha-adrenergic antagonist, a synthetic, a steroidal antimineralocorticoid agent; a combination of any of the foregoing; and a hypertension therapeutic agent formulated as a combination of agents, more optionally an angiotensin II receptor antagonist selected from the group consisting of losartan, val
• an oligomeric compound is provided according to the first aspect and/or a nucleic acid construct according to the second aspect as described above, for use in human or veterinary medicine or therapy.
• an oligomeric compound according to the first aspect and/or a nucleic acid construct according to the second aspect as described above may be used to treat, ameliorate and/or prevent a disease or disorder.
• One of the diseases may be an AGT-associated disease or disorder as discussed below. However, more diseases may be treated simultaneously by using muRNA constructs as described herein.
• Such diseases may be, for example, ApoB containing atherogenic Lipoprotein-associated diseases or disorders; Inflammatory signalling pathway-related diseases, such as related to IL-6, CRP and IL-11 ; Platelet aggregation and coagulation pathway-associated diseases; Diabetes; Obesity and metabolic syndrome; and/or other pro-atherogenic factor and modifiable risk factors for CVD-related diseases.
• Corresponding targets in particular in terms of dyslipidaemia, may be selected from the group consisting of APOC3, PCSK9, ANGPTL3, ANGPTL4, Lp(a), ANGPTL8 and ASGR1/2.
• AGT-associated disease or disorder may be selected from the group consisting of APOC3, PCSK9, ANGPTL3, ANGPTL4, Lp(a), ANGPTL8 and ASGR1/2.
• the disease or disorder may be a disease or disorder associated AGT or a disease or disorder requiring reduction of AGT expression.
• the disease or disorder is selected from the group consisting of high blood pressure, hypertension, borderline hypertension, primary hypertension, secondary hypertension isolated systolic or diastolic hypertension, pregnancy-associated hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, labile hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vasculopathy, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiac myopathy, nocturnal hypotension, glomerulosclerosis, coarctation of the aorta, aortic aneurism, ventricular fibrosis, heart failure, myocardial infarction, angina, stroke, renal disease
• the disease or disorder is further an APOC3-associated disease or disorder, or a disease or disorder requiring reduction of APOC3 expression levels, the disease or disorder advantageously being selected from dyslipidaemia including mixed dyslipidaemia; hyperchylomicronaemia including familial hyperchylomicronaemia; hypertriglyceridemia, advantageously severe hypertriglyceridemia and/or hypertriglyceridemia with blood triglyceride levels above 500 mg/dl; inflammation including low-grade inflammation; atherosclerosis; atherosclerotic cardiovascular diseases (ASCVD) including major adverse cardiovascular events (MACE) such as myocardial infarction, stroke and peripheral arterial disease; and pancreatitis including acute pancreatitis.
• dyslipidaemia including mixed dyslipidaemia
• hyperchylomicronaemia including familial hyperchylomicronaemia
• hypertriglyceridemia advantageously severe hypertriglyceridemia and/or hypertriglyceridemia with blood triglyceride levels above 500 mg/dl
• inflammation
• the disease or disorder is further a PCSK9- associated disease or disorder, or a disease or disorder requiring reduction of low-density lipoprotein (LDL) cholesterol, the disease or disorder advantageously being selected from dyslipidaemia including mixed dyslipidaemia, hypercholesterolemia, heterozygous familial hypercholesterolemia, non-familial hypercholesterolemia; atherosclerosis; and atherosclerotic cardiovascular disease (ASCVD) including myocardial infarction, stroke and peripheral arterial disease.
• dyslipidaemia including mixed dyslipidaemia, hypercholesterolemia, heterozygous familial hypercholesterolemia, non-familial hypercholesterolemia; atherosclerosis; and atherosclerotic cardiovascular disease (ASCVD) including myocardial infarction, stroke and peripheral arterial disease.
• dyslipidaemia including mixed dyslipidaemia, hypercholesterolemia, heterozygous familial hypercholesterolemia, non-familial hypercholesterolemia; atherosclerosis; and atheros
• a seventh aspect methods are provided for treating a disease or disorder by administering an oligomeric compound according the first aspect and/or a nucleic acid construct according to the second aspect, to an individual in need of treatment.
• the oligomeric compound and/or the nucleic acid construct may be administered subcutaneously or intravenously to the individual.
• an oligomeric compound according to the first aspect or a nucleic acid construct according to the second aspect may be used in research as a gene function analysis tool.
• an oligomeric compound according to the first aspect and/or a nucleic acid construct according to the second aspect may be used in the manufacture of a medicament for a treatment of a disease or disorder.
• nucleobase sequences of antisense and sense strands of the oligomeric compounds as well as of nucleobase sequences of single-stranded oligomeric compounds, and definitions of modified oligomeric compounds.
• the notation includes nucleobase sequence, sugar modifications, and, where applicable, modified phosphates.
• A represents adenine
• U represents uracil
• C represents cytosine
• G represents guanine.
• 5Phos represents a 5’ terminal phosphate group which is advantageous but not indispensable;
• m represents a methyl modification at the 2' position of the sugar of the underlying nucleoside;
• f represents a fluoro modification at the 2' position of the sugar of the underlying nucleoside;
• r indicates an unmodified (2'-OH) ribonucleotide;
• [Ps] or # represents a phosphorothioate inter-nucleoside linkage;
• I represents an inverted inter-nucleoside linkage, which can be either 3'-3', or 5'-5';
• 3xGalNAc represents a trivalent GalNAc.
• Tables 1 a and 1 b below show nucleobase sequences of antisense and sense strands of 100 oligomeric compounds in accordance with the Examples.
• Table 1a Nucleobase sequences of the antisense strands of 100 constructs
• nucleobase of the 3' terminal nucleotide of each of the sense strands presented within the table can be replaced by A.
• Table 2 below shows the nucleobase sequences of the 100 hairpin constructs as selected in accordance with the Examples.
• the nucleobase sequences are a direct fusion of the antisense sequences of Table 1 a with the corresponding sense sequences of Table 1 b.
• nucleobase of the 3' terminal nucleotide of each of the sense strands presented within the table can be replaced by A.
• Tables 3a to c below shows 100 antisense sequences, sense sequences and hairpins, respectively, with full modification information (modified sugars and, where applicable, modified phosphates).
• each of the above constructs may or may not have a phosphate modification at the 5' end group.
• each of the above constructs may or may not have a "3x GalNAc" coupled to the 3' end group.
• the constructs have a 3x GalNAc ligand, in particular a toothbrush ligand as defined herein.
• each of the above constructs may or may not have a phosphate modification at the 5' end group. Furthermore, and independently, each of the above constructs may or may not have a "3x GalNAc" coupled to the 3' end group.
• the constructs have a 3x GalNAc ligand, in particular a toothbrush ligand as defined herein.
• Particularly advantageous are constructs which in addition have a 5' phosphate, even though this is not a strict requirement, given that in the absence thereof, mammalian cells will add such phosphate in case it is absent from the molecule as administered.
• the term “A277” designates the sequence suitable for RNAi with APOC3, where the first number in the round brackets, i.e. 12 in the present case, designates the number of base pairs within a duplex region within a shRNA, and the second number in the round brackets, in this case 5, designates the number of nucleotides present in the hairpin loop of the shRNA. If there is no designation after the hyphen in the round brackets, it means that the loop consists of 5 nucleotides.
• Tables 4a to 4d below show nucleobase sequences and sugar-phosphate backbone modifications of antisense and sense strands of the 376 APOC3 constructs selected in accordance with the Examples. The disclosed 30 specific oligomeric compounds have been selected from these 376 constructs.
• Table 4a Nucleobase sequences of the APOC3 antisense strands of 376 constructs
• Table 5a Nucleobase sequences of the APOC3 antisense strands of 15 further constructs
• Table 5b Nucleobase sequences and sugar-phosphate backbone modifications of the APOC3 antisense strands of 15 further constructs
• Tables 6a to 6b below show nucleobase sequences and sugar-phosphate backbone modifications of 12 further APOC3 constructs.
• Example 9 the nucleobase sequences of antisense and sense strands of 27 specific oligomeric PCSK9-targeting compounds are given. These are also subject of specific embodiments disclosed further herein. Tables 7a and 7b below shows specific sugar-phosphate backbone modifications of PCSK9 antisense and sense strands of the 27 constructs.
• Table 7a Exemplary sugar-phosphate backbone modifications of PCSK9 antisense strands of the 27 constructs.
• Table 7b Exemplary sugar-phosphate backbone modifications of PCSK9 sense strands of the 27 constructs.
• Tables 8a to 8d below show nucleobase sequences and sugar-phosphate backbone modifications of PCSK9 antisense and sense strands of the 250 constructs selected in accordance with Example 8 below.
• the above disclosed 27 oligomeric PCSK9 compounds have been selected from these 250 constructs.
• Table 8b 250 modified PCSK9 antisense nucleobase sequences corresponding to Table 8a.
• Table 8c 250 PCSK9 unmodified sense nucleobase sequences corresponding to Table 8a.
• Table 8d 250 modified PCSK9 sense strands corresponding to the antisense strands of
• Tables 9a to 9d below show nucleobase sequences and sugar-phosphate backbone modifications of PCSK9 antisense and sense strands of a further 43 constructs.
• Table 9a 43 unmodified PCSK9 antisense nucleobase sequences.
• Table 9c 43 unmodified PCSK9 sense nucleobase sequences corresponding to Table 9a.
• Table 9d 43 modified PSCK9 modified sense strands corresponding to the strands of Table 9a.
• Table 10a shows the nucleobase sequences of PCSK9-targeting antisense portions (e.g. second nucleic acid portions).
• Table 10b shows the nucleobase sequences of APOC3-targeting antisense portions
• Table 11a shows the nucleobase sequences of PCSK9-targeting sense portions (third nucleic acid portions).
• Table 11b shows the nucleobase sequences of APOC3-targeting sense portions (fourth or sixth nucleic acid portions).
• Table 12a shows PCSK9-targeting antisense portions including modification information.
• Table 12b shows APOC3-targeting antisense portions including modification information.
• Table 13a shows PCSK9-targeting sense portions including modification information.
• Table 13b shows APOC3-targeting sense portions including modification information.
• Table 14a shows combination (APOC3 + PSCK9) linked first and fourth nucleic acid portions. Linking is direct to give rise to a single contiguous strand.
• Table 14b shows (combination (APOC3 + PCSK9) linked second and third nucleic acid portions. Linking is direct to give rise to a single contiguous strand.
• the 3' terminal nucleoside of the sense (passenger) strand can include any nucleobase that can be present in an RNA molecule, i.e., can be any of adenine (A), uracil (U), guanine (G) or cytosine (C), advantageously, however, the 3’ terminal residue is a nucleobase that is complementary to the 5' nucleobase of the antisense (guide) strand (first region as defined herein).
• RNAi constructs disclosed herein have been carried out using synthesis methods known to the person skilled in the art, such as synthesis methods disclosed in https://en.wikipedia.org/wiki/Oligonucleotide_synthesis ⁇ retrieved on 16 February 2022 ⁇ , where the methods disclosed on this website are incorporated by reference herein in their entirety .
• the only difference to the synthesis method disclosed in this reference is that GalNAc phosphoramidite immobilized on a support is used in the synthesis method during the first synthesis step.
• Oligomeric compounds targeting AGT were identified by bioinformatics analysis on human AGT mRNA sequence as given in RefSeq sequence ID NM_000029.3 100 compounds were selected for synthesis as mxRNA hairpins. Compounds were dissolved to 50uM in molecular biology grade water. Duplexes were annealed by heating at 95 °C for 5 minutes followed by gradual cooling to room temperature. mxRNAs were annealed by heating at 95 °C for 5 minutes followed by rapid cooling on ice.
• Human primary hepatocytes (5 donor pooled - Sekisui XenoTech, HPCH05+) were thawed immediately prior to experimentation and cultured in 1x complete Williams medium (Gibco, A1217601) supplemented with Hepatocytes plating supplement pack (Gibco, CM3000). FBS concentration was modified from manufacture recipe to a final 2.5% (as opposed to 5%) for compound stability.
• Human primary hepatocytes (5 donor pooled - Sekisui XenoTech, HPCH05+; 2 vials) of cells were thawed by pouring Hepatocyte pellets to warm, 45 ml of Sekisui OptiThaw Hepatocyte Media (K8000), spun at 250xg for 5 min, resuspended in 40 ml of complete 2X WEM 1x Complete WEM: 2.5% FBS, 1 pM Dexamethasone, Pen/Strep (100 U/mL /100 pg/mL), 4 pg/ml Human Insulin, 2 mM GlutaMAX, 15 mM HEPES, pH 7.4.) and counted.Cells were then plated in 50 pL of 2x complete WEM at 25,000 cells per well on 96 well type 1 rat tail Collagen plates and allowed to rest and attach for 5 hours before transfection.
• each 2 pM AGT targeting oligonucleoside compound was added to respective plated hepatocytes for a final concentration of 1 pM in a volume of 100uL 1x complete WEM.
• RNA samples were harvested and RNA isolated using the PureLink Pro 96 total RNA Purification Kit (ThermoFisher, 12173011 A) according to the manufacturer protocol.
• Harvested RNA was assayed for AGT expression via Taqman qPCR using the Luna Universal Probe One-Step RT-qPCR Kit (NEB, E3006).
• a qPCR assay was performed for each sample using a AGT TaqMan probe set (Hs01586213_m-FAM) multiplexed with a common GAPDH VIC probe (ThermoFisher, 4326317E). Thermocycling and data acquisition was performed with an Applied Biosystems Quantstudio 3/5 Real-Time PCR System.
• Example 2 Table 15 below shows IC50 values (in nM) for 30 selected constructs (from Table 3c) selected in accordance with the Examples. Seven serials of 5-fold dilution of these constructs starting at 10OOnM were prepared in basal WEM. Effect of different concentration of constructs on inhibition of AGT mRNA was determined using the protocol described above for the primary screen. Max % KD indicates the maximally achieved knock-down at 1000 nM with 0% being no knock-down and 100% full knock-down. M4K4 was used as reference. Percentage inhibition of construct at different concentration was calculated and the IC50 was determined using GraphPad Prism 9.0.
• Figs. 2a to 2h show the results for inhibiting AGT gene expression for fivefold dilution series for various oligomeric nucleoside compounds targeting AGT (from Table 3c).
• the TMPRSS6 construct used as a positive control, has the following modified structure: 5'vP[mA][fA][mC][fC][mA][fG][mA][fA][mG][fA][mA][fG][mC][fA][mG][fG][mU][fG][iN][fC][mU][fG][fC][ fU][mU][fC][mU][fU][mC][fU][mG][fG][mU][fU]#[3XGalNAc] (SEQ ID NO: 3587).
• HepG2 (ATCC cat. 85011430) cells were maintained by biweekly passing in EMEM supplemented with 10% FBS, 20 mM L-glutamine, 10 mM HEPES pH 7.2, 1 mM sodium pyruvate, 1x MEM non- essential amino acids, and 1x Pen/Strep (EMEM complete).
• Targets to APOC3 were identified by bioinformatic analysis on human APOC3 mRNA sequence as given in RefSeq sequence ID NM_000040, where inter alia it has been taken into consideration that constructs should target APOC3 mRNA irrespective of splice variants and isoforms. 376 targets were selected for synthesis as asymmetric duplexes (14 nucleotide sense strand, 19 nucleotide antisense strand). Compounds were dissolved to 50uM in molecular biology grade water and annealed by heating at 95C for 5 minutes followed by gradual cooling to room temperature.
• RNAiMax ThermoFisher
• 77 oligomeric compounds which exhibit at least 70% target knockdown when assessed with either probe. These 77 compounds are selected from SEQ ID Nos. 601-1352 above in Tables 4a and 4b.
• a yet narrower set of the best performing 30 APOC3 duplexes were tested in dose curves.
• HepG2 cells were collected by trypsinization and seeded in 96 well tissue culture plates at 10,000 cells per well in 50uL complete EMEM with 20% FBS and allowed to rest for 4 hours.
• Transfection complexes were formed by gently mixing 36 pmoles of each duplex in 180 uL OptiMEM with 2.16 uL RNAiMax in 180 uL OptiMEM to make 360 uL total complex. A two fold dilution series was then performed with basal OptiMEM.
• each dilution was added to respective triplicates of HepG2 cells to make a final dilution series of 50 nM down to 0.32 nM in a volume of 100uL, 50/50 EMEM/OptiMEM at 10% FBS.
• Table 16 shows IC50 values (in nM) for the 30 constructs selected in accordance with the Examples.
• Human primary hepatocytes (5 donor pooled - Sekisui XenoTech, HPCH05+) were thawed immediately prior to experimentation and cultured in 1x complete Williams medium (Gibco, A1217601) supplemented with Hepatocytes plating supplement pack (Gibco, CM3000). FBS concentration was modified from manufacture recipe to a final 2.5% (as opposed to 5%) for compound stability.
• 1x Complete WEM 2.5% FBS, 1 pM Dexamethasone, Pen/Strep (100 U/mL /100 pg/mL), 4 pg/ml Human Insulin, 2 mM GlutaMAX, 15 mM HEPES, pH 7.4).
• Hepatocytes were plated on Collagen I (rat tail) coated 96 well tissue culture plates (Gibco, A1142803).
• mice used in this study were human liver-uPA-SCID mice. About 80% of the hepatocytes of each mouse have been replaced by human hepatocytes. The skilled person is aware of ways of producing such mice; where at least some of these ways are shown and referenced in P. Meuleman and G. Leroux-Roels in Antiviral Res. 2008 Dec;80(3):231-8 which is incorporated herein by reference in its entirety.
• mice 36 human liver-uPA-SCID mice. Animals will be grouped by treatment type, dosage, and survival period. Each animal will be treated by subcutaneous injection of test material. Groups 1A and 1 B will have four animals receive a control dose of PBS. Groups
• 2A, 2B, 2C, 3A, 3B, and 3C will receive one dose (10 or 30 mg/kg) with four animals for each dose amount. All animals will be kept alive for 14 or 42 days. See study Table 18 below for details.
• A277(12-5) APOC3-targeting mxRNA construct (SEQ ID Nos. 2169, 2181)
• mice used for the study have to be properly considered.
• an estimated fraction of 20 to 25% of the cells of the humanized liver are sill murine.
• A28(14-4)mF does not target murine APOC3.
• the non-silenced murine APOC3 contributes to the observed levels of triglycerides and total cholesterol.
• the downregulation of these two blood fats in a (purely) human system is expected to exceed what has been observed in this study.
• RNAIMax ThermoFisher
• mice 40 PXB. Animals will be grouped by treatment type, dosage, and survival period. Each animal will be treated by subcutaneous injection of test material.
• Group 1A, 1 B, 1 C, and 1 D will have five animals and receive a single control dose of PBS.
• Group 2A, 2B, 2C, and 2D will have five animals and receive a single dose of AGT-27A at 30 mg/kg.

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