WO2024254589A9 - Produits et compositions - Google Patents

Produits et compositions

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Publication number
WO2024254589A9
WO2024254589A9 PCT/US2024/033246 US2024033246W WO2024254589A9 WO 2024254589 A9 WO2024254589 A9 WO 2024254589A9 US 2024033246 W US2024033246 W US 2024033246W WO 2024254589 A9 WO2024254589 A9 WO 2024254589A9
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WIPO (PCT)
Prior art keywords
sequence
optionally
antisense
oligomeric compound
nucleic acid
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PCT/US2024/033246
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English (en)
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WO2024254589A2 (fr
WO2024254589A3 (fr
Inventor
Dmitry Samarsky
Jack Wei
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Sirnaomics Inc
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Sirnaomics Inc
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Publication of WO2024254589A2 publication Critical patent/WO2024254589A2/fr
Publication of WO2024254589A3 publication Critical patent/WO2024254589A3/fr
Anticipated expiration legal-status Critical
Publication of WO2024254589A9 publication Critical patent/WO2024254589A9/fr
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/1137—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 against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin

Definitions

  • Nucleic acid products are provided that modulate, interfere with, and/or inhibit TMPRSS6 gene expression.
  • Methods, compounds, and compositions are provided for reducing expression of TMPRSS6 mRNA and protein in an animal. Such methods, compounds, and compositions are useful to treat, prevent, or ameliorate TMPRSS6-associated disorders or diseases such as iron overload or hemochromatosis.
  • Iron is an essential mineral, failure of its regulation, multiple blood transfusions, excessive intake, or disorders including genetic disorders may lead to iron overload - an excess of iron stored in the liver, heart and pancreas that can cause life-threatening conditions if left untreated. Iron overload occurs, for example, in patients suffering from hemochromatosis, an inherited disease.
  • TMPRSS6 Transmembrane protease, serine 6; also known as matriptase-2
  • matriptase-2 is an enzyme which is inter alia involved in iron ion homeostasis. It is highly expressed in the liver.
  • TMPRSS6 downregulates hepcidin, the key regulator of iron homeostasis. Since low levels of hepcidin correlate with iron overload, inhibiting the expression of TMPRSS6 is an approach for mitigating iron overload and its associated disorders and diseases.
  • Iron overload may contribute to the development of various disorders and diseases including diabetes, glucose intolerance, cardiovascular diseases, hepatic injury, and steatohepatitis, and may even be lethal
  • Double-stranded RNA capable of complementarily binding expressed mRNA has been shown to block gene expression (Fire et al., 1998, Nature. 1998 Feb 19; 391 (6669):806-1 1 and Elbashir et at, 2001, Nature. 2001 May 24; 41 1 (6836):494-88) by an RNA interference (RNAi) mechanism.
  • RNAi RNA interference
  • Short dsRNAs direct gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and have become a useful tool for studying gene function.
  • RNAi is mediated by the RNA-induced silencing complex (RISC), a sequence-specific, multi-component nuclease that destroys messenger RNAs homologous to the silencing trigger loaded into the RISC complex.
  • Interfering RNA iRNA
  • siRNAs small interfering RNA
  • asRNA antisense RNA
  • miRNA micro- RNA
  • nucleic acid construct comprising at least:
  • a first antisense sequence that is complementary to a first partial sequence of an RNA which is transcribed from a TMPRSS6 gene, wherein optionally being complementary allows for up to three mismatches;
  • a second antisense sequence that is complementary to a second partial sequence of the RNA which is transcribed from the TMPRSS6 gene or a different gene, wherein optionally being complementary allows for up to three mismatches, the second partial sequence being different from the first partial sequence;
  • the present disclosure is directed to an oligomeric compound capable of inhibiting expression of TMPRSS6, wherein the compound comprises an antisense sequence that is complementary to a partial sequence of an RNA transcribed from an TMPRSS6 gene, wherein optionally being complementary allows for up to three mismatches, wherein the antisense sequence is selected from the following sequences, or a portion thereof: sequences of Table 1a (SEQ ID NOs: 1 to 200), wherein the portion optionally has a length of at least 18 nucleosides.
  • the present disclosure is directed to a composition
  • a composition comprising a nucleic acid construct according to the first aspect and/or an oligomeric compound according to the second aspect, and a physiologically acceptable excipient.
  • the present disclosure is directed to pharmaceutical composition comprising a nucleic acid construct according to the first aspect and/or an oligomeric construct according to the second aspect.
  • the present disclosure is directed to the nucleic acid construct according to the first aspect and/or the oligomeric compound according to the second aspect, for use in human or veterinary medicine or therapy.
  • the present disclosure is directed to a nucleic acid construct according to the first aspect and/or an oligomeric compound according to the second aspect for use in a method of treating, ameliorating and/or preventing a disease or disorder.
  • the present disclosure is directed to a method of treating a disease or disorder comprising administration of a nucleic acid construct according to the first aspect and/or an oligomeric compound according to the second aspect, to an individual in need of treatment.
  • the present disclosure is directed to a use of a nucleic acid construct according to the first aspect and or an oligomeric compound according to the second aspect, for use in research as a gene function analysis tool
  • the present disclosure is directed to a use of a nucleic acid construct according to the first aspect and/or an oligomeric compound according to the second aspect in the manufacture of a medicament for a treatment of a disease or disorder.
  • nucleic acid constructs are as follows:
  • ligands e.g. delivery/targeting moieties such as GalNAc and or other carbohydrates, cholesterol, peptides, or small molecules, optionally attached via linkers
  • ligands e.g. delivery/targeting moieties such as GalNAc and or other carbohydrates, cholesterol, peptides, or small molecules, optionally attached via linkers
  • the constructs predominantly comprise chemically modified nucleotides (e.g. 2’F, 2’0Me, LNO, PNA, MOE, BNA, PMO, phosphorothioate, phosphorodithioate, etc.), mostly (but not only) to increase resistance to nucleases;
  • chemically modified nucleotides e.g. 2’F, 2’0Me, LNO, PNA, MOE, BNA, PMO, phosphorothioate, phosphorodithioate, etc.
  • the constructs contain “fragile” components (e.g. chemical linkers, unmodified nucleotides, etc.), which allow the constructs to disassemble upon exposure to certain biologic environments (e.g. exposure to extra- and/or intra-cellular fluids); particular examples could be (but not limited): a) cleavage of the oligo backbone by nucleases in the sites with nonmodified nucleotides; b) cleavage of the chemical linkage due to the change of pH (e.g. in endosomes);
  • fragmentile components e.g. chemical linkers, unmodified nucleotides, etc.
  • disassembly upon exposure to the certain biologic environments releases the components (e.g. the at least partially double-stranded agents capable of triggering RNA interference) to modulate (up- or down-regulate, optionally down-regulate) target gene expression in cells/organisms;
  • the components e.g. the at least partially double-stranded agents capable of triggering RNA interference
  • the constructs can be used to modulate, optionally down-regulate or silence gene expression, to study gene function, or to treat various diseases associated with the target genes to be down-regulated. Effects Achieved by the Disclosed Nucleic Acid Constructs and the Oligomeric Compounds
  • the oligomeric compounds according to the present disclosure significantly reduce TMPRSS6 gene expression, e.g. in vitro using 5-donor primary human hepatocytes, and can be achieved as shown in the examples.
  • the most inhibiting compounds surprisingly produce knockdowns of about 79% or more TMPRSS6 mRNA expression in vitro compared with a negative test.
  • the mxRNA compounds as shown in the examples, are at least capable of producing promising knockdownsTMPRSS6 expression in vitro relative to a negative test.
  • TMPRSS6 expression can be successfully reduced, the compounds have the potential of efficiently reducing the effects of TMPRSS6 overexpression and treating related diseases and/or disorders.
  • the mentioned effects are achieved by using nucleic acid according to the present disclosure for inhibiting TMPRSS6 gene expression in the form of mxRNA constructs having a reduced length of, e.g., 33 or 34 nucleosides compared to conventional shRNA molecules having greater lengths. This can, e g., make a synthesis of mxRNA molecules more cost and production efficient, because fewer units are needed.
  • TMPRSS6-targeting nucleic acid muRNA constructs disclosed herein also are capable of being used in conjunction with another RNA molecule targeting a different position or another gene, e.g., APOC3 or AGT, as part of a muRNA according to the present disclosure.
  • Figure 1 shows primary screens of 30 TMPRSS6 mxRNA sequences and their TMPRSS6 in vitro inhibition by certain mxRNA constructs of Table 3c.
  • Figure 2 shows a concentration dependence of 30 TMPRSS6 mxRNA sequences and their TMPRSS6 in vitro inhibition by certain mxRNA constructs of Table 3c.
  • excipient means any compound or mixture of compounds that is added to a composition as provided herein that is suitable for delivery of an oligomeric compound.
  • nucleoside means a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA) and modified nucleosides. Nucleosides may be linked to a phosphate moiety, phosphate- linked nucleosides also being referred to as "nucleotides”.
  • chemical modification means a chemical difference in a compound when compared to a naturally occurring counterpart.
  • Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications. In reference to an oligonucleotide, chemical modification does not include differences only in nucleobase sequence.
  • furanosyl means a structure comprising a 5-membered ring comprising four carbon atoms and one oxygen atom.
  • naturally occurring sugar moiety means a ribofuranosyl as found in naturally occurring RNA or a deoxyribofuranosyl as found in naturally occurring DNA
  • a "naturally occurring sugar moiety” as referred to herein is also termed as an "unmodified sugar moiety”.
  • a “naturally occurring sugar moiety” or an “unmodified sugar moiety” as referred to herein has a -H (DNA sugar moiety) or -OH (RNA sugar moiety) at the 2 -position of the sugar moiety, especially a -H (DNA sugar moiety) at the 2'-position of the sugar moiety.
  • sugar moiety means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside.
  • modified sugar moiety means a substituted sugar moiety or a sugar surrogate.
  • substituted sugar moiety means a furanosyl that has been substituted.
  • Substituted sugar moieties include but are not limited to furanosyls comprising substituents at the 2 -position , the 3'-position, the 5 -position and / or the 4-position.
  • Certain substituted sugar moieties are bicyclic sugar moieties.
  • 2'-substituted sugar moiety means a furanosyl comprising a substituent at the 2'- position other than H or OH. Unless otherwise indicated, a 2'-substituted sugar moiety is not a bicyclic sugar moiety (i e , the 2' -substituent of a 2'-substituted sugar moiety does not form a bridge to another atom of the furanosyl ring).
  • 2'-F nucleoside refers to a nucleoside comprising a sugar comprising fluorine at the 2' position. Unless otherwise indicated, the fluorine in a 2'-F nucleoside is in the ribo position (replacing the OH of a natural ribose).
  • Duplexes of uniformly modified 2'-fluorinated (ribo) oligonucleotides hybridized to RNA strands are not RNase H substrates while they are analogues retain RNase H activity.
  • sucrose surrogate means a structure that does not comprise a furanosyl and that replaces the naturally occurring sugar moiety of a nucleoside, such that the resulting nucleoside sub-units are capable of linking together and I or linking to other nucleosides to form an oligomeric compound which hybridizes to a complementary oligomeric compound.
  • Such structures include rings comprising a different number of atoms than furanosyl (e.g., 4, 6, or 7-membered rings); replacement of the oxygen of a furanosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both a change in the number of atoms and a replacement of the oxygen.
  • Such structures may also comprise substitutions corresponding to those described for substituted sugar moieties (e.g , 6-membered carbocyclic bicyclic sugar surrogates optionally comprising additional substituents).
  • Sugar surrogates also include more complex sugar replacements (e.g , the non-ring systems of peptide nucleic acid).
  • Sugar surrogates include without limitation morpholinos, cyclohexenyls and cyclohexitols.
  • bicyclic sugar moiety means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure.
  • the 4 to 7 membered ring is a sugar ring.
  • the 4 to 7 membered ring is a furanosyl.
  • the bridge connects the 2 '-carbon and the 4 '-carbon of the furanosyl.
  • nucleotide means a nucleoside further comprising a phosphate linking group.
  • linked nucleosides may or may not be linked by phosphate linkages and thus includes but is not limited to “linked nucleotides”.
  • linked nucleosides are nucleosides that are connected in a continuous sequence (i.e. no additional nucleosides are present between those that are linked).
  • nucleobase means a group of atoms that can be linked to a sugar moiety to create a nucleoside that may be incorporated into an oligonucleotide, and wherein 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.
  • modified nucleoside means a nucleoside comprising at least one chemical modification compared to naturally occurring RNA or DNA nucleosides. Modified nucleosides can comprise a modified sugar moiety and / or a modified nucleobase. As used herein, "bicyclic nucleoside” or"BNA” means a nucleoside comprising a bicyclic sugar moiety.
  • locked nucleic acid nucleoside or "LNA” means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH2-O-2'bridge.
  • 2 '-substituted nucleoside means a nucleoside comprising 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 comprising 2'-H furanosyl sugar moiety, as found in naturally occurring deoxyribonucleosides (DNA).
  • a 2'- deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e.g., uracil).
  • oligonucleotide means a compound comprising a plurality of linked nucleosides.
  • an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and I or unmodified deoxyribonucleosides (DNA) and I or one or more modified nucleosides.
  • modified oligonucleotide means an oligonucleotide comprising at least one modified nucleoside and / or at least one modified internucleoside linkage.
  • 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 comprising 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 comprising two or more substructures.
  • an oligomeric compound comprises an oligonucleotide, such as a modified oligonucleotide.
  • an oligomeric compound further comprises one or more conjugate groups and / or terminal groups and / or ligands
  • an oligomeric compound consists of an oligonucleotide.
  • an oligomeric compound comprises 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).
  • the structure of the sugar-phosphate backbone is not particularly limited and may or may not comprise 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, including the modification status of the sugars (unmodified, 2'-OMe modified, 2'-F modified etc.) and/or of the phosphates.
  • nucleic acid construct or “construct” refers to an assembly of two or more, such as four oligomeric compounds, the compounds being referred to as “antisense or sense sequences” in the context of the first aspect of the disclosure.
  • 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, and/or by non-covalent bonds such as hydrogen bonds, optionally hydrogen bonds between nucleobases such as Watson-Crick base pairing
  • a construct comprises four oligomeric compounds, wherein a first and a fourth compound or portion as well as a second and third compound or portion are connected covalently, respectively, thereby giving rise to two nucleic acid strands which nucleic acid strands are bound to each other by hydrogen bonds.
  • exemplary embodiments provide for an antisense region targeting an TMPRSS6 mRNA to be connected covalently with a sense region which is identical to a region of an TMPRSS6 mRNA, and of an antisense region complementary to the sense region to be connected covalently to a sense region which is complementary to an antisense region targeting an TMPRSS6 mRNA.
  • an optional construct of the disclosure 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, optionally full complementarity or 1 or 2 mismatches.
  • a muRNA is a 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.
  • 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 of an oligonucleotide.
  • a terminal group comprises one or more terminal group nucleosides.
  • 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.
  • conjugate linker or “linker” in the context of a conjugate group means a portion of a conjugate group comprising any atom or group of atoms and which covalently link an oligonucleotide to another portion of the conjugate group.
  • the point of attachment on the oligomeric compound is the 3 '-oxygen atom of the 3'-hydroxyl group of the 3' terminal nucleoside of the oligonucleotide.
  • the point of attachment on the oligomeric compound is the 5'-oxygen atom of the 5'-hydroxyl group of the 5' terminal nucleoside of the oligonucleotide.
  • the bond for forming attachment to the oligomeric compound is a cleavable bond.
  • such cleavable bond constitutes all or part of a cleavable moiety.
  • conjugate groups comprise a cleavable moiety (e.g. , a cleavable bond or cleavable nucleoside) and ligand portion that can comprise one or more ligands, such as a carbohydrate cluster portion, such as an N-Acetyl-Galactosamine, also referred to as "GalNAc", cluster portion.
  • the carbohydrate cluster portion is identified by the number and identity of the ligand
  • the carbohydrate cluster portion comprises 2 GalNAc groups.
  • the carbohydrate cluster portion comprises 3 GalNAc groups.
  • the carbohydrate cluster portion comprises 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 optional carbohydrate cluster has the following formula:
  • cleavable moiety means a bond or group that is cleaved under physiological conditions.
  • a cleavable moiety is cleaved inside a cell or sub-cellular compartments, such as an endosome or lysosome.
  • a cleavable moiety is cleaved by endogenous enzymes, such as nucleases.
  • a cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds.
  • a cleavable moiety is a phosphodiester linkage.
  • cleavable bond means any chemical bond capable of being broken.
  • 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
  • the carbohydrate is N-Acetyl-Galactosamine
  • strand means an oligomeric compound comprising linked nucleosides
  • single strand or “single-stranded” means an oligomeric compound comprising linked nucleosides that are connected in a continuous sequence without a break there between. Such single strands may include regions of sufficient self-complementarity so as to be form a stable selfduplex in a hairpin structure.
  • hairpin means a single stranded oligomeric compound that includes a duplex formed by base pairing between sequences in the strand that are self-complementary and opposite in directionality.
  • hairpin loop means an unpaired loop of linked nucleosides in a hairpin that is created as a result of hybridization of the self-complementary sequences. The resulting structure looks like a loop or a U-shape.
  • short hairpin RNA also denoted as shRNA
  • shRNA comprises 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 1 and/or (a) 5' overhang(s).
  • optionalal are blunt-ended constructs.
  • shRNA is more generic than "mxRNA", as defined below, and may include compounds in which the loop is not or not exclusively formed by a part 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 1 end of the sense strand via covalent bonding over several other groups.
  • Such direct linkage does not include a gap or nick.
  • directionality means the end-to-end chemical orientation of an oligonucleotide based on the chemical convention of numbering of carbon atoms in the sugar moiety meaning that there will be a 5'-end defined by the 5' carbon of the sugar moiety, and a 3'-end defined by the 3' carbon of the sugar moiety.
  • the respective strands run in opposite 5' to 3' directions to permit base pairing between them.
  • duplex means two or more complementary strand regions, or strands, of an oligonucleotide or oligonucleotides, hybridized together by way of non- covalent, sequence-specific interaction there between. Most commonly, the hybridization in the duplex will be between nucleobases adenine (A) and thymine (T), and I or (A) adenine and uracil (U), and I or guanine (G) and cytosine (C).
  • the duplex may be part of a single stranded structure, wherein self-complementarity leads to hybridization, or as a result of hybridization between respective strands in a double stranded construct.
  • double strand or “double stranded” means a pair of oligomeric compounds that are hybridized to one another.
  • a double-stranded oligomeric compound comprises a first and a second oligomeric compound.
  • expression means the process by which a gene ultimately results in a protein.
  • Expression includes, but is not limited to, transcription, post-transcriptional modification (e g , splicing, polyadenylation, addition of 5 '-cap), and translation.
  • transcription refers to the first of several steps of DNA based gene expression in which a target sequence of DNA is copied into RNA (especially mRNA) by the enzyme RNA polymerase. During transcription, a DNA sequence is read by an RNA polymerase, which produces a complementary, antiparallel RNA sequence called a primary transcript.
  • target sequence means a sequence to which an oligomeric compound is intended to hybridize to result in a desired activity with respect to TMPRSS6 expression. Oligonucleotides have sufficient complementarity to their target sequences to allow hybridization under physiological conditions.
  • nucleobase complementarity or “complementarity” when in reference to nucleobases means a nucleobase that is capable of base pairing with another nucleobase.
  • adenine (A) is complementary to thymine (T).
  • adenine (A) is complementary to uracil (U).
  • guanine (G) is complementary to cytosine (C).
  • complementary nucleobase means a nucleobase of an oligomeric compound that is capable of base pairing with a nucleobase of its target sequence.
  • nucleobases at a certain position of an oligomeric compound are 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 to be complementary at that nucleobase pair.
  • Nucleobases comprising certain modifications may maintain the ability to pair with a counterpart nucleobase and thus, are still capable of nucleobase complementarity.
  • non-complementary in reference to nucleobases means a pair of nucleobases that do not form hydrogen bonds with one another.
  • oligomeric compounds e.g. , linked nucleosides, oligonucleotides
  • complementary means the capacity of such oligomeric compounds or regions thereof to hybridize to a target sequence, or to a region of the oligomeric compound itself, through nucleobase complementarity.
  • Complementary oligomeric compounds need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated.
  • complementary oligomeric compounds or regions are complementary at 70% of the nucleobases (70% complementary).
  • complementary oligomeric compounds or regions are 80%> complementary.
  • complementary oligomeric compounds or regions are 90%> complementary.
  • complementary oligomeric compounds or regions are at least 95% complementary. In certain embodiments, complementary oligomeric compounds or regions are 100% complementary.
  • self-complementarity in reference to oligomeric compounds means a compound that may fold back on itself, creating a duplex as a result of nucleobase hybridization of internal complementary strand regions. Depending on how close together and I or how long the strand regions are, then the compound may form hairpin loops, junctions, bulges or internal loops.
  • mismatch means a nucleobase of an oligomeric compound that is not capable of pairing with a nucleobase at a corresponding position of a target sequence, or at a corresponding position of the oligomeric compound itself when the oligomeric compound hybridizes as a result of self-complementarity, when the oligomeric compound and the target sequence and / or self- complementary regions of the oligomeric compound, are aligned.
  • "allowing for up to three mismatches” means that 0, 1 , 2, or 3, optionally 1 , mismatch is present. Therefore, if a sequence is complementary to a target sequence and 1 mismatch is present, it might for example be that the sequence has a U at a certain position and a G or a C is at the corresponding position of the target sequence.
  • hybridization means the pairing of complementary oligomeric compounds (e.g., an oligomeric compound and its target sequence). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.
  • 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 comprises no mismatches or unhybridized nucleobases with respect to its target sequence or a self- complementary region of the oligomeric compound.
  • percent complementarity means the percentage of nucleobases of an oligomeric compound that are complementary to an equal-length portion of a target nucleic acid. Percent complementarity is calculated by dividing the number of nucleobases of the oligomeric compound that are complementary to nucleobases at corresponding positions in the target nucleic acid by the total length 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.
  • type of modification in reference to a nucleoside or a nucleoside of a “type” means the chemical modification of a nucleoside and includes modified and unmodified nucleosides.
  • nucleoside having a modification of a first type may be an unmodified nucleoside.
  • RNA nucleosides that are the same but for comprising different nucleobases are not differently modified.
  • nucleoside comprising a 2'-OMe modified sugar moiety and an unmodified adenine nucleobase and a nucleoside comprising a 2'-OMe modified sugar moiety and an unmodified thymine nucleobase are not differently modified.
  • RNA nucleosides having the same type of modification refers to modifications that are the same as one another, including absence of modifications.
  • two unmodified RNA nucleosides have “the same type of modification,” even though the RNA nucleosides are unmodified
  • Such nucleosides having the same type of modification may comprise different nucleobases.
  • region or regions mean a plurality of linked nucleosides that have a function or character as defined herein, in particular with reference to the claims and definitions as provided herein.
  • regions or portions comprise at least 10, at least 11 , at least 12 or at least 13 linked nucleosides.
  • regions can comprise 13 to 20 linked nucleosides, such as 13 to 16 or 18 to 20 linked nucleosides.
  • a first region as defined herein consists essentially of 18 to 20 nucleosides and a second region as defined herein consists essentially of 13 to 16 linked nucleosides.
  • pharmaceutically acceptable carrier or diluent means any substance suitable for use in administering to an animal
  • a pharmaceutically acceptable carrier or diluent is sterile saline.
  • such sterile saline is pharmaceutical grade saline.
  • substituted nucleoside and “substituent group,” means an atom or group that replaces the atom or group of a named parent compound.
  • a substituent of a modified nucleoside is any atom or group that differs from the atom or group found in a naturally occurring nucleoside (e.g. , a modified 2'- substituent is any atom or group at the 2 '-position of a nucleoside other than H or OH).
  • Substituent groups can be protected or unprotected.
  • compounds of the present disclosure have substituents at one or at more than one position of the parent compound.
  • Substituents may also be further substituted with other substituent groups and may be attached directly or via a linking group such as oxygen or an alkyl or hydrocarbyl group to a parent compound.
  • substituents can be present as the modification on the sugar moiety, for example a substituent present at the 2'-position of the sugar moiety.
  • groups amenable for use as substituents include without limitation, one or more of halo, hydroxyl, alkyl, alkenyl, alkynyl, acyl, carboxyl, alkoxy, alkoxyalkylene and amino substituents.
  • substituents as described herein can represent modifications directly attached to a ring of a sugar moiety (such as a halo, such as fluoro, directly attached to a sugar ring), or a modification indirectly linked to a ring of a sugar moiety by way of an oxygen linking atom that itself is directly linked to the sugar moiety (such as an alkoxyalkylene, such as methoxyethylene, linked to an oxygen atom, overall providing an MOE substituent as described herein attached to the 2'-position of the sugar moiety).
  • alkyl as used herein, means a saturated straight or branched monovalent Ci-e hydrocarbon radical.
  • methyl is the alkyl substituent at the 2'-position of the sugar moiety.
  • the alkyl group typically attaches to an oxygen linking atom at the 2'poisition of the sugar, therefore, overall providing an — O-alkyl substituent, such as an -OCH3 substituent, on a sugar moiety of an oligomeric compound as described herein.
  • alkylene means a saturated straight or branched divalent hydrocarbon radical of the general formula -CnH2n- where n is 1-6. Methylene or ethylene are examples of alkylenes.
  • alkenyl means a straight or branched unsaturated monovalent C2-6 hydrocarbon radical.
  • Ethenyl or propenyl moieties are examples of alkenyls as a substituent at the 2'-position of the sugar moiety.
  • the degree of unsaturation that is present in an alkenyl radical is the presence of at least one carbon to carbon double bond.
  • the alkenyl group typically attaches to an oxygen linking atom at the 2'-position of the sugar, therefore, overall providing a -Oalkenyl substituent, such as an -OCFECF CFE substituent, on a sugar moiety of an oligomeric compound a as described herein. This will be well understood be a person skilled in the art.
  • alkynyl means a straight or branched unsaturated C2-6 hydrocarbon radical.
  • Ethynyl is an example of an alkynyl as a substituent at the 2'-position of the sugar moiety.
  • the 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 an -O-alkynyl substituent on a sugar moiety of an oligomeric compound as described herein. This will be well understood be a person skilled in the art.
  • Carboxyl is a radical having a general formula -CO2H.
  • acyl means a radical formed by removal of a hydroxyl group from a carboxyl radical as defined herein and has the general Formula -C(O)-X where X is typically C1-6 alkyl.
  • alkoxy means a radical formed between an alkyl group, such as a C1-6 alkyl group, and an oxygen atom wherein 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 wherein 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.
  • 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 sequence (also referred to as the guide strand) and a sense sequence (also referred to the passenger strand).
  • the mxRNA comprises duplex region and a hairpin loop, wherein the mxRNA has an approximate length of about 33 or 34 nucleotides.
  • the duplex region comprises a region in which parts of the antisense sequence and substantially the entire sense sequence, typically 14 or 15 nucleotides of each strand, are base paired.
  • the hairpin loop connects both regions, i.e antisense region and sense region, of that duplex via e.g. a phosphate or a phosphorothioate linker, i.e. covalently, while the antisense sequence typically has a length of about 18 to 20 nucleotides and, therefore, forms the antisense duplex region and the loop.
  • the loop, of which the antisense sequence is part furthermore, connects the sense, forming the second strand of the loop, and the antisense sequence.
  • muRNA or “multi RNA” includes nucleic acid constructs comprising more than one, typically two, RNA sequences, i.e. first and second antisense sequence, targeting different regions of TMPRSS6 mRNA.
  • the targeting RNA sequences are also referred to as “antisense” or “guide” strands, while the respective passenger strands, i.e. first and second sense sequences being complementary to the first and second antisense sequence, respectively, are also included in the nucleic acid construct.
  • such muRNA are designed such that subsequent to in vivo administration, they are disassembled and the first and second antisense sequences are released.
  • muRNA A particular example for such muRNA is shown below, where (1 ) is the first antisense sequence, (2) is the first sense sequence being complementary to (1), (3) is the second antisense sequence being complementary to the second sense sequence, while (5) is a labile linker while (6) is a ligand, which will both be explained below.
  • GN designates a GalNAc moiety
  • SBS designates the fragile site which may be implemented as a nucleoside with a non-modified sugar
  • PC1 a is a PCSK9 siRNA sequence. This compound is known to be effective in PCSK9 gene knockdown and was used as negative control compound in the Examples section. This compound is to be considered as Inclisiran® analogues. It will also be understood that oligomeric compounds as described herein may have one or more nonhybridizing nucleosides at one or both ends of one or both strands (overhangs) and I or one or more internal non-hybridizing nucleosides (mismatches) provided there is sufficient complementarity to maintain hybridization under physiologically relevant conditions. Alternatively, oligomeric compounds as described herein may be blunt ended at least one end.
  • nucleic acid construct comprising at least:
  • a first antisense sequence that is complementary to a first partial sequence of an RNA which is transcribed from a TMPRSS6 gene, wherein optionally being complementary allows for up to three mismatches;
  • a second antisense sequence that is complementary to a second partial sequence of the RNA which is transcribed from the TMPRSS6 gene or a different gene, wherein optionally being complementary allows for up to three mismatches, the second partial sequence begin different from the first partial sequence;
  • the target gene different from the TMPRSS6 gene is selected from the group consisting of: a PCSK9 gene, an AGT gene, an APOC3 gene, an ANGPTL3 gene, an ANGPTL4 gene, an Lp(a) gene, an ANGPTL8 gene, an ASGR1/2 gene, and an HSD17B13 gene, further optionally an APOC3 gene.
  • the first/second antisense/sense sequences refer in their broadest sense to nucleobase sequences. In their narrower sense it is clear that these sequences may be composed of linked nucleosides or nucleotides. Complementarity is defined to allow for 0, 1 , 2 or 3 mismatches between an antisense sequence and a target region, whereas all other nucleobases are complementary to the target region.
  • 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 comprises, or is derived from, at least the first antisense sequence of (a), and (ii) the second nucleic acid targeting molecule is capable of modulating expression of the target gene of (b), and comprises, or is derived from, the second antisense sequence of (b).
  • the construct may be designed to disassemble such that the first and second discrete nucleic acid targeting molecules are respectively processed by independent RNAi-induced silencing complexes. Sequence features, labile functionality and structural features of the RNA molecules
  • the construct according to the first aspect and its aforementioned embodiments may at least comprise 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 comprise one or more unmodified nucleotides, wherein optionally the one or more unmodified nucleotides are part of the first and/or second antisense sequences, wherein more optionally the one or more unmodified nucleotides link the first antisense sequence and the second sense sequence and/or the second antisense sequence and the first sense sequence.
  • the one or more unmodified nucleotides of the labile functionality may 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 are respectively located within the construct so that subsequent to cleavage the first discrete nucleic acid targeting molecule comprises, or is derived from, the first nucleic acid duplex region, and the second discrete nucleic acid targeting molecule comprises, or is derived from, the second nucleic acid duplex region.
  • the first discrete nucleic acid targeting molecule comprises or consists of the first antisense sequence of (a) and the first sense sequence of (c)
  • the second discrete nucleic acid targeting molecule comprises or consists of the second antisense sequence of (b) and the second sense sequence of (d).
  • the first antisense sequence comprises at least 18, optionally 19, contiguous nucleotides allowing for up to three mismatches with a sequence being selected from Table 1a, wherein optionally the first antisense sequence is selected from SEQ ID NOs: 32, 94, 121 , 153, and 171 ;
  • the second antisense sequence comprises at least 18, optionally 19, contiguous nucleotides allowing for up to three mismatches with a sequence being selected from Table 1a, wherein optionally the second antisense sequence is selected from SEQ ID Nos. 32, 94, 121 , 153, and 171;
  • the first sense sequence comprises at least 11 , 12, 13, 14 or 15 contiguous nucleotides allowing for up to three mismatches with a sequence being complementary to the first antisense sequence of (a), wherein optionally the first sense sequence is selected from 15 contiguous nucleotides of a sequence being complementary to a sequence selected from SEQ ID NOs 32, 94, 121, 153, and 171 ; and/or
  • the second sense sequence comprises at least 11, 12, 13, 14 or 15 contiguous nucleotides allowing for up to three mismatches with a sequence being complementary to the second antisense sequence of (b), wherein optionally the second sense sequence is selected from 15 contiguous nucleotides of a sequence being complementary to a sequence selected from SEQ ID NOs 32, 94, 121, 153, and 171 , wherein further optionally the first antisense sequence is identical to the second antisense sequence and/or the first sense sequence is identical to the second sense sequence.
  • the first and the second sense sequence may alternatively be independently selected from Table 1c, such as from SEQ ID NOs 432, 494, 521 , 553 and 571 , wherein optionally at least 11 , more optionally 15, contiguous nucleotides out of the sequence in Table 1c may constitute the first and/or the second sense sequence.
  • the first and/or the second sense sequence comprises or consists of the first 15 contiguous nucleotides from the corresponding one selected from Table 1c, such as from SEQ ID NOs 432, 494, 521 , 553 and 571 , counted from the 3' terminus, wherein the last nucleotide at the 3' terminus of the sequence carries an adenine "A" base replacing the base indicated in Table 1c.
  • the first and second antisense sequence have identical sequences being selected from SEQ ID NOs: 32, 94, 121 , 153, and 171.
  • the first and the second sense sequences may be selected complementary sequences of SEQ ID NOs: 32, 94, 121 , 153, and 171 , each of the complementary sequences comprising at least 15 contiguous nucleotides, wherein the last nucleotide at the 3' terminus of the sequence comprising 15 contiguous nucleotides carries an adenine "A" base.
  • the first antisense sequence of (a) is directly or indirectly linked to the second sense sequence of (d) as a primary structure.
  • the first antisense sequence of (a) is selected from Table 1a and the second sense sequence of (d) optionally comprises 15 contiguous nucleotides being complementary to a corresponding part of the second antisense sequence of (b).
  • the second antisense sequence of (b) is directly or indirectly linked to the first sense sequence of (c) as a primary structure.
  • the second antisense sequence of (b) is selected from Table 1a and the first sense sequence of (c) optionally comprises at least 15 contiguous nucleotides being complementary to a corresponding part of the first antisense sequence of (a), wherein the last nucleotide at the 3' terminus of the at least 15 contiguous nucleotides may be an A.
  • sense sequences of the first and second sense sequence have a length of 15 nucleotides T o the extent the above specified entries of the sequence listing have a length of 14 nucleotides, a further nucleotide is to be added at the 5' end of the respective portion, the further nucleotide being complementary to nucleotide at position 15 of the corresponding antisense portion.
  • the construct may further comprise 1 to 8, optionally 2, additional antisense sequences that are respectively at least partially complementary to an additional 1 to 8 partial sequences of RNA transcribed from one or more target genes, which target genes may be the same or different to each other, and / or the same or different to the target genes defined in (a) and / or (b), and wherein each of the 1 to 8 additional antisense sequences respectively form additional duplex regions with respective passenger nucleic acid sequences that are respectively at least partially complementary therewith.
  • the second antisense sequence of (b), and the 1 to 8 additional antisense sequences are directly or indirectly linked to selected passenger nucleic acid sequences as respective primary structures.
  • the direct or indirect linking represents 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 optionally being single stranded, wherein further optionally the (iii) nucleic acid linker is an unmodified nucleotide.
  • the linking is direct, thereby giving rise to (a) contiguous strand(s).
  • (i) is/are 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, optionally 2, 3, 4 or 5 base pairs; and/or
  • (ii) is between the first antisense sequence of (a) and the second antisense sequence of (b).
  • the internucleotide bond involves at least one of the one or more unmodified nucleotides, wherein optionally cleavage occurs at the 3' position of (at least one of) the unmodified nucleotide(s).
  • the first antisense sequence of (a), and / or the second antisense sequence of (b), and / or the first sense sequence of (c), and / or the second sense sequence of (d), are respectively 7 to 25 nucleotides in length.
  • the first antisense sequence of (a) and/or the second antisense sequence of (b) have a length of 18 to 21 , more optionally 18 or 19, and yet more optionally 19 nucleotides.
  • the first antisense sequence of (a) and the second antisense sequence of (b) have a length of 19 nucleotides. It may be further optional that the first sense sequence of (c), and I or the second sense sequence of (d) have a length of 11 to 20, more optionally 13 to 16, and yet more optionally 14 or 15, most optionally 15 nucleotides.
  • the first nucleic portion of (a) and the second nucleic acid portion of (b) may have a length of 19 nucleotides and the third nucleic acid portion of (c) as well as the fourth nucleic acid portion of (b) may have a length of 15 nucleotides.
  • the unmodified nucleotide(s) is / are at any of position 18 to 25, more optionally at any of positions 18 to 21, and most optionally at position 19 and/or the 3' terminal position of the first antisense sequence of (a) and I or of the first sense sequence of (c).
  • the unmodified nucleotide is at position 19 of the first antisense sequence of (a) and/or the second antisense sequence of (b).
  • the nucleic acid linker portion is 1 to 8 nucleotides in length, optionally 2 to 7 or 3 to 6 nucleotides in length, more optionally about 4 or 5 and most optionally 4 nucleotides in length.
  • one, more of all of the duplex regions independently have a length of 10 to 19, more optionally 13 to 19, and yet more optionally 13, 14 or 15 base pairs, most optionally 15 base pairs, wherein optionally there is one mismatch within the duplex region.
  • the nucleic acid construct may be blunt ended.
  • the first antisense sequence is selected from Table 3a, in particular from Construct ID NO: 832, 894, 921 , 953, and 971 ;
  • the second antisense sequence is selected from Table 3a, in particular from Construct ID NO: 832, 894, 921 , 953, and 971 ;
  • the first sense sequence comprises at least 14, in particular 15, contiguous nucleotides being complementary to the corresponding part of the first antisense sequence;
  • the second sense sequence comprises at least 14, in particular 15, contiguous nucleotides being complementary to the corresponding part of the second antisense sequence, wherein optionally the first antisense sequence and the second antisense sequence are the same and/or the first sense sequence and the second sense sequence are the same.
  • the first antisense sequence is selected from Construct ID NO: 832, 894, 921, 953, and 971 selected from Table 3a;
  • the second antisense sequence is selected from Construct ID NO: 832, 894, 921, 953, and 971 selected from Table 3a and/or
  • the first sense sequence comprises Construct ID NO: 1032, 1094, 1121, 1153, or 1171 selected from Table 3b; and/or
  • the second sense sequence comprises Construct ID NO: 1032, 1094, 1121 , 1153, or 1171 selected from Table 3b.
  • the construct comprises two strands, wherein the first strand is selected from Table 2, in particular from SEQ ID NOs: 632, 694, 721, 753, and 771 , and the second strand is selected from Table 2, in particular from SEQ ID NOs: 632, 694, 721 , 753, and 771 , wherein optionally the first and the second strand have the same composition; or the first and second strands are selected from Table 3c, such as Construct ID NOs: 1232, 1294, 1321 , 1353, and 1371 , respectively, wherein optionally the first and the second strand are identical in composition.
  • the 3' terminal positions of the first antisense sequence is replaced with an unmodified nucleotide.
  • the target RNA is an mRNA or another RNA molecule.
  • the first antisense sequence of (a) has a greater number of linked nucleosides compared to the first sense sequence of (c), wherein optionally a ratio between a total number of linked nucleosides of the first antisense sequence of (a) and a total number of linked nucleosides of the first sense sequence of (c) ranges from about 19/16 to about 19/8, or from about 18/16 to about 18/8, wherein more optionally the ratio is 19/15 or 19/14, wherein the same may also apply for the second antisense strand and the second sense strand.
  • the first antisense sequence of (a) has a greater number of linked nucleosides compared to the first sense sequence of (c), wherein optionally a percentage of the total number of the first antisense sequence of (a) relative to the total number of nucleosides of the entire first strand encompassing the first antisense sequence of (a) and the second sense sequence of (d) ranges from about to about 55% to about 60%, optionally from about 55% to about 56%, the same may apply to the second antisense sequence of (b) and the first sense sequence of (c).
  • the total length of either strand of the construct is 30 to 35 nucleotides, optionally 34 nucleotides.
  • the construct is designed to disassemble such that the first and second discrete nucleic acid targeting molecules are respectively processed by independent RNAi-induced silencing complexes
  • the nucleic acid construct according to the first aspect and the aforementioned embodiments may further comprise one or more ligands.
  • one or more ligands are conjugated at the 3' region, optionally the 3' end, of any of (i) the first sense sequence of (c), and / or (ii) the second sense sequence of (d), and I or, to the extent present, the (iii) passenger nucleic acid sequences as defined previously herein.
  • one or more ligands are conjugated at one or more regions intermediate of the 5’ and 3’ regions of any of the sequences, optionally of first sense sequence of (c), and I or second sense sequence of (d), and / or the passenger nucleic acid sequences as defined previously herein. In certain embodiments, one or more ligands are conjugated at the 5' region, optionally the 5' end, or to the 3' region, optionally the 3' end, of any of the sequences.
  • 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 carbohydrates can be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide or polysaccharide.
  • the one or more carbohydrates comprise one or more hexose moieties.
  • the one or more hexose moieties are 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.
  • the hexose moiety may comprise two or three N-Acetyl-Galactosamine moieties.
  • the hexose moiety may comprise 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 nucleotide construct according to the first aspect of the present disclosure or its aforementioned embodiments may comprise one or more phosphorothioate or phosphorodithioate internucleotide linkages.
  • the nucleic acid construct may comprise 1 to 15 phosphorothioate or phosphorodithioate internucleotide linkages.
  • the nucleic acid construct may comprise phosphorothioate or phosphorodithioate internucleotide linkages between at least two adjacent nucleotides of the nucleic acid linker portion as defined previously herein.
  • the nucleic acid construct may comprise a phosphorothioate or phosphorodithioate internucleotide linkage between each adjacent nucleotide that is present in the nucleic acid linker portion.
  • the nucleic acid construct may comprise a phosphorothioate or phosphorodithioate internucleotide linkage linking: the first antisense sequence of (a) to the nucleic acid linker portion as defined in claim 16 (iii); and / or the second antisense sequence of (b) to the nucleic acid linker portion as defined in claim 16 (iii); and / or the first sense sequence of (c) to the nucleic acid linker portion as defined in claim 16 (iii) and / or the second sense sequence of (d) to the nucleic acid linker portion as defined in claim 16 (iii); and I or the 1 to 8 additional antisense sequences as defined in claims 14 or 15 to the nucleic acid linker portion as defined previously herein; and / or the passenger nucleic acid sequences as defined in claims 14 or 15 to the nucleic acid linker portion as defined previously herein.
  • At least one nucleotide of at least one of the following may be modified: the first antisense sequence of (a); and I or the second antisense sequence of (b); and / or the first sense sequence of (c); and / or the second sense sequence of (d); and / or to the extent present, the 1 to 8 additional antisense sequences as defined previously herein; and / or to the extent present, the passenger nucleic acid sequences as defined previously herein; and I or to the extent present, the nucleic acid linker portion as defined previously herein.
  • one or more of the odd numbered nucleotides starting from the 5’ region of one of the following are modified, and I or wherein one or more of the even numbered nucleotides starting from the 5’ region of one of the following are modified, wherein typically the modification of the even numbered nucleotides is a second modification that is different from the modification of odd numbered nucleotides: the first antisense sequence of (a); and / or the second antisense sequence of (b); and / or the first sense sequence of (c); and I or the second dense sequence of (d); and I or to the extent present, the 1 to 8 additional antisense sequences as defined previously herein; and / or to the extent present, the passenger nucleic acid sequences as defined previously herein.
  • a plurality of adjacent nucleotides of (i) the first antisense sequence of (a), and I or (ii) the second antisense sequence of (b), and I or (iii), to the extent present, the 1 to 8 additional antisense sequences as defined previously herein are modified by a common modification and/or, wherein a plurality of adjacent nucleotides of (i) the first sense sequence of (c), and / or (ii) the second sense sequence of (d), and I or (iii), to the extent present, the passenger nucleic acid sequences as defined previously herein, are modified by a common modification.
  • one or more of the odd numbered nucleotides starting from the 5’ region of one of the following are modified, and / or wherein one or more of the even numbered nucleotides starting from the 5’ region of one of the following are modified, wherein typically the modification of the even numbered nucleotides is a second modification that is different from the modification of odd numbered nucleotides: the first antisense sequence of (a); and I or the second antisense sequence of (b); and / or the first sense sequence of (c); and I or the second sense sequence of (d); and I or to the extent present, the 1 to 8 additional antisense sequences as defined previously herein; and I or to the extent present, the passenger nucleic acid sequences as defined previously herein.
  • 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 sequences 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.
  • one or more of the odd numbered nucleotides starting from the 3’ region of the first sense strand of (c) are modified by a modification that is different from the modification of odd numbered nucleotides starting from the 5’ region of the first antisense strand of (a); and / or wherein one or more of the odd numbered nucleotides starting from the 3’ region of the second sense strand of (d) are modified by a modification that is different from the modification of odd numbered nucleotides starting from the 5’ region of the second antisense strand of (b); and I or wherein one or more of the odd numbered nucleotides starting from the 3’ region of the passenger nucleic acid sequence as defined previously herein, to the extent present, are 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 antisense sequences as defined previously herein; and / or wherein one or more of the nucleotides of a nucle
  • one or more of the even numbered nucleotides starting from the 3’ region of: (i) the first sense sequence of (c), and / or (ii) the second sense sequence of (d), and / or (iii) the passenger nucleic acid sequences as defined previously herein, to the extent present, are modified by a modification that is different from the modification of odd numbered nucleotides starting from the 3’ region of these respective portions.
  • the 1 to 8 additional antisense sequences as defined previously herein is adjacent to at least one or more differently modified odd numbered nucleotides of these respective portions.
  • one or more of the odd numbered nucleotides starting from the 3’ region of the first sense strand of (c) are modified by a modification that is different from the modification of odd numbered nucleotides starting from the 5’ region of the first antisense strand of (a); and / or wherein one or more of the odd numbered nucleotides starting from the 3’ region of the second sense strand of (d) are modified by a modification that is different from the modification of odd numbered nucleotides starting from the 5’ region of the second antisense strand of (b); and I or wherein one or more of the odd numbered nucleotides starting from the 3’ region of the passenger nucleic acid sequence as defined previously herein, to the extent present, are 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 antisense sequences as defined previously herein; and / or wherein one or more of the nucleotides of a nucle
  • one or more of the even numbered nucleotides starting from the 3’ region of: (i) the first sense sequence of (c), and I or (ii) the second sense sequence of (d), and / or (iii) the passenger nucleic acid sequences as defined previously herein, to the extent present, are 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 antisense sequence of (a), and / or (ii) the second antisense sequence of (b), and / or (iii), to the extent present, the 1 to 8 additional antisense sequences as previously herein, is 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 first sense sequence of (c), and I or (ii) the second sense sequence of (d), and I or (iii), to the extent present, the passenger nucleic acid sequences as defined previously herein, is adjacent to at least one or more differently modified odd numbered nucleotides of these respective portions
  • a plurality of adjacent nucleotides of (i) the first antisense sequence of (a), and / or (ii) the second antisense sequence of (b), and / or (iii), to the extent present, the 1 to 8 additional antisense sequences as defined previously herein, are modified by a common modification.
  • a plurality of adjacent nucleotides of (i) the first sense sequence of (c), and I or (ii) the second sense sequence of (d), and I or (Hi) to the extent present, the passenger nucleic acid sequences as defined in previously herein, are modified by a common modification.
  • the plurality of adjacent commonly modified nucleotides are 2 to 4 adjacent nucleotides, optionally 3 or 4 adjacent nucleotides.
  • the plurality of adjacent commonly modified nucleotides are located in the 5’ region of (i) the first sense sequence of (c), and I or (ii) the second sense sequence of (d), and I or (iii), to the extent present, the passenger nucleic acid sequences as defined previously herein.
  • a plurality of adjacent commonly modified nucleotides are located in the nucleic acid linker portion as previously herein.
  • the one or more of the modified nucleotides of first antisense sequence of (a) do not have a common modification present in the corresponding nucleotide of the first sense sequence of (c) of the first duplex region; and I or one or more of the modified nucleotides of second antisense sequence of (b) do not have a common modification present in the corresponding nucleotide of the second sense sequence of (d) of the second duplex region; and I or one or more of the modified nucleotides of the 1 to 8 additional antisense sequences, to the extent present, as defined previously herein, do not have a common modification present in the corresponding nucleotide of the corresponding passenger nucleic acid sequences of the respective duplex regions.
  • the one or more of the modified nucleotides of the first antisense sequence of (a) are shifted by at least one nucleotide relative to a commonly modified nucleotide of the first sense sequence of (c); and I or one or more of the modified nucleotides of the second antisense sequence of (b) are shifted by at least one nucleotide relative to a commonly modified nucleotide of the second sense sequence of (d); and / or one or more of the modified nucleotides of the 1 to 8 additional antisense sequences, to the extent present, as defined previously herein are shifted by at least one nucleotide relative to a commonly modified nucleotide of the passenger nucleic acid sequences, to the extent present, as defined elsewhere herein
  • the modification and I or modifications are each and individually sugar, phosphate, or base modifications.
  • the modification is 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
  • LNA locked nucleic acid
  • S locked nucleic acid
  • cEt constrained ethyl
  • tricyclo-DNA tricyclo-DNA
  • morpholino unlocked nucleic acid
  • NDA unlocked nucleic acid
  • GNA glycol nucleic acid
  • HNA D-hexitol nucleic acid
  • CeNA cyclohexene nucleic acid
  • the 2' modified sugar is selected from 2'-O-alkyl modified sugar, 2'-O-methyl modified sugar, 2'-0-methoxyethyl modified sugar, 2'-O-ally I modified sugar, 2'-C-ally I 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, 2'-amino modified sugar, and 2'-O-methyl-4-pyridine modified sugar.
  • the base modification is any one of an abasic nucleotide and a non-natural base comprising nucleotide.
  • at least one modification is a 2'-O-methyl modification in a ribose moiety.
  • At least one modification is a 2'-F modification in a ribose moiety.
  • the nucleotides at any of positions 2 and 14 downstream from the first of (i) the first antisense sequence of (a); and I or (ii) the second antisense sequence of (b); and I or (iii), to the extent present, the 1 to 8 additional antisense sequences as previously herein; contain 2'-F modifications in ribose moieties.
  • nucleotides contain either 2'-O-methyl modifications or 2'-F modifications in ribose moieties, optionally with the exception of the unmodified nucleotide(s) as defined previously herein.
  • the remaining nucleotides contain 2'-O-methyl modifications in ribose moieties.
  • the one or more, optionally one, unmodified nucleotide represents any of the nucleotides of the nucleic acid linker portion as defined in claim 16 (iii), optionally the nucleotide of the nucleic acid linker portion as defined previously herein, that is adjacent to (i), the first sense sequence of (c); and I or (ii) the second sense sequence of (d); and I or (iii), to the extent present, the passenger nucleic acid sequence as defined previously herein.
  • small hairpin (shRNA) and mxRNA oligomeric compounds are small hairpin (shRNA) and mxRNA oligomeric compounds.
  • the present disclosure is related to an oligomeric compound capable of inhibiting expression of TMPRSS6, wherein the compound comprises an antisense sequence that is complementary to a partial sequence of an RNA transcribed from an TMPRSS6 gene, wherein optionally complementary allows for up to three mismatches, wherein the antisense sequence is selected from the following sequences, or a portion thereof: sequences of Table 1a (SEQ ID NOs: 1 to 200), wherein the portion optionally has a length of at least 18 nucleosides.
  • the antisense sequence and the sense sequence refer in their broadest sense to nucleobase sequences. In their narrower sense it is clear that these sequences may be composed of linked nucleosides or nucleotides Complementarity is defined to allow for 0, 1, 2 or 3 mismatches between an antisense sequence and a target region, whereas all other nucleobases are complementary to the target region.
  • the oligomeric compound further comprises at least a second region of linked nucleosides having a sense sequence that is at least partially complementary to the antisense sequence and is selected from the following sequences, or a portion thereof: sequences of Table 1b (SEQ ID NOs: 201 to 400), wherein the portion optionally has a length of at least 8, 9, 10 or 11, more optionally at least 10, nucleosides.
  • the antisense sequence is selected from the following sequences of Table 1a, or a portion thereof: SEQ ID NOs: SEQ ID NOs: 32, 94, 121, 153, and 171
  • the sense sequence is selected from the following sequences of T able 1 b, or a portion thereof: SEQ ID NOs: 232, 294, 321 , 353, and 371.
  • the antisense sequence is selected from the following sequences of Table 3a, or a portion thereof: Construct ID NO: 832, 894, 921, 953, and 971.
  • the sense sequence is selected from the following sequences of T able 3b, or a portion thereof: SEQ ID NOs: 1032, 1094, 1121, 1153, and 1171.
  • the antisense strand may consist of 18 to 35, optionally 18 to 20, more optionally 18 or 19, and yet more optionally 19 linked nucleosides.
  • the sense strand may consist of 10 to 35, optionally 10 to 20, more optionally 10 to 16, and yet more optionally 10 to 15, in particular 13, 14 or 15 linked nucleosides
  • the oligomeric compound comprises at least one complementary duplex region that comprises at least a portion of the antisense sequence directly or indirectly linked to at least a portion of the sense sequence, wherein optionally the duplex region has a length of 10 to 19, more optionally 12 to 19, and yet more optionally 12 to 15, in particular 14 or 15, base pairs, wherein optionally there is one mismatch within the duplex region.
  • each of the antisense sequence and the sense sequence has a 5’ to 3’ directionality thereby defining 5’ and 3’ regions respectively thereof.
  • the 5’ region of the antisense sequence is directly or indirectly linked to the 3’ region of the second region of linked nucleosides, for example by complementary base pairing, wherein optionally the 5' terminal nucleoside of the antisense sequence base pairs with the 3' terminal nucleoside of the sense sequence, wherein optionally the base of the 5' terminal nucleoside of the antisense sequence is U and the base of the 3' terminal nucleoside of the second region is A.
  • the 3’ region of the antisense sequence is directly or indirectly linked to the 5’ region of the sense sequence, wherein optionally the antisense sequence is directly and covalently linked to the sense sequence such as by a phosphate, a phosphorothioate, or a phosphorodithioate, wherein more optionally a 3' terminal nucleoside of the antisense sequence is directly and covalently linked to a 5' terminal nucleoside of the sense sequence by a phosphate, a phosphorothioate, or a phosphorod ithioate.
  • the compound consists of the antisense sequence and the sense sequence In certain embodiments there is an intervening nucleic acid sequence between the antisense and the sense sequence.
  • the oligomeric compound comprises or consists of a single strand comprising or consisting of the antisense sequence, the sense sequence and the intervening nucleic acid sequence, wherein at least a portion of the intervening nucleic acid sequence is directly or indirectly linked to at least a portion of the sense sequence so as to form the at least partially complementary duplex region.
  • the oligomeric compound comprises or consists of a single strand comprising or consisting of the antisense and the sense strand, wherein at least a portion of the antisense sequence is directly or indirectly linked to at least a portion of the sense sequence so as to form the at least partially complementary duplex region
  • the antisense and the sense sequence are directly adjacent on the single strand.
  • the antisense sequence has a greater number of linked nucleosides compared to the sense sequence, wherein optionally a ratio between a total number of linked nucleosides of the antisense sequence and a total number of linked nucleosides of the sense sequence ranges from about 19/15 to about 19/8, or from about 18/15 to about 18/8; and/or a percentage of the total number of linked nucleosides of the antisense sequence relative to the total number of nucleosides of the oligomeric compound ranges from about to about 55% to about 60%
  • the additional number of linked nucleosides of the first nucleoside region form a hairpin loop linking the first and second regions of linked nucleosides, wherein optionally a part of the first nucleobase sequence being complementary RN A transcribed from a TMPRSS6 gene forms the hairpin loop, wherein the loop comprises 2 to 5, optionally 4 or 5, nucleosides.
  • the single strand is selected from T able 2, in particular selected from the group consisting of SEQ IDs NO: 632, 694, 721 , 753, and 771.
  • the single strand is selected from Table 3c, in particular selected from Construct ID NOs: 1232, 1294, 1321 , 1353, and 1371.
  • a hairpin loop is present at the 3' region of the antisense sequence, wherein optionally one, two or more 3' terminal nucleosides of the antisense sequence, to the extent the nucleobases of the one, two or more 3' terminal nucleosides permit, fold back and form or contribute a part of the duplex region being on the same side of the duplex as the sense sequence.
  • the intervening nucleic acid sequence or a 3'-terminal portion optionally consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linked nucleosides, more optionally 4 or 5 nucleosides, of the antisense sequence and/or a 5'-terminal portion, optionally consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linked nucleosides, of the sense sequence form a hairpin loop.
  • the hairpin loop comprises 1 to 8, 2 to 7, 3 to 6, optionally 4 or 5 linked nucleosides
  • the oligomeric compound according to the second aspect further comprises one or more ligands.
  • the one or more ligands are conjugated to the sense sequence and/or the antisense sequence.
  • the one or more ligands are conjugated at the 3' region, optionally at the 3' terminal nucleoside of the sense sequence and/or of the antisense sequence, and/or to the 5' terminal nucleoside of the second region of linked nucleosides
  • 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 comprise one or more carbohydrates.
  • the one or more carbohydrates can be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide or polysaccharide.
  • the one or more carbohydrates comprise or consist of one or more hexose moieties.
  • the one or more hexose moieties are 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.
  • the one or more carbohydrates comprise one or more N-Acetyl- Galactosamine moieties.
  • the oligomeric compound comprises two or more N-Acetyl-Galactosamine moieties, optionally three.
  • the one or more ligands are attached to the oligomeric compound, optionally to the sense sequence thereof, in a linear configuration, or in a branched configuration, such as shown below In certain embodiments, the one or more ligands are attached to the oligomeric compound as a biantennary or triantennary configuration.
  • the oligomeric compound comprises internucleoside linkages and wherein at least one internucleoside linkage is a modified internucleoside linkage.
  • the modified internucleoside linkage is a phosphorothioate or phosphorodithioate internucleoside linkage.
  • the oligomeric compound comprises 1 to 16 phosphorothioate or phosphorodithioate internucleoside linkages
  • the oligomeric compound comprises 7, 8, 9 or 10 phosphorothioate or phosphorodithioate internucleoside linkages.
  • the oligomeric compound comprises one or more phosphorothioate or phosphorodithioate internucleoside linkages at the 5’ region of the antisense sequence.
  • the oligomeric compound comprises one or more phosphorothioate or phosphorodithioate internucleoside linkages at the 5’ region of the sense sequence, wherein optionally, the oligomeric compound comprises three phosphorothioate internucleoside linkages at three adjacent nucleosides at the 5' region.
  • the oligomeric compound comprises phosphorothioate or phosphorodithioate internucleoside linkages between at least two, optionally at least three, optionally at least four, optionally at least five, adjacent nucleosides of the hairpin loop, dependent on the number of nucleosides present in the hairpin loop.
  • the oligomeric compound comprises a phosphorothioate or phosphorodithioate internucleoside linkage between each adjacent nucleoside that is present in the hairpin loop.
  • the oligomeric compound according to the second aspect may at least comprise one nucleoside a modified sugar.
  • the modified sugar is selected from 2' modified sugars, a conformationally restricted nucleoside (CRN) sugar such as locked nucleic acid (LNA) sugar, (S)-constrained ethyl bicyclic nucleic acid, and constrained ethyl (cEt) sugar, tricyclo-DNA, morpholino, unlocked nucleic acid (UNA) sugar, glycol nucleic acid (GNA), D-hexitol nucleic acid (HNA), and cyclohexene nucleic acid (CeNA).
  • CRN conformationally restricted nucleoside
  • LNA locked nucleic acid
  • S locked nucleic acid
  • cEt constrained ethyl
  • tricyclo-DNA tricyclo-DNA
  • morpholino unlocked nucleic acid
  • UAA unlocked nucleic acid
  • GNA glycol nucleic acid
  • HNA D-hexitol nucleic acid
  • CeNA cyclohexene nucle
  • the 2' modified sugar is selected from 2'-O-alkyl modified sugar, 2'-O-methyl modified sugar, 2'-0-methoxyethyl modified sugar, 2'-O-ally I modified sugar, 2'-C-ally I 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 is a 2'-O-methyl modified sugar.
  • At least one modified sugar is a 2'-F modified sugar and, optionally, 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 of the antisense sequence do not contain 2'-O-methyl modifications.
  • the 3' terminal position of the sense sequence 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 antisense sequence contain 2'-F modifications.
  • sugars of the nucleosides of the sense strand which correspond in position to any of the nucleosides of the antisense strand at any of positions 11 to 13 downstream from the first nucleoside of the 5’ region of the antisense strand, 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 antisense sequence are modified, and I or wherein one or more of the even numbered nucleosides starting from the 5’ region of the antisense sequence are modified, wherein 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 sense sequence are modified by a modification that is different from the modification of odd numbered nucleosides of the antisense sequence
  • one or more of the even numbered nucleosides starting from the 3’ region of the sense sequence are modified by a modification that is different from the modification of even numbered nucleosides of the antisense sequence as defined previously herein.
  • At least one or more of the modified even numbered nucleosides of the antisense sequence is adjacent to at least one or more of the differently modified odd numbered nucleosides of the antisense sequence.
  • At least one or more of the modified even numbered nucleosides of the sense sequence is adjacent to at least one or more of the differently modified odd numbered nucleosides of the sense sequence.
  • sugars of one or more of the odd numbered nucleosides starting from the 5’ region of the antisense sequence are 2'-O-methyl modified sugars.
  • one or more of the even numbered nucleosides starting from the 3’ region of the antisense sequence are 2'-F modified sugars.
  • sugars of one or more of the odd numbered nucleosides starting from the 5’ region of the sense sequence are 2'-0 methyl modified sugars.
  • one or more of the even numbered nucleosides starting from the 5’ region of the sense sequence are 2'-F modified sugars
  • sugars of a plurality of adjacent nucleosides of the antisense sequence are modified by a common or different modification. In certain embodiments, sugars of a plurality of adjacent nucleosides of the sense sequence are modified by a common or different modification.
  • sugars of a plurality of adjacent nucleosides of the hairpin loop are modified by a common or different modification.
  • the common modification is a 2'-F modified sugar.
  • the common modification is a 2'-O-methyl modified sugar.
  • the plurality of adjacent 2'-O-methyl modified sugars are present in at least eight adjacent nucleosides of the antisense and/or sense sequence.
  • the plurality of adjacent 2'-O-methyl modified sugars are present in three or four adjacent nucleosides of the hairpin loop.
  • the hairpin loop comprises at least one nucleoside having a modified sugar.
  • the at least one nucleoside is adjacent to a nucleoside with a differently modified sugar, wherein optionally 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 antisense sequence and / or the sense sequence is 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
  • / or one or more nucleosides of antisense sequence and I or the sense sequence 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.
  • the oligomeric compound is blunt ended.
  • either the antisense or sense sequence has an overhang.
  • the oligomeric compound has a total length of about 25 to about 37 nucleosides, in particular about 33 or about 34 nucleosides.
  • a terminal nucleoside at a 5' position of the antisense sequence is selected from the group consisting of A, U, G and C, optionally U, and, wherein optionally, a terminal nucleoside at a 3' position of the sense region is replaced by a base being complementary to the base at the 5' position of the first region, optionally A.
  • the antisense and sense sequences are only being composed of nucleobases selected from the group consisting of A, U, G, C and not T.
  • the nucleosides do not contain a 2'-deoxy modification.
  • the third aspect of the present disclosure relates to a composition comprising a nucleic acid construct according to the first aspect and/or an oligomeric compound to the second aspect, and a physiologically acceptable excipient.
  • the fourth aspect of the present disclosure relates to a pharmaceutical composition comprising a nucleic acid construct according to the first aspect and/or an oligomeric compound according to the second aspect.
  • the pharmaceutical composition may further comprise a pharmaceutically acceptable excipient, diluent, antioxidant, and/or preservative.
  • nucleic acid construct according to the first aspect and/or the oligomeric compound according to the second aspect is the only pharmaceutically active agent.
  • the pharmaceutical composition is to be administered to patients or individuals which are statin-intolerant and/or for whom statins are contraindicated.
  • the further pharmaceutically active agent(s) is/are an RNAi agent which is directed to a target different from TMPRSS6 and/or a lipid-lowering agent distinct from the construct, wherein the lipid-lowering agent is optionally ezetimib; Vascepa; Vupanorsen; statins such as Rosuvastatin and Simvastatin; and/or fibrates such fenofibrate.
  • construct and the further pharmaceutically active agent(s) are to be administered concomitantly or in any order.
  • a fifth aspect of the present disclosure is related to the nucleic acid construct according to the first aspect and/or an oligomeric compound according to the second aspect for use in human or veterinary medicine or therapy; involving a step of administration of a therapeutically effective amount of the nucleic acid construct or the oligomeric compound to a patient or animal in need thereof
  • an administration of the nucleic acid construct according to the first aspect and/or an oligomeric compound according to the second aspect may be subcutaneously.
  • the present disclosure is directed to a nucleic acid construct according to the first aspect and/or an oligomeric compound according to the second aspect, for use in a method of treating, ameliorating and/or preventing a disease or disorder.
  • the disease or disorder is a TMPRSS6-associated disease or disorder requiring reduction of TMPRSS6 expression levels.
  • disease or disorder is associated with iron overload and/or a disorder of ineffective erythropoiesis.
  • the disease or disorder may be a TMPRSS6-associated disease or disorder, wherein the disease or disorder is selected from the group consisting of a TMPRSS6-associated disease or disorder; a disease or disorder associated with excess accumulation of iron and/or requiring reduction of iron levels such as transfusional iron overload, excess parenteral iron supplement, and excess dietary iron intake; a disease or disorder selected from blood disorders such as hemochromatosis, anaemia, thalassaemia, porphyria, and hemosiderosis; bone marrow failure syndromes and myelodysplasia; neurological disorders such as Parkinson's disease, Alzheimer's disease, and Friedreich's ataxia; and/or chronic liver diseases
  • the nucleic acid construct and/or the oligomeric compound is administered at a dose of about 0.05 mg/kg to about 50.0 mg/kg, optionally 0.05 mg/kg to about 30.0 mg/kg or 10 mg/kg to about 50 mg/kg of body weight of the human subject.
  • the administering results in a reduction of lipid levels, including triglyceride levels, cholesterol levels, insulin resistance, glucose levels or a combination thereof
  • TMPRSS6 knockdown renders it possible such compounds may be used in treating such diseases. This is because reducing TMPRSS6 levels is also at least credibly and plausibly connected with a reduction of triglyceride levels and/or cholesterol levels.
  • T ables show nucleobase sequences of antisense and sense strands of oligomeric compounds of the disclosure as well as of nucleobase sequences of single-stranded oligomeric compounds of the disclosure, and definitions of modified oligomeric compounds of the disclosure (the notation including nucleobase sequence, sugar modifications, and, where applicable, modified phosphates).
  • A represents adenine
  • U represents uracil
  • C represents cytosine
  • G represents guanine
  • P represents a terminal phosphate group which may or may not be present;
  • m represents a methyl modification at the 2' position of the sugar of the underlying nucleoside, wherein an accordingly modified nucleotide such as mG is sometimes displayed in brackets ([mG]);
  • f represents a fluoro modification at the 2' position of the sugar of the underlying nucleoside, wherein an accordingly modified nucleotide such as fG is sometimes displayed in brackets ([fG]);
  • r indicates an unmodified (2'-OH) ribonucleotide, wherein corresponding nucleotide such as rG is sometimes displayed in brackets ([rG]);
  • 3xGalNAc represents an optionally present trivalent GalNAc
  • Mono-GalNAc-PA represents an optional one of optionally three GalNAc bearing moieties, the assembly of three Mono-GalNAc-PA moieties also being referred to as "toothbrush", wherein the individual moieties are connected by phosphoramidates ("PA"); see the embodiments for an illustration.
  • Table 1a shows the nucleobase sequences of TMPRSS6-targeting antisense sequences (first and/or second antisense sequences of muRNA or antisense sequence of mxRNA).
  • nucleobase of the 3' terminal nucleotide of each of the sense strands presented within the table can be replaced by A
  • Table 1c shows a selection of specific 20mer sense sequences, which can be the basis for the first and/or the second sense sequence of muRNA, as well as their targeting regions.
  • nucleobase of the 3' terminal nucleotide of each of the sense strands presented within the table can be replaced by A
  • Table 2 shows the nucleobase sequences of TMPRSS6-targeting sequences (linked antisense and sense sequences for mxRNA).
  • each of the above constructs may or may not have a phosphate modification at the 5 1 end group.
  • the 3' terminus of the antisense sequence may be unmodified and not carry a phosphorothioate but a phosphate.
  • Table 3b shows TMPRSS6-targeting sense sequences (i.e. sense sequences for mxRNA) including sugar modification information.
  • each of the above constructs may or may not have a "3x GalNAc" coupled to the 3 1 end group.
  • constructs with a 3x GalNAc ligand in particular a toothbrush ligand as defined herein.
  • Table 3c shows linked TMPRSS6-targeting antisense and sense sequences including sugar modification information, wherein the linked TMPRSS6-targeting antisense and sense sequences may include the linked first antisense sequence and second sense sequence and/or the linked second antisense sequence and first sense sequence.
  • each of the above constructs may or may not have a phosphate modification at the 5 1 end group. Furthermore, and independently, each of the above constructs may or may not have a "3x GalNAc" coupled to the 3' end group
  • constructs with a 3x GalNAc ligand in particular a toothbrush ligand as defined herein.
  • Particularly optional 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 5 1 terminal nucleoside of the antisense (guide) strand can include any nucleobase that can be present in an RNA molecule, in other words can be any of adenine (A), uracil (U), guanine (G) or cytosine (C).
  • the scope of the present disclosure extends to sequences that correspond to those in the Tables above, and wherein the 3' terminal nucleoside of the sense (passenger) strand (second region as defined in the claims herein) can include any nucleobase that can be present in an RNA molecule, in other words can be any of adenine (A), uracil (U), guanine (G) or cytosine (C), optionally however a nucleobase that is complementary to the 5' nucleobase of the antisense (guide) strand (first region as defined in the claims herein).
  • A adenine
  • U uracil
  • G guanine
  • C cytosine
  • RNAi constructs e.g., muRNA constructs
  • syntheses of the RNAi constructs have been conducted 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 ⁇ , wherein the methods disclosed on this website are incorporated by reference herein.
  • the only difference from the synthesis method disclosed in the Wikipedia reference is that GalNAc phosphoramidite immobilized on a support is used in the synthesis method during the first synthesis step.
  • Example 1 Primary Screen
  • 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. Cells were cultured in 37 °C with 5% C0 2 .
  • each 2 pM compound was added to respective triplicates of the plated hepatocytes for a final concentration of 1 pM in a volume of 100uL 1x complete WEM. 72 hours post transfection, cells were harvested, and RNA isolated using the PureLink Pro 96 total RNA Purification Kit (ThermoFisher, 12173011 A) according to the manufacturer protocol.
  • TMPRSS6-targeting mxRNA constructs were tested in dose curves.
  • a seven step, fivefold dilution series of compounds was prepared in basal WEM from 2 pM to 0.000128 pM.
  • Table 4 shows IC50 values (maximum knock down value at 1000 nM in %) for specific constructs as a result of the dose response assay
  • the constructs correspond to the ones in Table 3c in view of their experimental denotation
  • the results of the dose response assay are also shown in Fig. 2
  • the IC50 data in the single- to double-digit nanomolar range demonstrate outstanding performance of numerous constructs as described herein
  • either of the targets of the double-targeting constructs is knocked down by each of a multitude of constructs.
  • the A28 construct, used as a negative control (also denoted as reference "NT" in the Figures), has the following modified structure:

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Abstract

L'invention concerne des produits d'acides nucléiques qui modulent, interfèrent avec ou inhibent l'expression du gène TMPRSS6, ainsi que des compositions contenant les constructions et des procédés pour leur utilisation.
PCT/US2024/033246 2023-06-09 2024-06-10 Produits et compositions Pending WO2024254589A2 (fr)

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