Products and compositions

Abstract

Nucleic acid products that modulate, interfere with, or inhibit PCSK9 gene expression are provided, together with compositions containing the constructs and methods for their use.

Description

[0001] Products and compositions

[0002] Related Applications

[0003] This application claims the benefit of and priority Provisional Patent Application No. 63 / 441 ,999, filed January 30, 2023, and, the contents of which are incorporated herein by reference in their entirety.

[0004] Sequence Listing

[0005] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML document, created on December 22, 2023, is named 4690_0087l_SL and is 19112 kilobytes in size.

[0006] Field

[0007] Nucleic acid products are provided that modulate, interfere with, and / or inhibit PCSK9 gene expression. Methods, compounds, and compositions are provided for reducing expression of PCSK9 mRNA and protein in an animal. Such methods, compounds, and compositions are useful to treat, prevent, or ameliorate PCSK9-associated disorders such as dyslipidaemia, more specifically hypercholesterinaemia, hypertriglyceridemia, hyperchylomicronaemia, and atherosclerotic cardiovascular disease (ASCVD).

[0008] Background

[0009] Cholesterol has multiple vital functions, including the maintenance of integrity and fluidity of cell membranes. It furthermore serves a precursor for biosynthetic pathways, including those leading to steroid hormones and vitamin D. Cholesterol is present in the blood, where it occurs mainly in two forms: as component in high density lipoproteins (HDL) and in low density lipoproteins (LDL). Often the HDL cholesterol is referred as "good" or beneficial, while LDL cholesterol at chronically elevated levels, presents a health risk and may lead to disease.

[0010] Proprotein convertase subtilisin / kexin type 9 (PCSK9) is a serine protease involved in lipid metabolism. PCSK9 reduces the number of LDL receptors on the surface of liver cells. As a consequence, elevated amounts and / or activity of PCSK9 entail higher blood levels of "bad" LDL cholesterol. This molecular and cellular function of PCSK9 has led to its recognition as a therapeutic target.

[0011] Triglycerides are esters of glycerol with three fatty acids. They serve as a storage form of fat and provide energy when broken down; they are transported via the bloodstream. Excess levels of blood triglycerides have been recognized early on as causative agents or bystanders of a range of disorders. More recent evidence suggests a causative role, partly in conjunction with elevated levels of cholesterol (especially LDL cholesterol) in ASCVD and disorders subsumed under this term or associated therewith. A more comprehensive list of disorders associated with elevated levels of triglycerides is given in the embodiments disclosed further below.

[0012] Disease

[0013] Chronically elevated, circulating cholesterol, in particular, in the form of LDL cholesterol, also referred to as hypercholesterolemia, is a recognized disorder in itself and may lead to disorders and diseases of the cardiovascular system. More specifically, hypercholesterolemia causes cholesterol deposition on the inner walls of blood vessels, which in turn may lead to partial or complete vascular occlusions, and elevating the risk of myocardial infarction, stroke and peripheral artery disease.

[0014] Chronically elevated circulating triglycerides, also referred to as hypertriglyceridemia, also is a recognized disorder and may contribute to disorders and diseases of the cardiovascular system, including inflammation.

[0015] Treatment

[0016] Established treatments for cardiovascular system-related disorders and diseases in subjects include among other things the administration of statins. Statins, however, frequently have negative side effects that outweigh their benefits, and certain patients are or can become statin-intolerant. Accordingly, there remains a need for therapies to treat lipid dysregulation and associated cardiovascular disorders and diseases, including PCSK9-associated disorders and diseases. Double-stranded RNA (dsRNA) able to complementarily bind 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):494-8) by an RNA interference (RNAi) mechanism. 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. Various types of interfering RNA (iRN A) such as siRNA, antisense RNA, and micro-RNA are oligonucleotides that bind to and inhibit the translation of mRNA into peptides and proteins, thus, silencing the targeted gene(s). Such genesilencing agents are becoming increasingly important for therapeutic applications in medicine. The discovery of potent gene-silencing agents with minimal, off-target effects is a complex process. Although algorithms can be used to design gene-silencing agents such as siRNA molecules, there are limitations. These include a failure of algorithms to account for the tertiary structure of target mRNA and for the involvement of RNA binding proteins (Watts and Corey. Journal of Pathology, 226:365- 379 (2012)). Accordingly, the design of siRNAs also may require additional methodologies For the development of pharmaceutical compositions containing these highly charged molecules, they should be capable of (i) being synthesized economically, (ii) being distributed to target tissues, (iii) entering cells, and (iv) functioning within acceptable limits of toxicity.

[0017] An aim of this disclosure is to provide oligomeric compounds such as siRNA molecules, and their pharmaceutical compositions and methods of their use in the treatment of lipid and cardiovascular disorders and diseases in mammals by modulating, in particular inhibiting, gene expression by RNAi. Summary

[0018] The following aspects are non-limiting.

[0019] According to a first aspect, the present disclosure is directed to a nucleic acid construct comprising at least:

[0020] (a) a first antisense sequence that is complementary to a first partial sequence of an RNA which is transcribed from a PCSK9 gene, wherein being complementary allows for up to three mismatches; (b) a second antisense sequence that is complementary to a second partial sequence of the RNA which is transcribed from the PCSK9 gene or a different gene, wherein being complementary allows for up to three mismatches, the second partial sequence being different from the first partial sequence;

[0021] (c) a first sense sequence that is at least partially complementary to the first antisense sequence of (a), so as to form a first nucleic acid duplex region therewith;

[0022] (d) a second sense sequence that is at least partially complementary to the second antisense sequence of (b), so as to form a second nucleic acid duplex region therewith.

[0023] According to a second aspect, the present disclosure is directed to an oligomeric compound capable of inhibiting expression of PCSK9, wherein the compound comprises an antisense sequence that is complementary to a partial sequence of an RNA transcribed from an PCSK9 gene, wherein 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 300) or Table 1c (SEQ ID NOs: 601 to 606), wherein the portion optionally has a length of at least 18 nucleosides According to a third aspect, the present disclosure is directed to 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.

[0024] According to a fourth aspect, 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.

[0025] According to a fifth 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.

[0026] According to a sixth aspect, 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.

[0027] According to a seventh aspect, 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.

[0028] According to an eighth aspect, 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.

[0029] According to a ninth aspect, 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.

[0030] Advantageous and / or exemplary features of constructs according to the present disclosure are as follows: 1 ) they contain multiple (2 or more) at least partially double-stranded agents capable of triggering RNA interference, tied together into a single nano-structure predominantly through complementary (Watson-Crick) interactions;

[0031] 2) optionally, other (e.g.) covalent bindings may be used to build the constructs and / or add various ligands (e.g. delivery / targeting moieties such as GalNAc and or other carbohydrates, cholesterol, peptides, or small molecules, optionally attached via linkers);

[0032] 3) 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;

[0033] 4) 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);

[0034] 5) 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-reg ulate) target gene expression in cells / organisms;

[0035] 6) 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.

[0036] Brief Description of the Figures

[0037] Figure 1 shows single dose screen of 30 PCSK9 duplexes and their PCSK9 in vitro inhibition by certain muRNA constructs of Table 3c

[0038] Figure 2 shows a concentration dependence of 30 PCSK9 duplexes and their PCSK9 in vitro inhibition by certain muRNA constructs of Table 3c.

[0039] Detailed Description

[0040] Provided are siRNA molecules, pharmaceutical compositions and methods of use for the treatment of lipid and cardiovascular disorders and diseases. Specifically, the disclosed siRNA molecules are administered to subjects in pharmaceutical compositions and inhibit and, thus, modulate, expression of the PCSK9 gene,

[0041] Further implementations of the present disclosure are described below by way of example only. These examples represent the advantageous ways of putting the disclosure into practice that are currently known to the applicant although they are not the only ways in which this could be achieved. Features of different aspects and implementations or embodiments may be combined as appropriate, as would be apparent to a skilled artisan.

[0042] Use of the nucleic acid constructs as gene-silencing compounds as described herein leads to a significant reduction of gene expression of PCSK9, e g., in vitro using 5-donor primary human hepatocytes, as shown in the examples disclosed below. The most inhibitory (gene-silencing) compounds surprisingly produce knockdowns of about 70% or more of PCSK9 mRNA expression in vitro compared with a negative test. Furthermore, the muRNA compounds, as shown in the examples, produce knockdowns of at least about 50% of PCSK9 expression in vitro relative to a negative test. The compounds can efficiently reduce the effects of PCSK9 overexpression and be used to treat related diseases and / or disorders.

[0043] Furthermore, in certain embodiments, the mentioned effects are achieved by using oligomeric compounds according to the present disclosure for inhibiting the expression of PCSK9 gene in the form of mxRNA constructs having a reduced length of, e.g., 33 nucleosides compared to conventional shRNA molecules having greater lengths. This can, e.g., make the synthesis of mxRNA molecules more cost- and production-efficient, because fewer units are needed.

[0044] In addition, PCSK9-targeting nucleic acid muRNA (gene-silencing) constructs disclosed herein may be used in conjunction with another, e.g., APOC3-targeting nucleic acid sequence disclosed herein as part of a muRNA according to the present disclosure as such positive interaction has been shown in another non-published application by the applicant for different PCSK9-targeting sequences using the same principle constructs Without wishing to be bound by a particular theory, the antisense strands targeting PCSK9 or other genes like APOC3, when being part of muRNA nucleic acid constructs according to the present disclosure, are also the active species in the respective gene knockdown. Thus, the muRNA constructs disclosed herein, including PCSK9-targeting antisense strands and antisense strands targeting different genes, are active in the respective target gene knockdown. Because it is assumed, without wishing to be bound by a particular theory, that the active species are the same for muRNA as for mxRNA molecules, if the respective targeting antisense strands are included, the experiments of the present disclosure also show that an muRNA molecule targeting PCSK9 using the strands disclosed herein is active not only in the muRNA form but also in the mxRNA form

[0045] The effects and technical advantages achieved by using the disclosed oligomeric (e.g., siRNA) compounds for inhibiting PCSK9 expression will become apparent in more detail below and in the examples.

[0046] Definitions

[0047] The following definitions apply to this entire disclosure. In many instances, the definitions, in addition to the respective definition as such, provide non-exhaustive listings of possible, optional or advantageous embodiments.

[0048] Unless specific definitions are provided, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. Certain such techniques and procedures may be found for example in "Carbohydrate Modifications in Antisense Research" Edited by Sangvi and Cook, American Chemical Society , Washington D.C., 1994; "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., 21stedition, 2005; and "Antisense Drug Technology, Principles, Strategies, and Applications" Edited by Stanley T. Crooke, CRC Press, Boca Raton, Florida; and Sambrook et al., "Molecular Cloning, A laboratory Manual," 2ndEdition, Cold Spring Harbor Laboratory Press, 1989, which are hereby incorporated by reference for any purpose. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout in the disclosure are incorporated by reference herein in their entirety.

[0049] Unless otherwise indicated, the following terms have the following meanings:

[0050] As used herein, "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.

[0051] As used herein, "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".

[0052] As used herein, "chemical modification" or "chemically modified" 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. As used herein, "furanosyl" means a structure comprising a 5-membered ring comprising four carbon atoms and one oxygen atom.

[0053] As used herein, "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

[0054] As used herein, "sugar moiety" means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside. As used herein, "modified sugar moiety" means a substituted sugar moiety or a sugar surrogate.

[0055] As used herein, "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.

[0056] As used herein, "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 21-substituent of a 2-substituted sugar moiety does not form a bridge to another atom of the furanosyl ring).

[0057] As used herein, "MOE" means -OCH2CH2OCH3. As used herein, "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.

[0058] As used herein the term "sugar 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 / 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.

[0059] As used herein, "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. In certain embodiments, the 4 to 7 membered ring is a sugar ring. In certain embodiments the 4 to 7 membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2 '-carbon and the 4 '-carbon of the furanosyl. As used herein, "nucleotide" means a nucleoside further comprising a phosphate linking group. As used herein, "linked nucleosides" may or may not be linked by phosphate linkages and thus includes, but is not limited to "linked nucleotides". As used herein, "linked nucleosides" are nucleosides that are connected in a continuous sequence (i.e no additional nucleosides are present between those that are linked)

[0060] As used herein, "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.

[0061] As used herein the terms, "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) As used herein, "modified nucleobase" means any nucleobase that is not a naturally occurring nucleobase.

[0062] As used herein, "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

[0063] As used herein, "locked nucleic acid nucleoside" or"LNA" means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH2-O-2'bridge.

[0064] As used herein, "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

[0065] As used herein, "deoxynucleoside" means a nucleoside comprising 2'-H furanosyl sugar moiety, as found in naturally occurring deoxyribonucleosides (DNA). In certain embodiments, a 2'- deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e.g. , uracil).

[0066] As used herein, "oligonucleotide" means a compound comprising a plurality of linked nucleosides In certain embodiments, an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and / or unmodified deoxyribonucleosides (DNA) and / or one or more modified nucleosides.

[0067] As used herein, "modified oligonucleotide" means an oligonucleotide comprising at least one modified nucleoside and / or at least one modified internucleoside linkage.

[0068] As used herein, "linkage" or "linking group" means a group of atoms that link together two or more other groups of atoms.

[0069] As used herein "internucleoside linkage" means a covalent linkage between adjacent nucleosides in an oligonucleotide.

[0070] As used herein "naturally occurring internucleoside linkage" means a 3' to 5' phosphodiester linkage. As used herein, "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.

[0071] As used herein, "terminal internucleoside linkage" means the linkage between the last two nucleosides of an oligonucleotide or defined region thereof.

[0072] As used herein, "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, thionoalky Iphosphotriester and boranophosphate.

[0073] As used herein, "internucleoside phosphorus linking group" means a phosphorus linking group that directly links two nucleosides.

[0074] As used herein, "oligomeric compound" means a polymeric structure comprising two or more substructures. In certain embodiments, an oligomeric compound comprises an oligonucleotide, such as a modified oligonucleotide. In certain embodiments, an oligomeric compound further comprises one or more conjugate groups and / or terminal groups and / or ligands. In certain embodiments, an oligomeric compound consists of an oligonucleotide. In certain embodiments, 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. In certain embodiments, oligomeric compounds may also include monomeric sugar moieties that are not linked to a heterocyclic base moiety, thereby providing abasic sites. Oligomeric compounds may be defined in terms of a nucleobase sequence only, i.e. , by specifying the sequence of A, G, C, U (or T). In such a case, the structure of the sugar-phosphate backbone is not particularly limited and may or may not comprise modified sugars and / or modified phosphates. On the other hand, 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.

[0075] As used herein, "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. In certain embodiments, optional is that 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. Owing to the covalent connection, what results is, strictly speaking, two compounds which are the two strands. Complementarity between the strands may be throughout, but is not necessarily so. In particular, exemplary embodiments provide for an antisense region targeting an PCSK9 mRNAto be connected covalently with a sense region which is identical to a region of an AGT mRNA (encoding angiotensinogen) , and of an antisense region complementary to the sense region to be connected covalently to a sense region which is complementary to the an antisense region targeting an PCSK9 mRNA. Since antisense and sense regions of the parent single-target-directed RNA molecules do not need to have the same length and optionally do not have the same length with antisense portions being longer than sense portions, 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 The term "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.

[0076] The term "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.

[0077] As used herein, "terminal group" means one or more atom attached to either, or both, the 3' end or the 5' end of an oligonucleotide. In certain embodiments, a terminal group comprises one or more terminal group nucleosides.

[0078] As used herein, "conjugate" or "conjugate group" means an atom or group of atoms bound to an oligonucleotide or oligomeric compound. In certain embodiments, a conjugate group links a ligand to a modified oligonucleotide or oligomeric compound. In general, 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 / or clearance properties

[0079] As used herein, "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. In certain embodiments, 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. In certain embodiments 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. In certain embodiments, the bond for forming attachment to the oligomeric compound is a cleavable bond. In certain such embodiments, such cleavable bond constitutes all or part of a cleavable moiety. In certain embodiments, 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. In certain embodiments, the carbohydrate cluster portion is identified by the number and identity of the ligand. For example, in certain embodiments, the carbohydrate cluster portion comprises 2 GalNAc groups. For example, in certain embodiments, the carbohydrate cluster portion comprises 3 GalNAc groups. In certain embodiments, 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:

[0080]

[0081] , wherein in the structural formula one, two, or three phosphodiester linkages can also be substituted by phosphorothioate linkages.

[0082] As used herein, "cleavable moiety" means a bond or group that is cleaved under physiological conditions. In certain embodiments, a cleavable moiety is cleaved inside a cell or sub-cellular compartments, such as an endosome or lysosome. In certain embodiments, a cleavable moiety is cleaved by endogenous enzymes, such as nucleases. In certain embodiments, a cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds. In certain embodiments, a cleavable moiety is a phosphodiester linkage.

[0083] As used herein, "cleavable bond" means any chemical bond capable of being broken.

[0084] As used herein, "carbohydrate cluster" means a compound having one or more carbohydrate residues attached to a linker group

[0085] As used herein, "modified carbohydrate" means any carbohydrate having one or more chemical modifications relative to naturally occurring carbohydrates.

[0086] As used herein, "carbohydrate derivative" means any compound which may be synthesized using a carbohydrate as a starting material or intermediate.

[0087] As used herein, "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 (0) 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 / or one or more mannose moieties. Advantageously, the carbohydrate is N-Acetyl-Galactosamine.

[0088] As used herein, "strand" means an oligomeric compound comprising linked nucleosides. As used herein, "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.

[0089] As used herein, "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.

[0090] As used herein, "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

[0091] In particular, short hairpin RNA, also denoted as 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' and / or (a) 5' overhang(s). Optional are blunt-ended constructs. The term "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. In particular, 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. More particularly, the antisense strand and the sense strand within the shRNA are directly linked, e g. by a phosphate or a phosphorothioate, or linked by a third portion of linked nucleosides forming the loop, which means that the 3' end of the antisense strand is linked to the 5' end of the sense strand via covalent bonding over several other groups. Such direct linkage does not include a gap or nick.

[0092] As used herein, "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. In a duplex or double stranded oligonucleotide, the respective strands run in opposite 51to 31directions to permit base pairing between them.

[0093] As used herein, "duplex", or also abbreviated as "dup", 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 / or (A) adenine and uracil (U), and / 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.

[0094] As used herein, "double strand" or "double stranded" means a pair of oligomeric compounds that are hybridized to one another In certain embodiments, a double-stranded oligomeric compound comprises a first and a second oligomeric compound As used herein, "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, polyadenlyation, addition of 5 '-cap), and translation.

[0095] As used herein, "transcription" or "transcribed" 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.

[0096] As used herein, "target sequence" means a sequence to which an oligomeric compound is intended to hybridize to result in a desired activity with respect to PCSK9 expression. Oligonucleotides have sufficient complementarity to their target sequences to allow hybridization under physiological conditions.

[0097] As used herein, "nucleobase complementarity" or "complementarity" when in reference to nucleobases means a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In both DNA and RNA, guanine (G) is complementary to cytosine (C). In certain embodiments, complementary nucleobase means a nucleobase of an oligomeric compound that is capable of base pairing with a nucleobase of its target sequence. For example, if a nucleobase at a certain position of an oligomeric compound is capable of hydrogen bonding with a nucleobase at a certain position of a target sequence, then 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.

[0098] As used herein, "non-complementary" in reference to nucleobases means a pair of nucleobases that do not form hydrogen bonds with one another.

[0099] As used herein, "complementary" in reference to oligomeric compounds (e.g., linked nucleosides, oligonucleotides) 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.

[0100] Complementary oligomeric compounds need not have nucleobase complementarity at each nucleoside Rather, some mismatches are tolerated. In certain embodiments, complementary oligomeric compounds or regions are complementary at 70% of the nucleobases (70% complementary). In certain embodiments, complementary oligomeric compounds or regions are 80%> complementary. In certain embodiments, complementary oligomeric compounds or regions are 90%> complementary. In certain embodiments, complementary oligomeric compounds or regions are at least 95% complementary. In certain embodiments, complementary oligomeric compounds or regions are 100% complementary.

[0101] As used herein, "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 / or how long the strand regions are, then the compound may form hairpin loops, junctions, bulges or internal loops.

[0102] As used herein, "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 / or how long the strand regions are, then the compound may form hairpin loops, junctions, bulges or internal loops.

[0103] As used herein, "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. For example "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.

[0104] As used herein, "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.

[0105] As used herein, "specifically hybridizes" means the ability of an oligomeric compound to hybridize to one nucleic acid site with greater affinity than it hybridizes to another nucleic acid site.

[0106] As used herein, "fully complementary" in reference to an oligomeric compound or region thereof means that each nucleobase of the 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. Thus, 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

[0107] As used herein, "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.

[0108] As used herein, "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.

[0109] As used herein, "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. For example, modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression. As used herein, "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.

[0110] Accordingly, unless otherwise indicated, a "nucleoside having a modification of a first type" may be an unmodified nucleoside.

[0111] As used herein, "differently modified" mean chemical modifications or chemical substituents that are different from one another, including absence of modifications. Thus, for example, a MOE nucleoside and an unmodified naturally occurring RNA nucleoside are "differently modified," even though the naturally occurring nucleoside is unmodified. Likewise, DNA and RNA oligonucleotides are "differently modified," even though both are naturally-occurring unmodified nucleosides. Nucleosides that are the same but for comprising different nucleobases are not differently modified. For example, a nucleoside comprising a 2'-0Me modified sugar moiety and an unmodified adenine nucleobase and a nucleoside comprising a 2'-0Me modified sugar moiety and an unmodified thymine nucleobase are not differently modified.

[0112] As used herein, "the same type of modifications" refers to modifications that are the same as one another, including absence of modifications. Thus, for example, two unmodified RNA nucleosides have "the same type of modification," even though the RNA nucleosides are unmodified. Such nucleosides having the same type modification may comprise different nucleobases.

[0113] As used herein, "region" or "regions", or "portion" or "portions", 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. Typically such regions or portions comprise at least 10, at least 11, at least 12 or at least 13 linked nucleosides. For example, such regions can comprise 13 to 20 linked nucleosides, such as 13 to 16 or 18 to 20 linked nucleosides. Typically 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.

[0114] As used herein, "pharmaceutically acceptable carrier or diluent" means any substance suitable for use in administering to an animal. Advantageously, the animal is a mammal, more advantageously, the animal is a human. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile saline. In certain embodiments, such sterile saline is pharmaceutical grade saline.

[0115] As used herein, "substituent" and "substituent group," means an atom or group that replaces the atom or group of a named parent compound. For example, 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. In certain embodiments, 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. Such substituents can be present as the modification on the sugar moiety, for example a substituent present at the 2 -position of the sugar moiety. Unless otherwise indicated, groups amenable for use as substituents include without limitation, one or more of halo, hydroxyl, alkyl, alkenyl, alky ny I, acyl, carboxyl, alkoxy, alkoxyalkylene and amino substituents. Certain 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).

[0116] As used herein, "alkyl," as used herein, means a saturated straight or branched monovalent Ci-s hydrocarbon radical. Advantageously, 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 -OCHs substituent, on a sugar moiety of an oligomeric compound as described herein. This will be well understood be a person skilled in the art.

[0117] As used 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.

[0118] As used herein, "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. As will be well understood in the art, 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 -OCH2CH=CH2 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.

[0119] As used herein, "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. As will be well understood in the art, 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.

[0120] As used herein, "carboxyl" is a radical having a general formula -CO2H.

[0121] As used herein, "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.

[0122] As used herein, "alkoxy" means a radical formed between an alkyl group, such as a Ci.@ 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. Examples of 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.

[0123] As used herein, 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'-pos iti on of the sugar, therefore, overall providing a - Oalkylenealkoxy substituent, such as an -OCH2CH2OCH3 substituent, on a sugar moiety of an oligomeric compound as described herein. This will be well understood by a person skilled in the art and is generally referred to as an MOE substituent as defined herein and as known in the art.

[0124] As used herein, "amino" includes primary, secondary and tertiary amino groups.

[0125] As used herein, "halo" and "halogen," mean an atom selected from fluorine, chlorine, bromine and iodine.

[0126] As used herein, the term "mxRNA" is in particular understood as defined in WO 2020 / 044186 A2, which is incorporated by reference herein in its entirety. In particular, 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 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.

[0127] As used herein, the term "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 PCSK9 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. In particular, such muRNA are designed such that subsequent to in vivo administration, they are disassembled and the first and second antisense sequences are released. 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.

[0128] Further miniaturization by shortening the sense regions leads to bulge in the central part of the molecule where the 3'-terminal regions of the two antisense regions face each other:

[0129] In the diagram above, "GN" designates a GalNAc moiety, and "SBS" designates the fragile site which may be implemented as a nucleoside with a non-modified sugar.

[0130] As used herein ""PC1a" is a PCSK9 siRNA sequence. This compound is known to be effective in PCSK9 gene knockdown and was used as positive control compound in the Examples section. This compound is to be considered as Inclisiran® analogues.

[0131] 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 / 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.

[0132] The term "comprising" is used herein to mean including the method steps or elements identified, but that such steps or elements do not comprise an exclusive list and as such there may be present additional steps or elements.

[0133] Further, to the extent that the term "includes" is used in either the detailed description or the claims, such term is intended to be inclusive in a manner similar to the term "comprising" as "comprising" is interpreted when employed as a transitional word in a claim.

[0134] The following aspects and embodiments are provided herein muRNA nucleic acid constructs

[0135] According to a first aspect, the present disclosure is directed to a nucleic acid construct comprising at least:

[0136] (a) a first antisense sequence that is complementary to a first partial sequence of an RNA which is transcribed from a PCSK9 gene, wherein complementary allows for up to three mismatches;

[0137] (b) a second antisense sequence that is complementary to a second partial sequence of the RNA which is transcribed from the PCSK9 gene or a different gene, wherein complementary allows for up to three mismatches, the second partial sequence begin different from the first partial sequence;

[0138] (c) a first sense sequence that is at least partially complementary to the first antisense sequence of (a), so as to form a first nucleic acid duplex region therewith;

[0139] (d) a second sense sequence that is at least partially complementary to the second antisense sequence of (b), so as to form a second nucleic acid duplex region therewith.

[0140] In particular, the target gene different from the PCSK9 gene is selected from the group consisting of: an AGT gene, an APOC3 gene, an ANGPTL3 gene, an ANGPTL4 gene, an Lp(a) gene, an ANGPTL 8 gene, and an ASGR1 / 2 gene, 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 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).

[0141] 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.

[0142] 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. Especially, 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. Optionally, the first discrete nucleic acid targeting molecule comprises or consists of the first antisense sequence of (a) and the first sense sequence of (c), and / or the second discrete nucleic acid targeting molecule comprises or consists of the second antisense sequence of (b) and the second sense sequence of (d).

[0143] In certain embodiments (a) the first antisense sequence comprises at least 18, optionally 19, contiguous nucleotides allowing for up to three mismatches with a sequence being selected from the group consisting of SEQ ID Nos. 1-300;

[0144] (b) the second antisense sequence comprises at least 18, optionally 19, contiguous nucleotides allowing for up to three mismatches with a sequence being selected from the group consisting of SEQ ID Nos. 1-300;

[0145] (c) the first sense sequence comprises at least 11 , optionally 15, contiguous nucleotides allowing for up to three mismatches with a sequence being selected from the group consisting of SEQ ID Nos. 301-600; and / or

[0146] (d) the first sense sequence comprises at least 11 , optionally 15, contiguous nucleotides allowing for up to three mismatches with a sequence being selected from the group consisting of SEQ ID Nos. 301-600, wherein 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.

[0147] Since the present inventors surprisingly found in several instances that outstanding performance in single-targeting molecules (such as mxRNAs) may be transferred to double-targeting molecules (such as muRNAs), any further sequences, in particular antisense sequences as disclosed in the above mentioned patent documents may serve as a basis for designing muRNAs of the present disclosure.

[0148] In certain embodiments, the first antisense sequence of (a) is directly or indirectly linked to the second sense sequence of (d) as a primary structure. In particular, the first antisense sequence of (a) is selected from Table 1c and the second sense sequence of (d) is selected from Table 1d.

[0149] In certain embodiments, the second antisense sequence of (b) is directly or indirectly linked to the first sense sequence of (c) as a primary structure. In particular, the second antisense sequence of (b) is selected from Table 1c and the first sense sequence of (c) is selected from Table 1d.

[0150] As stated herein below, it is optional that sense sequences of the first and second sense sequence have a length of 15 nucleotides. To 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.

[0151] In certain embodiments, the construct may further comprise 1 to 8, optionally 2, additional nucleic acid portions 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. In particular, 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.

[0152] In certain embodiments 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 (Hi) nucleic acid linker is an unmodified nucleotide. Especially, the linking is direct, thereby giving rise to (a) contiguous strand(s).

[0153] In certain embodiments, there exists some complementarity between the first antisense sequence of

[0154] (a) and the second antisense sequence of (b), or the first sense sequence of (c) and the second sense sequence of (d). Optionally the complementarity

[0155] (i) is / are 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, optionally 2, 3, 4 or 5 base pairs; and / or

[0156] (ii) is between the first antisense sequence of (a) and the second antisense sequence of (b).

[0157] In certain embodiments, the internucleotide bond involves at least one of the one or more unmodified nucleotides, wherein optionally cleavage occurs at the 31position of (at least one of) the unmodified nucleotide(s)

[0158] In certain embodiments, the first antisense sequence of (a), and / or the second antisense sequence of

[0159] (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. Optionally, 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. In optional embodiments, 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 / 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.

[0160] In certain embodiments, 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.

[0161] In certain embodiments, 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 / or of the first sense sequence of (c).

[0162] In certain embodiments, the unmodified nucleotide is at position 19 of the first antisense sequence of (a) and / or the second antisense sequence of (b).

[0163] In certain embodiments, wherein 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.

[0164] In certain embodiments, 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.

[0165] In certain embodiments, the nucleic acid construct may be blunt ended.

[0166] In certain embodiments

[0167] (a) the first antisense sequence is selected from Table 3a;

[0168] (b) the second antisense sequence is selected from Table 3a;

[0169] (c) the first sense sequence is selected from Table 3b; and / or

[0170] (d) the second sense sequence is selected from Table 3b, 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. More optionally, the nucleic acid construct has a first antisense sequence which is identical to the second antisense sequence and a first sense sequence which is identical to the second sense sequence.

[0171] In certain embodiments

[0172] (a) the first antisense sequence is selected from Construct ID NO: 42, 56, 259, 261 , 43 or 35 in Table 3a;

[0173] (b) the second antisense sequence is selected from Construct ID NO: 42, 56, 259, 261, 43 or 35 in Table 3a and / or

[0174] (c) the first sense sequence is selected from Construct ID NO: 342, 356, 559, 561 , 343 or 335 in Table 3b; and / or

[0175] (d) the second sense sequence is selected from Construct ID NO: 342, 356, 559, 561 , 343 or 335 in Table 3b.

[0176] In certain embodiments, the construct comprises two strands, wherein the first strand is selected from Table 2, and the second strand is selected from Table 2, 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 NO: 642, 657, 859, 861 , 643 and 635, respectively, wherein optionally the first and the second strand are identical in composition.

[0177] In certain embodiments, the 31terminal positions of the first antisense sequence is replaced with an unmodified nucleotide.

[0178] In certain embodiments 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); and / or to the extent present, the 1 to 8 additional antisense sequences as defined in previously herein; and / or to the extent present, the passenger nucleic acid portions as defined previously herein; has an overhang.

[0179] In certain embodiments, the target RNA is an mRNA or another RNA molecule.

[0180] In certain embodiments, 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. .

[0181] In certain embodiments, 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)

[0182] In certain embodiments, the total length of either strand of the construct is 30 to 35 nucleotides, optionally 34 nucleotides.

[0183] In certain embodiments, 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.

[0184] Specific effects of the nucleic acid constructs according to the first aspect are shown in the Examples. Ligands

[0185] The nucleic acid construct according to the first aspect and the aforementioned embodiments may further comprise one or more ligands.

[0186] In certain embodiments, the first antisense sequence of (a), and / or the second antisense sequence of ( b), and / or the first sense sequence of (c) , and / or second sense sequence of (d), and / or, to the extent present, the 1 to 8 additional nucleic acid portions as defined in claims 14 and 15, and / or the passenger nucleic acid portions as defined previously herein, respectively have a 5’ to 3’ directionality thereby defining 5’ and 3’ regions thereof.

[0187] In certain embodiments, one or more ligands are conjugated at the 31region, optionally the 31end, of any of (i) the first sense sequence of (c), and / or (ii) the second sense sequence of (d), and / or, to the extent present, the (iii) passenger nucleic acid sequences as defined previously herein.

[0188] In certain embodiments, 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 / or second sense sequence of (d), and / or the passenger nucleic acid sequences as defined previously herein.

[0189] In certain embodiments, one or more ligands are conjugated at the 5' region, optionally the 5' end, or to the 31region, optionally the 31end, of any of the sequences.

[0190] In certain embodiments, 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. In an optional embodiment, the one or more carbohydrates can be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide or polysaccharide. In a more optional embodiment, the one or more carbohydrates comprise one or more hexose moieties. Especially, 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 / or one or more mannose moieties. The hexose moiety may be comprise two or three N-Acetyl-Galactosamine moieties. In particular, the hexose moiety may comprise three N-Acetyl-Galactosamine moieties.

[0191] In certain embodiments, the one or more ligands may be attached in a linear configuration, or in a branched configuration. Optionally, wherein 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. Optionally, the ligand may have the following structure:

[0192] Internucleoside linkages

[0193] 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.

[0194] In certain embodiments, the nucleic acid construct may comprise 1 to 15 phosphorothioate or phosphorodithioate internucleotide linkages.

[0195] In certain embodiments, comprises one or more phosphorothioate or phosphorodithioate internucleotide linkages at one or more of the 5’ and / or 3’ regions of 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), and / or the 1 to 8 additional nucleic acid portions as defined previously herein, and / or the passenger nucleic acid portions as defined previously herein.

[0196] In certain embodiments, 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 in previously herein.

[0197] In certain embodiments, 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.

[0198] In certain embodiments, the nucleic acid construct may comprises a phosphorothioate or phosphorodithioate internucleotide linkage linking: the first antisense sequence of (a) to the nucleic acid linker portion as defined in claim 12 (iii); and / or the second antisense sequence of (b) to the nucleic acid linker portion as defined in claim 12 (iii); and / or the first sense sequence of (c) to the nucleic acid linker portion as defined in claim 12 (iii) and / or the second sense sequence of (d) to the nucleic acid linker portion as defined in claim 12 (iii); and / or the 1 to 8 additional antisense sequences as defined in claims 9 or 10 to the nucleic acid linker portion as defined previously herein; and / or the passenger nucleic acid portions as defined in claims 10 or 11 to the nucleic acid linker portion as defined previously herein.

[0199] Modifications

[0200] In the nucleic acid construct according to the first aspect of the present disclosure and its aforementioned embodiments, at least one nucleotide of at least one of the following may be modified: 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); and / or to the extent present, the 1 to 8 additional nucleic acid portions as defined previously herein; and / or to the extent present, the passenger nucleic acid sequences as defined previously herein; and / or to the extent present, the nucleic acid linker portion as defined previously herein.

[0201] In an optional embodiment, 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 / or the second antisense sequence of (b); and / or the first sense sequence of (c); and / or the second dense 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.

[0202] In certain embodiments, 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 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 / or (iii), to the extent present, the passenger nucleic acid portions as defined previously herein, are modified by a common modification.

[0203] In certain embodiments, 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 / 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 nucleic acid portions as defined previously herein; and / or to the extent present, the passenger nucleic acid portions as defined previously herein.

[0204] In certain embodiments, one or more of the even numbered nucleotides starting from the 3’ region of: (i) the third nucleic acid portion of (c), and / or (ii) the fourth nucleic acid portion of (d), and / or (iii) the passenger nucleic acid portions as defined previously herein, to the extent present, may be modified by a modification that is different from the modification of odd numbered nucleotides starting from the 3’ region of these respective portions.

[0205] In certain embodiments, 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 / 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 nucleic acid linker portion as defined previously herein , to the extent present, are modified by a modification that (i) is different from the modification of an adjacent nucleotide of the 3’ region of the first antisense sequence of (a); and / or (ii) is different from the modification of an adjacent nucleotide of the 3’ region of the antisense sequence of (b); and / or is different from the modification of an adjacent nucleotide of the 3’ region of the 1 to 8 additional antisense sequences, to the extent present, as defined previously herein.

[0206] In certain embodiments, 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.

[0207] In certain embodiments, wherein 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 defined previously herein, is adjacent to at least one or more differently modified odd numbered nucleotides of these respective portions. In certain embodiments, 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 / 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 nucleic acid linker portion as defined previously herein, to the extent present, are modified by a modification that (i) is different from the modification of an adjacent nucleotide of the 3’ region of the first antisense sequence of (a); and / or (ii) is different from the modification of an adjacent nucleotide of the 3’ region of the antisense sequence of (b); and / or is different from the modification of an adjacent nucleotide of the 3’ region of the 1 to 8 additional antisense sequences, to the extent present, as defined previously herein.

[0208] In certain embodiments, one or more of the even numbered nucleotides starting from the 3’ region of:

[0209] (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.

[0210] In certain embodiments 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.

[0211] In certain embodiments, at least one or more of the modified even numbered nucleotides of (i) the first sense sequence of (c), and / or (ii) the second sense sequence of (d), and / 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.

[0212] In certain embodiments, 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. In certain embodiments, a plurality of adjacent nucleotides of (i) the first sense sequence of (c), and / or

[0213] (ii) the second sense sequence of (d), and / or (iii), to the extent present, the passenger nucleic acid sequences as defined in previously herein, are modified by a common modification.

[0214] In certain embodiments, the plurality of adjacent commonly modified nucleotides are 2 to 4 adjacent nucleotides, optionally 3 or 4 adjacent nucleotides.

[0215] In certain embodiments, the plurality of adjacent commonly modified nucleotides are located in the 5’ region of (i) the first sense sequence of (c), and / or (ii) the second sense sequence of (d), and / or (iii), to the extent present, the passenger nucleic acid sequences as defined previously herein.

[0216] In certain embodiments, a plurality of adjacent commonly modified nucleotides are located in the nucleic acid linker portion as previously herein.

[0217] In certain embodiments, 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 / 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 / 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. In certain embodiments, 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 / 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 in claim 14 or 15.

[0218] In certain embodiments, the modification and / or modifications are each and individually sugar, phosphate, or base modifications.

[0219] In certain embodiments, 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).

[0220] In certain embodiments, the 21modified sugar is selected from 2 -O-alkyl modified sugar, 2'-O-methyl modified sugar, 2'-O-methoxyethyl modified sugar, 2 -O-allyl 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.

[0221] In certain embodiments, the base modification is any one of an abasic nucleotide and a non-natural base comprising nucleotide.

[0222] In certain embodiments, at least one modification is a 2'-O-methyl modification in a ribose moiety. In certain embodiments, at least one modification is a 2'-F modification in a ribose moiety.

[0223] In certain embodiments, the nucleotides at any of positions 2 and 14 downstream from the first nucleotide of the 5’ region 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; do not contain 2'-O-methyl modifications in ribose moieties.

[0224] In certain embodiments one, two or all three nucleotides of (i) the first sense sequence of (c); and / or (ii) the second sense sequence of (d); and / or (iii), to the extent present, the passenger nucleic acid portions as defined previously herein; that respectively correspond in position to any of the nucleotides at any of positions 11 to 13 downstream from the first nucleotide of the 5’ region of (i) the first antisense sequence of (a); and / or (ii) the second antisense sequence of (b); and / or (iii) the 1 to 8 additional antisense sequence, to the extent present, as defined previously herein; do not contain 2'- O-methyl modifications in ribose moieties.

[0225] In certain embodiments, the nucleotides at any of positions 2 and 14 downstream from the first 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; contain 2'-F modifications in ribose moieties.

[0226] In certain embodiments, one, two or all three nucleotides of (i) the first sense sequence of (c); and or (ii) the second sense sequence of (d); and / or (iii), to the extent present, the passenger nucleic acid sequences as defined previously herein; that respectively correspond in position to any of the nucleotides at any of positions 11 to 13 downstream from the first nucleotide of the 5’ region of (i) the first 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; contain 2'-F modifications in ribose moieties.

[0227] In certain embodiments, all remaining 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.

[0228] In certain embodiments, the remaining nucleotides contain 2'-O-methyl modifications in ribose moieties.

[0229] In certain embodiments, 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 (iii) that is adjacent to (i) the first sense sequence of (c); and or (ii) the second sense sequence of (d); and / or (iii), to the extent present, the passenger nucleic acid sequence as defined previously herein.

[0230] Small hairpin (shRNA) and mxRNA oligomeric compounds

[0231] According to a second aspect, the present disclosure is related to an oligomeric compound capable of inhibiting expression of PCSK9, wherein the compound comprises an antisense sequence that is complementary to a partial sequence of an RNA transcribed from an PCSK9 gene, wherein 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 300) or Table 1c (SEQ ID NOs: 601 to 606), 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 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.

[0232] In certain embodiments 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 1 b (SEQ ID NOs: 301 to 600) or Table 1d (SEQ ID NOs: 607 to 612), wherein the portion optionally has a length of at least 8, 9, 10 or 11, more optionally at least 10, nucleosides.

[0233] In certain embodiments the antisense sequence is selected from the following sequences, or a portion thereof: SEQ ID NOs: 601 , 602, 603, 604, 605 and 606, in particular 605.

[0234] In certain embodiments, the sense sequence is selected from the following sequences, or a portion thereof: SEQ ID NOs: 607, 608, 609, 610, 611 and 612.

[0235] In certain embodiments, the antisense sequence is selected from the following sequences of Table 3a, or a portion thereof: Construct ID NO: 42, 56, 259, 261 , 43 and 35.

[0236] In certain embodiments, the sense sequence is selected from the following sequences, or a portion thereof: SEQ ID NOs: 342, 356, 559, 561 , 343 and 335.

[0237] Lengths and molecular features of the oligomeric compounds according to the second aspect 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. In addition, 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.

[0238] In certain embodiments, 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.

[0239] In certain embodiments each of the antisense sequence and the sense sequence has a 5’ to 3’ directionality thereby defining 5’ and 3’ regions respectively thereof.

[0240] In certain embodiments, 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 51terminal nucleoside of the antisense sequence is U and the base of the 3' terminal nucleoside of the second region is A.

[0241] In certain embodiments, 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 phosphorodithioate

[0242] In certain embodiments, 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.

[0243] In certain embodiments, 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.

[0244] In certain embodiments, 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.

[0245] In certain embodiments, the antisense and the sense sequence are directly adjacent on the single strand.

[0246] In certain embodiments, 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%. In certain embodiments, 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 RNA transcribed from a PCSK9 gene forms the hairpin loop, wherein the loop comprises 2 to 5, optionally 4 or 5, nucleosides.

[0247] In certain embodiments, the single strand is selected from Table 2, in particular selected from the group consisting of SEQ IDs NO: 613, 614, 615, 616, 617, and 618.

[0248] In certain embodiments, the single strand is selected from Table 3c, in particular from Construct ID NOs: 642, 656, 859, 861 , 643 and 635.

[0249] In certain embodiments, 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.

[0250] In certain embodiments, 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.

[0251] In certain embodiments, the hairpin loop comprises 1 to 8, 2 to 7, 3 to 6, optionally 4 or 5 linked nucleosides.

[0252] Ligands

[0253] In certain embodiments, the oligomeric compound according to the second aspect further comprises one or more ligands.

[0254] In certain embodiments the one or more ligands, in particular two or more or three ligands, are conjugated to the sense sequence and / or the antisense sequence. In certain embodiments, 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.

[0255] In certain embodiments, 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.

[0256] In certain embodiments, the one or more ligands comprise one or more carbohydrates.

[0257] In certain embodiments, the one or more carbohydrates can be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide or polysaccharide.

[0258] In certain embodiments, the one or more carbohydrates comprise or consist of one or more hexose moieties.

[0259] In certain embodiments, 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 / or one or more mannose moieties.

[0260] In certain embodiments, the one or more carbohydrates comprise one or more N-Acetyl- Galactosamine moieties.

[0261] In certain embodiments, the oligomeric compound comprises two or more N-Acetyl-Galactosamine moieties, optionally three.

[0262] In certain embodiments, 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

[0263] In certain embodiments, the one or more ligands are attached to the oligomeric compound as a biantennary or triantennary configuration.

[0264] Intemucleoside linkages

[0265] In certain embodiments of the second aspect of the present disclosure, the oligomeric compound comprises internucleoside linkages and wherein at least one internucleoside linkage is a modified internucleoside linkage. In certain embodiments, the modified internucleoside linkage is a phosphorothioate or phosphorodithioate internucleoside linkage.

[0266] In certain embodiments, the oligomeric compound comprises 1 to 16 phosphorothioate or phosphorodithioate internucleoside linkages.

[0267] In certain embodiments, the oligomeric compound comprises 7, 8, 9 or 10 phosphorothioate or phosphorodithioate internucleoside linkages.

[0268] In certain embodiments, the oligomeric compound comprises one or more phosphorothioate or phosphorodithioate internucleoside linkages at the 5’ region of the antisense sequence.

[0269] In certain embodiment, 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.

[0270] In certain embodiments, 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.

[0271] In certain embodiments, the oligomeric compound comprises a phosphorothioate or phosphorodithioate internucleoside linkage between each adjacent nucleoside that is present in the hairpin loop.

[0272] Modifications

[0273] The oligomeric compound according to the second aspect may at least comprise one nucleoside a modified sugar.

[0274] In certain embodiments, 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).

[0275] In certain embodiments, the 2 modified sugar is selected from 2'-O-alkyl modified sugar, 2'-O-methyl modified sugar, 2'-O-methoxyethyl modified sugar, 2 -O-allyl 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.

[0276] In certain embodiments, at least one modified sugar is a 2'-O-methyl modified sugar.

[0277] In certain embodiments, at least one modified sugar is a 2'-F modified sugar and, optionally, at most 16 or 17 sugars are 2'-F modified sugars.

[0278] In certain embodiments, the sugar is ribose.

[0279] In certain embodiments, 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.

[0280] In certain embodiments, the 3' terminal position of the sense sequence does not contain a 2'-O-methyl modification. In certain embodiments, 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.

[0281] In certain embodiments, sugars of the nucleosides of the sense strand, that 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.

[0282] In certain embodiments, the 3' terminal nucleoside of the second region of linked nucleosides contains a 2'-F modification.

[0283] In certain embodiments, one or more of the odd numbered nucleosides starting from the 5’ region of the antisense sequence are modified, and / 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.

[0284] In certain embodiments 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.

[0285] In certain embodiments, 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.

[0286] In certain embodiments, 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.

[0287] In certain embodiments, 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.

[0288] In certain embodiments, 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.

[0289] In certain embodiments, one or more of the even numbered nucleosides starting from the 3’ region of the antisense sequence are 2'-F modified sugars.

[0290] In certain embodiments, 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.

[0291] In certain embodiments, one or more of the even numbered nucleosides starting from the 5’ region of the sense sequence are 2'-F modified sugars.

[0292] In certain embodiments, sugars of a plurality of adjacent nucleosides of the antisense sequence are modified by a common or different modification.

[0293] In certain embodiments, sugars of a plurality of adjacent nucleosides of the sense sequence are modified by a common or different modification.

[0294] In certain embodiments, sugars of a plurality of adjacent nucleosides of the hairpin loop are modified by a common or different modification.

[0295] In certain embodiments, the common modification is a 2'-F modified sugar. In certain embodiments, the common modification is a 2 -O-methyl modified sugar.

[0296] In certain embodiments, the plurality of adjacent 2'-O-methyl modified sugars are present in at least eight adjacent nucleosides of the antisense and / or sense sequence.

[0297] In certain embodiments, the plurality of adjacent Z-O-methyl modified sugars are present in three or four adjacent nucleosides of the hairpin loop.

[0298] In certain embodiments, the hairpin loop comprises at least one nucleoside having a modified sugar. In certain embodiments, 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.

[0299] In certain embodiments, the modified sugar is a 2'-O-methyl modified sugar, and the differently modified sugar is a 2'-F modified sugar.

[0300] In certain embodiments, 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 31carbon of the sugar of the adjacent nucleoside, and / or one or more nucleosides of antisense sequence and / 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

[0301] In certain embodiments, the oligomeric compound is blunt ended.

[0302] In certain embodiment, either the antisense and sense sequence has an overhang

[0303] In certain embodiments, the oligomeric compound has a total length of about 25 to about 37 nucleosides, in particular about 33 or about 34 nucleosides.

[0304] In certain embodiments, a terminal nucleoside at a 51position 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.

[0305] In certain embodiments, the antisense and sense sequences are only being composed of nucleobases selected from the group consisting of A, U, G, C and not T.

[0306] In certain embodiments, the nucleosides do not contain a 2'-deoxy modification.

[0307] Compositions and pharmaceutical compositions including shRNA, mxRNA and / or muRNA oligomeric constructs

[0308] 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.

[0309] 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.

[0310] The pharmaceutical composition may further comprise a pharmaceutically acceptable excipient, diluent, antioxidant, and / or preservative.

[0311] Alternatively, the nucleic acid construct according to the first aspect and / or the oligomeric compound according to the second aspect is the only pharmaceutically active agent. In certain embodiments, the pharmaceutical composition is to be administered to patients or individuals which are statin-intolerant and / or for whom statins are contraindicated.

[0312] In certain embodiments the pharmaceutical composition furthermore comprises one or more further pharmaceutically active agents.

[0313] In certain embodiments, the further pharmaceutically active agent(s) is / are an RNAi agent which is directed to a target different from PCSK9 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.

[0314] In certain embodiments, the construct and the further pharmaceutically active agent(s) are to be administered concomitantly or in any order.

[0315] Diseases to be treated by shRNA, mxRNA and / or muRNA oligomeric compounds and further uses 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.

[0316] In particular, 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.

[0317] According to a sixth aspect, 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.

[0318] PCSK9-associated disease or disorder

[0319] In particular, the disease or disorder is a PCSK9-associated disease or disorder requiring reduction of PCSK9 expression levels.

[0320] The disease or disorder may be a PCSK9-associated disease or disorder, or a disease or disorder requiring reduction of low-density lipoprotein (LDL) cholesterol, the disease or disorder optionally being selected from dyslipidemia including mixed dyslipidemia, hypercholesterolemia, heterozygous familial hypercholesterolemia, non-familial hypercholesterolemia; atherosclerosis; and atherosclerotic cardiovascular disease (ASCVD) including myocardial infarction, stroke and peripheral arterial disease.

[0321] The fact that the Example compounds show PCSK9 knockdown renders it possible such compounds may be used in treating such diseases.

[0322] Structure and sequences of the constructs

[0323] The following Tables 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).

[0324] The notation used is common in the art and as the following meaning: A represents adenine;

[0325] U represents uracil; C represents cytosine;

[0326] G represents guanine.

[0327] 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]);

[0328] (ps), #, [#], or * represents a phosphorothioate inter-nucleoside linkage; i represents an inverted inter-nucleoside linkage, which can be either 3'-3', or 5'-5'; vp represents vinyl phosphonate; mvp represents methyl vinyl phosphonate;

[0329] 3xGalNAc represents an optionally present trivalent GalNAc; and

[0330] 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.

[0331] Table 1a below shows the nucleobase sequences of PCSK9-targeting antisense sequences (first and / or second antisense sequences).

[0332] Note = In particular, the first nucleobase at the 5' terminus in each of the above constructs may be substituted by U. Table 1 b below shows the nucleobase sequences of PCSK9 sense sequences (first and / or second sense sequences).

[0333] Note = In particular, the nucleobase of the 31terminal nucleotide of each of the sense strands presented within the table can be replaced by A.

[0334] Table 1c below shows a selection of specific antisense sequences, which can be the first and / or the second antisense sequence, as well as their targeting regions.

[0335] Table 1d below shows a selection of specific sense sequences, which can be the first and / or the second sense sequence. Table 2 below shows the nucleobase sequences of PCSK9-targeting sense sequences (linked antisense and sense sequences). Table 3a shows PCSK9-targeting antisense sequences (i.e., first and / or second antisense sequences) including sugar modification information.

[0336] Note = each of the above constructs may or may not have a phosphate modification at the 5' end group.

[0337] Table 3b shows PCSK9-targeting sense sequences (i.e., first and / or second sense sequences) including sugar modification information.

[0338]

[0339]

[0340]

[0341]

[0342]

[0343] Note = each of the above constructs may or may not have a "3x GalNAc" coupled to the 31end group.

[0344] Optional are constructs with a 3x GalNAc ligand, in particular a toothbrush ligand as defined herein.

[0345] Table 3c shows linked PCSK9-targeting antisense and sense sequences including sugar modification information, wherein the linked PCSK9- 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.

[0346]

[0347]

[0348]

[0349] Note = each of the above constructs may or may not have a phosphate modification at the 5' end group. Furthermore, and independently, each of the above constructs may or may not have a "3x GalNAc" coupled to the 31end group. Optional are 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.

[0350] Specific notes about the nomenclature in Tables 3a to 3c: fN: 2'-Fluoro residues mN: 2'-O-methyl residues Ps: phosphorothioate p, Phos: phosphate

[0351] (GalNAc): Sirnaomics mono-GalNAc building block

[0352] The scope of the present disclosure extends to sequences that correspond to those in the Tables above, and wherein the 51terminal nucleoside of the antisense (guide) strand (first 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). Additionally, the scope of the present disclosure extends to sequences that correspond to those in the Tables above, and wherein the 31terminal 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).

[0353] While the methods are shown and described as being a series of acts that are performed in a particular sequence, it is to be understood and appreciated that the methods are not limited by the order of the sequence. For example, some acts can occur in a different order than what is described herein. In addition, an act can occur concurrently with another act. Further, in some instances, not all acts may be required to implement a method described herein.

[0354] The order of the steps of the methods described herein is exemplary, but the steps may be carried out in any suitable order, or simultaneously where appropriate. Additionally, steps may be added or substituted in, or individual steps may be deleted from any of the methods without departing from the scope of the subject matter described herein Aspects of any of the Examples described above may be combined with aspects of any of the other Examples described to form further Examples.

[0355] It will be understood that the above description of an optional embodiment is given by way of example only and that various modifications may be made by those skilled in the art. What has been described above includes Examples of one or more embodiments. It is, of course, not possible to describe every conceivable modification and alteration of the above compounds, compositions or methods for purposes of describing the aforementioned aspects, but one of ordinary skill in the art can recognize that many further modifications and permutations of various aspects are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the scope of the appended claims.

[0356] Examples

[0357] The following Examples illustrate certain embodiments of the present disclosure and are not limiting. Moreover, where specific embodiments are provided, the inventors have contemplated generic application of those specific embodiments. For example, disclosure of a construct having a specific motif or modification patterns provides reasonable support for additional constructs having the same or similar motif or modification patterns.

[0358] The syntheses of the RNAi constructs, e.g , muRNA constructs, disclosed herein have been carried out using synthesis methods known to the person skilled in the art, such as synthesis methods disclosed in https: / / en.wikipedia.org / wiki / Oligonucleotide_synthesis {retrieved on 16 February 2022}, wherein the methods disclosed on this website are incorporated by reference herein in their entirety. The only difference to the synthesis method disclosed in this reference is that GalNAc phosphoramidite immobilized on a support is used in the synthesis method during the first synthesis step.

[0359] Example 1 :

[0360] Materials and Methods

[0361] Cell Culture:

[0362] 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.

[0363] Compounds preparation

[0364] A seven step, five-fold dilution series of each compound of T able 3c was prepared in basal WEM from 2 pM to 0.000128 pM.

[0365] Transfection:

[0366] On the day of transfection, primary human hepatocytes were thawed in 45mL of human OptiThaw (Sekisui XenoTech, K8000) and centrifuged down at 200g for 5 minutes. Cells were resuspended in 2x complete WEM and counted. Cells were then plated in 50 pL of 2x complete WEM at 25,000 cells per well on 96 well type 1 rat tail Collagen plates and allowed to rest and attach for four hours before transfection. After rest, 50 pL of each dilution was added to respective triplicates of the plated hepatocytes in a final dilution of 1 pM during the primary screening or in series of dilution from 1 pM down to 0.000064 pM in a volume of 100uL 1x complete WEM during the dose response assay. qPCR:

[0367] 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. Harvested RNA was assayed for ApoC3 and AGT expression via Taqman qPCR using the Luna Universal Probe One-Step RT-qPCR Kit (NEB, E3006). A qPCR assay was performed for each sample using a PCSK9 TaqMan probe set (Hs00906501_g1-FAM ) multiplexed with a common GAPDH VIC probe (ThermoFisher, 4326317E). Thermocycling and data acquisition was performed with an Applied Biosystems QuantStudio 3 / 5 Real-Time PCR System.

[0368] Results Table 4a below shows the PCSK9 expression for a single dose screen of 30 PCSK9 duplex compounds. Table 4b below shows IC50 values (maximum knock down value at 1000 nM in %) for specific constructs selected in accordance with the Examples The numbers in the second column correspond to the construct numbering in Tables 3c. The results of Table 4a are also visualized in Fig.

[0369] 1 and the results of T able 4b (in the form of dose curves) are visualized in Fig. 2. Table 4a:

[0370] Table 4b: The I C50 data in the single- to double-digit nanomolar range demonstrate outstanding performance of numerous constructs as described herein. Of note, either target of the double-targeting constructs is knocked down by each of a multitude of constructs

[0371] The TMPRSS6 construct, used as a negative control (also denoted as reference "NT" in the Figures)PCS, has the following modified structure:

[0372] 5'vP[mA][fA][mC][fC][mA][fG][mA][fA][mG][fA][mA][fG][mC][fA][mG][fG][mU][fG][iN][fC][mU][fG][fC][fU][ mU][fC][mU][fU][mC][fU][mG][fG][mU][fU]#[3XGalNAc] (SEQ ID NO: 1860).

Claims

Claims1. A nucleic acid construct comprising at least:(a) a first antisense sequence that is complementary to a first partial sequence of an RNA which is transcribed from a PCSK9 gene, wherein being complementary allows for up to three mismatches;(b) a second antisense sequence that is complementary to a second partial sequence of the RNA which is transcribed from the PCSK9 gene or a different gene, wherein being complementary allows for up to three mismatches, the second partial sequence being different from the first partial sequence;(c) a first sense sequence that is at least partially complementary to the first antisense sequence of (a), so as to form a first nucleic acid duplex region therewith;(d) a second sense sequence that is at least partially complementary to the second antisense sequence of (b), so as to form a second nucleic acid duplex region therewith.

2. The construct according to claim 1 , wherein the construct is 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).

3. The construct according to claim 1 or 2, wherein 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.

4. The construct according to any one of claims 1 to 3, which further comprises at least 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.

5. The construct according to claim 4, wherein the labile functionality comprises 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.

6. The construct according to claim 5, wherein the one or more unmodified nucleotides of the labile functionality represent one or more cleavage positions within the construct whereby subsequent to in vivo administration the construct is cleaved at the one or more cleavage positions so as to yield the at least first and second discrete nucleic acid targeting molecules.

7. The construct according to claim 6, wherein 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.

8. The construct according to claim 7, wherein the first discrete nucleic acid targeting molecule comprises or consists of the first antisense sequence of (a) and the first sense sequence of (c), and / or the second discrete nucleic acid targeting molecule comprises or consists of the second antisense sequence of (b) and the second sense sequence of (d).

9. The construct according to any one of the preceding claims, wherein(a) the first antisense sequence comprises at least 18, optionally 19, contiguous nucleotides allowing for up to three mismatches with a sequence being selected from the group consisting of SEQ ID Nos. 1-300;(b) the second antisense sequence comprises at least 18, optionally 19, contiguous nucleotides allowing for up to three mismatches with a sequence being selected from the group consisting of SEQ ID Nos. 1-300;(c) the first sense sequence comprises at least 11 , optionally 15, contiguous nucleotides allowing for up to three mismatches with a sequence being selected from the group consisting of SEQ ID Nos. 301-600; and / or(d) the first sense sequence comprises at least 11 , optionally 15, contiguous nucleotides allowing for up to three mismatches with a sequence being selected from the group consisting of SEQ ID Nos. 301-600, wherein 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.

10. The construct according to any of the preceding claims, wherein the first antisense sequence of (a) is directly or indirectly linked to the second sense sequence of (d) as a primary structure.

11. The construct according to claim 10, wherein the first antisense sequence of (a) is selected from the group consisting of SEQ ID Nos. 601-606and the second sense sequence of (d) is selected from the group consisting of SEQ ID Nos. 607-612.

12. The construct according to any of the preceding claims, wherein the second antisense sequence of (b) is directly or indirectly linked to the first sense sequence of (c) as a primary structure.

13. The construct according to claim 10 to 12, wherein the second antisense sequence of (b) is selected from the group consisting of SEQ ID Nos. 601-606and the first sense sequence of (c) is selected from the group consisting of SEQ ID Nos. 607-612.

14. The construct according to any of claims 1 to 9, 12 or 13, that further comprises 1 to 8, optionally 2, additional nucleic acid portions 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.

15. The construct according to claim 14, wherein 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.

16. The construct according to any of claims 10, 11 or 15, wherein 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.

17. The construct according to claim 16 (i), wherein the linking is direct, thereby giving rise to (a) contiguous strand(s).

18. The construct of any one of the preceding claims, especially of claim 16 (i), wherein there exists some complementarity between the first antisense sequence of (a) and the second antisense sequence of (b), or the first sense sequence of (c) and the second sense sequence of (d).

19. The construct according to claim 18, wherein the complementarity(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).

20. The construct according to claim 16 (i) to 19, as dependent on claim 4, wherein the internucleotide bond involves at least one of the one or more unmodified nucleotides, wherein optionally cleavage occurs at the 31position of (at least one of) the unmodified nucleotide(s).

21. The construct according to any of the preceding claims, wherein 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.

22. The construct according to claim 21 , wherein 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.

23. The construct according to claim 21 or 22, wherein the first sense sequence of (c), and / 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.

24. The construct according to any one of claims 21 to 23, wherein 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 31terminal position of the first antisense sequence of (a) and / or of the first sense sequence of (c).

25. The construct according to claim 24, wherein the unmodified nucleotide is at position 19.

26. The construct according to any of claims 17 to 19 or 21 to 23 as dependent on claim 16 (iii), wherein 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.

27. The construct according to any one of claims 21 to 26, wherein 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.

28. The construct according to any of claims 1 to 27, which further comprises one or more ligands.

29. The construct according to any one of claims 1 to 28, wherein the first antisense sequence of (a), and / or the second antisense sequence of (b), and / or the first sense sequence of (c), and / or second sense sequence of (d), and / or, to the extent present, the 1 to 8 additional nucleic acid portions as defined in claims 14 and 15, and / or the passenger nucleic acid portions as defined in claims 14 or 15, respectively have a 5’ to 3’ directionality thereby defining 5’ and 3’ regions thereof.

30. The construct according to any one of claims 28 or 29, wherein one or more ligands are conjugated at the 3' region, optionally the 31end, of any of (i) the first sense sequence of (c), and / or (ii) the second sense sequence of (d), and / or, to the extent present, the (iii) passenger nucleic acid sequences as defined in claims 14 or 15.

31. The construct according to any one of claims 28 to 30, wherein 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 / or second sense sequence of (d), and / or the passenger nucleic acid sequences as defined in claims 14 or 1532. The construct of any one of claims 28 to 31 , wherein one or more ligands are conjugated at the 51region, optionally the 51end, or to the 31region, optionally the 31end, of any of the sequences33. The construct according to any of claims 28 to 32, wherein 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.

34. The construct according to claim 33, wherein the one or more carbohydrates can be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide or polysaccharide.

35. The construct according to claim 34, wherein the one or more carbohydrates comprise one or more hexose moieties.

36. The construct of claim 35, wherein 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 / or one or more mannose moieties.

37. The construct according to claim 36, which comprises two or three N-Acetyl-Galactosamine moieties.

38. The construct according to any of claims 28 to 37, wherein the one or more ligands are attached in a linear configuration, or in a branched configuration.

39. The construct according to claim 38, wherein the one or more ligands are attached as a biantennary or triantennary configuration, or as a configuration based on single ligands at different positions.

40. The construct according to claim 37 or 38, wherein the ligand has the following structure:

41. The construct according to any of the preceding claims, which further comprises one or more phosphorothioate or phosphorodithioate internucleotide linkages.

42. The construct according claim 41 , which comprises 1 to 15 phosphorothioate or phosphorodithioate internucleotide linkages.

43. The construct according to claim 41 or 42, which comprises one or more phosphorothioate or phosphorodithioate internucleotide linkages at one or more of the 5’ and / or 3’ regions of 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), and / or the 1 to 8 additional nucleic acid portions as defined in claims 9 or 10, and / or the passenger nucleic acid portions as defined in claims 9 or 10.

44. The construct according to any of claims 41 to 43, which comprises phosphorothioate or phosphorodithioate internucleotide linkages between at least two adjacent nucleotides of the nucleic acid linker portion as defined in claim 16 (iii).

45. The construct according to any of claim 44, which comprises a phosphorothioate or phosphorodithioate internucleotide linkage between each adjacent nucleotide that is present in the nucleic acid linker portion.

46. The construct according to any of claims 41 to 45, which comprises a phosphorothioate or phosphorodithioate internucleotide linkage linking: the first antisense sequence of (a) to the nucleic acid linker portion as defined in claim 12 (iii); and / or the second antisense sequence of (b) to the nucleic acid linker portion as defined in claim 12 (iii); and / or the first sense sequence of (c) to the nucleic acid linker portion as defined in claim 12 (iii) and / or the second sense sequence of (d) to the nucleic acid linker portion as defined in claim 12 (iii); and / or the 1 to 8 additional antisense sequences as defined in claims 9 or 10 to the nucleic acid linker portion as defined in claim 16 (iii); and / or the passenger nucleic acid portions as defined in claims 10 or 11 to the nucleic acid linker portion as defined in claim 12 (iii).

47. The construct according to any of claims 1 to 46, wherein at least one nucleotide of at least one of the following is modified: 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); and / or to the extent present, the 1 to 8 additional antisense sequences as defined in claims 14 or 15; and / or to the extent present, the passenger nucleic acid sequences as defined in claims 14 or 15; and / or to the extent present, the nucleic acid linker portion as defined in claim 16 (iii).

48. The construct according to claim 47, wherein 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 / or the second antisense sequence of (b); and / or the first sense sequence of (c); and / or the second dense sequence of (d); and / or to the extent present, the 1 to 8 additional antisense sequences as defined in claims 14 or 15; and / or to the extent present, the passenger nucleic acid sequences as defined in claims 14 or 15.

49. The construct according to claim 47, wherein 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 in claims 10 or 11 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 / or (iii), to the extent present, the passenger nucleic acid portions as defined in claims 15 or 16, are modified by a common modification.

50. The construct according to claim 47, wherein 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 / 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 nucleic acid portions as defined in claims 14 or 15; and / or to the extent present, the passenger nucleic acid portions as defined in claims 14 or 15.

51. The construct according to any of claims 47 to 50, wherein one or more of the odd numbered nucleotides starting from the 3’ region of the first sense strand of (c) are modified by a modificationthat 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 / or wherein one or more of the odd numbered nucleotides starting from the 3’ region of the passenger nucleic acid sequence as defined in claims 14 or 15, 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 in claims 14 or 15; and / or wherein one or more of the nucleotides of a nucleic acid linker portion as defined in claims 16 (iii), to the extent present, are modified by a modification that (i) is different from the modification of an adjacent nucleotide of the 3’ region of the first antisense sequence of (a); and / or (ii) is different from the modification of an adjacent nucleotide of the 3’ region of the antisense sequence of (b); and / or is different from the modification of an adjacent nucleotide of the 3’ region of the 1 to 8 additional antisense sequences, to the extent present, as defined in claims 14 or 15.

52. The construct according to any of claims 47 to 51 , wherein 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 in claims 14 or 15, 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.53 The construct according to any of claims 47 to 52, wherein 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 defined in claims 14 or 15, is adjacent to at least one or more differently modified odd numbered nucleotides of these respective portions.

54. The construct according to any of claims 47 to 53, wherein at least one or more of the modified even numbered nucleotides of (i) the first sense sequence of (c), and / or (ii) the second sense sequence of (d), and / or (iii), to the extent present, the passenger nucleic acid sequences as defined in claims 14 or 15, is adjacent to at least one or more differently modified odd numbered nucleotides of these respective portions.

55. The construct according to any of claims 47 to 54, wherein 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 in claims 14 or 15, are modified by a common modification.

56. The construct according to any of claims 47 to 55, wherein a plurality of adjacent nucleotides of (i) the first sense sequence of (c), and / or (ii) the second sense sequence of (d), and / or (iii), to the extent present, the passenger nucleic acid sequences as defined in claims 14 or 15, are modified by a common modification57. The construct according to claim 55 or 56, wherein the plurality of adjacent commonly modified nucleotides are 2 to 4 adjacent nucleotides, optionally 3 or 4 adjacent nucleotides.

58. The construct according to claim 57, wherein the plurality of adjacent commonly modified nucleotides are located in the 5’ region of (i) the first sense sequence of (c), and / or (ii) the second sense sequence of (d), and / or (iii), to the extent present, the passenger nucleic acid sequences as defined in claims 14 or 15.

59. The construct according to any one of claims 55 to 58, wherein a plurality of adjacent commonly modified nucleotides are located in the nucleic acid linker portion as defined in claim 16 (iii).

60. The construct according to any of claims 47 to 59, wherein 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 / 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 / or one or more of the modified nucleotides of the 1 to 8 additional antisense sequences, to the extent present, as defined in claim 14 or 15, do not have a common modification present in the corresponding nucleotide of the corresponding passenger nucleic acid sequences of the respective duplex regions.

61. The construct according to any of claims 47 to 60, wherein 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 / 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 in claim 14 or 15 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 in claim 14 or 15.

62. The construct according to any of claims 47 to 61 , wherein the modification and / or modifications are each and individually sugar, phosphate, or base modifications.

63. The construct according to claim 61 , where 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).

64. The construct according to claim 63, wherein the 2' modified sugar is selected from 2-O-alkyl modified sugar, 2'-O-methyl modified sugar, 2'-0-methoxyethyl modified sugar, 2 -O-allyl modified sugar, 2 -C-allyl modified sugar, 2'-deoxy modified sugar such as 2 -deoxy ribose, 2'-F modified sugar, 2'-arabino-fluoro modified sugar, 2'-O-benzyl modified sugar, 2'-amino modified sugar, and 2'-O- methyl-4-pyridine modified sugar.

65. The construct according to any of claims 62 to 64, wherein the base modification is any one of an abasic nucleotide and a non-natural base comprising nucleotide.

66. The construct according to any of claims 47 to 65, wherein at least one modification is a 2'-O- methyl modification in a ribose moiety.

67. The construct according to any of claims 47 to 66, wherein at least one modification is a 2'-F modification in a ribose moiety.

68. The construct according to any of claims 47 to 67 wherein the nucleotides at any of positions 2 and 14 downstream from the first nucleotide of the 5’ region 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 in claim 14 or 15; do not contain 2'-O-methyl modifications in ribose moieties.

69. The construct according to any of claims 47 to 68, wherein one, two or all three nucleotides of (i) the first sense sequence of (c); and / or (ii) the second sense sequence of (d); and / or (iii), to the extent present, the passenger nucleic acid portions as defined in claim 14 or 15; that respectively correspond in position to any of the nucleotides at any of positions 11 to 13 downstream from the first nucleotide of the 5’ region of (i) the first antisense sequence of (a); and / or (ii) the second antisense sequence of (b); and / or (iii) the 1 to 8 additional antisense sequence, to the extent present, as defined in claim 14 or 15; do not contain 2'-O-methyl modifications in ribose moieties.

70. The construct according to claim 68 or 69, wherein the nucleotides at any of positions 2 and 14 downstream from the first 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 in claim 14 or 15; contain 2'-F modifications in ribose moieties71. The construct according to any of claims 68 to 70, wherein one, two or all three nucleotides of (i) the first sense sequence of (c); and or (ii) the second sense sequence of (d); and / or (iii), to the extent present, the passenger nucleic acid sequences as defined in claims 14 or 15; that respectively correspond in position to any of the nucleotides at any of positions 11 to 13 downstream from the first nucleotide of the 5’ region of (i) the first 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 in claim 14 or 15; contain 2'-F modifications in ribose moieties72. The construct according to any one of claims 67 to 71, wherein all remaining nucleotides contain either 2'-O-methyl modifications or 2'-F modifications in ribose moieties, optionally with the exception of the unmodified nucleotide(s) in accordance with claim 5.

73. The construct according to claim 72, wherein the remaining nucleotides contain 2'-O-methyl modifications in ribose moieties.

74. The construct according to claim 72 or 73, wherein 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 in claim 16 (iii) that is adjacent to (i) the first sense sequence of (c); and or (ii) the second sense sequence of (d); and / or (iii), to the extent present, the passenger nucleic acid sequence as defined in claim 14 or 15.

75. The construct of any one of the preceding claims, wherein(a) the first antisense sequence is selected from the group consisting of SEQ ID Nos. 619-926 and 1859;(b) the second antisense sequence is selected from the group consisting of SEQ ID Nos 619- 926 and 1859;(c) the first sense sequence is selected from the group consisting of SEQ ID Nos. 927-1226; and / or(d) the second sense sequence is selected from the group consisting of SEQ ID Nos. 927-1226, 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.

76. The construct according to any one of the preceding claims, wherein(a) the first antisense sequence is selected from the group consisting of SEQ ID Nos. 653, 660- 661 , 674, 885 and 887;(b) the second antisense sequence is selected from Construct ID NO: the group consisting of SEQ ID Nos. 653, 660-661 , 674, 885 and 887and / or(c) the first sense sequence is selected from the group consisting of SEQ ID Nos. 961 , 968, 969, 982, 1185 and 1187; and / or(d) the second sense sequence is selected from the group consisting of SEQ ID Nos. 961 , 968, 969, 982, 1185 and 1187.

77. The construct of any one of the preceding claims wherein the construct comprises two strands, wherein the first strand is selected from the group consisting of SEQ ID Nos 613-618, and the second strand is selected from the group consisting of SEQ ID Nos. 613-618, wherein optionally the first and the second strand have the same composition; or the first and second strands are selected from the group consisting of SEQ ID Nos 1227-1858,, such as SEQ ID Nos. 1261, 1268, 1269, 1282, 1473, and 1475, respectively, wherein optionally the first and the second strand are identical in composition.

78. The construct according to any one of claims 75 to 77, wherein the 31terminal positions of the first antisense sequence is replaced with an unmodified nucleotide.

79. The construct according to any one of the preceding claims, wherein 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); and / or to the extent present, the 1 to 8 additional antisense sequences as defined in claim 14 or 15; and / or to the extent present, the passenger nucleic acid portions as defined in claim 14 or 15; has an overhang.

80. The construct according to any one of the preceding claims, wherein the target RNA is an mRNA or an other RNA molecule.

81. The construct according to any one of the preceding claims, wherein 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. .

82. The construct according to any one of the preceding claims, wherein 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)83. The construct according to any one of claims 34 to 39, wherein the total length of either strand of the construct is 30 to 35 nucleotides, optionally 34 nucleotides.

84. The construct according to any one of the preceding claims, wherein 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.

85. An oligomeric compound capable of inhibiting expression of PCSK9, wherein the compound comprises an antisense sequence that is complementary to a partial sequence of an RNA transcribed from an PCSK9 gene, wherein being complementary allows for up to three mismatches, and wherein the antisense sequence is selected from the group consisting of, or a portion thereof: SEQ ID NOs: 1 to 300 and SEQ ID NOs: 601 to 606), wherein the portion optionally has a length of at least 18 nucleosides.

86. The oligomeric compound according to claim 85, which further comprises at least a second region of linked nucleosides having an sense sequence that is at least partially complementary to the first nucleobase sequence and is selected from the group consisting of, or a portion thereof: SEQ ID NOs: 301 to 600 and SEQ ID NOs: 607 to 612, wherein the portion optionally has a length of at least 8, 9, 10 or 11, more optionally at least 10, nucleosides.

87. The oligomeric compound according to claim 85 or 86, wherein the antisense sequence is selected from the group consisting of, or a portion thereof: SEQ ID NOs: 601, 602, 603, 604, 605 and 606.

88. The oligomeric compound according to claim 87, wherein the sense sequence is selected from the group consisting of, or a portion thereof: SEQ ID NOs: 607, 608, 609, 610, 611 and 61289. The oligomeric compound according to any of claims 85 to 88, wherein the antisense sequence is selected from the group consisting of, or a portion thereof: SEQ ID Nos 653, 660, 661, 674, 885 and 887.

90. The oligomeric compound according to claim 89, wherein the sense sequence is selected from the group consisting of, or a portion thereof: SEQ ID NOs: 961, 968, 969, 982, 1185 and 1187.

91. The oligomeric compound according to any of claims 85 to 90, wherein the antisense sequence consists of 18 to 35, optionally 18 to 20, more optionally 18 or 19, and yet more optionally 19 linked nucleosides.

92. The oligomeric compound according to any of claims 86 to 91 , wherein the second region of linked nucleosides consists essentially 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.

93. The oligomeric compound according to any of claims 86 to 91 , which 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.

94. The oligomeric compound according to claim 93, wherein each of the antisense sequence and the sense sequence has a 5’ to 3’ directionality thereby defining 5’ and 3’ regions respectively thereof.

95. The oligomeric compound according to claim 94, wherein 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 51terminal 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.

96. The oligomeric compound according to claim 94 or 95, wherein 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 51terminal nucleoside of the sense sequence by a phosphate, a phosphorothioate, or a phosphorodithioate.

97. The oligomeric compound according to any of claims 85 to 96, which further comprises one or more ligands98. The oligomeric compound according to claim 13, wherein the one or more ligands, in particular two or more or three ligands, are conjugated to the sense sequence and / or the antisense sequence.

99. The oligomeric compound according to claim 98, as dependent on claim 94, wherein the one or more ligands are conjugated at the 31region, optionally at the 31terminal nucleoside of the sense sequence and / or of the antisense sequence, and / or to the 51terminal nucleoside of the second region of linked nucleosides.

100. The oligomeric compound according to any of claims 97 to 99, wherein 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.

101. The oligomeric compound according to claim 100, wherein the one or more ligands comprise one or more carbohydrates.

102. The oligomeric compound according to claim 101, wherein the one or more carbohydrates can be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide or polysaccharide103. The oligomeric compound according to claim 102, wherein the one or more carbohydrates comprise or consist of one or more hexose moieties.

104. The oligomeric compound according to claim 103, wherein 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 / or one or more mannose moieties.

105. The oligomeric compound according to claim 104, wherein the one or more carbohydrates comprise one or more N-Acetyl-Galactosamine moieties.

106. The oligomeric compound according to claim 105, which comprises two or more N-Acetyl- Galactosamine moieties, optionally three.

107. The oligomeric compound according to any of claims 97 to 106, wherein 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.

108. The oligomeric compound according to claim 107, wherein the one or more ligands are attached to the oligomeric compound as a biantennary or triantennary configuration.

109. The oligomeric compound according to any one of claims 85 to 108, wherein the compound consists of the antisense sequence and the sense sequence.

110. The oligomeric compound according to any one of claims 85 to 109, wherein there is an intervening nucleic acid sequence between the antisense and the sense sequence.

111. The oligomeric compound according to claim 110, wherein 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.

112. The oligomeric compound according to any one of claim 93 to 109, wherein 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.

113. The oligomeric compound according to claim 112, wherein the antisense and the sense sequence are directly adjacent on the single strand.

114. The oligomeric compound according to claim 28 or 29, wherein 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%.

115. The oligomeric compound of claim 114, whereby 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 RNA transcribed from a PCSK9 gene forms the hairpin loop, wherein the loop comprises 2 to 5, optionally 4 or 5, nucleosides.

116. The oligomeric compound according to any one of the preceding claims, wherein the single strand is selected from the group consisting of SEQ ID Nos 613-618.

117. The oligomeric compound according to claim 116, wherein the single strand is selected from the group consisting of SEQ ID Nos. 1227-1858, in particular, SEQ ID Nos. 1261, 1268, 1269, 1282, 1473 and 1475118. The oligomeric compound according to claim 117, as dependent on claim 96, whereby a hairpin loop is present at the 31region 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.

119. The oligomeric compound according to any one of claims 85 to 118, wherein 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.

120. The oligomeric compound according to any one of claims 85 to 119, wherein the hairpin loop comprises 1 to 8, 2 to 7, 3 to 6, optionally 4 or 5 linked nucleosides.

121. The oligomeric compound according to any of claims 85 to 120, which comprises internucleoside linkages and wherein at least one internucleoside linkage is a modified internucleoside linkage.

122. The oligomeric compound according to claim 121, wherein the modified internucleoside linkage is a phosphorothioate or phosphorodithioate internucleoside linkage.

123. The oligomeric compound according to claim 122, which comprises 1 to 16 phosphorothioate or phosphorodithioate internucleoside linkages.

124. The oligomeric compound according to claim 123, which comprises 7, 8, 9 or 10 phosphorothioate or phosphorodithioate internucleoside linkages.

125. The oligomeric compound according to any of claims 112 to 124, as dependent on claim 94, which comprises one or more phosphorothioate or phosphorodithioate internucleoside linkages at the 5’ region of the antisense sequence.

126. The oligomeric compound according to any of claims 112 to 125, as dependent on claim 94, which 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 51region.

127. The oligomeric compound according to any of claims 112 to 126, as dependent on any one of claims 112 to 116, which 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.

128. The oligomeric compound according to claim 127, which comprises a phosphorothioate or phosphorodithioate internucleoside linkage between each adjacent nucleoside that is present in the hairpin loop.

129. The oligomeric compound according to any of claims 85 to 128, wherein at least one nucleoside comprises a modified sugar.

130. The oligomeric compound according to claim 129, wherein the modified sugar is selected from 21modified 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).

131. The oligomeric compound according to claim 130, wherein the 2' modified sugar is selected from 2-O-alkyl modified sugar, 2'-O-methyl modified sugar, 2'-O-methoxyethyl modified sugar, 2'-O- allyl modified sugar, 2'-C-allyl modified sugar, 2'-deoxy modified sugar such as 2'-deoxy ribose, 2'-F modified sugar, 2-arabino-fluoro modified sugar, 2'-O-benzyl modified sugar, and 2'-O-methyl-4- pyridine modified sugar132. The oligomeric compound according to claim 131, wherein at least one modified sugar is a 2'- O-methyl modified sugar.

133. The oligomeric compound according to claim 131 or 132, wherein at least one modified sugar is a 2'-F modified sugar and, optionally, at most 16 or 17 sugars are 2'-F modified sugars134. The oligomeric compound according to any of claims 48-51, wherein the sugar is ribose.

135. The oligomeric compound according to any of claims 132-134, as dependent on claim 94, wherein 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.

136. The oligomeric compound of any one of claims 132 to 135, wherein the 31terminal position of the sense sequence does not contain a 2'-O-methyl modification.

137. The oligomeric compound according to any one of claims 132 to 136, wherein 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.

138. The oligomeric compound according to any of claims 136 to 137, wherein sugars of the nucleosides of the sense strand, that 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.

139. The oligomeric compound of claim 55 or 56, wherein the 3' terminal nucleoside of the second region of linked nucleosides contains a 2'-F modification.

140. The oligomeric compound according to any of claims 136 to 139, as dependent on claim 94, wherein one or more of the odd numbered nucleosides starting from the 5’ region of the antisense sequence are modified, and / 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.

141. The oligomeric compound according to claim 140, wherein 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.

142. The oligomeric compound according to claim 140 or 141 , wherein 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 according to claim 133.

143. The oligomeric compound according to any of claims 140-142, wherein 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.

144. The oligomeric compound according to any of claims 140 to 143, wherein 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 sequence145. The oligomeric compound according to any of claims 140 to 144, wherein 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.

146. The oligomeric compound according to any of claims 140 to 145, wherein one or more of the even numbered nucleosides starting from the 3’ region of the antisense sequence are 2'-F modified sugars.

147. The oligomeric compound according to any of claims 140 to 146, wherein 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.

148. The oligomeric compound according to any of claims 140 to 147, wherein one or more of the even numbered nucleosides starting from the 5’ region of the sense sequence are 2'-F modified sugars.

149. The oligomeric compound according to any of claims 129 to 148, wherein sugars of a plurality of adjacent nucleosides of the antisense sequence are modified by a common or different modification.

150. The oligomeric compound according to any of claims 129 to 149, wherein sugars of a plurality of adjacent nucleosides of the sense sequence are modified by a common or different modification.

151. The oligomeric compound according to any of claims 140 to 150, as dependent on any one of claims 112 to 115, wherein sugars of a plurality of adjacent nucleosides of the hairpin loop are modified by a common or different modification.

152. The oligomeric compound according to any of claims 149 to 151, wherein the common modification is a 2'-F modified sugar.

153. The oligomeric compound according to any of claims 149 to 151, wherein the common modification is a 2'-O-methyl modified sugar.

154. The oligomeric compound according to claim 153, wherein the plurality of adjacent 2'-O- methyl modified sugars are present in at least eight adjacent nucleosides of the antisense and / or sense sequence.

155. The oligomeric compound according to claim 154, wherein the plurality of adjacent 2'-O- methyl modified sugars are present in three or four adjacent nucleosides of the hairpin loop.

156. The oligomeric compound according to claim 130, as dependent on any one of claims 115 to 117, wherein the hairpin loop comprises at least one nucleoside having a modified sugar.

157. The oligomeric compound according to claim 156, wherein 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.

158. The oligomeric compound according to claim 157, wherein the modified sugar is a 2'-O- methyl modified sugar, and the differently modified sugar is a 2'-F modified sugar.

159. The oligomeric compound according to any of claims 85 to 158, wherein 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 31carbon of its sugar and the 3' carbon of the sugar of the adjacent nucleoside, and / or one or more nucleosides of antisense sequence and / 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.

160. The oligomeric compound according to any of claims 85 to 159, which is blunt ended161. The oligomeric compound according to any of claims 85 to 159, wherein either the antisense and sense sequence has an overhang.

162. The oligomeric compound according to any one of claims 85 to 161 , wherein the oligomeric compound has a total length of about 25 to about 37 nucleosides, in particular about 33 or about 34 nucleosides.

163. The oligomeric compound according to any one of claims 94 to 162, wherein 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 31position of the sense regionis replaced by a base being complementary to the base at the 5' position of the first region, optionally A.

164. The oligomeric compound according to any one of claims 85 to 163, wherein the antisense and sense sequences are only being composed of nucleobases selected from the group consisting of A, U, G, C and not T.

165. The oligomeric compound according to any one of claims 85 to 164, wherein the nucleosides do not contain a 2'-deoxy modification.

166. A composition comprising a construct according to any one of claims 1 to 84 and / or an oligomeric compound according to any one of claims 85 to 165, and a physiologically acceptable excipient.

167. A pharmaceutical composition comprising a nucleic acid construct according to any of claims 1 to 84 and / or an oligomeric compound according to any of claims 85 to 165168. The pharmaceutical composition of claim 167, further comprising a pharmaceutically acceptable excipient, diluent, antioxidant, and / or preservative169. The pharmaceutical composition of claim 167 or 168, wherein the nucleic acid construct according to any one of claims 1 to 84 and / or the oligomeric compound according to any one of claims 85 to 165 is the only pharmaceutically active agent.

170. The pharmaceutical composition of claim 166, wherein the pharmaceutical composition is to be administered to patients or individuals which are statin-intolerant and / or for whom statins are contraindicated.

171. The pharmaceutical composition of claim 166 or 167, wherein the pharmaceutical composition furthermore comprises one or more further pharmaceutically active agents.

172. The pharmaceutical composition of claim 169, wherein the further pharmaceutically active agent(s) is / are an RNAi agent which is directed to a target different from PCSK9 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 fenofibrate173. The pharmaceutical composition of claim 168 or 170, wherein the construct and the further pharmaceutically active agent(s) are to be administered concomitantly or in any order.

174. A nucleic acid construct according to any of claims 1 to 84 and / or an oligomeric compound according to any one of claims 85 to 165 for use in human or veterinary medicine or therapy.

175. A nucleic acid construct according to any of claims 1 to 84 and / or an oligomeric compound according to any one of claims 85 to 165, for use in a method of treating, ameliorating and / or preventing a disease or disorder.

176. The nucleic acid construct and / or the oligomeric compound for use of claim 175, wherein the disease or disorder is a PCSK9-associated disease or disorder requiring reduction of PCSK9 expression levels.

177. The nucleic acid construct and / or the oligomeric compound for use of claim 175 or 176, wherein the disease or disorder is a PCSK9-associated disease or disorder, or a disease or disorder requiring reduction of low-density lipoprotein (LDL) cholesterol, the disease or disorder optionallybeing selected from dyslipidemia including mixed dyslipidemia, hypercholesterolemia, heterozygous familial hypercholesterolemia, non-familial hypercholesterolemia; atherosclerosis; and atherosclerotic cardiovascular disease (ASCVD) including myocardial infarction, stroke and peripheral arterial disease.

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