WO2024129874A1

WO2024129874A1 - Products and compositions

Info

Publication number: WO2024129874A1
Application number: PCT/US2023/083855
Authority: WO
Inventors: Dmitry Samarsky; Jack Wei
Original assignee: Sirnaomics, Inc.
Priority date: 2022-12-13
Filing date: 2023-12-13
Publication date: 2024-06-20

Abstract

Provided are products, and compositions, and their uses. In particular, provided are nucleic acid products that modulate, in particular interfere with or inhibit TTR gene expression.

Description

Products and Compositions

Cross-Reference to Related Applications

This application claims priority to U.S. Provisional Patent Application Serial No. 63/432,267, filed December 13, 2022, the disclosure of which is hereby incorporated by reference in its entirety.

Sequence Listing

The instant application contains a Sequence Listing submitted electronically in ST.26 (XML) format and hereby incorporated by reference in its entirety. The XML file, created on December 5, 2023, is named 4690_0086I_SL.xml and is 4,275,275 bytes in size.

Field

Nucleic acid compounds, compositions and methods of their manufacture and use are provided that modulate, in particular interfere with or inhibit, transthyretin (TTR) gene expression in mammals. Such methods, compounds, and compositions are useful in the treatment, prevention, or amelioration of TTR-associated diseases or disorders.

Background

Transthyretin (TTR, for “transports thyroxine and retinol”) is a serum/plasma and cerebrospinal fluid protein responsible for the transport of thyroxine and retinol. TTR is synthesized primarily by the liver and the choroid plexus of the brain. Transthyretin synthesized in the liver is secreted into the blood. TTR is a 55 kDa homotetramer with a dimer of dimers quaternary structure.

Disease

TTR misfolding and aggregation are known to be associated with amyloidosis, such as senile systemic amyloidosis, familial amyloid polyneuropathy and familial amyloid cardiomyopathy. TTR mutations accelerate the process of TTR amyloid formation and are the most important risk factor for the development of clinically significant TTR amyloidosis (also called ATTR (amyloidosis-transthyretin type) or transthyretin-mediated amyloidosis). Treatment

Double-stranded RNA (dsRNA) are able to complementarity bind expressed mRNA has been shown to be able 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 a mechanism that has been termed RNA interference (RNAi). Short dsRNAs direct genespecific, post-transcriptional silencing in many organisms, including vertebrates, and have become a useful tool for studying gene function. RNAi is mediated by the RNA-induced silencing complex (RISC), a sequence-specific, multi-component nuclease that destroys messenger RNAs homologous to the silencing trigger loaded into the RISC complex. Interfering RNA (iRNA), such as siRNAs, antisense RNA, and micro-RNA are oligonucleotides that prevent the formation of proteins by gene-silencing i.e. inhibiting gene translation of the protein through degradation of mRNA molecules. Gene-silencing agents are becoming increasingly important for therapeutic applications in medicine.

According to Watts and Corey in the Journal of Pathology (2012; Vol 226, p 365-379) there are algorithms that can be used to design nucleic acid silencing triggers, but all of these have severe limitations. It may take various experimental methods to identify potent siRNAs, as algorithms do not take into account factors such as tertiary structure of the target mRNA or the involvement of RNA binding proteins. Therefore, the discovery of a potent nucleic acid silencing trigger with minimal off-target effects is a complex process. For the pharmaceutical development of these highly charged molecules it is necessary that they can be synthesised economically, distributed to target tissues, enter cells and function within acceptable limits of toxicity.

Patisiran is an example for a siRNA-based treatment for transthyretin-related hereditary amyloidosis. However, the expected costs of such treatment of up to $450,000 per year are high (“Rare-Disease Treatment from Alnylam to Cost $450,000 a Year”. Bloomberg.com. 10 August 2018. Retrieved 11 August 2018).

Therefore, there is a need for further compounds and treatments being capable of efficiently reducing the adverse effects discussed herein in connection with production of transthyretin, such as amyloidosis, and overcoming the aforementioned disadvantages.

Summary

The aforementioned problem of providing compounds and treatments having the potential of efficiently reducing the adverse effects discussed herein in connection with production of TTR, such as amyloidosis, above is solved by the present disclosure.

The following aspects of the present disclosure are reflected in the independent claims. According to a first aspect, the present disclosure is directed to an oligomeric compound having the potential of inhibiting expression of transthyretin (TTR), wherein the compound comprises at least a first region of linked nucleosides having at least a first nucleobase sequence that is at least partially complementary to at least a portion of RNA transcribed from a TTR gene, wherein the first nucleobase sequence is selected from the following sequences, or a portion thereof: sequences of Table la (SEQ ID NOs: 1 to 100), wherein the portion optionally has a length of at least 18 nucleosides. Particularly optional embodiments according to the first aspect of the present disclosure relate to optimized hairpin RNAs (referred to as mxRNAs); for further details see the embodiments and their discussion further below.

Furthermore, and as disclosed further below, the disclosure also relates to double-stranded RNAs (dsRNAs). Deviant from mxRNAs, dsRNAs lack a loop connecting antisense and sense portions and therefore comprise two strands. The two strands are not covalently connected to each other, but form a duplex region where base pairing occurs.

According to a second aspect, the present disclosure is directed to a composition comprising an oligomeric compound according to the first aspect and a physiologically acceptable excipient.

According to a third aspect, the present disclosure is directed to a pharmaceutical composition comprising an oligomeric compound according to the first aspect.

According to a fourth aspect, the present disclosure is directed to an oligomeric compound according to the first aspect for use in human or veterinary medicine or therapy.

According to a fifth aspect, the present disclosure is directed to an oligomeric compound according to the first aspect for use in a method of treating a disease or disorder.

According to a sixth aspect, the present disclosure is directed to a method of treating a disease or disorder comprising administration of an oligomeric compound according to the first aspect to an individual in need of treatment.

According to a seventh aspect, the present disclosure is directed to a use of an oligomeric compound according to the first aspect for use in research as a gene function analysis tool. According to an eight aspect, the present disclosure is directed to a use of an oligomeric compound according to the first aspect in the manufacture of a medicament for a treatment of a disease or disorder.

Effects achieved by the oligomeric compounds

Due to the use of the oligomeric compounds according to the present disclosure, a significant reduction of gene expression of TTR, e.g, in vitro using primary human hepatocytes, can be achieved as e.g. shown in the examples disclosed herein. The most inhibiting compounds surprisingly produce knockdowns of more than 90% TTR mRNA expression in vitro. Furthermore, the compounds, as, e.g., shown in the examples, are at least capable of producing knockdowns of at least 50% of TTR expression in vitro. As TTR expression can be successfully reduced, the compounds have the potential of efficiently reducing the negative effects in terms of TTR misfolding/aggregation/mutation for treating amyloidosis. These results were confirmed by in vivo tests in humanized liver mice models where knockdowns of TTR mRNA expression of over 90% were achieved.

Furthermore, it was surprisingly found that, in certain embodiments, the mentioned effects are achieved by using oligomeric compounds according to the present disclosure for inhibiting the expression of TTR 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 a synthesis of mxRNA molecules more cost and production efficient, because less units are needed.

For certain oligomeric compounds according to the present disclosure, being in the form of mxRNA constructs for inhibiting the expression of TTR gene, it was surprisingly found out that the aforementioned effects can be achieved by using short sense strands within the mxRNA having a length of optionally 14 nucleosides which is shorter than the length of the sense strands in conventional shRNA molecules.

The effects and technical advantages achieved by using the disclosed embodiments for oligomeric compounds for inhibiting TTR expression will become apparent in more detail in the detailed description and the examples.

Brief Description of the Drawings

Figure 1 shows single dose curves of certain TTR mxRNA compounds of the present disclosure and their activity in inhibiting TTR gene expression (primary screening).

Figure 2a shows dose curves of 43 TTR mxRNA compounds and their activity in inhibiting TTR gene expression (secondary screening).

Figure 2b shows dose curves of 5 TTR mxRNA compounds and their activity in inhibiting TTR gene expression (second batch)

Figure 3 shows the treatment schedule and the test and control groups for evaluating mxRNA in humanized liver mice models (in vivo) for the inhibition of TTR mRNA expression. Figure 4 shows results of TTR mRNA expression in Liver Tissues in vivo.

Detailed Description

Further embodiments (items) of the present disclosure are described below by way of example only. These examples represent some of the most 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.

It will be understood that the benefits and advantages described herein may relate to one embodiment or may relate to several embodiments. The embodiments are not limited to those that solve any or all of the stated disadvantages or those that have any or all of the stated benefits and advantages. Embodiments labelled "optional" are not intended to limit the scope of the claims but to show optional embodiments of the present disclosure.

Features of different aspects and embodiments of the disclosure may be combined as appropriate, as would be apparent to a skilled person, and may be combined with any of the aspects of the disclosure.

Definitions

The following definitions pertain to the disclosure throughout. In many instances, the definitions, in addition to the respective definition as such, provide non-exhaustive listings of possible implementations which amount to optional embodiments.

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., 21st edition, 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,” 2nd Edition, 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.

Unless otherwise indicated, the following terms have the following meanings:

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.

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”. The structural features and/or the lengths of oligomeric compounds or nucleic acid constructs disclosed herein is expressed in terms of “nucleosides” or “nucleotides”.

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 intemucleoside 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.

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”. In particular, such 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 2¹- position of the sugar moiety'.

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.

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.

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 (z.e., the 2' -substituent of a 2' -substituted sugar moiety does not form a bridge to another atom of the furanosyl ring).

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 the analogues retain RNase H activity.

As used herein the term “sugar surrogate” means a structure that does not comprise a furanosyl and that is capable of replacing the naturally occurring sugar moiety of a nucleoside, such that the resulting nucleoside sub-units are capable of linking together and I or linking to other nucleosides to form an oligomeric compound which is capable of hybridizing 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 morpholines, cyclohexenyls and cyclohexitols.

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).

As used herein, “nucleobase” means a group of atoms that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into an oligonucleotide, and 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.

As used herein the terms, “unmodified nucleobase” or “naturally occurnng 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.

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.

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

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.

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).

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 I or unmodified deoxyribonucleosides (DNA) and I or one or more modified nucleosides.

As used herein, “modified oligonucleotide” means an oligonucleotide comprising at least one modified nucleoside and / or at least one modified intemucleoside linkage.

Optional modified intemucleoside linkages are those, which confer increased stability as compared to the naturally occurring phosphodiesters. “Stability” refers in particular to stability against hydrolysis including enzyme-catalyzed hydrolysis, enzymes including exonucleases and endonucleases.

Optional positions for such modified intemucleoside linkages include the termini and the hairpin loop of single-stranded oligomeric compounds of the disclosure. For example, the intemucleoside linkages connecting first and second nucleoside and second and third nucleoside counting from the 5' terminus, and/or the intemucleoside linkages connecting first and second nucleoside and second and third nucleoside counting from the 3' terminus are modified. In addition, a linkage connecting the terminal nucleoside of the 3' terminus with a ligand, such as GalNAc, may be modified.

As discussed above, optional positions are in the hairpin loop of the single-stranded oligomeric compounds. In particular, all linkages, all but one linkages or the majority of linkages in the hairpin loop are modified. As used herein, “linkages in the hairpin loop” designates the linkages between nucleosides, which are not engaged in base pairing. For example, in a hairpin loop consisting of five nucleosides, there are four linkages between nucleosides which are not engaged in base pairing. Optionally, the term “linkages in the hairpin loop” also extends to the linkages connecting the stem to the loop, i.e., those linkages which connect a base-paired nucleoside to a non-based paired nucleoside. Generally, there are two such positions in hairpins and mxRNAs in accordance with the disclosure.

Most optional is that modified intemucleoside linkages are at both termini and in the hairpin loop.

As used herein, “linkage” or “linking group” means a group of atoms that link together two or more other groups of atoms.

As used herein “intemucleoside linkage” means a covalent linkage between adjacent nucleosides in an oligonucleotide.

As used herein “naturally occurring intemucleoside linkage” means a 3' to 5' phosphodiester linkage.

As used herein, “modified intemucleoside linkage,” means any intemucleoside linkage other than a naturally occurring intemucleoside linkage. In particular, a “modified intemucleoside linkage” as referred to herein can include a modified phosphorous linking group such as a phosphorothioate or phosphorodithioate intemucleoside linkage.

As used herein, “terminal intemucleoside linkage” means the linkage between the last two nucleosides of an oligonucleotide or defined region thereof.

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, thionoalkylphosphotriester and boranophosphate. As used herein, “intemucleoside phosphorus linking group” means a phosphorus linking group that directly links two nucleosides.

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 I or terminal groups and I 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, in particular the modification status of the sugars (unmodified, 2'-0Me modified, 2'-F modified etc.) and/or of the phosphates. An mxRNA is one non-limiting example for an oligomeric compound.

As used herein, “nucleic acid construct” or “construct” refers to an assembly of two or more, such as four oligomeric compounds. The oligomeric compounds may be connected to each other by covalent bonds such phosphodiester bonds as they occur in naturally occurring nucleic acids or modified versions thereof as disclosed herein, or by non-covalent bonds such as hydrogen bonds, optionally hydrogen bonds between nucleobases such as Watson-Crick base pairing. In certain embodiments, optional is that a construct comprises four oligomeric compounds, two of which are connected covalently, thereby giving rise to two nucleic acid strands which nucleic acid strands are bound to each other by hydrogen bonds. Complementarity between the strand may be throughout, but is not necessarily so. In particular, exemplary embodiments provide for an antisense strand targeting a first region of TTR mRNA to be connected covalently with a sense strand of another TTR-targeting double stranded RNA molecule, and of the antisense strand of the TTR mRNA-targeting double stranded RNA molecule to be connected covalently to a sense strand of the other TTR mRNA-targeting double stranded RNA molecule. Since antisense and sense strands 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 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.

As used herein, “terminal group” means one or more atom attached to either, or both, the 3 ' end or the 5' end, also called “terminus” of an oligonucleotide. In certain embodiments, a terminal group comprises one or more terminal group nucleosides, whereas a “terminal nucleoside” is only one nucleotide at the respective end (5' end or 3' end).

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 I or clearance properties.

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 and this is particularly optional. 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:

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

As used herein, “cleavable moiety” means a bond or group that is capable of being 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.

As used herein, “cleavable bond” means any chemical bond capable of being broken.

As used herein, “carbohydrate cluster” means a compound having one or more carbohydrate residues attached to a linker group.

As used herein, “modified carbohydrate” means any carbohydrate having one or more chemical modifications relative to naturally occurring carbohydrates.

As used herein, “carbohydrate derivative” means any compound which may be synthesized using a carbohydrate as a starting material or intermediate.

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 I or one or more mannose moieties. A particularly optional carbohydrate is N-Acetyl-Galactosamine.

As used herein, “strand” means an oligomeric compound compnsing 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 capable of forming a stable self-duplex in a hairpin structure.

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 sei f-complementary and opposite in directionality.

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.

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 cany (a) 3' and/or (a) 5¹ 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 out 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.

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 3¹ carbon of the sugar moiety. In a duplex or double stranded oligonucleotide, the respective strands run in opposite 5¹ to 3¹ directions to permit base pairing between them. 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 I or (A) adenine and uracil (U), and I or guanine (G) and cytosine (C). The duplex may be part of a single stranded structure, wherein self-complementarity leads to hybridization, or as a result of hybridization between respective strands in a double stranded construct.

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, polyadenylation, addition of 5 '-cap), and translation.

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 enzy me RNA polymerase. During transcription, a DNA sequence is read by an RNA polymerase, which produces a complementary, antiparallel RNA sequence called a primary transcript.

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 TTR expression. Oligonucleotides have sufficient complementarity to their target sequences to allow hybridization under physiological conditions.

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

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.

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.

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.

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 I or self-complementary regions of the oligomeric compound, are aligned.

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.

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.

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. 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.

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.

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. Accordingly, unless otherwise indicated, a “nucleoside having a modification of a first type” may be an unmodified nucleoside.

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.

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.

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.

As used herein, “pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administering to an animal. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile saline. In certain embodiments, such sterile saline is pharmaceutical grade saline.

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', in particular 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, alkynyl, 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').

As used herein, “alkyl,” as used herein, means a saturated straight or branched monovalent Cl -6 hydrocarbon radical, with methyl being a most optional alkyl as a 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 a -Oalkyl substituent, such as an - 0CH3 substituent, on a sugar moiety of an oligomeric compound according to the present disclosure. This will be well understood be a person skilled in the art.

As used herein, “alkylene” means a saturated straight or branched divalent hydrocarbon radical of the general formula -CnFbn- where n is 1-6. Methylene or ethylene are optional alkylenes.

As used herein, “alkenyl” means a straight or branched unsaturated monovalent C2-6 hydrocarbon radical, with ethenyl or propenyl being most optional 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 according to the present disclosure. This will be well understood be a person skilled in the art.

As used herein, “alkynyl” means a straight or branched unsaturated C2-6 hydrocarbon radical, with ethynyl being a most optional 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 according to the present disclosure. This will be well understood be a person skilled in the art.

As used herein, “carboxyl” is a radical having a general formula -CO2H.

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 ty pically Cl -6 alkyl.

As used herein, “alkoxy” means a radical formed between an alkyl group, such as a Cl -6 alkyl group, and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group either to a parent molecule (such as at the 2'-position of a sugar moiety), or to another group such as an alkylene group as defined herein. 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. 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'-position of the sugar, therefore, overall providing a -O-alkylenealkoxy substituent, such as an -OCH2CH2OCH3 substituent, on a sugar moiety of an oligomeric compound according to the present disclosure. 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.

As used herein, “amino” includes primary, secondary and tertiary amino groups.

As used herein, “halo” and “halogen,” mean an atom selected from fluorine, chlorine, bromine, and iodine.

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 portion (also referred to as the guide strand) and a sense portion (also referred to the passenger strand). The mxRNA comprises duplex region and a hairpin loop, wherein the mxRNA has approximate length of about 34 nucleotides. The duplex region comprises a region in which parts of the antisense portion and substantially the entire sense portion, typically 14 or 15 nucleotides of each strand, are base-paired. The hairpin loop connects both regions, i.e. antisense region and sense region, of that duplex via e.g. a phosphate or a phosphorothioate linker, i.e. covalently, while the antisense portion 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 portion is part, furthermore connects the sense, forming the second strand of the loop, and the antisense portion.

The term “transthyretin” or abbreviated “TTR”, also known as prealbumin or thyroxine- binding prealbumin, is used in its common sense and denotes a transport protein in the plasma and cerebrospinal fluid that transports the thy roid hormone T4 and retinol to the liver. 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 nucleic acid portions, targeting different regions of TTR mRNA; or one region of TTR mRNA and an mRNA region of another target molecule. The targeting RNA sequences are also referred to as “antisense” or “guide” strands, while the respective passenger strands, i.e. third and fourth nucleic acid portions being complementary to the first and second portion, 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 nucleic acid portions are released. A particular example for such muRNA is shown below, where (1) is the first nucleic acid portion, (2) is the third nucleic acid portion being complementary to (1), (3) is the second nucleic acid portion being complementary to the fourth nucleic acid portion, while (5) is a labile linker while (6) is a ligand, which will both be explained below.

It will also be understood that oligomeric compounds as described herein may have one or more non-hybridizing nucleosides at one or both ends of one or both strands (overhangs) and I or one or more internal non-hybridizing nucleosides (mismatches) provided there is sufficient complementarity to maintain hybridization under physiologically relevant conditions. Alternatively, oligomeric compounds as described herein may be blunt ended at least one end.

As used herein, 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 molecule.

As used herein, APOC3 is referred to apolipoprotein C3 which is secreted by the liver and the small intestine. It can be found on triglyceride-rich lipoproteins including very low density lipoproteins (VLDL) and chylomicrons. It is involved in the negative regulation of lipid catabolism, especially triglyceride catabolism, and of the clearance of VLDL, LDL and HDL lipoproteins. A molecular function of APOC3 is the inhibition of lipoprotein lipase and of hepatic lipase.

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.

20

SUBSTITUTE SHEET (RULE 26) 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.

Small hairpin (shRN A) and mxRNA oligomeric compounds

According to a first aspect, the present disclosure is directed to an oligomeric compound capable of inhibiting expression of transthyretin (TTR), wherein the compound comprises at least a first region of linked nucleosides having at least a first nucleobase sequence that is at least partially complementary to at least a portion of RNA transcribed from an TTR gene, wherein the first nucleobase sequence is selected from the following sequences, or a portion thereof: sequences of Table la (SEQ ID NOs: 1 to 100), wherein the portion optionally has a length of at least 18 nucleosides. In particular, the 5' terminal nucleoside of the first nucleobase sequence can contain U instead of A; or U instead of G; or U instead of C, respectively.

In certain embodiments, the oligomeric compound further may comprise at least a second region of linked nucleosides having at least a second nucleobase sequence that is at least partially complementary to the first nucleobase sequence and is selected from the following sequences, or a portion thereof: sequences of Table lb (SEQ ID NOs: 101 to 200), wherein the portion optionally has a length of at least 8, 9, 10 or 11, more optionally at least 10, nucleosides. In particular, the 3' terminal nucleoside of the second nucleobase sequence may contain an A instead of U, G or C, respectively; and more particularly the nucleobase A as a complementary nucleobase to the 5' terminal nucleoside of the first nucleobase sequence. The first region of linked nucleosides is also referred to as antisense region or guide region/strand, and the second region of linked nucleosides is referred to as sense region or passenger region/strand. As disclosed in optional embodiments below, the two regions may be located on the same RNA strand, optionally in an adjacent manner. This gives rise to hairpin molecules, also referred to as mxRNAs. On the other hand, the two regions may be located on separate strands, which gives rise to double-stranded RNAs (dsRNAs), wherein optionally each strand consists of the respective region.

Without wishing to be bound by theory, it is assumed that the disclosed embodiments for oligomeric compounds set forth above including the first region of linked nucleosides and the second region of linked nucleosides are, during the process of RNA interference, incorporated into the RNA-induced silencing complex (RISC). The RISC assembly then binds and degrades the target mRNA. Specifically, this is accomplished when the guide strand pairs with a complementary sequence in a TTR mRNA molecule and induces cleavage by Ago2, a catalytic component of the RISC. For that reason, as the expression of TTR is inhibited, it is believed effects correlating with TTR misfolding/aggregation/mutation, is inhibited too.

In certain embodiments the first nucleobase sequence is selected from the following sequences, or a portion thereof SEQ ID NOs: 58, 64, 48, 19, 50, 36, 85, 23, 28, 30, 38, 91, 98, 69, 86, 41, 37, 51, 47, 67, 33, 46, 78, 45, 25, 60, 44, 9, 73, 32, 68, 53, 31, 66, 11, 1, 2, 3, 4, 5, 6, 7, and 8. Optionally the first nucleobase sequence is selected from the following sequences, or a portion thereof: SEQ ID NOs: 36, 58, 50, 46, 98, and 67.

In an optional embodiment thereof, wherein the second nucleobase sequence is selected from the following sequences, or a portion thereof: SEQ ID NOs: 158, 164, 148, 119, 150, 136, 185, 123, 128, 130, 138, 191, 198, 169, 186, 141, 137, 151, 147, 167, 133, 146, 178, 145, 125, 160, 144, 109, 173, 132, 168, 153, 131, 166, 111, 101, 102, 103, 104, 105, 106, 107, and 108. Optionally the second nucleobase sequence is selected from the following sequences, or a portion thereof: SEQ ID NOs: 136, 158, 150, 146, 198, and 167.

Lengths and molecular features of the oligomeric compounds according to the first aspect The first region of linked nucleosides, e.g. 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 second region of linked nucleosides, e.g. the sense strand, may consist of 10 to 35, optionally 10 to 20, more optionally 10 to 16, and yet more optionally 10 to 15, in particular 13, 14 or 15 linked nucleosides.

The oligomeric compound including the first and second regions of linked nucleosides may comprise at least one complementary duplex region that comprises at least a portion of the first region of linked nucleosides directly or indirectly linked to at least a portion of the second region of linked nucleosides, 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.

In certain embodiments, each of the first and second regions of linked nucleosides has a 5’ to 3’ directionality thereby defining 5’ and 3’ regions respectively thereof.

In the oligomeric compound having the first and second regions of linked nucleosides having a 5' to 3' di recti onalily. the 5’ region of the first region of linked nucleosides may be directly or indirectly linked to the 3’ region of the second region of linked nucleosides, for example by complementary base pairing, wherein optionally the 5' terminal nucleoside of the first nucleoside region base pairs with the 3' terminal nucleoside of the second nucleoside region, wherein optionally the base of the 5' terminal nucleoside of the first region is U and the base of the 3' terminal nucleoside of the second region is A.

In the aforementioned embodiments, the 3’ region of the first region of linked nucleosides may be directly or indirectly linked to the 5’ region of the second region of linked nucleosides, wherein optionally the first nucleoside region is directly and covalently linked to the second nucleoside region such as by a phosphate, a phosphorothioate, or a phosphorodithioate, wherein more optionally a 3' terminal nucleoside of the first region of linked nucleosides is directly and covalently linked to a 5' terminal nucleoside of the second region of linked nucleosides by a phosphate, a phosphorothioate, or a phosphorodithioate. It is particularly optional that the 3' terminal nucleoside of the first region is directly linked to the 5' terminal nucleoside of the second region via a phosphorothioate intemucleoside linkage.

This amounts to the formation of a single oligonucleotide comprising or consisting of the two regions being directly fused to each other. Owing to the base pairing as defined in the previous embodiment, such oligonucleotide will assume a hairpin configuration. Optimized hairpins, especially in terms of size, are the subject of further embodiments below.

In certain embodiments, the oligomeric compound may consist of the first region of linked nucleosides and the second region of linked nucleosides.

Each of the regions may constitute a separate strand, thereby giving rise to a double-stranded RNA (dsRNA). Particularly optional dsRNAs of the disclosure are those with a length of the first strand of 19 nucleosides and a length of the second region of 14 or 15, optionally 14 nucleosides. When used for defining the length of a region or strand, the terms "nucleoside" and “nucleotide” (sometimes abbreviated “nt”) are used equivalently.

In the alternative, and as stated above, the two regions may be fused together, giving rise to a hairpin.

In certain embodiments, there may be an intervening third region of linked nucleosides between the first and the second region.

In optional embodiments, the oligomeric compound comprises or consists of a single strand comprising or consisting of the first, the third, and the second nucleoside regions, wherein at least a portion of the first nucleoside region is directly or indirectly linked to at least a portion of the second nucleoside region so as to form the at least partially complementary duplex region.

In other words, the oligomeric compound comprises a single strand comprising the first and second nucleoside regions, wherein at least a portion of the first nucleoside region is directly or indirectly linked to at least a portion of the second nucleoside region so as to form the at least partially complementary duplex region. As noted above, the third region is optional. In certain embodiments, the oligomeric compound may comprise or may consist of a single strand comprising or consisting of the first and second regions of linked nucleosides, wherein at least a portion of the first region of linked nucleosides is directly or indirectly linked to at least a portion of the second region of linked nucleosides so as to form the at least partially complementary duplex region.

In the oligomeric compound, which may comprise or may consist of a single strand, the first and the second nucleoside regions are directly adjacent on the single strand.

In certain embodiments, the first nucleoside region may have a greater number of linked nucleosides compared to the second nucleoside region.

Optionally, a ratio between a total number of linked nucleosides of the first nucleoside region, e.g., the antisense strand, and a total number of linked nucleosides of the second nucleoside region, e.g, the sense strand, ranges from about 19/15 to about 19/8 or from about 18/15 to about 18/8. In particularly optional embodiments, the ratio is 19/15, 19/14, 19/13, 18/15, 18/14 or 18/13, most optionally 19/14 or 19/15.

Alternatively or in addition, a percentage of the total number of linked nucleosides of the first nucleoside region, e.g. the antisense strand, relative to the total number of nucleosides of the oligomeric compound may range from about to about 55% to about 60%. In particularly optional embodiments, the percentage may range from 57% to about 59.5%, most optionally the percentage is about 57.6% or about 59.4%.

Without wishing to be bound by theory, it is assumed that the ratio and/or percentages as mentioned above provides a suitable ratio/percentage of the number of nucleotides in the mxRNA to be processed by the RISC complex as mentioned above, and therefore, for being effective in TTR knockdown.

In the oligomeric compound having a greater number of linked nucleotides in the first region than in the second region, the additional number of linked nucleosides of the first nucleoside region, which exceed the number of linked nucleotides of the second region, form a hairpin loop linking the first and second regions of linked nucleosides, wherein optionally a part of the first nucleobase sequence of the first nucleobase sequence being complementary RNA transcribed from an TTR gene forms the hairpin loop, wherein the loop comprises 2 to 5, optionally 4 or 5, nucleosides. In particular, all, optionally 4 or 5 nucleosides, of the nucleosides forming the hairpin loop are complementary to the RNA transcribed from the TTR gene. Such compounds are also referred to as hairpins or mxRNAs herein. Owing to the second region being shorter as compared to the first region, the compound is optimized in terms of size (or miniaturized) as compared to a conventional siRNA which has two regions of comparable length.

Optionally, the loop has 4 or 5 linked nucleosides. Particularly optional is a length of the first region of 19 nucleosides, of the second region of 14 nucleosides, and of the hairpin loop of 5 nucleosides, wherein the 5 nucleosides in the hairpin are the 5 3 '-terminal nucleosides of the first region. Such molecular architecture of a hairpin or mxRNA of the disclosure is also designated “14-5-14” herein.

In certain embodiments, an oligomeric single strand as disclosed earlier herein, can be selected from Table 2, in particular selected from the group consisting of SEQ IDs NO: 236, 258, 250, 246, 298, and 267 wherein optionally the 5' terminal nucleoside of the first region of linked nucleosides is substituted by an U as the nucleobase, and the 3' terminal nucleoside of the second region of linked nucleosides is substituted by an A as the nucleobase.

In particular embodiments, the single strand is selected from Table 3c, in particular from Construct ID NOs: 536, 558, 550, 546, 598 and 567, wherein optionally the 5' terminal nucleoside of the first region of linked nucleosides is substituted by an U as the nucleobase, and the 5' terminal nucleoside of the second region of linked nucleosides is substituted by an A as the nucleobase.

In certain embodiments a hairpin loop as described earlier herein may be present at the 3' region of the first region of linked nucleosides, wherein optionally one, two or more 3' terminal nucleosides of the first nucleobase sequence, to the extent the nucleobases of the one, two or more 3' terminal nucleosides permit, fold back and form or contribute to the second region of linked nucleoside.

This is a structural design also referred to as “spill-over”. It is only possible in those cases where there is self-complementarity between the nucleobases at the 3 '-terminal end of the region of the guide sequence comprised in the duplex and the very 3'-terminal nucleobases of the same guide sequence. For example, this could implemented as a 14-5-14 design, thereby allowing for further miniaturization. The first “14” refers to the region of the guide sequence involved in the duplex, 5 is the length of the loop which is also formed by the guide sequence, and the second 14 refers to the second region of the duplex and is formed by one nucleobase of the guide sequence and 13 nucleobases of the passenger region in 5' to 3' direction. As such, a length of the guide sequence of 19 nucleosides is maintained, but the passenger sequence is shortened to 13 nucleosides. In certain embodiments, in case a third nucleoside region as described earlier herein, the third nucleoside region and optionally a 3'-terminal portion, optionally consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linked nucleosides, of the first nucleoside region and/or a 5'-terminal portion, optionally consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linked nucleosides, of the second nucleoside region may form a hairpin loop.

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

The oligomeric compounds according to the first aspect disclosed herein may be blunt ended. In the oligomeric compounds according to the first aspect disclosed herein, either the first or second nucleoside region may have an overhang.

In the oligomeric compounds according to the first aspect disclosed herein, the first region may be selected from the sequences of Table 3 a, or a portion thereof, in particular from Construct ID NOs: 336, 358, 350, 346, 398, and 367.

In the oligomeric compounds according to the first aspect disclosed herein the second region may be selected from the sequences of Table 3b, or a portion thereof, especially a portion having a length of 14 nucleosides, in particular from Construct ID NOs: 436, 458, 450, 446, 498, and 467.

The oligomeric compound may have a total length of about 25 to about 35 nucleosides, in particular about 33 or about 34 nucleosides.

In certain embodiments, a terminal nucleoside at a 5' position of the first region has a nucleobase 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 second region is substituted by a base being complementary to the base at the 5' position of the first region, optionally A.

In certain embodiments, wherein the nucleobase sequences within the oligomeric compounds may only be composed of nucleobases selected from the group consisting of A, U, G, C and not T.

Ligands

The oligomeric compounds may comprise one or more ligands.

The one or more ligands, in particular two or more or three ligands, may be conjugated to the second region of linked nucleosides and/or the first region of linked nucleosides.

The one or more ligands may be conjugated at the 3' region, optionally at the 3' terminal nucleoside of the second region of linked nucleosides and/or of the first region of linked nucleosides, and/or to the 5' terminal nucleoside of the second region of linked nucleosides. In particular, the ligands may be conjugated to the 3' terminal nucleoside. The one or more ligands are any cell directing moiety, such as lipids, carbohydrates, aptamers, vitamins and I or peptides that bind cellular membrane or a specific target on cellular surface.

The one or more ligands may comprise one or more, in particular three, carbohydrates.

The one or more, in particular three, carbohydrates can be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide or polysaccharide.

The one or more carbohydrates may comprise or consist of one or more, in particular three, hexose moieties.

The one or more, in particular three, hexose moieties are one or more galactose moieties, one or more lactose moieties, one or more, in particular three, N-Acetyl-Galactosamine moieties, and I or one or more mannose moieties.

The one or more carbohydrates may comprise one or more, in particular three, N-Acetyl- Galactosamine moieties.

Alternatively, the one or more carbohydrates may comprise two or more N-Acetyl- Galactosamine moieties, optionally three.

The one or more ligands are attached to the oligomeric compound, optionally to the second region of linked nucleosides thereof, in a linear configuration, or in a branched configuration. A particularly optional ligand is the following, also referred to as “toothbrush”:

Without wishing to be bound by a particular theory, it is assumed that due to such ligand the target tissue, i.e. the liver where TTR is produced, can be selectively targeted so the oligomeric compounds can exhibit their inhibition of TTR gene more efficiently.

27

SUBSTITUTE SHEET (RULE 26) The one or more, in particular three, ligands may be attached to the oligomeric compound as a biantennary or triantennary configuration.

The one or more ligands as discussed above are optionally attached to the 3' terminal nucleoside of the second region of linked nucleosides.

Internucleoside linkages

The oligomeric compound according to the first aspect disclosed herein may comprise intemucleoside linkages and wherein at least one intemucleoside linkage is a modified intemucleoside linkage.

The modified intemucleoside linkage may be a phosphorothioate or phosphorodithioate intemucleoside linkage.

The oligomeric compound according to the first aspect disclosed herein may comprise 1 to 16 phosphorothioate or phosphorodithioate intemucleoside linkages.

Optionally modified intemucleoside linkages are subject of the optional embodiments, which follow. Certain modified intemucleoside linkages are known in the art and described in, for example, Hu et al., Signal Transduction and Targeted Therapy (2020)5: 101.

The oligomeric compound may comprise 7, 8, 9 or 10 phosphorothioate or phosphorodithioate intemucleoside linkages. The one or more phosphorothioate or phosphorodithioate intemucleoside linkages may present at the 5’ region of the first region of linked nucleosides, wherein optionally, the oligomeric compound comprises three phosphorothioate intemucleoside linkages at three adjacent nucleosides at the 5' region.

In addition, the oligomeric compound may comprise phosphorothioate or phosphorodithioate intemucleoside 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. Particularly, the oligomeric compound may comprise a phosphorothioate or phosphorodithioate intemucleoside linkage between each adjacent nucleoside that is present in the hairpin loop.

Modifications

In the oligomeric compound according to the first aspect of the present disclosure, at least one nucleoside comprises a modified sugar.

The modified sugar may be 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). Optionally modified sugars are subject of the optional embodiments, which follow. Certain modified sugars are known in the art and described in, for example, Hu et al., Signal Transduction and Targeted Therapy (2020)5:101.

The 2' modified sugar may be selected from 2'-O-alkyl modified sugar, 2'-O-methyl modified sugar, 2'-O-methoxy ethyl modified sugar, 2'-O-ally 1 modified sugar, 2'-C-allyl modified sugar, 2'-deoxy modified sugar such as 2'-deoxy ribose, 2'-F modified sugar, 2'-arabino-fluoro modified sugar, 2'-O-benzyl modified sugar, and 2'-O-methyl-4-pyridine modified sugar. At least one modified sugar may be a 2'-O-methyl modified sugar.

At least one modified sugar may be a 2'-F modified sugar and, optionally, at most 16 or 17 sugars are 2'-F modified sugars. Optionally, the sugar is ribose.

In the oligomeric compound according to the first aspect disclosed herein, sugars of the nucleosides at any of positions 2 and 14 downstream from the first nucleoside of the 5’ region of the first region of linked nucleosides, do not contain 2'-O-methyl modifications.

In certain embodiments, the 3' terminal position of the second region of linked nucleosides 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 first region of linked nucleosides contain 2'-F modifications.

In certain embodiments, sugars of the nucleosides of the second region of linked nucleosides that correspond in position to any of the nucleosides of the first region of linked nucleosides at any of positions 11 to 13 downstream from the first nucleoside of the 5’ region of the first region of linked nucleosides contain 2'-F modifications.

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

In certain embodiments, one or more of the odd numbered nucleosides starting from the 5’ region of the first region of linked nucleosides may be modified, and I or wherein one or more of the even numbered nucleosides starting from the 5’ region of the first region of linked nucleosides may be modified, wherein typically the modification of the even numbered nucleosides is a second modification that is different from the modification of odd numbered nucleosides.

In certain embodiments, one or more of the odd numbered nucleosides starting from the 3’ region of the second region of linked nucleosides may be modified by a modification that is different from the modification of odd numbered nucleosides of the first region of linked nucleosides. In certain embodiments, one or more of the even numbered nucleosides starting from the 3’ region of the second region of linked nucleosides are modified by a modification that is different from the modification of even numbered nucleosides of the first region of linked nucleoside.

In certain embodiments, at least one or more of the modified even numbered nucleosides of the first region of linked nucleosides is adjacent to at least one or more of the differently modified odd numbered nucleosides of the first nucleoside region.

In certain embodiments, at least one or more of the modified even numbered nucleosides of the second nucleoside region is adjacent to at least one or more of the differently modified odd numbered nucleosides of the second region of linked nucleosides.

In certain embodiments, sugars of one or more of the odd numbered nucleosides starting from the 5’ region of the first region of nucleosides may be 2'-O-methyl modified sugars.

In certain embodiments, one or more of the even numbered nucleosides starting from the 3’ region of the first region of linked nucleosides may be 2'-F modified sugars.

In certain embodiments, sugars of one or more of the odd numbered nucleosides starting from the 5’ region of the second region of linked nucleosides may be 2'-0 methyl modified sugars. In certain embodiments, one or more of the even numbered nucleosides starting from the 5’ region of the second region of linked nucleosides may be 2'-F modified sugars.

In certain embodiments, sugars of a plurality of adjacent nucleosides of the first nucleoside region may be modified by a common or different modification.

In certain embodiments, sugars of a plurality of adjacent nucleosides of the second nucleoside region may be modified by a common or different modification.

In certain embodiments, sugars of a plurality of adjacent nucleosides of the hairpin loop may be modified by a common or different modification. The common modification may be a 2'-F modified sugar.

Alternatively, the common modification may be a 2'-O-methyl modified sugar.

The plurality of adjacent 2'-O-methyl modified sugars may be present in at least eight adjacent nucleosides of the first and I or second nucleoside regions. The plurality of adjacent 2'-O-methyl modified sugars may be present in three or four adjacent nucleosides of the hairpin loop.

In certain embodiments, wherein the hairpin loop, as disclosed earlier herein, may comprise 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.

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

In certain embodiments one or more nucleosides of the first region of linked nucleosides and / or the second region of linked nucleosides may be an inverted nucleoside and is attached to an adjacent nucleoside via the 3' carbon of its sugar and the 3' carbon of the sugar of the adjacent nucleoside, and / or one or more nucleosides of the first region of linked nucleosides and I or the second region of linked nucleosides is an inverted nucleoside and is attached to an adjacent nucleoside via the 5' carbon of its sugar and the 5' carbon of the sugar of the adjacent nucleoside.

In certain embodiments, the nucleosides of the oligomeric compound do not contain a 2'- deoxy modification in which -OH has been substituted by -H.

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

According to a second aspect, the present disclosure is directed to a composition comprising an oligomeric compound according to the first aspect of the present disclosure and a physiologically acceptable excipient.

According to a third aspect, the present disclosure is directed to pharmaceutical composition comprising an oligomeric compound according to the first aspect of the present disclosure. The pharmaceutical composition may further comprise a pharmaceutically acceptable excipient, diluent, antioxidant, and/or preservative.

The oligomeric compound according to the first aspect and/or the construct according to the second aspect may be the only pharmaceutically active agent(s).

Alternatively, the pharmaceutical composition furthermore comprises one or more further pharmaceutically active agents. The further pharmaceutically active agent(s) is/are (an) agent(s) which target non-TTR-related diseases or TTR-related diseases, wherein the further pharmaceutically active agent(s) is/are optionally selected from the group consisting of Eplontersen, Vutrisiran, Inotersen and Patisiran. Optionally the oligomeric compound and/or the nucleic acid construct; and the further pharmaceutically active agent(s) are to be administered concomitantly or in any order. Diseases to be treated by shRNA, mxRNA and/or muRNA oligomeric compounds and further uses According to a fourth aspect, the present disclosure is directed to an oligomeric compound according to the first aspect of the present disclosure, for use in human or veterinary medicine or therapy.

According to a fifth aspect, the present disclosure is directed to an oligomeric compound according to the first aspect of the present disclosure, for use in a method of treating, ameliorating and/or preventing a disease or disorder.

One of the diseases may be a TTR-associated disease or disorder as discussed below. TTR-associated disease or disorder

The disease or disorder may be a disease or disorder associated TTR or a disease or disorder requiring reduction of TTR expression.

In particular, the disease or disorder is selected from the group consisting of transthyretin- mediated amyloidosis, in particular hereditary transthyretin-mediated amyloidosis or hereditary transthyretin-mediated amyloidosis with polyneuropathy or hereditary transthyretin-mediated amyloidosis with cardiomyopathy.

According to a sixth aspect, the present disclosure is directed to a method of treating a disease or disorder comprising administration of an oligomeric compound according the first aspect of the present disclosure, to an individual in need of treatment.

The oligomeric compound and/or the nucleic acid construct may be administered subcutaneously or intravenously to the individual.

According to a seventh aspect, the present disclosure is directed to a use of an oligomeric compound according to the first aspect for use in research as a gene function analysis tool. According to an eighth aspect, the present disclosure is directed to a use of an oligomeric compound according to the first aspect in the manufacture of a medicament for a treatment of a disease or disorder.

Constructs and sequences of the disclosed embodiments for oligomeric compounds

The following Tables show nucleobase sequences of antisense and sense strands of oligomeric compounds of the disclosure as well as of nucleobase sequences of singlestranded 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).

The notation used is common in the art and as the following meaning:

A represents adenine;

U represents uracil; C represents cytosine;

G represents guanine.

5Phos represents a 5’ terminal phosphate group which is optional but not indispensable; m represents a methyl modification at the 2' position of the sugar of the underlying nucleoside; f represents a fluoro modification at the 2' position of the sugar of the underlying nucleoside; r indicates an unmodified (2'-OH) ribonucleotide;

[Ps] or # represents a phosphorothioate inter-nucleoside linkage; i represents an inverted inter-nucleoside linkage, which can be either 3'-3', or 5'-5'; 3xGalNAc represents an optionally present trivalent GalNAc.

Tables la and lb below show nucleobase sequences of antisense and sense strands of 100 oligomeric compounds in accordance with the Examples.

Table la: Nucleobase sequences of the antisense strands of 100 constructs

Mote = In particular, the first nucleobase at the 5' terminus in each of the above constructs may be substituted by U.

Table lb: Nucleobase sequences of the sense strands of 100 constructs

Note = In particular, the nucleobase of the 3' terminal nucleotide of each of the sense strands presented within the table may be substituted by A.

Table 2 below shows the nucleobase sequences of the 100 hairpin constructs of the disclosure as selected in accordance with the Examples. The nucleobase sequences are a direct fusion of the antisense sequences of Table la with the corresponding sense sequences of Table lb.

Table 2: Nucleobase sequences of the 100 constructs in which the sense and the antisense sequences of tables la and lb are combined.

Note = In particular, the nucleobase of the 3' terminal nucleotide of each of the sense strands presented within the table can be replaced by A.

Tables 3a to 3c below show 100 antisense sequences, sense sequences and hairpins of the disclosure, respectively, with full modification information (modified sugars and, where applicable, modified phosphates). Table 3a: Modified antisense constructs

41

SUBSTITUTE SHEET (RULE 26)

42

SUBSTITUTE SHEET (RULE 26)

43

SUBSTITUTE SHEET (RULE 26)

44

SUBSTITUTE SHEET (RULE 26)

45

SUBSTITUTE SHEET (RULE 26)

46

SUBSTITUTE SHEET (RULE 26)

47

SUBSTITUTE SHEET (RULE 26)

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

Table 3b: Modified sense constructs

48

SUBSTITUTE SHEET (RULE 26)

49

SUBSTITUTE SHEET (RULE 26)

50

SUBSTITUTE SHEET (RULE 26)

51

SUBSTITUTE SHEET (RULE 26)

52

SUBSTITUTE SHEET (RULE 26)

Note = each of the above constructs may or may not have a “3x GalNAc” coupled to the 3' end group. Optional are constructs with a 3x GalNAc ligand, in particular a toothbrush ligand as defined herein.

Table 3c: Modified hairpin constructs

53

SUBSTITUTE SHEET (RULE 26)

54

SUBSTITUTE SHEET (RULE 26)

55

SUBSTITUTE SHEET (RULE 26)

56

SUBSTITUTE SHEET (RULE 26)

57

SUBSTITUTE SHEET (RULE 26)

58

SUBSTITUTE SHEET (RULE 26)

59

SUBSTITUTE SHEET (RULE 26)

60

SUBSTITUTE SHEET (RULE 26)

61

SUBSTITUTE SHEET (RULE 26)

Mote = each of the above constructs may or may not have a phosphate modification at the 5' end group. Furthermore, and independently, each of the above constructs may or may not have a “3x GalNAc” coupled to the 3' end group. 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.

Specific notes about the nomenclature in Tables 3a to 3c: fN: 2'-Fluoro residues mN: 2'-O-methyl residues

Ps: phosphorothioate p, Phos: phosphate

(GalNAc): Simaomics mono-GalNAc building block

Negative Control

The TMPRSS6 construct, used as a negative control in the examples, has the following modified structure:

5'vP[mA] [fA] [mC] [fC] [mA] [fG] [mA] [fA] [mG] [fA] [mA] [fG] [mC] [fA] [mG] [fG] [mU] [fG] [i N] [fC] [mU] [fG] [fC] [fU] [mU] [fC] [mU] [fU] [mC] [fU] [mG] [fG] [mU] [fU] # [3XGalNAc] (SEQ ID NO: 601). (Construct ID No. 601)

It should also be noted that the scope of the present disclosure extends to sequences that correspond to those in the Tables above, and wherein the 5' terminal 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 3'

62

SUBSTITUTE SHEET (RULE 26) 5.1.8 SDS*: TBD

5.1.9 Appearance: Clear Liquid

5.1.10 Dose Information: See Table 5

5.1.11 Residual Test Article Storage: None

5.2 Test Drug 2:

5.2. 1 Identification: TTR-58 (see Table 3c for structure; Construct ID NO: 558)

5.2.2 Manufacturer: Simaomics

5.2.3 Description: GalNAc-mxRNA targeting human Transthyretin (TTR) mRNA

5.2.4 Lot/Batch Number: Will be recorded on study materials form.

5.2.5 Expiration Date: Will be recorded on study materials form.

5.2.6 Storage Temperature: 4°C

5.2.7 Bio-Hazard Status: None

5.2.8 SDS*: TBD

5.2.9 Appearance: Clear Liquid

5.2.10 Dose Information: See Table 5

5.2. 11 Residual Test Article Storage: None

6 RESULTS

The study results are shown in Fig. 4, and in Table 6 below.

Table 6: Results of in vivo studies

The results show that high knock downs in TTR mRNA expression in liver tissues are achieved by the disclosed embodiments of the compounds.

Claims

1. An oligomeric compound capable of inhibiting expression of transthyretin (TTR), comprising: at least a first region of linked nucleosides having at least a first nucleobase sequence that is at least partially complementary to at least a portion of RNA transcribed from an TTR gene, wherein the first nucleobase sequence is selected from the group consisting of, or a portion thereof: SEQ ID NOs: 1 to 100, wherein the portion optionally has a length of at least 18 nucleosides.

2. The oligomeric compound according to claim 1, which further comprises at least a second region of linked nucleosides having at least a second nucleobase 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: 101 to 200, wherein the portion optionally has a length of at least 8, 9, 10 or 11, more optionally at least 10, nucleosides.

3. The oligomeric compound according to claim 1 or 2, wherein the first nucleobase sequence is selected from the group consisting of, or a portion thereof: SEQ ID NOs: 58, 64, 48, 19, 50, 36, 85, 23, 28, 30, 38, 91, 98, 69, 86, 41, 37, 51, 47, 67, 33, 46, 78, 45, 25, 60, 44, 9, 73, 32, 68, 53, 31, 66, 11, 1, 2, 3, 4, 5, 6, 7, and 8.

4. The oligomeric compound according to claim 3, wherein the second nucleobase sequence is selected from the group consisting of, or a portion thereof: SEQ ID NOs: 158, 164, 148, 119, 150, 136, 185, 123, 128, 130, 138, 191, 198, 169, 186, 141, 137, 151, 147, 167, 133, 146, 178, 145, 125, 160, 144, 109, 173, 132, 168, 153, 131, 166, 111, 101, 102, 103, 104, 105, 106, 107, and 108.

5. The oligomeric compound according to any of claims 1 to 4, wherein the first nucleobase sequence is selected from the group consisting of, or a portion thereof: SEQ ID NOs: 36, 58, 50, 46, 98, and 67, optionally 58 or 50.

6. The oligomeric compound according to claim 5, wherein the second nucleobase sequence is selected from the group consisting of, or a portion thereof: SEQ ID NOs: 136, 158, 150, 146, 198, and 167, optionally 158 or 150.

7. The oligomeric compound according to any of claims 1 to 6, wherein the first region of linked nucleosides consists essentially of 18 to 35, optionally 18 to 20, more optionally 18 or 19, and yet more optionally 19 linked nucleosides.

8. The oligomeric compound according to any of claims 2 to 7, 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.

9. The oligomeric compound according to any of claims 2 to 8, further comprising at least one complementary duplex region that comprises at least a portion of the first region of linked nucleosides directly or indirectly linked to at least a portion of the second region of linked nucleosides, 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.

10. The oligomeric compound according to claim 9, wherein each of the first and second regions of linked nucleosides has a 5’ to 3’ directionality thereby defining 5’ and 3’ regions respectively thereof.

11. The oligomeric compound according to claim 10, wherein the 5 ’ region of the first region of linked nucleosides 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 first nucleoside region base pairs with the 3' terminal nucleoside of the second nucleoside region, wherein optionally the base of the 5' terminal nucleoside of the first region is U and the base of the 3' terminal nucleoside of the second region is A.

12. The oligomeric compound according to claim 10 or 11, wherein the 3’ region of the first region of linked nucleosides is directly or indirectly linked to the 5’ region of the second region of linked nucleosides, wherein optionally the first nucleoside region is directly and covalently linked to the second nucleoside region such as by a phosphate, a phosphorothioate, or a phosphorodithioate, wherein more optionally a 3' terminal nucleoside of the first region of linked nucleosides is directly and covalently linked to a 5' terminal nucleoside of the second region of linked nucleosides by a phosphate, a phosphorothioate, or a phosphorodithioate.

13. The oligomeric compound according to any of claims 1 to 12, which further comprises one or more ligands.

14. The oligomeric compound according to claim 13, wherein the one or more ligands, in particular, at least two ligands, are conjugated to the second region of linked nucleosides and/or the first region of linked nucleosides.

15. The oligomeric compound according to claim 14, as dependent on claim 10, wherein the one or more ligands are conjugated at the 3' region, optionally at the 3' terminal nucleoside of the second region of linked nucleosides and/or of the first region of linked nucleosides, and/or to the 5' terminal nucleoside of the second region of linked nucleosides.

16. The oligomeric compound according to any of claims 13 to 15, 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.

17. The oligomeric compound according to claim 16, wherein the one or more ligands comprise one or more carbohydrates.

18. The oligomeric compound according to claim 17, wherein the one or more carbohydrates can be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide or polysaccharide.

19. The oligomeric compound according to claim 18, wherein the one or more carbohydrates comprise one or more hexose moieties.

20. The oligomeric compound according to claim 19, 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 I or one or more mannose moieties.

21. The oligomeric compound according to claim 20, wherein the one or more carbohydrates comprise one or more N-Acetyl-Galactosamine moieties.

22. The oligomeric compound according to claim 21, comprising two or more N-Acetyl- Galactosamine moieties, optionally three.

23. The oligomeric compound according to any of claims 13 to 22, wherein the one or more ligands are attached to the oligomeric compound, optionally to the second region of linked nucleosides thereof, in a linear configuration, or in a branched configuration.

24. The oligomeric compound according to claim 23, wherein the one or more ligands are attached to the oligomeric compound as a biantennary or triantennary configuration.

25. The oligomeric compound according to any one of claims 1 to 24, wherein the compound comprises the first region of linked nucleosides and the second region of linked nucleosides.

26. The oligomeric compound according to any one of claims 1 to 24, wherein there is an intervening third region of linked nucleosides between the first and the second region.

27. The oligomeric compound according to claim 26, wherein the oligomeric compound comprises a single strand comprising the first, the third, and the second nucleoside regions, wherein at least a portion of the first nucleoside region is directly or indirectly linked to at least a portion of the second nucleoside region so as to form the at least partially complementary duplex region.

28. The oligomeric compound according to any one of claim 9 to 25, wherein the oligomeric compound comprises a single strand comprising the first and second regions of linked nucleosides, wherein at least a portion of the first region of linked nucleosides is directly or indirectly linked to at least a portion of the second region of linked nucleosides so as to form the at least partially complementary duplex region.

29. The oligomeric compound according to claim 28, wherein the first and the second nucleoside regions are directly adjacent on the single strand.

30. The oligomeric compound according to claim 28 or 29, wherein the first nucleoside region has a greater number of linked nucleosides compared to the second nucleoside region, wherein optionally a ratio between a total number of linked nucleosides of the first nucleoside region and a total number of linked nucleosides of the second nucleoside region 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 first nucleoside region relative to the total number of nucleosides of the oligomeric compound ranges from about to about 55% to about 60%.

31. The oligomeric compound of claim 30, 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 TTR gene forms the hairpin loop, wherein the loop comprises 2 to 5, optionally 4 or 5, nucleosides.

32. 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. 201-300, and optionally is selected from the group consisting of SEQ ID NOs: 236, 258, 250, 246, 298, and 267, in particular 250 or 258, wherein optionally the 5' terminal nucleoside of the first region of linked nucleosides is substituted by a U as the nucleobase, and the 3' terminal nucleoside of the second region of linked nucleosides is substituted by an A as the nucleobase.

33. The oligomeric compound according to claim 32, wherein the single strand is selected from the group consisting of SEQ ID Nos. 501-600, optionally from SEQ ID / Construct ID NOs: 536, 558, 550, 546, 598 and 567, in particular 550 and 558, wherein optionally the 5' terminal nucleoside of the first region of linked nucleosides is substituted by an U as the nucleobase, and the 3' terminal nucleoside of the second region of linked nucleosides is substituted by an A as the nucleobase.

34. The oligomeric compound according to claim 33, as dependent on claim 10, whereby the hairpin loop is present at the 3' region of the first region of linked nucleosides, wherein optionally one, two or more 3' terminal nucleosides of the first nucleobase sequence, to the extent the nucleobases of the one, two or more 3' terminal nucleosides permit, fold back and form or contribute to the second region of linked nucleoside.

35. The oligomeric compound of claim 26 or 27, wherein the third nucleoside region and optionally a 3'-terminal portion, optionally consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linked nucleosides, of the first nucleoside region and/or a 5'-terminal portion, optionally consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linked nucleosides, of the second nucleoside region form a hairpin loop.

36. The oligomeric compound according to any one of claims 29 to 31, wherein the hairpin loop comprises 1 to 8, 2 to 7, 3 to 6, optionally 4 or 5 linked nucleosides.

37. The oligomeric compound according to any of claims 1 to 36, comprising intemucleoside linkages and wherein at least one intemucleoside linkage is a modified intemucleoside linkage.

38. The oligomeric compound according to claim 37, wherein the modified intemucleoside linkage is a phosphorothioate or phosphorodithioate intemucleoside linkage.

39. The oligomeric compound according to claim 38, further comprising 1 to 16 phosphorothioate or phosphorodithioate intemucleoside linkages.

40. The oligomeric compound according to claim 39, further comprising 7, 8, 9 or 10 phosphorothioate or phosphorodithioate intemucleoside linkages.

41. The oligomeric compound according to any of claims 38 to 40, as dependent on claim 10, further comprising one or more phosphorothioate or phosphorodithioate intemucleoside linkages at the 5’ region of the first region of linked nucleosides.

42. The oligomeric compound according to any of claims 38 to 41, as dependent on claim 10, further comprising one or more phosphorothioate or phosphorodithioate intemucleoside linkages at the 5’ region of the second region of linked nucleosides, wherein optionally, the oligomeric compound comprises three phosphorothioate intemucleoside linkages at three adjacent nucleosides at the 5' region.

43. The oligomeric compound according to any of claims 38 to 42, as dependent on any one of claims 30 to 32, further comprising phosphorothioate or phosphorodithioate intemucleoside 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.

44. The oligomeric compound according to claim 43, further comprising a phosphorothioate or phosphorodithioate intemucleoside linkage between each adjacent nucleoside that is present in the hairpin loop.

45. The oligomeric compound according to any of claims 1 to 44, wherein at least one nucleoside comprises a modified sugar.

46. The oligomeric compound according to claim 45, wherein 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).

47. The oligomeric compound according to claim 46, wherein the 2' modified sugar is selected from 2'-O-alkyl modified sugar, 2'-O-methyl modified sugar, 2'-O-methoxy ethyl 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-pyndine modified sugar.

48. The oligomeric compound according to claim 47, wherein at least one modified sugar is a 2'-O-methyl modified sugar.

49. The oligomeric compound according to claim 47 or 48, wherein at least one modified sugar is a 2'-F modified sugar and, optionally, at most 16 or 17 sugars are 2'-F modified sugars.

50. The oligomeric compound of claim 48 or 49, wherein the sugar is ribose.

51. The oligomeric compound according to any of claims 48 to 50, as dependent on claim 10, wherein sugars of the nucleosides at any of positions 2 and 14 downstream from the first nucleoside of the 5’ region of the first region of linked nucleosides, do not contain 2'-O- methyl modifications.

52. The oligomeric compound of any one of claims 48 to 51, wherein the 3' terminal position of the second region of linked nucleosides does not contain a 2'-O-methyl modification.

53. The oligomeric compound according to any one of claims 48 to 52, wherein sugars of the nucleosides at any of positions 2 and 14 downstream from the first nucleoside of the 5’ region of the first region of linked nucleosides, contain 2'-F modifications.

54. The oligomeric compound according to any of claims 52 to 53, wherein sugars of the nucleosides of the second region of linked nucleosides, that correspond in position to any of the nucleosides of the first region of linked nucleosides at any of positions 11 to 13 downstream from the first nucleoside of the 5’ region of the first region of linked nucleosides, contain 2'-F modifications.

55. The oligomeric compound of claim 53 or 54, wherein the 3' terminal nucleoside of the second region of linked nucleosides contains a 2'-F modification.

56. The oligomeric compound according to any of claims 52 to 55, as dependent on claim 10, wherein one or more of the odd numbered nucleosides starting from the 5' region of the first region of linked nucleosides are modified, and I or wherein one or more of the even numbered nucleosides starting from the 5’ region of the first region of linked nucleosides 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.

57. The oligomeric compound according to claim 56, wherein one or more of the odd numbered nucleosides starting from the 3’ region of the second region of linked nucleosides are modified by a modification that is different from the modification of odd numbered nucleosides of the first region of linked nucleosides.

58. The oligomeric compound according to claim 56 or 57, wherein one or more of the even numbered nucleosides starting from the 3’ region of the second region of linked nucleosides are modified by a modification that is different from the modification of even numbered nucleosides of the first region of linked nucleoside according to claim 51.

59. The oligomeric compound according to any of claims 56 to 58, wherein at least one or more of the modified even numbered nucleosides of the first region of linked nucleosides is adjacent to at least one or more of the differently modified odd numbered nucleosides of the first nucleoside region.

60. The oligomeric compound according to any of claims 56 to 59, wherein at least one or more of the modified even numbered nucleosides of the second nucleoside region is adjacent to at least one or more of the differently modified odd numbered nucleosides of the second region of linked nucleosides.

61. The oligomeric compound according to any of claims 56 to 60, wherein sugars of one or more of the odd numbered nucleosides starting from the 5’ region of the first region of nucleosides are 2'-O-methyl modified sugars.

62. The oligomeric compound according to any of claims 56 to 61, wherein one or more of the even numbered nucleosides starting from the 3’ region of the first region of linked nucleosides are 2'-F modified sugars.

63. The oligomeric compound according to any of claims 56 to 62, wherein sugars of one or more of the odd numbered nucleosides starting from the 5’ region of the second region of linked nucleosides are 2'-0 methyl modified sugars.

64. The oligomeric compound according to any of claims 56 to 63, wherein one or more of the even numbered nucleosides starting from the 5’ region of the second region of linked nucleosides are 2'-F modified sugars.

65. The oligomeric compound according to any of claims 45 to 64, wherein sugars of a plurality of adjacent nucleosides of the first nucleoside region are modified by a common or different modification.

66. The oligomeric compound according to any of claims 45 to 65, wherein sugars of a plurality of adjacent nucleosides of the second nucleoside region are modified by a common or different modification.

67. The oligomeric compound according to any of claims 56 to 66, as dependent on any one of claims 30 to 33, wherein sugars of a plurality of adjacent nucleosides of the hairpin loop are modified by a common or different modification.

68. The oligomeric compound according to any of claims 65 to 67, wherein the common modification is a 2'-F modified sugar.

69. The oligomeric compound according to any of claims 65 to 67, wherein the common modification is a 2'-O-methyl modified sugar.

70. The oligomeric compound according to claim 69, wherein the plurality of adjacent 2'- O-methyl modified sugars are present in at least eight adjacent nucleosides of the first and I or second nucleoside regions.

71. The oligomeric compound according to claim 70, wherein the plurality of adjacent 2'- O-methyl modified sugars are present in three or four adjacent nucleosides of the hairpin loop.

72. The oligomeric compound according to claim 46, as dependent on any one of claims 31 to 35, wherein the hairpin loop comprises at least one nucleoside having a modified sugar.

73. The oligomeric compound according to claim 72, 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.

74. The oligomeric compound according to claim 73, wherein the modified sugar is a 2'- O-methyl modified sugar, and the differently modified sugar is a 2'-F modified sugar.

75. The oligomeric compound according to any of claims 1 to 74, wherein one or more nucleosides of the first region of linked nucleosides and / or the second region of linked nucleosides is an inverted nucleoside and is attached to an adjacent nucleoside via the 3' carbon of its sugar and the 3' carbon of the sugar of the adjacent nucleoside, and I or one or more nucleosides of the first region of linked nucleosides and / or the second region of linked nucleosides is an inverted nucleoside and is attached to an adjacent nucleoside via the 5' carbon of its sugar and the 5' carbon of the sugar of the adjacent nucleoside.

76. The oligomeric compound according to any of claims 1 to 75, which is blunt ended.

77. The oligomeric compound according to any of claims 1 to 76, wherein either the first or second nucleoside region has an overhang.

78. The oligomeric compound the oligomeric compound according to any one of claims 1 to 77, wherein the first region is selected from the the group consisting of SEQ ID Nos. 301- 400, or a portion thereof, in particular from SEQ ID / Construct ID NOs: 336, 358, 350, 346, 398, and 367, optionally 358 and 350, or a portion thereof

79. The oligomeric compound according to any one of claims 1 to 78, wherein the second region is selected from the group consisting of SEQ ID Nos. 401-500, or a portion thereof, SEQ ID I Construct ID NOs: 436, 458, 450, 446, 498, and 467, optionally 458 and 450, or a portion thereof, wherein the portion optionally has a length of 14 nucleosides.

80. The oligomeric compound according to any one of claims 1 to 79, wherein the oligomeric compound has a total length of about 25 to about 37 nucleosides, in particular about 33 or about 34 nucleosides.

81. The oligomeric compound according to any one of claims 10 to 80, wherein a terminal nucleoside at a 5' position of the first region has a nucleobase 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 second region is substituted by a base being complementary to the base at the 5' position of the first region, optionally A.

82. The oligomeric compound according to any one of claims 1 to 81, wherein the nucleobase sequences are selected from the group consisting of A, U, G, and C and not T.

83. The oligomeric compound according to any one of claims 1 to 82, wherein none of the nucleosides has a 2'-deoxy modification.

84. A composition comprising an oligomeric compound according to any of claims 1 to 83 and a physiologically acceptable excipient.

85. A pharmaceutical composition comprising an oligomeric compound according to any of claims 1 to 83.

86. The pharmaceutical composition of claim 85, further comprising a pharmaceutically acceptable excipient, diluent, antioxidant, and/or preservative.

87. The pharmaceutical composition of claim 85 or 86, wherein the oligomeric compound according to any one of claims 1 to 83 is the only pharmaceutically active agent.

88. The pharmaceutical composition of claim 85 or 86. wherein the pharmaceutical composition further comprises one or more pharmaceutically active agents.

89. The pharmaceutical composition of claim 88, wherein the one or more pharmaceutically active agent(s) is/are (an) agent(s) that target(s) non-TTR-related diseases or TTR-related diseases, wherein the one or more pharmaceutically active agent(s) optionally are selected from the group consisting of Eplontersen, Vutrisiran, Inotersen and Patisiran.

90. The pharmaceutical composition of claim 85 to 89, wherein the oligomeric compound and the one or more pharmaceutically active agent(s) are to be administered concomitantly or in any order.

91. An oligomeric compound according to any of claims 1 to 83 for use in human or veterinary medicine or therapy.

92. An oligomeric compound according to any of claims 1 to 83 for use in a method of treating, ameliorating and/or preventing a disease or disorder.

93. The compound for use of claim 92, wherein the disease or disorder is a disease or disorder associated TTR or a disease or disorder requiring reduction of TTR expression.

94. The compound for use of claim 93, wherein the disease or disorder is selected from the group consisting of transthyretin-mediated amyloidosis, in particular hereditary transthyretin-mediated amyloidosis or hereditary transthyretin-mediated amyloidosis with polyneuropathy or hereditary transthyretin-mediated amyloidosis with cardiomyopathy, familial amyloidotic polyneuropathy, familial amyloidotic cardiomyopathy, or leptomeningeal/CNS (Central Nervous System) amyloidosis.

95. A method of treating a disease or disorder comprising administration of an oligomeric compound according to any of claims 1 to 83 to an individual in need of treatment.

96. The method according to claim 95, wherein the oligomeric compound and/or the nucleic acid construct is administered subcutaneously or intravenously to the individual.

97. Use of an oligomeric compound according to any of claims 1 to 83 for use in research as a gene function analysis tool.

98. Use of an oligomeric compound according to any of claims 1 to 83 in the manufacture of a medicament for a treatment of a disease or disorder.