Nucleic acids for inhibiting expression of inhbe in a cell

Abstract

The invention relates to double-stranded nucleic acid molecules that interfere with or inhibit inhibin beta E subunit (INHBE) gene expression (also called activin beta E-chain). It further relates to therapeutic uses of such inhibition such as for the treatment of INHBE-mediated diseases, disorders or syndromes, such as cardiovascular disease, Type 2 diabetes, NAFLD or obesity.

Description

[0001] Nucleic acids for inhibiting expression of INHBE in a cell Field of the invention

[0002] The invention relates to double-stranded nucleic acid molecules that interfere with or inhibit expression of Inhibin subunit beta E (INHBE), also called Activin subunit beta-E. It further relates to new, safe and effective therapeutic uses of such inhibition such as for the treatment of INHBE mediated diseases, disorders or syndromes, such as cardiovascular disease, Type 2 diabetes, non-alcoholic fatty liver disease (NAFLD) or obesity.

[0003] Background

[0004] Double-stranded RNAs (dsRNA) able to bind through complementary base pairing to expressed mRNAs have been shown to block gene expression (Fire et al., 1998, Nature. 1998 Feb 19;391 (6669):806-11 and Elbashir et al., 2001, Nature. 2001 May 24;411 (6836):494-8) by a mechanism that has been termed “RNA interference (RNAi)”. Short dsRNAs direct gene specific, post-transcriptional silencing in many organisms, including vertebrates, and have become a useful tool for studying gene function. RNAi is mediated by the RNA induced silencing complex (RISC), a sequence-specific, multi-component nuclease that degrades messenger RNAs having sufficient complementary or homology to the silencing trigger loaded into the RISC complex. Interfering RNAs such as siRNAs, antisense RNAs, and micro RNAs, 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.

[0005] 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.

[0006] Elevated waist to hip ratios as a surrogate measurement for visceral adiposity are leading risk factors for cardiovascular diseases and metabolic diseases, like type 2 diabetes, dyslipidemia and MAFLD and MASH. Carriers of one allelic form of INHBE loss of function (“LOF”) show favourable fat distribution, favourable metabolic profile and protection from type 2 diabetes (Akbari et al., 2022 Nat. Commun. 2022 Aug 23;13(1):4844.). Similarly, Deaton et al reported that carriers of INHBE LOF variants are protected from abdominal obesity and have a lower risk for type 2 diabetes and coronary artery disease (Deaton et al., 2022, Nat. Commun. 2022 Jul 27;13(1):4319. doi: 10.1038 / s41467-022-31757-8.). Furthermore, elevated INHBE expression in the liver is positively correlated with body mass index in humans and with insulin resistance (Sugiyava et al., 2018 PLoS One. 2018 Mar 29; 13(3):e0194798.).

[0007] INHBE encodes the circulating factor Inhibin beta E subunit also called Activin beta E subunit, which is predominantly expressed by hepatocytes and upregulated upon fasting. It is also elevated in obese individuals with insulin resistance, individuals with fatty liver, MAFLD or MASH or with ALD (Cao et al., Biochem Biophys Res Commun. 2023 Dec 17;686:149180; Akbari et al., 2022 Nat Commun. 2022 Aug 23;13(1):4844.) Inhibin E is a member of the Tgf-p family and it is proposed to bind to the Activin receptor, ACVR1C, expressed in adipose tissues, were it can attenuate lipolysis and excessive lipid break down, preserving fat mass during fasting (Adam et al., 2023 Proc Natl Acad Sci U S A. 2023 Aug 8;120(32):e2309967120), Griffin et al., 2023, MOLECULAR METABOLISM 78 (2023) 101830. doi: 10.1016 / j.molmet.2023.101830. PMCID: PMC10656223).

[0008] Preclinical mechanistic studies have pointed towards RNA interference-mediated off-target effects that can be a driver of hepatotoxicity for GalNAc-siRNA conjugates. These off-target effects can be driven by binding of the RISC-loaded siRNA to off-target transcripts mediated through base pairing between the seed region of the siRNA guide strand (nucleotides 2-8) and complementary site(s) in the 3'-untranslated region of mRNAs. This noncatalytic mechanism essentially mimics the post-transcriptional silencing by endogenous miRNAs and can lead to translational repression and / or mRNA destabilization at suprapharmacological levels of RISC-loaded siRNA with reductions in mRNA levels accounting for most (66% to >90%) of the post-transcriptional repression mediated by mammalian miRNAs (Schlegel et al., Nucleic Acid Research 2022; 50(12), 6656-6670).

[0009] WO 2024187190 discloses double-stranded ribonucleic acid (dsRNA) targeting an INHBE gene, and methods of using the dsRNA to inhibit expression of INHBE.

[0010] WO 2022132666 discloses methods of treating a subject having metabolic disorders and / or cardiovascular diseases, methods of identifying subjects having an increased risk of developing a metabolic disorder and / or a cardiovascular disease, and methods of detecting human Inhibin Subunit Beta E variant nucleic acid molecules and variant polypeptides. W02023003922 discloses RNAi agents, e.g., double stranded RNA (dsRNA) agents, targeting a metabolic disorder-associated target gene, e.g., inhibin subunit beta E (INHBE), activin A receptor type 1C (ACVR1C), perilipin-1 (PLIN1), phosphodiesterase 3B (PDE3B), or inhibin subunit beta C (INHBC) gene.

[0011] W02023044094 discloses modulators, e.g., double stranded RNA (dsRNA) agents, antisense polynucleotide agents, antibodies, guideRNAs that affect ADAR editing, or guideRNAs that effect CRISPR editing, that modulate, e.g., inhibit, the expression and / or activity of inhibin subunit beta E (INHBE).

[0012] WO 2024179573 discloses siRNA targeting inhibin βE, an siRNA conjugate, and medical application thereof; and specifically relates to an siRNA targeting INHBE, an siRNA conjugate, a composition, and a medical use thereof.

[0013] Accordingly, it is an object of the present invention to provide new, safe and effective compounds and (pharmaceutical) compositions for the treatment of INHBE-mediated diseases, disorders or syndromes, such as metabolic-ssociated fatty liver disease (MAFLD), metabolic dysfunction-associated steatohepatitis (MASH), alcoholic liver disease (ALD), type 2 diabetes, cardiovascular disease, coronary artery disease, dyslipidemia, obesity and hypertension.

[0014] Summary of the invention

[0015] One aspect of the invention is a double-stranded nucleic acid for inhibiting expression of Activin beta E, also referred to as Inhibin beta E (INHBE), wherein the nucleic acid comprises a first strand and a second strand, wherein the unmodified equivalent of the first strand sequence comprises a sequence of at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the first strand sequences shown in Table 1, Table 5a, Table 5b, Table 2, or in Table 5c.

[0016] The nucleic acids described herein are thus double-stranded nucleic acids capable of inhibiting expression of INHBE, preferably in a cell, and may find use as a therapeutic agent or diagnostic agent, e.g., in associated therapeutic or diagnostic methods, respectively.

[0017] The nucleic acid of the invention comprises or consists of a first strand and a second strand, and the first strand typically comprises sequences sufficiently complementary to INHBE mRNA, so as to mediate RNA interference. One aspect relates to a composition comprising a nucleic acid as disclosed herein and a solvent (preferably water) and / or a delivery vehicle and / or a physiologically acceptable excipient and / or a carrier and / or a salt and / or a diluent and / or a buffer and / or a preservative.

[0018] One aspect relates to a composition comprising a nucleic acid as disclosed herein and another therapeutic agent selected from, e.g., an oligonucleotide, a small molecule, a monoclonal antibody, a polyclonal antibody and a peptide.

[0019] One aspect relates to a nucleic acid or a composition comprising the nucleic acid as disclosed herein for use as a therapeutic agent or diagnostic agent, e.g., in associated methods.

[0020] One aspect relates to a nucleic acid or a composition comprising the nucleic acid as disclosed herein for use in the prophylaxis or treatment of a disease, disorder or syndrome.

[0021] One aspect relates to the use of a nucleic acid or a composition comprising the nucleic acid as disclosed herein in the prophylaxis or treatment of a disease, disorder or syndrome.

[0022] One aspect relates to the use of a nucleic acid or a composition comprising the nucleic acid as disclosed herein in the preparation of a medicament for the prophylaxis or treatment of a disease, disorder or syndrome.

[0023] One aspect relates to a composition as disclosed herein for use as a medicament.

[0024] One aspect relates to a method of prophylaxis or treatment of a disease, disorder or syndrome comprising administering a pharmaceutically effective dose or amount of a nucleic acid or of a composition as disclosed herein to a subject in need of treatment. Preferably, the nucleic acid or composition is administered to the subject subcutaneously, intravenously or by oral, rectal, pulmonary, intramuscular or intraperitoneal administration.

[0025] Detailed description of the invention

[0026] The present invention relates to a nucleic acid which is double-stranded and which comprises a sequence homologous to an expressed RNA transcript of INHBE, and compositions thereof. These nucleic acids, conjugates thereof, and compositions comprising them, may be used in the prophylaxis and treatment of a variety of diseases, disorders and syndromes in which reduced expression of the Inhibin subunit beta E (INHBE) gene product is desirable. One aspect of the invention is a double-stranded nucleic acid for inhibiting expression of Inhibin beta E (INHBE), wherein the nucleic acid comprises a first strand and a second strand, wherein the unmodified equivalent of the first strand sequence comprises a sequence of at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the first strand sequences shown in Table 5a, Table 1, Table 5b, Table 2, or in Table 5c.

[0027] The unmodified equivalent of the first strand sequence may comprise a sequence of at least 15 contiguous nucleotides from anyone of the first strand sequences shown in Table 5a, Table 1, Table 5b, Table 2, or in Table 5c. The unmodified equivalent of the first strand sequence may comprise a sequence of at least 16 contiguous nucleotides from any one of the first strand sequences shown in Table 5a, Table 1, Table 5b, Table 2, or in Table 5c. The unmodified equivalent of the first strand sequence may comprise a sequence of at least 17 contiguous nucleotides from any one of the first strand sequences shown in Table 5a, Table 1, Table 5b, Table 2, or in Table 5c. The unmodified equivalent of the first strand sequence may comprise a sequence of at least 18 contiguous nucleotides from any one of the first strand sequences shown in Table 5a, Table 1, Table 5b, Table 2, or in Table 5c.

[0028] An aspect of the invention is a double-stranded nucleic acid for inhibiting expression of INHBE, preferably in a cell, wherein the nucleic acid comprises a first strand and a second strand, wherein the unmodified equivalent of the first strand sequence comprises a sequence of at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the first strand sequences shown in Table 5a. These nucleic acids among others have the advantage of being active in various species that are relevant for pre-clinical and clinical development and / or of having few relevant off-target effects. Having few relevant off-target effects means that a nucleic acid specifically inhibits the intended target and does not significantly inhibit other genes or inhibits only one or few other genes at a therapeutically acceptable level.

[0029] The first strand may otherwise be referred to as an anti-sense strand (AS). The second strand may otherwise be referred to as a sense strand (SS).

[0030] In the context of the present invention, inhibition of expression of INHBE will be understood to mean that the nucleic acid is capable of reducing the level of expression of INHBE.

[0031] Alternatively, especially if the nucleic acid or conjugated nucleic acid of the invention is administered to a subject, the level of inhibition can be measured in a different group of cells or in a tissue or an organ or in a body fluid such as blood or plasma. For example, the unmodified equivalent of the first strand sequence may comprise a sequence of at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the first strand sequences listed in Table 1:

[0032] Table 1

[0033] First strand Second strand sequence (SEQ sequence (SEQ ID No.) ID No.)

[0034] 200 201

[0035] 226 227

[0036] 296 297

[0037] 292 293

[0038] 378 379

[0039] 382 18

[0040] 387 184

[0041] 388 186

[0042]

[0043] For example, the unmodified equivalent of the first strand sequence may comprise a sequence of at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably all 19 contiguous nucleotides differing by no more than 3 nucleotides, preferably differing by no more than 2 nucleotides, more preferably differing by no more than 1 nucleotide, and most preferably not differing by any nucleotide from any one of the first strand sequences listed in Table 1 or in Table 5a.

[0044] The unmodified equivalent of the first strand sequence may comprise a sequence of at least 15 contiguous nucleotides from any one of the first strand sequences listed in Table 1 or in Table 5a. The unmodified equivalent of the first strand sequence may comprise preferably at least 16 contiguous nucleotides, more preferably at least 17 contiguous nucleotides, yet more preferably at least 18 contiguous nucleotides and most preferably all 19 contiguous nucleotides not differing by any nucleotide from any one of the first strand sequences listed in Table 1 or in Table 5a.

[0045] Preferably, the unmodified equivalent of the first strand sequence of the nucleic acid consists of one of the first strand sequences shown in Table 1 or in Table 5a. The sequence may however be modified by a number of nucleic acid modifications that do not change the identity of the nucleotide. For example, modifications of the backbone or ribose sugar residues of the nucleic acid do not change the identity of the nucleotide because the base itself remains the same as in the reference sequence. It, will, however, be appreciated that when a nucleotide is modified, it may be referred to as a “modified nucleotide”. For example, the unmodified equivalent of the first strand sequence of the nucleic acid may consist of one of the first strand sequences shown in Table 1 or in Table 5a, optionally modified by one or more of said nucleic acid modifications.

[0046] A nucleic acid that comprises a sequence according to a reference sequence herein means that the nucleic acid comprises a sequence of contiguous nucleotides in the order as defined in the reference sequence. The term “reference sequence”, as used herein, will typically be understood to mean a sequence which is found in, for example, at least one of the Tables disclosed herein, such as Table 5a, Table 1, Table 5b, Table 2, or in Table 5c.

[0047] When reference is made herein to a reference sequence comprising, consisting essentially of, or consisting of nucleotides, this reference is not limited to the sequence with unmodified nucleotides. The same reference also encompasses the same nucleotide sequence in which one, several, such as two, three, four, five, six, seven or more, including all, nucleotides are modified by modifications such as, e.g., 2’-OMe, 2’-F, a ligand, a linker, a 3’ end or 5’ end modification or of any other modification. It also refers to sequences in which two or more nucleotides are linked to each other by the natural phosphodiester linkage or by any other linkage such as a phosphorothioate or a phosphorodithioate linkage.

[0048] A double-stranded nucleic acid is a nucleic acid in which the first strand and the second strand hybridise to each other over at least part of their lengths and are therefore capable of forming a duplex region under physiological conditions, such as in PBS at 37°C at a concentration of 1 pM of each strand. The first and second strand are preferably able to hybridise to each other and therefore to form a duplex region over a region of at least 15 contiguous nucleotides, preferably 16, 17, 18 or 19 contiguous nucleotides. The first strand and the second strand may form a duplex region of from 17-25 nucleotides in length. For example, the first and second strand may be able to hybridise to each other and therefore to form a duplex region over a region of at least 19 contiguous nucleotides. The first strand and the second strand may form a duplex region of 19 contiguous nucleotides in length. The duplex region may consist of 17-25 contiguous nucleotide base pairs.

[0049] This duplex region comprises nucleotide base parings between the two strands, preferably based on Watson-Crick base pairing and / or wobble base pairing (such as GU base pairing). All the nucleotides of the two strands within a duplex region do not have to base pair to each other to form a duplex region. A certain number of mismatches, deletions or insertions between the nucleotide sequences of the two strands are acceptable. Overhangs on either end of the first or second strand or unpaired nucleotides at either end of the double-stranded nucleic acid are also possible. The double-stranded nucleic acid is preferably a stable double-stranded nucleic acid under physiological conditions, and preferably has a melting temperature (Tm) of 45°C or more, preferably 50°C or more, and more preferably 55°C or more for example in PBS at a concentration of 1 pM of each strand.

[0050] The duplex region (formed between the first and the second strand) may consist of one and only one duplex region.

[0051] A stable double-stranded nucleic acid under physiological conditions is a double-stranded nucleic acid that has a Tm of 45°C or more, preferably 50°C or more, and more preferably 55°C or more, for example in PBS at a concentration of 1 pM of each strand.

[0052] The first strand and the second strand are preferably capable of forming a duplex region (i.e., are complementary to each other) over i) at least a portion of their lengths, preferably over at least 15 contiguous nucleotides of both of their lengths, ii) over the entire length of the first strand, iii) over the entire length of the second strand or iv) over the entire length of both the first and the second strand. Strands being complementary to each other over a certain length means that the strands are able to base pair to each other, either via Watson-Crick or wobble base pairing, over that length. Each nucleotide of the length does not necessarily have to be able to base pair with its counterpart in the other strand over the entire given length as long as a stable double-stranded nucleotide under physiological conditions can be formed. It is however, preferred, in certain embodiments, if each nucleotide of the length can base pair with its counterpart in the other strand over the entire given length.

[0053] A certain number of mismatches, deletions or insertions between the first strand and the target sequence, or between the first strand and the second strand can be tolerated in the context of the nucleic acids according to the present invention and even have the potential in certain cases to increase RNA interference (e.g., inhibition) activity.

[0054] The inhibition activity of the nucleic acids according to the present invention relies on the formation of a duplex region between all or a portion of the first strand and a portion of a target nucleic acid. The portion of the target nucleic acid that forms a duplex region with the first strand, defined as beginning with the first base pair formed between the first strand and the target sequence and ending with the last base pair formed between the first strand and the target sequence, inclusive, is the target nucleic acid sequence or simply, target sequence. The duplex region formed between the first strand and the second strand need not be the same as the duplex region formed between the first strand and the target sequence. That is, the second strand may have a sequence different from the target sequence; however, the first strand must be able to form a duplex structure with both the second strand and the target sequence, at least under physiological conditions.

[0055] The complementarity between the first strand and the target sequence may be perfect (i.e., 100% identity with no nucleotide mismatches or insertions or deletions in the first strand as compared to the target sequence).

[0056] The complementarity between the first strand and the complementary sequence of the target sequence may range from about 75% to about 100%. More specifically, the complementarity may be at least 75%, 80%, 85%, 90% or 95% and intermediate values, provided a nucleic acid is capable of reducing or inhibiting the expression of INHBE.

[0057] A nucleic acid having less than 100% complementarity between the first strand and the target sequence may be able to reduce the expression of INHBE to the same level as a nucleic acid having perfect complementarity between the first strand and target sequence. Alternatively, it may be able to reduce expression of INHBE to a level that is 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the level of reduction achieved by the nucleic acid with perfect complementarity.

[0058] A nucleic acid of the present disclosure may be an isolated nucleic acid.

[0059] A nucleic acid of the present disclosure may be a nucleic acid wherein:

[0060] (a) the unmodified equivalent of the first strand sequence comprises a sequence differing by no more than 3 nucleotides from any one of the first strand sequences of Table 1 or Table 5a, and optionally wherein the unmodified equivalent of the second strand sequence comprises a sequence differing by no more than 3 nucleotides from the corresponding second strand sequence of Table 1 or Table 5a;

[0061] (b) the unmodified equivalent of the first strand sequence comprises a sequence differing by no more than 2 nucleotides from any one of the first strand sequences of Table 1 or Table 5a, and optionally wherein the unmodified equivalent of the second strand sequence comprises a sequence differing by no more than 2 nucleotides from the corresponding second strand sequence of Table 1 or Table 5a;

[0062] (c) the unmodified equivalent of the first strand sequence comprises a sequence differing by no more than 1 nucleotide from any one of the first strand sequences of Table 1 or Table 5a, and optionally wherein the unmodified equivalent of the second strand sequence comprises a sequence differing by no more than 1 nucleotide from the corresponding second strand sequence of Table 1 or Table 5a;

[0063] (d) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2 to 17 from the 5’ end of any one of the first strand sequences with a given SEQ ID No. shown in Table 1 or Table 5a, and optionally wherein the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 3 to 18 from the 5’ end of the corresponding second strand sequence with a given SEQ ID No. shown in Table 1 or Table 5a;

[0064] (e) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2 to 18 from the 5’ end of any one of the first strand sequences with a given SEQ ID No. shown in Table 1 or Table 5a, and optionally wherein the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 2 to 18 from the 5’ end of the corresponding second strand sequence with a given SEQ ID No. shown in Table 1 or Table 5a;

[0065] (f) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2 to 19 from the 5’ end of any one of the first strand sequences with a given SEQ ID No. shown in Table 1 or Table 5a, and optionally wherein the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 1 to 18 from the 5’ end of the corresponding second strand sequence with a given SEQ ID No. shown in Table 1 or Table 5a;

[0066] (g) the unmodified equivalent of the first strand sequence comprises a sequence of any one of the first strand sequences with a given SEQ ID No. shown in Table 1 or Table 5a, and optionally wherein the unmodified equivalent of the second strand sequence comprises a sequence of the corresponding second strand sequence with a given SEQ ID No. shown in Table 1 or Table 5a;

[0067] (h) the unmodified equivalent of the first strand sequence consists essentially of any one of the first strand sequences with a given SEQ ID No. shown in Table 1 or Table 5a, and optionally wherein the unmodified equivalent of the second strand sequence consists essentially of the sequence of the corresponding second strand sequence with a given SEQ ID No. shown in Table 1 or Table 5a;

[0068] (i) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 1 to 19 from the 5’ end of any one of the first strand sequences with a given SEQ ID No. shown in Table 1 or Table 5a, wherein said unmodified equivalent of the first strand sequence further comprises 1 (nucleotide 20 counted from the 5'end), 2 (nucleotides 20 and 21), 3 (nucleotides 20, 21 and 22), 4 (nucleotides 20, 21, 22 and 23), 5 (nucleotides 20, 21, 22, 23 and 24) or 6 (nucleotides 20, 21, 22, 23, 24 and 25) additional nucleotide(s) at the 3'end of any one of the first strand sequences with a given SEQ ID No. shown in Table 1 or Table 5a; and optionally wherein the unmodified equivalent of the second strand sequence comprises or consists essentially of or consists of a sequence of the corresponding second strand sequence with a given SEQ ID No. shown in Table 1 or Table 5a;

[0069] (j) the unmodified equivalent of the first strand sequence consists of a sequence 25, 24, 23, 22, 21 or 20 nucleotides in length, wherein nucleotides 1-19 from the 5’ end of the unmodified equivalent of the first strand sequence correspond to nucleotides 1 to 19 from the 5’ end of any one of the first strand sequences with a given SEQ ID No. shown in T able 5a and wherein the unmodified equivalent of the first strand sequence has a further 1 (nucleotide 20 counted from the 5'end), 2 (nucleotides 20 and 21), 3 (nucleotides 20, 21 and 22), 4 (nucleotides 20, 21, 22 and 23), 5 (nucleotides 20, 21, 22, 23 and 24) or 6 (nucleotides 20, 21, 22, 23, 24 and 25) additional nucleotide(s) at the 3'end of any one of the first strand sequences with a given SEQ ID No. shown in Table 1 or Table 5a, and optionally wherein the unmodified equivalent of the second strand sequence comprises or consists essentially of or consists of a sequence of the corresponding second strand sequence with a given SEQ ID No. shown in Table 1 or Table 5a;

[0070] (k) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 1 to 19 from the 5’ end of any one of the first strand sequences with a given SEQ ID No. shown in Table 1 or Table 5a, wherein said unmodified equivalent of the first strand sequence further comprises 1 (nucleotide 20 counted from the 5'end), 2 (nucleotides 20 and 21), 3 (nucleotides 20, 21 and 22), 4 (nucleotides 20, 21, 22 and 23), 5 (nucleotides 20, 21, 22, 23 and 24) or 6 (nucleotides 20, 21, 22, 23, 24 and 25) additional nucleotide(s) at the 3'end of any one of the first strand sequences with a given SEQ ID No. shown in Table 1 or Table 5a; and wherein said unmodified equivalent of the first strand sequence comprises, consists essentially of or consists of a contiguous region of from 17-25 nucleotides in length, preferably of from 18-24 nucleotides in length, complementary to the INHBE transcript of SEQ ID NO. 837; and optionally wherein the unmodified equivalent of the second strand sequence comprises or consists essentially of or consists of a sequence of the corresponding second strand sequence with a given SEQ ID No. shown in Table 1 or Table 5a;

[0071] (l) the unmodified equivalent of the first strand sequence consists of a sequence 25, 24, 23, 22, 21 or 20 nucleotides in length, wherein nucleotides 1-19 from the 5’ end of the unmodified equivalent of the first strand sequence correspond to nucleotides 1 to 19 from the 5’ end of any one of the first strand sequences with a given SEQ ID No. shown in Table 5a and wherein the unmodified equivalent of the first strand sequence has a further 1 (nucleotide 20 counted from the 5'end), 2 (nucleotides 20 and 21), 3 (nucleotides 20, 21 and 22), 4 (nucleotides 20, 21, 22 and 23), 5 (nucleotides 20, 21, 22, 23 and 24) or 6 (nucleotides 20, 21, 22, 23, 24 and 25) additional nucleotide(s) at the 3'end of any one of the first strand sequences with a given SEQ ID No. shown in Table 1 or Table 5a, and wherein said unmodified equivalent of the first strand sequence comprises, consists essentially of or consists of a contiguous region of from 17-25 nucleotides in length, preferably of from 18-24 nucleotides in length, complementary to the INHBE transcript of SEQ ID NO. 837; and optionally wherein the unmodified equivalent of the second strand sequence comprises or consists essentially of or consists of a sequence of the corresponding second strand sequence with a given SEQ ID No. shown in Table 1 or Table 5a;

[0072] (m) the unmodified equivalent of the first strand sequence consists of any one of the first strand sequences with a given SEQ ID No. shown in Table 1 or Table 5a, and optionally wherein the unmodified equivalent of the second strand sequence consists of the sequence of the corresponding second strand sequence with a given SEQ ID No. shown in Table 1 or Table 5a;

[0073] (n) the unmodified equivalent of the first strand and the unmodified equivalent of the second strand of any one of the nucleic acid molecules of subsections (a) to (m) above are present on a single strand wherein the unmodified equivalent of the first strand and the unmodified equivalent of the second strand are able to hybridise to each other and to thereby form a double-stranded nucleic acid with a duplex region of 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; or

[0074] (o) the unmodified equivalent of the first strand and the unmodified equivalent of the second strand of any one of the nucleic acid molecules of subsections (a) to (m) above are on two separate strands that are able to hybridise to each other and to thereby form a double-stranded nucleic acid with a duplex region of 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.

[0075] In (k) or (I), the unmodified equivalent of the first strand sequence may comprise, consist essentially of or consist of a contiguous region of from 19-25 nucleotides in length complementary to the INHBE transcript of SEQ ID NO. 837. In (k) or (I), the unmodified equivalent of the first strand sequence may comprise, consist essentially of or consist of a contiguous region of from 20-25 nucleotides in length complementary to the INHBE transcript of SEQ ID NO. 837, preferably of from 20-24 nucleotides in length, complementary to the INHBE transcript of SEQ ID NO. 837.

[0076] By a “corresponding” second strand is meant a second strand present in the same duplex as a given first strand in Table 5a, 5b or 5c, or listed as a corresponding second strand sequence in Table 1 or Table 2, as the case may be. That is to say, a first strand and its corresponding second strand are designated as the “A” and “B” strands, respectively, of a duplex having a given Duplex ID in Tables 5a, 5b or 5c, or are described as such in Tables 1 and 2.

[0077] In one aspect, if the 5’-most nucleotide of the first strand is a nucleotide other than A or U, this nucleotide is replaced by an A or U. Preferably, if the 5’-most nucleotide of the first strand is a nucleotide other than U, this nucleotide is replaced by U, and more preferably by U with a 5’ vinylphosphonate.

[0078] When a nucleic acid of the invention does not comprise the entire sequence of a reference first strand and / or second strand sequence (as for example given in Tables 1, 2, 5a, 5b or 5c), or one or both strands differ from the corresponding reference sequence by one, two or three nucleotides, this nucleic acid preferably retains at least 30%, more preferably at least 50%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, yet more preferably at least 95% and most preferably at least 100% of the INHBE inhibition activity compared to the inhibition activity of the corresponding nucleic acid that comprises the entire first strand and second strand reference sequences in a comparable experiment.

[0079] Nucleic acids that are capable of hybridising under physiological conditions are nucleic acids that are capable of forming base pairs, preferably Watson-Crick or wobble base-pairs, between at least a portion of the opposed nucleotides in the strands so as to form at least a duplex region. Such a double-stranded nucleic acid is preferably a stable double-stranded nucleic acid under physiological conditions (for example in PBS at 37°C at a concentration of 1 pM of each strand), meaning that under such conditions, the two strands stay hybridised to each other. The Tm of the double-stranded nucleotide is preferably 45°C or more, preferably 50°C or more and more preferably 55°C or more.

[0080] One aspect of the present invention relates to a nucleic acid for inhibiting expression of INHBE, wherein the nucleic acid comprises a first sequence of at least 15, preferably at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably all 19 contiguous nucleotides differing by no more than 3 nucleotides, preferably no more than 2 nucleotides, more preferably no more than 1 nucleotide and most preferably not differing by any nucleotide from any of the first strand unmodified equivalent sequences of Table 5a, or of Table 1, the first sequence being able to hybridise to a target gene transcript (such as an mRNA) under physiological conditions. Preferably, the nucleic acid further comprises a second sequence of at least 15, preferably at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably all contiguous nucleotides differing by no more than 3 nucleotides, preferably no more than 2 nucleotides, more preferably no more than 1 nucleotide and most preferably not differing by any nucleotide from any of the corresponding second strand unmodified equivalent sequences of Table 5a, or of Table 1, the second sequence being able to hybridise to the first sequence under physiological conditions and preferably the nucleic acid being an siRNA that is capable of inhibiting INHBE expression via the RNAi pathway.

[0081] One aspect relates to any double-stranded nucleic acid as disclosed in Tables 1, 2, 5a, 5b or 5c, each of which may be referred to by a given Duplex ID (e.g., EU1000, EU1001, etc.),, provided that the double-stranded nucleic acid is able to inhibit expression of INHBE. These nucleic acids are all siRNAs. Inhibition occurs through targeted degradation of mRNA transcripts of the target gene after transcription. The siRNA forms part of the RISC complex. The RISC complex specifically targets the target RNA by sequence complementarity of the first (antisense) strand with the target RNA sequence.

[0082] One aspect relates to a double-stranded nucleic acid that is capable of inhibiting expression of INHBE, preferably in a cell, for use as a therapeutic or diagnostic agent, e.g., in associated therapeutic or diagnostic methods, wherein the nucleic acid preferably comprises or consists of a first strand and a second strand and preferably wherein the first strand comprises sequences sufficiently complementary to an INHBE mRNA so as to mediate RNA interference.

[0083] Certain of the nucleic acids described herein are capable of inhibiting the expression of INHBE. Inhibition may be complete, i.e., 0% remaining expression. Inhibition of INHBE expression may be partial, i.e., it may be 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more, or intermediate values of inhibition of the level of INHBE expression in the absence of a nucleic acid of the invention. The level of inhibition may be measured by comparing a treated sample with an untreated (Ut) sample or with a sample treated with a control such as for example a siRNA that does not target INHBE. Inhibition may be measured by measuring INHBE mRNA and / or protein levels or levels of a biomarker or indicator that correlates with INHBE presence or activity. It may be measured in cells that may have been treated in vitro with a nucleic acid described herein. Alternatively, or in addition, inhibition may be measured in cells, such as hepatocytes, or tissue, such as liver tissue, or an organ, such as the liver, or in a body fluid such as blood, serum, lymph or any other body part or fluid that has been taken from a subject previously treated with a nucleic acid disclosed herein. Preferably, inhibition of INHBE expression is determined by comparing the INHBE mRNA level measured in INHBE-expressing cells after at least 24 or 48 hours, for example after 7 or 14 days of in vitro treatment with a double-stranded RNA disclosed herein under ideal conditions (see the examples for appropriate concentrations and conditions) to the INHBE mRNA level measured in control cells that were untreated or mock treated or treated with a control double-stranded RNA under the same conditions.

[0084] One aspect of the present invention relates to a nucleic acid, wherein the first strand and the second strand are present on a single strand of a nucleic acid that loops around so that the first strand and the second strand are able to hybridise to each other and to thereby form a double-stranded nucleic acid with a duplex region.

[0085] Preferably, the first strand and the second strand of the nucleic acid are separate strands. The two separate strands are preferably each 17-25 nucleotides in length, more preferably 18-25 nucleotides in length. The two strands may be of the same or different lengths. The first strand may be 17-25 nucleotides in length, preferably it may be 18-24 nucleotides in length, it may be 18, 19, 20, 21, 22, 23 or 24 nucleotides in length. Most preferably, the first strand is 19 nucleotides in length. The second strand may independently be 17-25 nucleotides in length, preferably it may be 18-24 nucleotides in length, it may be 18, 19, 20, 21, 22, 23 or 24 nucleotides in length. More preferably, the second strand is 18 or 19 or 20 nucleotides in length, and most preferably it is 19 nucleotides in length.

[0086] Preferably, the first strand and the second strand of the nucleic acid form a duplex region of 17-25 nucleotides in length. More preferably, the duplex region is 18-24 nucleotides in length. The duplex region may be 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In the most preferred embodiment, the duplex region is 18 or 19 nucleotides in length. The duplex region is defined here as the region between and including the 5’-most nucleotide of the first strand that is base paired to a nucleotide of the second strand to the 3’-most nucleotide of the first strand that is base paired to a nucleotide of the second strand. The duplex region may comprise nucleotides in either or both strands that are not base-paired to a nucleotide in the other strand. It may comprise one, two, three or four such nucleotides on the first strand and / or on the second strand. However, preferably, the duplex region consists of 17-25 contiguous nucleotide base pairs. That is to say that it preferably comprises 17-25 contiguous nucleotides on both of the strands that all base pair to a nucleotide in the other strand. More preferably, the duplex region consists of 18 or 19 contiguous nucleotide base pairs, most preferably 18.

[0087] In each of the embodiments disclosed herein, the nucleic acid may be blunt ended at both ends; have an overhang at one end and a blunt end at the other end; or have an overhang at both ends. The nucleic acid may have an overhang at one end and a blunt end at the other end. In such embodiments, the nucleic acid may be blunt ended at the end with the 5’ end of the first strand and the 3’ end of the second strand or at the 3’ end of the first strand and the 5’ end of the second strand.

[0088] The nucleic acid may comprise an overhang at a 3’ or 5’ end. The nucleic acid may have a 3’ overhang on the first strand. The nucleic acid may have a 3’ overhang on the second strand. The nucleic acid may have a 5’ overhang on the first strand. The nucleic acid may have a 5’ overhang on the second strand. The nucleic acid may have an overhang at both the 5’ end and 3’ end of the first strand. The nucleic acid may have an overhang at both the 5’ end and 3’ end of the second strand. The nucleic acid may have a 5’ overhang on the first strand and a 3’ overhang on the second strand. The nucleic acid may have a 3’ overhang on the first strand and a 5’ overhang on the second strand. The nucleic acid may have a 3’ overhang on the first strand and a 3’ overhang on the second strand. The nucleic acid may have a 5’ overhang on the first strand and a 5’ overhang on the second strand.

[0089] An overhang at the 3’ end or 5’ end of the second strand or the first strand may consist of 1, 2, 3, 4 or 5 nucleotides in length. Optionally, an overhang may consist of 1 or 2 nucleotides, which may or may not be modified.

[0090] In one embodiment, the 5’ end of the first strand is a single-stranded overhang of one, two or three nucleotides, preferably of one nucleotide.

[0091] Nucleic acid modifications

[0092] Nucleic acids discussed herein include unmodified RNA as well as RNA which has been modified, e.g., to improve efficacy or stability. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as those which occur in nature, for example as occur naturally in the human body. The term “modified nucleotide” as used herein refers to a nucleotide in which one or more of the components of the nucleotide, namely the sugar, base, and phosphate moiety, is / are different from those which occur in nature. The term “modified nucleotide” also refers in certain cases to molecules that are not nucleotides in the strict sense of the term because they lack, or have a substitute of, an essential component of a nucleotide, such as the sugar, base or phosphate moiety. A nucleic acid comprising such modified nucleotides is still to be understood as being a nucleic acid, even if one or more of the nucleotides of the nucleic acid has been replaced by a modified nucleotide that lacks, or has a substitution of, an essential component of a nucleotide.

[0093] Modifications of the nucleic acid of the present invention generally provide a powerful tool in overcoming potential limitations including, but not limited to, in vitro and in vivo stability and bioavailability inherent to native RNA molecules. The nucleic acids according to the invention may be modified by chemical modifications. Modified nucleic acids can also minimise the possibility of inducing interferon activity in humans. Modifications can further enhance the functional delivery of a nucleic acid to a target cell. Preferably, the modified nucleic acids of the present invention may comprise one or more chemically modified ribonucleotides of either or both of the first strand or the second strand. A ribonucleotide may comprise a chemical modification of the base, sugar or phosphate moieties. The ribonucleic acid may be modified by substitution with or insertion of analogues of nucleic acids or bases.

[0094] Throughout the description of the invention, “same or common modification” means the same modification to any nucleotide, be that A, G, C or U modified with a group such as a methyl group (2’-OMe) or a fluoro group (2’-F). For example, 2'-F-dll, 2'-F-dA, 2'-F-dC, 2'-F-dG are all considered to be the same or common modification, as are 2’-OMe-rll, 2’-OMe-rA; 2’-OMe-rC; 2’-OMe-rG. In contrast, a 2’-F modification is a different modification compared to a 2’-OMe modification.

[0095] Preferably, at least one nucleotide of the first and / or second strand of the nucleic acid is a modified nucleotide, preferably a non-naturally occurring nucleotide such as a 2’-F modified nucleotide.

[0096] A modified nucleotide can be a nucleotide with a modification of the ribose sugar group. The 2' hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents.

[0097] The 2' hydroxyl group (OH) of the ribose sugar can be replaced by a group -X-R, wherein X is selected from O, NR', S or SiR'2;

[0098] R is one of C2-C6 alkyl, substituted C2-C6 alkyl, C₆-C₈ aryl and substituted C₆-C₈ aryl; each R' is independently selected from one or more of H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₆-C₈ aryl and substituted C₆-C₈ aryl;

[0099] the substituted C₂-C₆ alkyl or substituted C₆-C₈ aryl refers to a group formed by substituting one or more hydrogen atoms on C₂-C₆ alkyl or C₆-C₈ aryl with a substituent, and the substituent is each independently selected from one or more of the following substituents: C₁-C₃ alkyl, C₆-C₈ aryl, C₁-C₃ alkoxy, halogen, oxo and sulfanylidene.

[0100] Examples of “oxy”-2' hydroxyl ribose sugar group modifications include alkoxy or aryloxy (OR, e.g., R=H, alkyl (such as methyl), cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR; “locked” nucleic acids (LNA) in which the 2' hydroxyl is connected, e.g., by a methylene bridge, to the 4' carbon of the same ribose sugar; O-AMINE (AMINE=NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, or polyamino) and aminoalkoxy, O(CH2)nAMINE, (e.g., AMINE=NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, or polyamino).

[0101] Other examples of “oxy”-2' hydroxyl ribose sugar group modifications include a groupselected from 2'-0-methoxyethyl, 2'-O-allyl, 2'-C-allyl, 2'-0-2-N-methylamino-2-oxoethyl, 2'-O-2-N, N-dimethylaminoethyl, 2'-0-3-aminopropyl or 2'-0-2,4-dinitrophenyl.

[0102] “Deoxy” modifications include hydrogen, halogen, amino (e.g., NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); NH(CH2CH2NH)nCH2CH2-AMINE (AMINE=NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino), — NHC(O)R (R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino functionality. Other substituents of certain embodiments include 2-methoxyethyl, 2'-OCH3, 2-O-allyl, 2-C-allyl, and 2'-fluoro.

[0103] The ribose sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose sugar. Thus, a modified nucleotide may contain a sugar such as arabinose.

[0104] Modified nucleotides can also include “abasic” sugars, which lack a nucleobase at C – 1′. These abasic sugars can further contain modifications at one or more of the constituent sugar atoms.

[0105] The 2' modifications may be used in combination with one or more phosphate internucleoside linker modifications (e.g., phosphorothioate or phosphorodithioate). One or more nucleotides of a nucleic acid of the present invention may be modified. The nucleic acid may comprise at least one modified nucleotide. The modified nucleotide may be in the first strand. The modified nucleotide may be in the second strand. The modified nucleotide may be in the duplex region. The modified nucleotide may be outside the duplex region, i.e., in a single-stranded region. The modified nucleotide may be on the first strand and may be outside the duplex region. The modified nucleotide may be on the second strand and may be outside the duplex region. The 3’-terminal nucleotide of the first strand may be a modified nucleotide. The 3’-terminal nucleotide of the second strand may be a modified nucleotide. The 5’-terminal nucleotide of the first strand may be a modified nucleotide. The 5’-terminal nucleotide of the second strand may be a modified nucleotide.

[0106] A nucleic acid of the invention may have 1 modified nucleotide or a nucleic acid of the invention may have about 2-4 modified nucleotides, or a nucleic acid may have about 4-6 modified nucleotides, about 6-8 modified nucleotides, about 8-10 modified nucleotides, about 10-12 modified nucleotides, about 12-14 modified nucleotides, about 14-16 modified nucleotides about 16-18 modified nucleotides, about 18-20 modified nucleotides, about 20-22 modified nucleotides, about 22-24 modified nucleotides, about 24-26 modified nucleotides or about 26-28 modified nucleotides. In each case the nucleic acid comprising said modified nucleotides retains at least 50% of its activity as compared to the same nucleic acid but without said modified nucleotides or vice versa. The nucleic acid may retain 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% and intermediate values of its activity as compared to the same nucleic acid but without said modified nucleotides, or may have more than 100% of the activity of the same nucleic acid without said modified nucleotides.

[0107] The modified nucleotide may be a purine or a pyrimidine. At least half of the purines may be modified. At least half of the pyrimidines may be modified. All of the purines may be modified. All of the pyrimidines may be modified. The modified nucleotides may be selected from the group consisting of a 3’ terminal deoxy thymine (dT) nucleotide, a 2’-O-methyl (2’-OMe) modified nucleotide, a 2’ modified nucleotide, a 2’ deoxy modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2’ amino modified nucleotide, a 2’ alkyl modified nucleotide, a 2’-deoxy-2’-fluoro (2’-F) modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a nucleotide comprising a 5’-phosphorothioate group, a nucleotide comprising a 5' phosphate or 5' phosphate mimic and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.

[0108] The nucleic acid may comprise a nucleotide comprising a modified base, wherein the base is selected from 2-aminoadenosine, 2, 6-diaminopurine, inosine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g., 5-methylcytidine), 5-alkyluridine (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine), 6-azapyrimidine, 6-alkylpyrimidine (e.g. 6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5’-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1 -methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid and 2-thiocytidine.

[0109] Many of the modifications described herein and that occur within a nucleic acid will be repeated within a polynucleotide molecule, such as a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases, the modification will occur at all of the possible positions / nucleotides in the polynucleotide but in many cases it will not. A modification may only occur at a 3' or 5' terminal position, may only occur in a terminal region, such as at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double-strand region, a single-strand region, or in both. A modification may occur only in the double-strand region of a nucleic acid of the invention or may only occur in a single-strand region of a nucleic acid of the invention. A phosphorothioate or phosphorodithioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4 or 5 nucleotides of a strand, or may occur in duplex and / or in single-strand regions, particularly at termini. The 5' end and / or 3’ end may be phosphorylated.

[0110] Stability of a nucleic acid of the invention may be increased by including particular bases in overhangs, or by including modified nucleotides, in single-strand overhangs, e.g., in a 5' or 3' overhang, or in both. Purine nucleotides may be included in overhangs. All or some of the bases in a 3' or 5' overhang may be modified. Modifications can include the use of modifications at the 2' OH group of the ribose sugar, the use of deoxyribonucleotides, instead of ribonucleotides, and modifications in the phosphate group, such as phosphorothioate or phosphorodithioate modifications. Overhangs need not be homologous with the target sequence. Nucleases can hydrolyse nucleic acid phosphodiester bonds. However, chemical modifications to nucleic acids can confer improved properties, and, can render oligoribonucleotides more stable to nucleases.

[0111] Modified nucleic acids, as used herein, can include one or more of:

[0112] (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and / or of one or more of the linking phosphate oxygens (referred to as linking even if at the 5’ and 3’ terminus of the nucleic acid of the invention);

[0113] (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar;

[0114] (iii) replacement of the phosphate moiety with “dephospho” linkers;

[0115] (iv) modification or replacement of a naturally occurring base;

[0116] (v) replacement or modification of the ribose sugar-phosphate backbone; and

[0117] (vi) modification of the 3' end or 5' end of the first strand and / or the second strand, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, e.g., a fluorescently labelled moiety, to either the 3' or 5' end of one or both strands.

[0118] The terms “replacement”, “modification” and “alteration” indicate a difference from a naturally occurring molecule.

[0119] Specific modifications are discussed in more detail below.

[0120] The nucleic acid may comprise one or more nucleotides on the second and / or first strands that are modified. Alternating nucleotides may be modified, to form modified nucleotides.

[0121] “Alternating” as described herein means to occur one after another in a regular way. In other words, alternating means to occur in turn repeatedly. For example, if one nucleotide is modified, the next contiguous nucleotide is not modified and the following contiguous nucleotide is modified and so on. One nucleotide may be modified with a first modification, the next contiguous nucleotide may be modified with a second modification and the following contiguous nucleotide is modified with the first modification and so on, where the first and second modifications are different. Unless specified otherwise, the term “alternating” as used herein will be understood to mean completely alternating. In other words, the alternating pattern spans the entirety of the relevant strand, for example the first strand. For example, completely alternating may comprise all odd numbered nucleotides comprising a first modification, and all even numbered nucleotides not being modified. Alternatively, completely alternating may comprise all odd numbered nucleotides comprising a first modification, and all even numbered nucleotides comprising a second modification. Completely alternating may comprise all even numbered nucleotides comprising a first modification, and all odd numbered nucleotides comprising a second modification.

[0122] Some representative modified nucleic acid sequences of the present invention are shown in the examples. These examples are meant to be representative and not limiting.

[0123] In one aspect of the nucleic acid, at least nucleotides 2 and 14 of the first strand are modified, preferably by a first common modification, the nucleotides being numbered consecutively starting with nucleotide number 1 at the 5’ end of the first strand. The first modification is preferably 2’-F.

[0124] In one aspect, at least one, several or preferably all the even-numbered nucleotides of the first strand are modified, preferably by a first common modification, the nucleotides being numbered consecutively starting with nucleotide number 1 at the 5’ end of the first strand. The first modification is preferably 2’-F.

[0125] In one aspect, at least one, several or preferably all the odd-numbered nucleotides of the first strand are modified, the nucleotides being numbered consecutively starting with nucleotide number 1 at the 5’ end of the first strand. Preferably, they are modified by a second modification. This second modification is preferably different from the first modification if the nucleic acid also comprises a first modification, for example of nucleotides 2 and 14 or of all the even-numbered nucleotides of the first strand. The first modification is preferably any 2’ ribose sugar modification that is of the same size or smaller in volume than a 2’-OH group, or a locked nucleic acid (LNA), or an unlocked nucleic acid (UNA), ora 2'-Fluoroarabino Nucleic Acid (FANA) modification. A 2’ ribose sugar modification that is of the same size or smaller in volume than a 2’-OH group can for example be a 2’-F, 2’-H, 2’-halo, or 2’-NH2. The second modification is preferably any 2’ ribose sugar modification that is larger in volume than a 2’-OH group. A 2’ ribose sugar modification that is larger in volume than a 2’-OH group can for example be a 2’-OMe, 2’-O-MOE (2’-0-methoxyethyl), 2’-O-allyl or 2’-O-alkyl, with the proviso that the nucleic is capable of reducing the expression of the target gene to at least the same extent as the same nucleic acid without the modification(s) under comparable conditions. The first modification is preferably 2’-F and / or the second modification is preferably 2’-OMe.

[0126] In the context of this disclosure, the size or volume of a substituent, such as a 2’ ribose sugar modification, is preferably measured as the van der Waals volume. In one aspect, at least one, several or preferably all the nucleotides of the second strand in a position corresponding to an even-numbered nucleotide of the first strand are modified, preferably by a third modification. Preferably in the same nucleic acid nucleotides 2 and 14 or all the even numbered nucleotides of the first strand are modified with a first modification. In addition, or alternatively, the odd-numbered nucleotides of the first strand are modified with a second modification. Preferably, the third modification is different from the first modification and / or the third modification is the same as the second modification. The first modification is preferably any 2’ ribose sugar modification that is of the same size or smaller in volume than a 2’-OH group, or a locked nucleic acid (LNA), or an unlocked nucleic acid (UNA), or a 2'-Fluoroarabino Nucleic Acid (FANA) modification. A 2’ ribose sugar modification that is of the same size or smaller in volume than a 2’-OH group can for example be a 2’-F, 2’-H, 2’-halo, or 2’-NH2. The second and / or third modification is preferably any 2’ ribose sugar modification that is larger in volume than a 2’-OH group. A 2’ ribose sugar modification that is larger in volume than a 2’-OH group can for example be a 2’-OMe, 2’-O-MOE (2’-0-methoxyethyl), 2’-O-allyl or 2’-O-alkyl, with the proviso that the nucleic is capable of reducing the expression of the target gene to at least the same extent as the same nucleic acid without the modification(s) under comparable conditions. The first modification is preferably 2’-F and / or the second and / or third modification is / are preferably 2’-OMe. The nucleotides on the first strand are numbered consecutively starting with nucleotide number 1 at the 5’ end of the first strand.

[0127] A nucleotide of the second strand that is in a position corresponding, for example, to an even-numbered nucleotide of the first strand is a nucleotide of the second strand that is base-paired to an even-numbered nucleotide of the first strand.

[0128] In one aspect, at least one, several or preferably all the nucleotides of the second strand in a position corresponding to an odd-numbered nucleotide of the first strand are modified, preferably by a fourth modification. Preferably in the same nucleic acid nucleotides 2 and 14 or all the even numbered nucleotides of the first strand are modified with a first modification. In addition, or alternatively, the odd-numbered nucleotides of the first strand are modified with a second modification. In addition, or alternatively, all the nucleotides of the second strand in a position corresponding to an even-numbered nucleotide of the first strand are modified with a third modification. The fourth modification is preferably different from the second modification and preferably different from the third modification and the fourth modification is preferably the same as the first modification. The first and / or fourth modification is preferably any 2’ ribose sugar modification that is of the same size or smaller in volume than a 2’-OH group, or a locked nucleic acid (LNA), or an unlocked nucleic acid (UNA), or a 2'-Fluoroarabino Nucleic Acid (FANA) modification. A 2’ ribose sugar modification that is of the same size or smaller in volume than a 2’-OH group can for example be a 2’-F, 2’-H, 2’-halo, or 2’-NH2. The second and / or third modification is preferably any 2’ ribose sugar modification that is larger in volume than a 2’-OH group. A 2’ ribose sugar modification that is larger in volume than a 2’-OH group can for example be a 2’-OMe, 2’-O-MOE (2’-0-methoxyethyl), 2’-O-allyl or 2’-O-alkyl, with the proviso that the nucleic is capable of reducing the expression of the target gene to at least the same extent as the same nucleic acid without the modification(s) under comparable conditions. The first and / or the fourth modification is / are preferably a 2’-OMe modification and / or the second and / or third modification is / are preferably a 2’-F modification. The nucleotides on the first strand are numbered consecutively starting with nucleotide number 1 at the 5’ end of the first strand.

[0129] In one aspect of the nucleic acid, the nucleotide / nucleotides of the second strand in a position corresponding to nucleotide 11 or nucleotide 13 or nucleotides 11 and 13 or nucleotides 11- 13 of the first strand is / are modified by a fourth modification. Preferably, all the nucleotides of the second strand other than the nucleotide / nucleotides in a position corresponding to nucleotide 11 or nucleotide 13 or nucleotides 11 and 13 or nucleotides 11 -13 of the first strand is / are modified by a third modification. Preferably in the same nucleic acid nucleotides 2 and 14 or all the even numbered nucleotides of the first strand are modified with a first modification. In addition, or alternatively, the odd-numbered nucleotides of the first strand are modified with a second modification. The fourth modification is preferably different from the second modification and preferably different from the third modification and the fourth modification is preferably the same as the first modification. The first and / or fourth modification is preferably any 2’ ribose sugar modification that is of the same size or smaller in volume than a 2’-OH group, or a locked nucleic acid (LNA), or an unlocked nucleic acid (UNA), or a 2'-Fluoroarabino Nucleic Acid (FANA) modification. A 2’ ribose sugar modification that is of the same size or smaller in volume than a 2’-OH group can for example be a 2’-F, 2’-H, 2’-halo, or 2’-NH2. The second and / or third modification is preferably any 2’ ribose sugar modification that is larger in volume than a 2’-OH group. A 2’ ribose sugar modification that is larger In volume than a 2’-OH group can for example be a 2’-OMe, 2’-O-MOE (2’-0-methoxyethyl), 2’-O-allyl or 2’-O-alkyl, with the proviso that the nucleic is capable of reducing the expression of the target gene to at least the same extent as the same nucleic acid without the modification(s) under comparable conditions. The first and / or the fourth modification is / are preferably a 2’-OMe modification and / or the second and / or third modification is / are preferably a 2’-F modification. The nucleotides on the first strand are numbered consecutively starting with nucleotide number 1 at the 5’ end of the first strand. In one aspect of the nucleic acid, all the even-numbered nucleotides of the first strand are modified by a first modification, all the odd-numbered nucleotides of the first strand are modified by a second modification, all the nucleotides of the second strand in a position corresponding to an even-numbered nucleotide of the first strand are modified by a third modification, all the nucleotides of the second strand in a position corresponding to an odd-numbered nucleotide of the first strand are modified by a fourth modification, wherein the first and / or fourth modification is / are 2’-F and / or the second and / or third modification is / are 2’-OMe.

[0130] In one aspect of the nucleic acid, all the even-numbered nucleotides of the first strand are modified by a first modification, all the odd-numbered nucleotides of the first strand are modified by a second modification, all the nucleotides of the second strand in positions corresponding to nucleotides 11-13 of the first strand are modified by a fourth modification, all the nucleotides of the second strand other than the nucleotides corresponding to nucleotides 11-13 of the first strand are modified by a third modification, wherein the first and fourth modification are 2’-F and the second and third modification are 2’-OMe. In one embodiment in this aspect, the 3’ terminal nucleotide of the second strand is an inverted RNA nucleotide (i.e., the nucleotide is linked to the 3’ end of the strand through its 3’ carbon, rather than through its 5’ carbon as would normally be the case). When the 3’ terminal nucleotide of the second strand is an inverted RNA nucleotide, the inverted RNA nucleotide is preferably an unmodified nucleotide in the sense that it does not comprise any modifications compared to the natural nucleotide counterpart. Specifically, the inverted RNA nucleotide is preferably a 2’-OH nucleotide. Preferably, in this aspect when the 3’ terminal nucleotide of the second strand is an inverted RNA nucleotide, the nucleic acid is blunt-ended at least at the end that comprises the 5’ end of the first strand.

[0131] One aspect of the present invention is a nucleic acid as disclosed herein for inhibiting expression of the INHBE gene, preferably in a cell, wherein said first strand includes modified nucleotides or unmodified nucleotides at a plurality of positions in order to facilitate processing of the nucleic acid by RISC.

[0132] In one aspect, “facilitate processing by RISC” means that the nucleic acid can be processed by RISC, for example any modification present will permit the nucleic acid to be processed by RISC and preferably, will be beneficial to processing by RISC, suitably such that siRNA activity can take place.

[0133] One aspect of the present invention is a nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5’ end of the first strand are not modified with a 2’- OMe modification, and the nucleotide / nucleotides on the second strand which corresponds to position 11 or position 13 or positions 11 and 13 or positions 11, 12 and 13 of the first strand is / are not modified with a 2’-OMe modification (in other words, they are not modified or are modified with a modification other than 2’-OMe).

[0134] In one aspect, the nucleotide on the second strand which corresponds to position 13 of the first strand is the nucleotide that forms a base pair with position 13 (from the 5’ end) of the first strand.

[0135] In one aspect, the nucleotide on the second strand which corresponds to position 11 of the first strand is the nucleotide that forms a base pair with position 11 (from the 5’ end) of the first strand.

[0136] In one aspect, the nucleotide on the second strand which corresponds to position 12 of the first strand is the nucleotide that forms a base pair with position 12 (from the 5’ end) of the first strand.

[0137] For example, in a 19-mer nucleic acid which is double-stranded and blunt ended, position 13 (from the 5’ end) of the first strand would pair with position 7 (from the 5’ end) of the second strand. Position 11 (from the 5’ end) of the first strand would pair with position 9 (from the 5’ end) of the second strand. This nomenclature may be applied to other positions of the second strand.

[0138] In one aspect, in the case of a partially complementary first and second strand, the nucleotide on the second strand that “corresponds to” a position on the first strand may not necessarily form a base pair if that position is the position in which there is a mismatch, but the principle of the nomenclature still applies. Mismatches can occur at position 1 and / or position 19 of the nucleic acid.

[0139] One aspect is a nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5’ end of the first strand are not modified with a 2’-OMe modification, and the nucleotides on the second strand which correspond to position 11, or 13, or 11 and 13, or 11-13 of the first strand are modified with a 2'-F modification.

[0140] One aspect is a nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5’ end of the first strand are modified with a 2'-F modification, and the nucleotides 1

[0141] on the second strand which correspond to position 11, or 13, or 11 and 13, or 11 -13 of the first strand are not modified with a 2’-OMe modification.

[0142] One aspect is a nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5’ end of the first strand are modified with a 2'-F modification, and the nucleotides on the second strand which correspond to position 11, or 13, or 11 and 13, or 11 -13 of the first strand are modified with a 2'-F modification.

[0143] One aspect is a nucleic acid as disclosed herein wherein greater than 50% of the nucleotides of the first and / or second strand comprise a 2’-OMe modification, such as greater than 55%, 60%, 65%, 70%, 75%, 80%, or 85%, or more, of the first and / or second strand comprise a 2’-OMe modification, preferably measured as a percentage of the total nucleotides of both the first and second strands.

[0144] One aspect is a nucleic acid as disclosed herein wherein greater than 50% of the nucleotides of the first and / or second strand comprise a naturally occurring RNA modification, such as wherein greater than 55%, 60%, 65%, 70%, 75%, 80%, or 85% or more of the first and / or second strands comprise such a modification, preferably measured as a percentage of the total nucleotides of both the first and second strands. Suitable naturally occurring modifications include, as well as 2’-OMe, other 2’ sugar modifications, in particular a 2’-H modification resulting in a DNA nucleotide.

[0145] One aspect is a nucleic acid as disclosed herein comprising no more than 20%, such as no more than 15% such as no more than 10%, of nucleotides which have 2' modifications that are not 2’-OMe modifications on the first and / or second strand, preferably as a percentage of the total nucleotides of both the first and second strands.

[0146] One aspect is a nucleic acid as disclosed herein, wherein the number of nucleotides in the first and / or second strand with a 2’-modification that is not a 2’-OMe modification is no more than 7, more preferably no more than 5, and most preferably no more than 3.

[0147] One aspect is a nucleic acid as disclosed herein comprising no more than 20%, (such as no more than 15% or no more than 10%) of 2’-F modifications on the first and / or second strand, preferably as a percentage of the total nucleotides of both strands. One aspect is a nucleic acid as disclosed herein, wherein the number of nucleotides in the first and / or second strand with a 2’-F modification is no more than 7, more preferably no more than 5, and most preferably no more than 3.

[0148] One aspect is a nucleic acid as disclosed herein, wherein all nucleotides are modified with a 2’-OMe modification except positions 2 and 14 from the 5’ end of the first strand and the nucleotides on the second strand which correspond to position 11, or 13, or 11 and 13, or 11-13 of the first strand. Preferably the nucleotides that are not modified with 2’-OMe are modified with fluoro at the 2’ position (2’-F modification).

[0149] In certain embodiments, a preferred aspect is a nucleic acid as disclosed herein wherein all nucleotides of the nucleic acid are modified at the 2’ position of the ribose sugar. Preferably these nucleotides are modified with a 2’-F modification where the modification is not a 2’-OMe modification.

[0150] In one aspect the nucleic acid is modified on the first strand with alternating 2’-OMe modifications and 2-F modifications, and positions 2 and 14 (starting from the 5’ end) are modified with 2’-F. Preferably, alternating will be understood to mean alternating over the complete strand.

[0151] Preferably the second strand is modified with 2’-F modifications at nucleotides on the second strand which correspond to position 11, or 13, or 11 and 13, or 11-13 of the first strand. Preferably the second strand is modified with 2’-F modifications at positions 11-13 counting from the 3’ end starting at the first position of the complementary (double-stranded) region, and the remaining modifications are naturally occurring modifications, preferably 2’-OMe. The complementary region at least in this case starts at the first position of the second strand that has a corresponding nucleotide in the first strand, regardless of whether the two nucleotides are able to base pair to each other.

[0152] In one aspect of the nucleic acid, each of the nucleotides of the first strand and of the second strand is a modified nucleotide.

[0153] The term “odd numbered” as described herein means a number not divisible by two. Examples of odd numbers are 1, 3, 5, 7, 9, 11 and so on. The term “even numbered” as described herein means a number which is evenly divisible by two. Examples of even numbers are 2, 4, 6, 8, 10, 12, 14 and so on. Unless specifically stated otherwise, herein the nucleotides of the first strand are numbered consecutively starting with nucleotide number 1 at the 5’ end of the first strand. Nucleotides of the second strand are numbered consecutively starting with nucleotide number 1 at the 3’ end of the second strand.

[0154] One or more nucleotides on the first and / or second strand may be modified, to form modified nucleotides. One or more of the odd-numbered nucleotides of the first strand may be modified. One or more of the even-numbered nucleotides of the first strand may be modified by at least a second modification, wherein the at least second modification is different from the modification on the one or more odd nucleotides. At least one of the one or more modified even numbered-nucleotides may be adjacent to at least one of the one or more modified odd-numbered nucleotides.

[0155] A plurality of odd-numbered nucleotides in the first strand may be modified in the nucleic acid of the invention. A plurality of even-numbered nucleotides in the first strand may be modified by a second modification. The first strand may comprise adjacent nucleotides that are modified by a common modification. The first strand may also comprise adjacent nucleotides that are modified by a second different modification (i.e., the first strand may comprise nucleotides that are adjacent to each other and modified by a first modification as well as other nucleotides that are adjacent to each other and modified by a second modification that is different to the first modification).

[0156] One or more of the odd-numbered nucleotides of the second strand (wherein the nucleotides are numbered consecutively starting with nucleotide number 1 at the 3’ end of the second strand) may be modified by a modification that is different to the modification of the odd-numbered nucleotides on the first strand (wherein the nucleotides are numbered consecutively starting with nucleotide number 1 at the 5’ end of the first strand) and / or one or more of the even-numbered nucleotides of the second strand may be modified by the same modification of the odd-numbered nucleotides of the first strand. At least one of the one or more modified even-numbered nucleotides of the second strand may be adjacent to the one or more modified odd-numbered nucleotides. A plurality of odd-numbered nucleotides of the second strand may be modified by a common modification and / or a plurality of even-numbered nucleotides may be modified by the same modification that is present on the first stand odd-numbered nucleotides. A plurality of odd-numbered nucleotides on the second strand may be modified by a modification that is different from the modification of the first strand odd-numbered nucleotides. The second strand may comprise adjacent nucleotides that are modified by a common modification, which may be a modification that is different from the modification of the odd-numbered nucleotides of the first strand.

[0157] In some aspects of the nucleic acid of the invention, each of the odd-numbered nucleotides in the first strand and each of the even-numbered nucleotides in the second strand may be modified with a common modification and, each of the even-numbered nucleotides may be modified in the first strand with a different modification and each of the odd-numbered nucleotides may be modified in the second strand with the different modification.

[0158] The nucleic acid of the invention may have the modified nucleotides of the first strand shifted by at least one nucleotide relative to the unmodified or differently modified nucleotides of the second strand.

[0159] In certain aspects, one or more or each of the odd numbered-nucleotides may be modified in the first strand and one or more or each of the even-numbered nucleotides may be modified in the second strand. One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification. One or more or each of the even-numbered nucleotides may be modified in the first strand and one or more or each of the even-numbered nucleotides may be modified in the second strand. One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification. One or more or each of the odd-numbered nucleotides may be modified in the first strand and one or more of the odd-numbered nucleotides may be modified in the second strand by a common modification. One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification. One or more or each of the even-numbered nucleotides may be modified in the first strand and one or more or each of the odd-numbered nucleotides may be modified in the second strand by a common modification. One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification.

[0160] The nucleic acid of the invention may comprise single- or double-stranded constructs that comprise at least two regions of alternating modifications in one or both of the strands. These alternating regions can comprise up to about 12 nucleotides but preferably comprise from about 3 to about 10 nucleotides. The regions of alternating nucleotides may be located at the termini of one or both strands of the nucleic acid of the invention. The nucleic acid may comprise from 4 to about 10 nucleotides of alternating nucleotides at each of the termini (3’ and 5') and these regions may be separated by from about 5 to about 12 contiguous unmodified or differently or commonly modified nucleotides. The odd numbered nucleotides of the first strand may be modified with a first modification and the even numbered nucleotides may be modified with a second modification. The second strand may comprise adjacent nucleotides that are modified with a common modification, which may be the same as the modification of the odd-numbered nucleotides of the first strand. The second strand may comprise adjacent nucleotides that are modified with a common modification, which may be the same as the modification of the even-numbered nucleotides of the first strand. One or more nucleotides of the second strand may be modified with the second modification. One or more nucleotides with the second modification may be adjacent to each other and to nucleotides having a modification that is the same as the modification of the odd-numbered nucleotides of the first strand. The first strand may also comprise phosphorothioate linkages between the two nucleotides at the 3’ end and at the 5’ end or a phosphorodithioate linkage between the two nucleotides at the 3’ end. The second strand may comprise a phosphorothioate or phosphorodithioate linkage between the two nucleotides at the 5’ end. The second strand may also be conjugated to a ligand at the 5’ end.

[0161] The nucleic acid of the invention may comprise a first strand comprising adjacent nucleotides that are modified with a common modification. One or more such nucleotides may be adjacent to one or more nucleotides which may be modified with a second modification. One or more nucleotides with the second modification may be adjacent. The second strand may comprise adjacent nucleotides that are modified with a common modification, which may be the same as one of the modifications of one or more nucleotides of the first strand. One or more nucleotides of the second strand may also be modified with the second modification. One or more nucleotides with the second modification may be adjacent. The first strand may also comprise phosphorothioate linkages between the two nucleotides at the 3’ end and at the 5’ end or a phosphorodithioate linkage between the two nucleotides at the 3’ end. The second strand may comprise a phosphorothioate or phosphorodithioate linkage between the two nucleotides at the 3’ end. The second strand may also be conjugated to a ligand at the 5’ end. The nucleotides numbered from 5' to 3' on the first strand and 3' to 5' on the second strand, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 and 25 may be modified by a modification on the first strand. The nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modified by a second modification on the first strand. The nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 may be modified by a modification on the second strand. The nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modified by a second modification on the second strand. Nucleotides are numbered for the sake of the nucleic acid of the present invention from 5' to 3' on the first strand and 3' to 5' on the second strand. The nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modified by a modification on the first strand. The nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 may be modified by a second modification on the first strand. The nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 may be modified by a modification on the second strand. The nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modified by a second modification on the second strand.

[0162] Clearly, if the first and / or the second strand are shorter than 25 nucleotides in length, such as 19 nucleotides in length, there are no nucleotides numbered 20, 21, 22, 23, 24 and 25 to be modified. The skilled person understands the description above to apply to shorter strands, accordingly.

[0163] One or more modified nucleotides on the first strand may be paired with modified nucleotides on the second strand having a common modification. One or more modified nucleotides on the first strand may be paired with modified nucleotides on the second strand having a different modification. One or more modified nucleotides on the first strand may be paired with unmodified nucleotides on the second strand. One or more modified nucleotides on the second strand may be paired with unmodified nucleotides on the first strand. In other words, the alternating nucleotides can be aligned on the two strands such as, for example, all the modifications in the alternating regions of the second strand are paired with identical modifications in the first strand or alternatively the modifications can be offset by one nucleotide with the common modifications in the alternating regions of one strand pairing with dissimilar modifications (i.e. a second or further modification) in the other strand. Another option is to have dissimilar modifications in each of the strands.

[0164] The modifications on the first strand may be shifted by one nucleotide relative to the modified nucleotides on the second strand, such that common modified nucleotides are not paired with each other.

[0165] The modification and / or modifications may each and individually be selected from the group consisting of 3' terminal deoxy thymine, 2'-OMe, a 2' deoxy modification, a 2' amino modification, a 2' alkyl modification, a morpholino modification, a phosphoramidate modification, 5'-phosphorothioate group modification, a 5' phosphate or 5' phosphate mimic modification and a cholesteryl derivative or a dodecanoic acid bisdecylamide group modification and / or the modified nucleotide may be any one of a locked nucleotide, an abasic nucleotide or a non-natural base comprising nucleotide. At least one modification may be 2'-OMe and / or at least one modification may be 2'-F. Further modifications as described herein may be present on the first and / or second strand.

[0166] The nucleic acid of the invention may comprise an inverted RNA nucleotide at one or several of the strand ends. Such inverted nucleotides provide stability to the nucleic acid. Preferably, the nucleic acid comprises at least an inverted nucleotide at the 3’ end of the first and / or the second strand and / or at the 5’ end of the second strand. More preferably, the nucleic acid comprises an inverted nucleotide at the 3’ end of the second strand. Most preferably, the nucleic acid comprises an inverted RNA nucleotide at the 3’ end of the second strand and this nucleotide is preferably an inverted A. An inverted nucleotide is a nucleotide that is linked to the 3’ end of a nucleic acid through its 3’ carbon, rather than its 5’ carbon as would normally be the case or is linked to the 5’ end of a nucleic acid through its 5’ carbon, rather than its 3’ carbon as would normally be the case. The inverted nucleotide is preferably present at an end of a strand not as an overhang but opposite a corresponding nucleotide in the other strand. Accordingly, the nucleic acid is preferably blunt-ended at the end that comprises the inverted RNA nucleotide. An inverted RNA nucleotide being present at the end of a strand preferably means that the last nucleotide at this end of the strand is the inverted RNA nucleotide. A nucleic acid with such a nucleotide is stable and easy to synthesise. The inverted RNA nucleotide is preferably an unmodified nucleotide in the sense that it does not comprise any modifications compared to the natural nucleotide counterpart. Specifically, the inverted RNA nucleotide is preferably a 2’-OH nucleotide.

[0167] Nucleic acids of the invention may comprise one or more nucleotides modified at the 2’ position with a 2’-H, and therefore having a DNA nucleotide within the nucleic acid. Nucleic acids of the invention may comprise DNA nucleotides at positions 2 and / or 14 of the first strand counting from the 5’ end of the first strand. Nucleic acids may comprise DNA nucleotides on the second strand which correspond to position 11, or 13, or 11 and 13, or 11-13 of the first strand.

[0168] In one aspect there is no more than one DNA nucleotide per nucleic acid of the invention.

[0169] Nucleic acids of the invention may comprise one or more LNA nucleotides. Nucleic acids of the invention may comprise LNA nucleotides at positions 2, and / or 14 of the first strand counting from the start of the double stranded region of the first strand. Certain nucleic acids of the invention may comprise an LNA nucleotide at one of the five most 3-terminal positions of the double stranded region of the first strand, preferably at the penultimate position of the double stranded region at the 3’ end of the first strand. Inclusion of a LNA modification alone or in combination with other modifications can significantly enhance RNAi knockdown activity and stability. Examples provided hereunder clearly demonstrate that the presence of a LNA in penultimate position in the first (antisense) strand leads to improved activity and stability of the molecule.

[0170] Abbreviations for LNA building blocks used in here (LT, LA, LC, LG) refer to the structures depicted below.

[0171]

[0172] Some representative modified nucleic acid sequences of the present invention are shown in the examples. These examples are meant to be representative and not limiting.

[0173] In certain preferred embodiments, the nucleic acid may comprise a first modification and a second or further modification which are each and individually selected from the group comprising 2'-OMe modification and 2'-F modification. The nucleic acid may comprise a modification that is 2'-OMe that may be a first modification, and a second modification that is 2'-F. The nucleic acid of the invention may also include a phosphorothioate or phosphorodithioate modification and / or a deoxy modification which may be present in or between the terminal 2 or 3 nucleotides of each or any end of each or both strands.

[0174] In one aspect of the nucleic acid, at least one nucleotide of the first and / or second strand is a modified nucleotide, wherein if the first strand comprises at least one modified nucleotide: (i) at least one or both of the nucleotides 2 and 14 of the first strand is / are modified by a first modification; and / or

[0175] (ii) at least one, several, or all the even-numbered nucleotides of the first strand is / are modified by a first modification; and / or

[0176] (iii) at least one, several, or all the odd-numbered nucleotides of the first strand is / are modified by a second modification; and / or

[0177] wherein if the second strand comprises at least one modified nucleotide: (iv) at least one, several, or all the nucleotides of the second strand in a position corresponding to an even-numbered nucleotide of the first strand is / are modified by a third modification; and / or

[0178] (v) at least one, several, or all the nucleotides of the second strand in a position corresponding to an odd-numbered nucleotide of the first strand is / are modified by a fourth modification; and / or

[0179] (vi) at least one, several, or all the nucleotides of the second strand in a position corresponding to nucleotide 11 or nucleotide 13 or nucleotides 11 and 13 or nucleotides 11-13 of the first strand is / are modified by a fourth modification; and / or

[0180] (vii) at least one, several, or all the nucleotides of the second strand in a position other than the position corresponding to nucleotide 11 or nucleotide 13 or nucleotides 11 and 13 or nucleotides 11-13 of the first strand is / are modified by a third modification; wherein the nucleotides on the first strand are numbered consecutively starting with nucleotide number 1 at the 5’ end of the first strand;

[0181] wherein the modifications are preferably at least one of the following:

[0182] (a) the first modification is preferably different from the second and from the third modification;

[0183] (b) the first modification is preferably the same as the fourth modification;

[0184] (c) the second and the third modification are preferably the same modification;

[0185] (d) the first modification is preferably a 2’-F modification;

[0186] (e) the second modification is preferably a 2’-OMe modification;

[0187] (f) the third modification is preferably a 2’-OMe modification; and / or

[0188] (g) the fourth modification is preferably a 2’-F modification; and

[0189] wherein optionally the nucleic acid is conjugated to a ligand.

[0190] One aspect is a double-stranded nucleic acid for inhibiting expression of INHBE, preferably in a cell, wherein the nucleic acid comprises a first strand and a second strand, wherein the unmodified equivalent of the first strand sequence comprises a sequence of at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the first strand sequences shown in Table 5a, or in Table 1, wherein all the even-numbered nucleotides of the first strand are modified by a first modification, all the odd-numbered nucleotides of the first strand are modified by a second modification, all the nucleotides of the second strand in a position corresponding to an even-numbered nucleotide of the first strand are modified by a third modification, all the nucleotides of the second strand in a position corresponding to an odd-numbered nucleotide of the first strand are modified by a fourth modification, wherein the first and fourth modification are 2’-F and the second and third modification are 2’-OMe. One aspect is a double-stranded nucleic acid for inhibiting expression of INHBE, preferably in a cell, wherein the nucleic acid comprises a first strand and a second strand, wherein the unmodified equivalent of the first strand sequence comprises a sequence of at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the first strand sequences shown in Table 5a, or in Table 1, wherein all the even-numbered nucleotides of the first strand are modified by a first modification, all the odd-numbered nucleotides of the first strand are modified by a second modification, all the nucleotides of the second strand in positions corresponding to nucleotides 11-13 of the first strand are modified by a fourth modification, all the nucleotides of the second strand other than the nucleotides corresponding to nucleotides 11-13 of the first strand are modified by a third modification, wherein the first and fourth modification are 2’-F and the second and third modification are 2’-OMe.

[0191] The 3' and 5' ends of an oligonucleotide can be modified. Such modifications can be at the 3' end or the 5' end or both ends of the molecule. They can include modification or replacement of an entire terminal phosphate or of one or more of the atoms of the phosphate group. For example, the 3' and 5' ends of an oligonucleotide can be conjugated to other functional molecular entities such as labelling moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based, e.g., on sulfur, silicon, boron or ester). The functional molecular entities can be attached to the ribose sugar through a phosphate group and / or a linker. The terminal atom of the linker can connect to or replace the linking atom of the phosphate group or the C-3' or C-5' O, N, S or C group of the ribose sugar. Alternatively, the linker can connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNAs). These spacers or linkers can include e.g., — (CH2)n—, — (CH2)nN —, — (CH2)nO —, — (CH2)nS —, — (CH2CH2O)nCH2CH2O — (e.g., n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents. The 3' end can be an — OH group.

[0192] Other examples of terminal modifications include dyes, intercalating agents (e.g., acridines), cross-linkers (e.g., psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases, EDTA, lipophilic carriers (e.g., cholesterol, cholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, 1,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles).

[0193] Terminal modifications can also be useful for monitoring distribution, and in such cases the groups to be added may include fluorophores, e.g., fluorescein or an Alexa dye. Terminal modifications can also be useful for enhancing uptake, useful modifications for this include cholesterol. Terminal modifications can also be useful for cross-linking an RNA agent to another moiety.

[0194] Terminal modifications can be added for a number of reasons, including to modulate activity or to modulate resistance to degradation. Terminal modifications useful for modulating activity include modification of the 5' end with phosphate or phosphate analogues. Nucleic acids of the invention, on the first or second strand, may be 5' phosphorylated or include a phosphoryl analogue at the 5' prime terminus. 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5'-monophosphate ((HO)2(O)P— 0-5'); 5'-diphosphate ((HO)2(O)P— O— P(HO)(O)— 0-5'); 5'-triphosphate ((HO)2(O)P — O — (HO)(O)P — O — P(HO)(O) — 0-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-O-5'-(HO)(O)P— O— (HO)(O)P— O— P(HO)(O)— 0-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N — 0-5'-(HO)(O)P — O — (HO)(O)P — O — P(HO)(O) — 0-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)P — 0-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P — 0-5'), 5'-phosphorothiolate ((HO)2(O)P — S-5'); any additional combination of oxygen / sulfur replaced monophosphate, diphosphate and triphosphates (e.g., 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2(O)P — NH-5', (HO)(NH2)(O)P — 0-5'), 5'-alkylphosphonates (alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O) — 0-5'-(wherein R is an alkyl), (OH)2(O)P-5'-CH2-), 5' vinylphosphonate, 5'-alkyletherphosphonates (alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O) — 0-5'- (wherein R is an alkylether)).

[0195] Certain moieties may be linked to the 5' terminus of the first strand or the second strand. These include abasic ribose sugar moiety, abasic deoxyribose moiety, modifications abasic ribose sugar and abasic deoxyribose moieties including 2'-0 alkyl modifications; inverted abasic ribose sugar and abasic deoxyribose moieties and modifications thereof, C6-imino-Pi; a mirror nucleotide including L-DNA and L-RNA; 5'0Me nucleotide; and nucleotide analogues including 4',5'-methylene nucleotide; 1-(P-D-erythrofuranosyl)nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; alpha-nucleotide; threo-pentofuranosyl nucleotide; acyclic 3', 4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5'-5'-inverted abasic moiety; 1,4-butanediol phosphate; 5'-amino; and bridging or non-bridging methylphosphonate and 5'-mercapto moieties.

[0196] In each sequence described herein, a C-terminal “-OH” moiety may be substituted for a C-terminal “-NH2” moiety, and vice-versa.

[0197] The invention also provides a nucleic acid according to any aspect of the invention described herein, wherein the first strand has a terminal 5’ (E)-vinylphosphonate nucleotide at its 5’ end. This terminal 5’ (E)-vinylphosphonate nucleotide is preferably linked to the second nucleotide in the first strand by a phosphodiester linkage. Preferably, the terminal 5’ (E)-vinylphosphonate (“vp”) nucleotide is a uridine (“vp-U”).

[0198] The first strand of the nucleic acid may comprise formula (I):

[0199] (vp)-N(po)[N(po)]n- (I)

[0200] where ‘(vp)-’ is the 5’ (E)-vinylphosphonate, N’ is a nucleotide, ‘po’ is a phosphodiester linkage, and n is from 1 to (the total number of nucleotides in the first strand - 2), preferably wherein n is from 1 to (the total number of nucleotides in the first strand -3), more preferably wherein n is from 1 to (the total number of nucleotides in the first strand -4).

[0201] Preferably, the terminal 5’ (E)-vinylphosphonate nucleotide is an RNA nucleotide, preferably a (vp)-U.

[0202] A terminal 5’ (E)-vinylphosphonate nucleotide is a nucleotide wherein the phosphate group at the 5’-end of the ribose sugar has been replaced with a E-vinylphosphonate group:

[0203]

[0204] Nucleotides with a phosphate at the 5’-end of the ribose sugar

[0205]

[0206] Nucleotide with a terminal 5' (E)-vinylphosphonate

[0207] at the 5’-end of the ribose sugar.

[0208] In one aspect, the first strand has a terminal 5’ (E)-vinylphosphonate nucleotide at its 5’ end, the terminal 5’ (E)-vinylphosphonate nucleotide is linked to the second nucleotide in the first strand by a phosphodiester linkage and the first strand comprises a) more than 1 phosphodiester linkage; b) phosphodiester linkages between at least the terminal three 5’ nucleotides and / or c) phosphodiester linkages between at least the terminal four 5’ nucleotides.

[0209] In one aspect, the first strand and / or the second strand of the nucleic acid comprises at least one phosphorothioate (ps) and / or at least one phosphorodithioate (ps2) linkage between two nucleotides.

[0210] In one aspect, the first strand and / or the second strand of the nucleic acid comprises more than one phosphorothioate and / or more than one phosphorodithioate linkage.

[0211] In one aspect, the first strand and / or the second strand of the nucleic acid comprises a phosphorothioate or phosphorodithioate linkage between the terminal two 3’ nucleotides or phosphorothioate or phosphorodithioate linkages between the terminal three 3’ nucleotides. Preferably, the linkages between the other nucleotides in the first strand and / or the second strand are phosphodiester linkages.

[0212] In one aspect, the first strand and / or the second strand of the nucleic acid comprises a phosphorothioate linkage between the terminal two 5’ nucleotides or a phosphorothioate linkages between the terminal three 5’ nucleotides. In one aspect, the nucleic acid of the present invention comprises one or more phosphorothioate or phosphorodithioate modifications on one or more of the terminal ends of the first and / or the second strand. Optionally, each or either end of the first strand may comprise one or two or three phosphorothioate or phosphorodithioate modified nucleotides (internucleoside linkage). Optionally, each or either end of the second strand may comprise one or two or three phosphorothioate or phosphorodithioate modified nucleotides (internucleoside linkage).

[0213] In one aspect, the nucleic acid comprises a phosphorothioate linkage between the terminal two or three 3’ nucleotides and / or 5’ nucleotides of the first and / or the second strand. Preferably, the nucleic acid comprises a phosphorothioate linkage between each of the terminal three 3’ nucleotides and the terminal three 5’ nucleotides of the first strand and of the second strand. Preferably, all remaining linkages between nucleotides of the first and / or of the second strand are phosphodiester linkages.

[0214] In one aspect, the nucleic acid comprises a phosphorodithioate linkage between each of the two, three or four terminal nucleotides at the 3’ end of the first strand and / or comprises a phosphorodithioate linkage between each of the two, three or four terminal nucleotides at the 3’ end of the second strand and / or a phosphorodithioate linkage between each of the two, three or four terminal nucleotides at the 5’ end of the second strand and comprises a linkage other than a phosphorodithioate linkage between the two, three or four terminal nucleotides at the 5’ end of the first strand.

[0215] In one aspect, the nucleic acid comprises a phosphorothioate linkage between the terminal three 3’ nucleotides and the terminal three 5’ nucleotides of the first strand and of the second strand. Preferably, all remaining linkages between nucleotides of the first and / or of the second strand are phosphodiester linkages.

[0216] In one aspect, the nucleic acid:

[0217] (i) has a phosphorothioate linkage between the terminal three 3’ nucleotides and the terminal three 5’ nucleotides of the first strand;

[0218] (ii) is conjugated to a triantennary ligand either on the 3’ end nucleotide or on the 5’ end nucleotide of the second strand;

[0219] (iii) has a phosphorothioate linkage between the terminal three nucleotides of the second strand at the end opposite to the one conjugated to the triantennary ligand; and

[0220] (iv) optionally all remaining linkages between nucleotides of the first and / or of the second strand are phosphodiester linkages. In one aspect, the nucleic acid:

[0221] (i) has a terminal 5’ (E)-vinylphosphonate nucleotide at the 5’ end of the first strand;

[0222] (ii) has a phosphorothioate linkage between the terminal three 3’ nucleotides on the first and second strand and between the terminal three 5’ nucleotides on the second strand or it has a phosphorodithioate linkage between the terminal two 3’ nucleotides on the first and second strand and between the terminal two 5’ nucleotides on the second strand; and

[0223] (iii) optionally all remaining linkages between nucleotides of the first and / or of the second strand are phosphodiester linkages.

[0224] The use of a phosphorodithioate linkage in the nucleic acid of the invention reduces the variation in the stereochemistry of a population of nucleic acid molecules compared to molecules comprising a phosphorothioate in that same position. Phosphorothioate linkages introduce chiral centres and it is difficult to control which non-linking oxygen is substituted for sulphur. The use of a phosphorodithioate ensures that no chiral centre exists in that linkage and thus reduces or eliminates any variation in the population of nucleic acid molecules, depending on the number of phosphorodithioate and phosphorothioate linkages used in the nucleic acid molecule.

[0225] In one aspect, the nucleic acid comprises a phosphorodithioate linkage between the two terminal nucleotides at the 3’ end of the first strand and a phosphorodithioate linkage between the two terminal nucleotides at the 3’ end of the second strand and a phosphorodithioate linkage between the two terminal nucleotides at the 5’ end of the second strand and comprises a linkage other than a phosphorodithioate linkage between the two, three or four terminal nucleotides at the 5’ end of the first strand. Preferably, the first strand has a terminal 5’ (E)-vinylphosphonate nucleotide at its 5’ end. This terminal 5’ (E)-vinylphosphonate nucleotide is preferably linked to the second nucleotide in the first strand by a phosphodiester linkage. Preferably, all the linkages between the nucleotides of both strands other than the linkage between the two terminal nucleotides at the 3’ end of the first strand and the linkages between the two terminal nucleotides at the 3’ end and at the 5’ end of the second strand are phosphodiester linkages.

[0226] In one aspect, the nucleic acid comprises a phosphorothioate linkage between each of the three terminal 3’ nucleotides and / or between each of the three terminal 5’ nucleotides on the first strand, and / or between each of the three terminal 3’ nucleotides and / or between each of the three terminal 5’ nucleotides of the second strand when there is no phosphorodithioate linkage present at that end. No phosphorodithioate linkage being present at an end means that the linkage between the two terminal nucleotides, or preferably between the three terminal nucleotides of the nucleic acid end in question are linkages other than phosphorodithioate linkages.

[0227] In one aspect, all the linkages of the nucleic acid between the nucleotides of both strands other than the linkage between the two terminal nucleotides at the 3’ end of the first strand and the linkages between the two terminal nucleotides at the 3’ end and at the 5’ end of the second strand are phosphodiester linkages.

[0228] Other phosphate linkage modifications are possible.

[0229] The phosphate linker can also be modified by replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at a terminal oxygen. Replacement of the non-linking oxygens with nitrogen is possible.

[0230] The phosphate groups can also individually be replaced by non-phosphorus containing connectors.

[0231] Examples of moieties which can replace the phosphate group include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. In certain embodiments, replacements may include the methylenecarbonylamino and methylenemethylimino groups.

[0232] The phosphate linker and ribose sugar may be replaced by nuclease resistant nucleotides. Examples include the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates. In certain embodiments, PNA surrogates may be used.

[0233] In one aspect, the nucleic acid, which is preferably an siRNA that inhibits expression of INHBE, preferably via RNAi, and preferably in a cell, comprises one or more or all of:

[0234] (i) a modified nucleotide;

[0235] (ii) a modified nucleotide other than a 2’-OMe modified nucleotide at positions 2 and 14 from the 5’ end of the first strand with a given SEQ ID No., preferably a 2’-F modified nucleotide; (iii) each of the odd-numbered nucleotides of the first strand as numbered starting from one at the 5’ end of the first strand with a given SEQ ID No. are 2’-OMe modified nucleotides; (iv) each of the even-numbered nucleotides of the first strand as numbered starting from one at the 5’ end of the first strand with a given SEQ ID No. are 2’-F modified nucleotides; (v) the second strand nucleotide corresponding to position 11 and / or 13 or 11 -13 of the first strand with a given SEQ ID No. is modified by a modification other than a 2’-OMe modification, preferably wherein one or both or all of these positions comprise a 2’-F modification;

[0236] (vi) an inverted nucleotide, preferably a 3’-3’ linkage at the 3’ end of the second strand with a given SEQ ID No.;

[0237] (vii) one or more phosphorothioate linkages;

[0238] (viii) one or more phosphorodithioate linkages; and / or

[0239] (ix) the first strand with a given SEQ ID No. has a terminal 5’ (E)-vinylphosphonate nucleotide at its 5’ end, in which case the terminal 5’ (E)-vinylphosphonate nucleotide is preferably a uridine and is preferably linked to the second nucleotide in the first strand by a phosphodiester linkage.

[0240] A nucleic acid of the present disclosure may comprise a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the first strand sequences with a given SEQ ID No. shown in Table 2 or Table 5b.

[0241] A nucleic acid of the present disclosure may be a nucleic acid wherein:

[0242] (a) the first strand sequence comprises a sequence differing by no more than 3 nucleotides from any one of the first strand sequences of Table 2 or Table 5b, and optionally wherein the second strand sequence comprises a sequence differing by no more than 3 nucleotides from the corresponding second strand sequence of Table 2 or Table 5b; (b) the first strand sequence comprises a sequence differing by no more than 2 nucleotides from any one of the first strand sequences of Table 2 or Table 5b, and optionally wherein the second strand sequence comprises a sequence differing by no more than 2 nucleotides from the corresponding second strand sequence of Table 2 or Table 5b; (c) the first strand sequence comprises a sequence differing by no more than 1 nucleotide from any one of the first strand sequences of Table 2 or Table 5b, and optionally wherein the second strand sequence comprises a sequence differing by no more than 1 nucleotide from the corresponding second strand sequence of Table 2 or Table 5b; (d) the first strand sequence comprises a sequence corresponding to nucleotides 2 to 17 from the 5’ end of any one of the first strand sequences with a given SEQ ID No. shown in Table 2 or Table 5b, and optionally wherein the second strand sequence comprises a sequence corresponding to nucleotides 3 to 18 from the 5’ end of the corresponding second strand sequence with a given SEQ ID No. shown in Table 2 or Table 5b;

[0243] (e) the first strand sequence comprises a sequence corresponding to nucleotides 2 to 18 from the 5’ end of any one of the first strand sequences with a given SEQ ID No. shown in Table 2 or Table 5b, and optionally wherein the second strand sequence comprises a sequence corresponding to nucleotides 2 to 18 from the 5’ end of the corresponding second strand sequence with a given SEQ ID No. shown in Table 2 or Table 5b (f) the first strand sequence comprises a sequence corresponding to nucleotides 2 to 19 from the 5’ end of any one of the first strand sequences with a given SEQ ID No. shown in Table 2 or Table 5b, and optionally wherein the second strand sequence comprises a sequence corresponding to nucleotides 1 to 18 from the 5’ end of the corresponding second strand sequence with a given SEQ ID No. shown in Table 2 or Table 5b;

[0244] (g) the first strand sequence comprises a sequence of any one of the first strand sequences with a given SEQ ID No. shown in Table 2 or Table 5b, and optionally wherein the second strand sequence comprises a sequence of the corresponding second strand sequence with a given SEQ ID No. shown in Table 2 or Table 5b; or

[0245] (h) the first strand sequence consists essentially of any one of the first strand sequences with a given SEQ ID No. shown in Table 2 or Table 5b, and optionally wherein the second strand sequence consists essentially of the sequence of the corresponding second strand sequence with a given SEQ ID No. shown in Table 2 or Table 5b; or

[0246] (i) the first strand sequence comprises a sequence corresponding to nucleotides 1 to 19 from the 5’ end of any one of the first strand sequences with a given SEQ ID No. shown in Table 2 or Table 5b, wherein said first strand sequence further comprises 1 (nucleotide 20 counted from the 5'end), 2 (nucleotides 20 and 21), 3 (nucleotides 20, 21 and 22), 4 (nucleotides 20, 21, 22 and 23), 5 (nucleotides 20, 21, 22, 23 and 24) or 6 (nucleotides 20, 21, 22, 23, 24 and 25) additional nucleotide(s) at the 3'end of any one of the first strand sequences with a given SEQ ID No. shown in Table 2 or Table 5b; and optionally wherein the second strand sequence comprises or consists essentially of or consists of a sequence of the corresponding second strand sequence with a given SEQ ID No. shown in Table 2 or Table 5b;

[0247] (j) the first strand sequence consists of a sequence 25, 24, 23, 22, 21 or 20 nucleotides in length, wherein nucleotides 1-19 from the 5’ end of the first strand sequence correspond to nucleotides 1 to 19 from the 5’ end of any one of the first strand sequences with a given SEQ ID No. shown in Table 2 or Table 5b and wherein the first strand sequence has a further 1 (nucleotide 20 counted from the 5'end), 2 (nucleotides 20 and 21), 3 (nucleotides 20, 21 and 22), 4 (nucleotides 20, 21, 22 and 23), 5 (nucleotides 20, 21, 22, 23 and 24) or 6 (nucleotides 20, 21, 22, 23, 24 and 25) additional nucleotide(s) at the 3'end of any one of the first strand sequences with a given SEQ ID No. shown in Table 2 or Table 5b,

[0248] and optionally wherein the second strand sequence comprises or consists essentially of or consists of a sequence of the corresponding second strand sequence with a given SEQ ID No. shown in Table 2 or Table 5b;

[0249] (k) the first strand sequence comprises a sequence corresponding to nucleotides 1 to 19 from the 5’ end of any one of the first strand sequences with a given SEQ ID No. shown in Table 2 or Table 5b, wherein said first strand sequence further comprises 1 (nucleotide 20 counted from the 5'end), 2 (nucleotides 20 and 21), 3 (nucleotides 20, 21 and 22), 4 (nucleotides 20, 21, 22 and 23), 5 (nucleotides 20, 21, 22, 23 and 24) or 6 (nucleotides 20, 21, 22, 23, 24 and 25) additional nucleotide(s) at the 3'end of any one of the first strand sequences with a given SEQ ID No. shown in Table 2 or Table 5b; and wherein said first strand sequence comprises, consists essentially of or consists of a contiguous region of from 17-25 nucleotides in length, preferably of from 18-24 nucleotides in length, complementary to the INHBE transcript of SEQ ID NO. 837; and optionally wherein the second strand sequence comprises or consists essentially of or consists of a sequence of the corresponding second strand sequence with a given SEQ ID No. shown in Table 2 or Table 5b;

[0250] (l) the first strand sequence consists of a sequence 25, 24, 23, 22, 21 or 20 nucleotides in length, wherein nucleotides 1-19 from the 5’ end of the first strand sequence correspond to nucleotides 1 to 19 from the 5’ end of any one of the first strand sequences with a given SEQ ID No. shown in Table 2 or Table 5b and wherein the first strand sequence has a further 1 (nucleotide 20 counted from the 5'end), 2 (nucleotides 20 and 21), 3 (nucleotides 20, 21 and 22), 4 (nucleotides 20, 21, 22 and 23), 5 (nucleotides 20, 21, 22, 23 and 24) or 6 (nucleotides 20, 21, 22, 23, 24 and 25) additional nucleotide(s) at the 3'end of any one of the first strand sequences with a given SEQ ID Nos. shown in Table 2 or Table 5b, and wherein said first strand sequence comprises, consists essentially of or consists of a contiguous region of from 17-25 nucleotides in length, preferably of from 18-24 nucleotides in length, complementary to the INHBE transcript of SEQ ID NO. 837; and

[0251] optionally wherein the second strand sequence comprises or consists essentially of or consists of a sequence of the corresponding second strand sequence with a given SEQ ID No. shown in Table 2 or Table 5b;

[0252] (m) the first strand sequence consists of any one of the first strand sequences with a given SEQ ID No. shown in Table 2 or Table 5b, and optionally wherein the second strand sequence consists of the sequence of the corresponding second strand sequence with a given SEQ ID No. shown in Table 2 or Table 5b;

[0253] (n) the first strand and the second strand of any one of the nucleic acid molecules of subsections (a) to (m) above are present on a single strand wherein the first strand and the second strand are able to hybridise to each other and to thereby form a doublestranded nucleic acid with a duplex region of 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length; or

[0254] (o) the first strand and the second strand of any one of the nucleic acid molecules of subsections (a) to (m) above are on two separate strands that are able to hybridise to each other and to thereby form a double-stranded nucleic acid with a duplex region of 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.

[0255] In (k) or (I), the first strand sequence may comprise, consist essentially of or consist of a contiguous region of from 19-25 nucleotides in length complementary to the INHBE transcript of SEQ ID NO. 837. In (k) or (I), the first strand sequence may comprise, consist essentially of or consist of a contiguous region of from 20-25 nucleotides in length complementary to the INHBE transcript of SEQ ID NO. 837, preferably of from 20-24 nucleotides in length, complementary to the INHBE transcript of SEQ ID NO. 837.

[0256] A nucleic acid of the present disclosure may comprise a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the first strand sequences with a given SEQ ID No. shown in Table 2.

[0257] Table 2

[0258] First strand Second strand sequence sequence (SEQ ID No.) (SEQ ID No.)

[0259] 792 793

[0260] 832 793

[0261] 796 797

[0262] 833 801

[0263] 810 811

[0264] 800 801

[0265] 808 809

[0266] 816 817

[0267] 830 777

[0268] 831 791

[0269]

[0270] A nucleic acid of the present disclosure may comprise a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 17 contiguous nucleotides differing by no more than 3 nucleotides from any one of the first strand sequences with a given SEQ ID No. shown in Table 2.

[0271] A nucleic acid of the present disclosure may comprise a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 17 contiguous nucleotides differing by no more than 2 nucleotides from any one of the first strand sequences with a given SEQ ID No. shown in Table 2.

[0272] A nucleic acid of the present disclosure may comprise a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 17 contiguous nucleotides differing by no more than 1 nucleotide from any one of the first strand sequences with a given SEQ ID No. shown in Table 2.

[0273] A nucleic acid of the present disclosure may comprise a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 18 contiguous nucleotides differing by no more than 3 nucleotides from any one of the first strand sequences with a given SEQ ID No. shown in Table 2.

[0274] A nucleic acid of the present disclosure may comprise a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 18 contiguous nucleotides differing by no more than 2 nucleotides from any one of the first strand sequences with a given SEQ ID No. shown in Table 2.

[0275] A nucleic acid of the present disclosure may comprise a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 18 contiguous nucleotides differing by no more than 1 nucleotide from any one of the first strand sequences with a given SEQ ID No. shown in Table 2.

[0276] A nucleic acid of the present disclosure may comprise a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 19 contiguous nucleotides differing by no more than 3 nucleotides from any one of the first strand sequences with a given SEQ ID No. shown in Table 2. A nucleic acid of the present disclosure may comprise a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 19 contiguous nucleotides differing by no more than 2 nucleotides from any one of the first strand sequences with a given SEQ ID No. shown in Table 2.

[0277] A nucleic acid of the present disclosure may comprise a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 19 contiguous nucleotides differing by no more than 1 nucleotide from any one of the first strand sequences with a given SEQ ID No. shown in Table 2.

[0278] A nucleic acid of the present disclosure may comprise a first strand and a second strand, wherein the first strand sequence consists essentially of, or consists of a sequence from any one of the first strand sequences with a given SEQ ID No. shown in Table 2.

[0279] All the features of the nucleic acids can be combined with all other aspects of the invention disclosed herein.

[0280] Heterolooous moieties

[0281] The nucleic acids of the invention may be conjugated to a heterologous moiety. Such nucleic acids may otherwise be referred to as conjugates or conjugated nucleic acids. A heterologous moiety is any moiety which is not a nucleic acid molecule capable of inhibiting expression of INHBE. A heterologous moiety may be, or may comprise, a peptide (or polypeptide), a saccharide (or polysaccharide), a lipid, a different nucleic acid, or any other suitable molecule. Any given nucleic acid may be conjugated to a plurality of heterologous moieties, which may be the same or different.

[0282] An individual heterologous moiety may itself comprise one or more functional moieties (such as targeting agents as described in more detail below), each optionally covalently associated to the nucleic acid via a linker.

[0283] A heterologous moiety, or the functional component thereof, may serve for example to modulate bioavailability or pharmacokinetics. For example, it may increase half-life in vivo. Alternatively, a heterologous moiety (or the functional component thereof) may comprise a targeting agent. Efficient delivery of oligonucleotides, in particular double-stranded nucleic acids of the invention, to cells in vivo is important and requires specific targeting and substantial protection from the extracellular environment, particularly serum proteins. One method of achieving specific targeting is to conjugate a targeting agent to the nucleic acid, wherein the targeting agent helps in targeting the nucleic acid to a target cell which has a cell surface receptor that binds to the targeting agent.

[0284] In this context, the term “receptor” is used to include any molecule on the surface of a target cell capable of binding to the targeting agent, and should not be taken to imply any particular function for the cell surface receptor. The targeting agent may be regarded as a “ligand” for the cell surface receptor. The terms “targeting agent” and “ligand” may be used interchangeably. Again, this terminology should not be taken to imply any particular function for the targeting agent or the cell surface receptor, or any particular relationship between the two molecules other than the ability of one to bind to the other.

[0285] Thus, the targeting agent may be any moiety having affinity for the chosen receptor. It may, for example, be an affinity protein (such as an antibody or a fragment thereof having affinity for the chosen receptor), an aptamer, or any other suitable moiety. In some embodiments, the targeting agent may be a physiological ligand for the receptor.

[0286] Binding between the targeting agent and the receptor may promote uptake of the conjugated nucleic acid by the target cell, e.g., via internalisation of the receptor, or any other suitable mechanism. Thus, appropriate ligands for the desired receptor molecules may be used as targeting agents in order for the conjugated nucleic acids to be taken up by the target cells by mechanisms such as different receptor-mediated endocytosis pathways or functionally analogous processes. In other embodiments, a ligand which can mediate internalization of the nucleic acid into a target cell by mechanisms other than receptor mediated endocytosis may alternatively be conjugated to a nucleic acid of the invention for cell or tissue specific targeting.

[0287] One example of a ligand that mediates receptor mediated endocytosis is the GalNAc moiety described herein, which has high affinity to the asialoglycoprotein receptor complex (ASGP-R). The ASGP-R complex is composed of varying ratios of multimers of membrane ASGR1 and ASGR2 receptors, which are highly abundant on hepatocytes. One of the first disclosures of the use of triantennary cluster glycosides as conjugated ligands was in US patent number US 5,885,968. Conjugates having three GalNAc ligands and comprising phosphate groups are known and are described in Dubber et al. (Bioconjug. Chem. 2003 Jan-Feb;14(1):239-46.). The ASGP-R complex shows a 50-fold higher affinity for N-Acetyl-D-Galactosamine (GalNAc) than D-Gal. The ASGP-R complex recognizes specifically terminal β-galactosyl subunits of glycosylated proteins or other oligosaccharides (Weigel, P. H. et. al., Biochim. Biophys. Acta. 2002 Sep 19;1572(2-3):341 -63) and can be used for delivering a drug to the liver’s hepatocytes expressing the receptor complex by covalent coupling of galactose or galactosamine to the drug substance (lshibashi, S.; et. al., J Biol. Chem. 1994 Nov 11;269(45):27803-6). Furthermore, the binding affinity can be significantly increased by the multi-valency effect, which is achieved by the repetition of the targeting moiety (Biessen EA, et al., J Med Chem.

[0288] 1995 Apr 28;38(9):1538-46).

[0289] The ASGP-R complex is a mediator for an active uptake of terminal β-galactosyl containing glycoproteins to the cell’s endosomes. Thus, the ASGPR is highly suitable for targeted delivery of drug candidates conjugated to such ligands like, e.g., nucleic acids into receptor-expressing cells (Akinc et al., Mol Ther. 2010 Jul;18(7):1357-64).

[0290] More generally the ligand can comprise a saccharide that is selected to have an affinity for at least one type of receptor on a target cell. In particular, the receptor is on the surface of a mammalian liver cell, for example, the hepatic asialoglycoprotein receptor complex described before (ASGP-R).

[0291] The saccharide may be selected from N-acetyl galactosamine, mannose, galactose, glucose, glucosamine and fucose. The saccharide may be N-acetyl galactosamine (GalNAc). The heterologous moiety may comprise a plurality of such saccharides, e.g., two or especially three such saccharides, e.g. three GalNAc groups.

[0292] A heterologous moiety may therefore comprise (i) one or more functional components, and (ii) a linker, wherein the linker conjugates the functional components to a nucleic acid as defined in any preceding aspects. The linker may be a monovalent structure or bivalent or trivalent or tetravalent branched structure. The nucleotides may be modified as defined herein.

[0293] The functional components may therefore be ligands (or targeting agents). Where multiple functional components are present, they may be the same or different. Where the functional components are ligands, they may be saccharides, and may therefore be (or comprise) GalNAc.

[0294] In one aspect, the nucleic acid is conjugated to a heterologous moiety comprising a compound of formula (II):

[0295] [S-X1-P-X2]3-A-X3- (II)

[0296] wherein: S represents a functional component, e.g., a ligand, such as a saccharide, preferably wherein the saccharide is N-acetyl galactosamine;

[0297] X1represents C3-C6 alkylene or (-CH2-CH2-O)m(-CH2)2- wherein m is 1, 2, or 3;

[0298] P is a phosphate or modified phosphate, preferably a thiophosphate;

[0299] X2is alkylene or an alkylene ether of the formula (-CH2)n-O-CH2- where n = 1- 6;

[0300] A is a branching unit;

[0301] X3represents a bridging unit;

[0302] wherein a nucleic acid according to the present invention is conjugated to X3via a phosphate or modified phosphate, preferably a thiophosphate.

[0303] In formula (II), the branching unit “A” preferably branches into three in order to accommodate three saccharide ligands. The branching unit is preferably covalently attached to the remaining tethered portions of the ligand and the nucleic acid. The branching unit may comprise a branched aliphatic group comprising groups selected from alkyl, amide, disulphide, polyethylene glycol, ether, thioether and hydroxyamino groups. The branching unit may comprise groups selected from alkyl and ether groups.

[0304] The branching unit A may have a structure selected from:

[0305]

[0306] wherein each Ai independently represents O, S, C=O or NH; and each n independently represents an integer from 1 to 20.

[0307] The branching unit may have a structure selected from:

[0308]

[0309] wherein each Ai independently represents O, S, C=O or NH; and each n independently represents an integer from 1 to 20.

[0310] The branching unit may have a structure selected from:

[0311]

[0312] wherein Ai is O, S, C=O or NH; and each n independently represents an integer from 1 to 20. The branching unit may have the structure:

[0313] V

[0314]

[0315] The branching unit may have the structure:

[0316]

[0317] The branching unit may have the structure:

[0318]

[0319] Alternatively, the branching unit A may have a structure selected from:

[0320] A1= O, NR1, C(R1)2A2= NR2A1= 0, NR1, C(R1)2A2= NR2

[0321]

[0322] n = 1 to 4 n = 1 to 4

[0323] wherein:

[0324] R1is hydrogen or C1-C10 alkylene;

[0325] and R2is C1-C10 alkylene. Optionally, the branching unit consists of only a carbon atom.

[0326] The “X3” portion is a bridging unit. The bridging unit is linear and is covalently bound to the branching unit and the nucleic acid.

[0327] X3may be selected from -C1-C20 alkylene-, -C2-C20 alkenylene-, an alkylene ether of formula -(C1-C20 alkylene)-0-(Ci-C2o alkylene)-, -C(0)-Ci-C2o alkylene-, -C0-C4 alkylene(Cy)Co-C4 alkylene- wherein Cy represents a substituted or unsubstituted 5 or 6 membered cycloalkylene, arylene, heterocyclylene or heteroarylene ring, -C1-C4 alkylene-NHC(O)-Ci-C4 alkylene-, -Ci-04 alkylene-C(O)NH-Ci-C4 alkylene-, -C1-C4 alkylene-SC(O)-Ci-C4 alkylene-, -C1-C4 alkylene-C(O)S-Ci-C4 alkylene-, -C1-C4 alkylene-OC(O)-Ci-C4 alkylene-, -C1-C4 alkylene-C(O)O-Ci-C4 alkylene-, and -Ci-Ce alkylene-S-S-Ci-Ce alkylene-.

[0328] X3may be an alkylene ether of formula -(C1-C20 alkylene)-O-(Ci-C2o alkylene)-. X3may be an alkylene ether of formula -(C1-C20 alkylene)-O-(C4-C2o alkylene)-, wherein said (C4-C20 alkylene) is linked to Z. X3may be selected from the group consisting of -CH2-O-C3H6-, -CH2-O-C4H8-, -CH2-O-C6H12- and -CH2-O-C8H16-, especially -CH2-O-C4H8-, -CH2-O-C6H12- and -CH2-O-C8H16-, wherein in each case the -CH2- group is linked to A.

[0329] In one aspect, the nucleic acid is conjugated to a heterologous moiety of formula (III):

[0330] [S-X1-P-X2]3-A-X3- (III)

[0331] wherein:

[0332] S represents a functional component, e.g. a ligand, such as a saccharide, preferably GalNAc;

[0333] X1represents C3-C6 alkylene or (-CH2-CH2-O)m(-CH2)2- wherein m is 1, 2, or 3;

[0334] P is a phosphate or modified phosphate, preferably a thiophosphate;

[0335] X2is Ci-Cs alkylene;

[0336] A is a branching unit selected from:

[0337] A1= O, NH A1= O, NH A2= NH, CH2, O

[0338]

[0339] n = 1 to 4 n = 1 to 4

[0340] X3is a bridging unit;

[0341] wherein a nucleic acid according to the present invention is conjugated to X3via a phosphate or a modified phosphate, preferably a thiophosphate. The branching unit A may have the structure:

[0342]

[0343] The branching unit A may have the structure:

[0344] X X

[0345] 0— \ / —0

[0346] O - ' HN—

[0347]

[0348] , wherein X3is attached to the nitrogen atom.

[0349] X3may be C1-C20 alkylene. Preferably, X3is selected from the group consisting of -C3H6-, -C4H8-, -C6H12- and -C8H16-, especially -C4H8-, -C6H12- and -C8H16-.

[0350] In one aspect, the nucleic acid is conjugated to a ligand comprising a compound of formula (IV):

[0351] [S-X1-P-X2]3-A-X3- (IV)

[0352] wherein:

[0353] S represents a functional component, e.g., a ligand, such as a saccharide, preferably GalNAc;

[0354] X1represents C3-C6 alkylene or (-CH2-CH2-O)m(-CH2)2- wherein m is 1, 2, or 3;

[0355] P is a phosphate or modified phosphate, preferably a thiophosphate;

[0356] X2is an alkylene ether of formula -C3H6-O-CH2-;

[0357] A is a branching unit;

[0358] X3is an alkylene ether of formula selected from the group consisting of -CH2-O-CH2-, - CH2-O-C2H4-, -CH2-O-C3H6-, -CH2-O-C4H8-, -CH2-O-C5H10-, -CH2-O-C6Hi2-, -CH2-O- C7H14-, and -CH2-O-C8H16-, wherein in each case the -CH2- group is linked to A, and wherein X3is conjugated to a nucleic acid according to the present invention by a phosphate or modified phosphate, preferably a thiophosphate.

[0359] The branching unit may comprise carbon. Preferably, the branching unit is a carbon.

[0360] X3may be selected from the group consisting of -CH2-O-C4H8-, -CH2-O-C5H10-, -CH2-O-C6H12-, -CH2-O-C7H14-, and -CH2-O-C8H16-. Preferably, X3is selected from the group consisting of -CH2-O-C4H8-, -CH2-O-C6H12- and -CH2-O-C8H16. X1may be (-CH2-CH2-O)(-CH2)2-. X1may be (-CH2-CH2-O)2(-CH2)2-. X1may be (-CH2-CH2-O)3(-CH2)2-. Preferably, X1is (-CH2-CH2-O)2(-CH2)2-. Alternatively, X1represents C3-C6 alkylene. X1may be propylene. X1may be butylene. X1may be pentylene. X1may be hexylene. Preferably the alkyl is a linear alkylene. In particular, X1may be butylene.

[0361] X2represents an alkylene ether of formula -C3H6-O-CH2- i.e. C3 alkoxy methylene, or -CH2CH2CH2OCH2-.

[0362] For any of the above aspects, when P represents a modified phosphate group, P can be represented by:

[0363]

[0364] wherein Y1and Y2each independently represent =0, =S, -O’, -OH, -SH, -BH3, -OCH2CO2, -OCH2CO2RX, -OCH2C(S)ORX, and -ORX, wherein Rxrepresents Ci-Ce alkyl and wherein ~ indicates attachment to the remainder of the compound.

[0365] By modified phosphate it is meant a phosphate group wherein one or more of the non-linking oxygens is replaced. Examples of modified phosphate groups include phosphorothioate, phosphorodithioates, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulphur. One, each or both non-linking oxygens in the phosphate group can be independently any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl).

[0366] The phosphate can also be modified by replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at a terminal oxygen. Replacement of the non-linking oxygens with nitrogen is possible.

[0367] For example, Y1may represent -OH and Y2may represent =0 or =S; or

[0368] Y1may represent -O' and Y2may represent =0 or =S;

[0369] Y1may represent =O and Y2may represent -CH3, -SH, -ORX, or -BH3

[0370] Y1may represent =S and Y2may represent -CH3, ORXor -SH.

[0371] It will be understood by the skilled person that in certain instances there will be delocalisation between Y1and Y2. Preferably, the modified phosphate group is a thiophosphate group. Thiophosphate groups include bithiophosphate (i.e., where Y1represents =S and Y2represents -S-) and monothiophosphate (i.e., where Y1represents -O-and Y2represents =S, or where Y1represents =O and Y2represents -S-). Preferably, P is a monothiophosphate. The inventors have found that conjugates having thiophosphate groups in replacement of phosphate groups have improved potency and duration of action in vivo.

[0372] P may also be an ethylphosphate (i.e. where Y1represents =O and Y2represents OCH2CH3).

[0373] The ligand, e.g., saccharide, may be selected to have an affinity for at least one type of receptor on a target cell. In particular, the receptor is on the surface of a mammalian liver cell, for example, the hepatic asialoglycoprotein receptor complex (ASGP-R).

[0374] For any of the above or below aspects, the saccharide may be selected from N-acetyl with one or more of galactosamine, mannose, galactose, glucose, glucosamine and fructose. Typically, a ligand to be used in the present invention may include N-acetyl galactosamine (GalNAc). Preferably the compounds of the invention may have 3 ligands, which will each preferably include N-acetyl galactosamine.

[0375] " GalNAc" refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose, commonly referred to in the literature as N-acetyl galactosamine. Reference to “GalNAc” or “N-acetyl galactosamine” includes both the p- form: 2-(Acetylamino)-2-deoxy-p -D-galactopyranose and the a-form: 2-(Acetylamino)-2-deoxy-a-D- galactopyranose. In certain embodiments, both the p-form: 2-(Acetylamino)-2-deoxy-p-D-galactopyranose and a-form: 2-(Acetylamino)-2-deoxy-a-D-galactopyranose may be used interchangeably. Preferably, the compounds of the invention comprise the p-form, 2-(Acetylamino)-2-deoxy-p-D-galactopyranose.

[0376] 2-(Acetylamino)-2-deoxy-D-galactopyranose

[0377]

[0378] NHAc 2-(Acetylamino)-2-deoxy-p-D-galactopyranose

[0379] HO

[0380]

[0381] 2-(Acetylamino)-2-deoxy-a-D-galactopyranose

[0382] In one aspect, the nucleic acid is a conjugated nucleic acid, wherein the nucleic acid is conjugated to a heterologous moiety with one of the following structures, which may be referred to as “triantennary ligands” for ease of reference:

[0383]

[0384]

[0385]

[0386]

[0387]

[0388] wherein Z is any nucleic acid as defined herein. In certain embodiments, the nucleic acid Z is conjugated to the triantennary ligand via the phosphate or thiophosphate group which links the triantennary ligand to the 3’ or 5’ position of the ribose sugar, particularly to the 3’ or 5’ position of the ribose sugar, of the terminal nucleotide of said nucleic acid Z.

[0389] In certain embodiments, the heterologous moiety (“triantennary ligand”) is conjugated to the 3' position of the ribose sugar of the terminal nucleotide of the second (sense) strand of Z (which is also referred to as strand “B” in Tables 5a, 5b, 5c).

[0390] In other embodiments, the heterologous moiety (“triantennary ligand”) is conjugated to the 5' position of the ribose sugar of the terminal nucleotide of the second (sense) strand of Z (which is also referred to as strand “B” in Tables 5a, 5b, 5c).

[0391] In other embodiments, the heterologous moiety (“triantennary ligand”) is conjugated to the 3' position of the ribose sugar of the terminal nucleotide of the first (antisense) strand of Z (which is also referred to as strand “A” in Tables 5a, 5b, 5c).

[0392] Preferably, the nucleic acid is a conjugated nucleic acid, wherein the nucleic acid is conjugated to a triantennary ligand with one of the following structures:

[0393]

[0394]

[0395] wherein Z is any nucleic acid as defined herein.

[0396] In a preferred embodiment, the nucleic acid Z is conjugated to the triantennary ligand via the phosphate or thiophosphate group which links the triantennary ligand to the 3’ or 5’ position of the ribose sugar of the terminal nucleotide of said nucleic acid Z. Preferably, the triantennary ligand” is conjugated to the 5' position of the ribose sugar of the terminal nucleotide of the second (sense) strand of Z (which is also referred to as strand “B” in Tables 5a, 5b, 5c).

[0397] A heterologous moiety of formula (II), (III) or (IV) or any one of the triantennary ligands disclosed herein can be attached at the 3’-end of the first (antisense) strand and / or at any of the 3’ and / or 5’ end of the second (sense) strand. The nucleic acid can comprise more than one heterologous moiety of formula (II), (III) or (IV) or any one of the triantennary ligands disclosed herein. However, a single heterologous moiety of formula (II), (III) or (IV) or any one of the triantennary ligands disclosed herein is preferred because a single such moiety is sufficient for efficient targeting of the nucleic acid to the target cells. Preferably in that case, at least the last two, preferably at least the last three and more preferably at least the last four nucleotides at the end of the nucleic acid to which the ligand is attached are linked by a phosphodiester linkage.

[0398] Preferably, the 5’-end of the first (antisense) strand is not attached to a heterologous moiety, since attachment at this position can potentially interfere with the biological activity of the nucleic acid.

[0399] A nucleic acid with a single heterologous moiety (e.g., of formula (II), (III) or (IV) or any one of the triantennary ligands disclosed herein) at the 5’ end of a strand is easier and therefore cheaper to synthesise than the same nucleic acid with the same group at the 3’ end. Preferably therefore, a single heterologous moiety (e.g., of any of formulae (II), (III) or (IV) or any one of the triantennary ligands disclosed herein) is covalently attached to (conjugated with) the 5’ end of the second strand of the nucleic acid.

[0400] In one aspect, the first strand of the nucleic acid is a compound of formula (V):

[0401] wherein b is

[0402]

[0403] preferably 0 or 1; and

[0404] the second strand is a compound of formula (VI):

[0405]

[0406] wherein:

[0407] c and d are independently preferably 0 or 1;

[0408] Zi and Z2are respectively the first and second strand of the nucleic acid;

[0409] Y is independently O or S;

[0410] n is independently 0, 1, 2 or 3; and

[0411] Liis a linker to which a ligand is attached, wherein Liis the same or different in formulae (V) and (VI), and is the same or different within formulae (V) and (VI) when Li is present more than once within the same formula, wherein Li is preferably of formula (VII); and wherein b + c + d is preferably 2 or 3.

[0412] Preferably, Li in formulae (V) and (VI) is of formula (VII):

[0413] . GalNAc

[0414] X

[0415] I

[0416] w.,

[0417] -W5-V-W3- (VII)

[0418]

[0419] wherein:

[0420] L is selected from the group comprising, or preferably consisting of:

[0421] -(CH2)r-C(O)-, wherein r = 2-12;

[0422] -(CH2-CH2-O)S-CH2-C(O)-, wherein s = 1-5;

[0423] -(CH2)t-CO-NH-(CH2)t-NH-C(O)-, wherein t is independently 1-5;

[0424] -(CH2)U-CO-NH-(CH2)U-C(O)-, wherein u is independently 1-5; and -(CH2)V-NH-C(O)-, wherein v is 2-12; and

[0425] wherein the terminal C(O), if present, is attached to X of formula (VII), or if X is absent, to W1 of formula (VII), or if W1 is absent, to V of formula (VII);

[0426] W1, W3and W5are individually absent or selected from the group comprising, or preferably consisting of:

[0427] -(CH2)r-, wherein r = 1-7;

[0428] -(CH2)S-O-(CH2)S-, wherein s is independently 0-5;

[0429] -(CH2)t-S-(CH2)t-, wherein t is independently 0-5;

[0430] X is absent or is selected from the group comprising, or preferably consisting of: NH, NCH3or NC2H5;

[0431] V is selected from the group comprising, or preferably consisting of: C

[0432]

[0433] H, N,

[0434] wherein B, if present, is a modified or natural nucleobase.

[0435] In one aspect, the first strand is a compound of formula (VIII)

[0436] " GalNAc GalNAc

[0437]

[0438] wherein b is preferably 0 or 1; and

[0439] the second strand is a compound of formula (IX):

[0440]

[0441] wherein c and d are independently preferably 0 or 1;

[0442] wherein:

[0443] Zi and Z2are respectively the first and second strand of the nucleic acid;

[0444] Y is independently O or S;

[0445] Ri is H or methyl;

[0446] n is independently preferably 0, 1, 2 or 3; and

[0447] L is the same or different in formulae (VIII) and (IX), and is the same or different within formulae (VIII) and (IX) when L is present more than once within the same formula, and is selected from the group comprising, or preferably consisting of:

[0448] -(CH2)r-C(O)-, wherein r = 2-12;

[0449] -(CH2-CH2-O)S-CH2-C(O)-, wherein s = 1-5;

[0450] -(CH2)t-CO-NH-(CH2)t-NH-C(O)-, wherein t is independently 1-5;

[0451] -(CH2)U-CO-NH-(CH2)U-C(O)-, wherein u is independently 1-5; and

[0452] -(CH2)V-NH-C(O)-, wherein v is 2-12; and

[0453] wherein the terminal C(O), if present, is attached to the NH group (of the linker, not of the targeting ligand);

[0454] and wherein b + c + d is preferably 2 or 3. In one aspect, the first strand of the nucleic acid is a compound of formula (X):

[0455] wherein b is pre

[0456]

[0457] ferably 0 or 1; and

[0458] the second strand is a compound of formula (XI):

[0459]

[0460] wherein:

[0461] c and d are independently preferably 0 or 1;

[0462] Zi and Z2are respectively the first and second RNA strand of the nucleic acid;

[0463] Y is independently O or S;

[0464] n is independently preferably 0, 1, 2 or 3; and

[0465] L2is the same or different in formulae (X) and (XI) and is the same or different in moieties bracketed by b, c and d, and is selected from the group comprising, or preferably consisting of:

[0466] n is 0 and L2is:

[0467] i H

[0468] -N.. Gal N Ac

[0469]

[0470] ' F

[0471] and the terminal OH group is absent such that the following moiety is formed:

[0472] GalNAc — L 0— P - O-r

[0473] \

[0474] HN— I

[0475]

[0476] OH

[0477] wherein:

[0478] F is a saturated branched or unbranched (such as unbranched) C1-8alkyl (e.g., C1-6alkyl) chain wherein one of the carbon atoms is optionally replaced with an oxygen atom provided that said oxygen atom is separated from another heteroatom (e.g., an O or N atom) by at least 2 carbon atoms;

[0479] L is the same or different in formulae (X) and (XI) and is selected from the group comprising, or preferably consisting of:

[0480] -(CH2)r-C(O)-, wherein r = 2-12;

[0481] -(CH2-CH2-O)S-CH2-C(O)-, wherein s = 1-5;

[0482] -(CH2)t-CO-NH-(CH2)t-NH-C(O)-, wherein t is independently 1-5;

[0483] -(CH2)U-CO-NH-(CH2)U-C(O)-, wherein u is independently 1-5; and

[0484] -(CH2)V-NH-C(O)-, wherein v is 2-12; and

[0485] wherein the terminal C(O), if present, is attached to the NH group (of the linker, not of the targeting ligand);

[0486] and wherein b + c + d is preferably 2 or 3.

[0487] In one aspect, b is 0, c is 1 and d is 1; b is 1, c is 0 and d is 1; b is 1, c is 1 and d is 0; or b is 1, c is 1 and d is 1 in any of the nucleic acids of formulae (V) and (VI) or (VIII) and (IX) or (X) and (XI). Preferably, b is 0, c is 1 and d is 1; b is 1, c is 0 and d is 1; or b is 1, c is 1 and d is 1. Most preferably, b is 0, c is 1 and d is 1.

[0488] In one aspect, Y is O in any of the nucleic acids of formulae (V) and (VI) or (VIII) and (IX) or (X) and (XI). In another aspect, Y is S. In a preferred aspect, Y is independently selected from O or S in the different positions in the formulae.

[0489] In one aspect, Ri is H or methyl in any of the nucleic acids of formulae (VIII) and (IX). In one aspect, Ri is H. In another aspect, Ri is methyl.

[0490] In one aspect, n is 0, 1, 2 or 3 in any of the nucleic acids of formulae (V) and (VI) or (VIII) and (IX) or (X) and (XI). Preferably, n is 0.

[0491] Examples of F moieties in any of the nucleic acids of formulae (X) and (XI) include (CH2)1-6e.g. (CH2)1-4e.g. CH2, (CH2)4, (CH2)5or (CH2)6, or CH2O(CH2)2-3, e.g. CH2O(CH2)CH3.

[0492] In one aspect, L2in formulae (X) and (XI) is:

[0493] H

[0494] . GalNAc

[0495]

[0496] In one aspect, L2is: H

[0497] N.. GalNAc

[0498] L'

[0499] In one aspect, L2is:

[0500] / Lx

[0501] N GalNAc

[0502] H

[0503]

[0504] In one aspect, L2is:

[0505] L.

[0506] GalNAc

[0507] In one aspect, n is 0 and L2is:

[0508] H

[0509] N. GalNAc

[0510]

[0511] and the terminal OH group is absent such that the following moiety is formed:

[0512] GalNAc

[0513] \

[0514] L— NH

[0515] \ Y

[0516] ' - < O— P II — O-;! —

[0517]

[0518] OH 5

[0519] wherein Y is O or S.

[0520] In one aspect, L in the nucleic acids of formulae (V) and (VI) or (VIII) and (IX) or (X) and (XI), is selected from the group comprising, or preferably consisting of:

[0521] -(CH2)r-C(O)-, wherein r = 2-12;

[0522] -(CH2-CH2-O)S-CH2-C(O)-, wherein s = 1-5;

[0523] -(CH2)t-CO-NH-(CH2)t-NH-C(O)-, wherein t is independently 1-5;

[0524] -(CH2)U-CO-NH-(CH2)U-C(O)-, wherein u is independently 1-5; and

[0525] -(CH2)V-NH-C(O)-, wherein v is 2-12;

[0526] wherein the terminal C(O) is attached to the NH group.

[0527] Preferably, L is -(CH2)r-C(O)-, wherein r = 2-12, more preferably r = 2-6 even more preferably, r = 4 or 6 e.g. 4.

[0528] Preferably, L is:

[0529]

[0530] Within the moiety bracketed by b, c and d, L2in the nucleic acids of formulae (X) and (XI) is typically the same. Between moieties bracketed by b, c and d, L2may be the same or different. In an embodiment, L2in the moiety bracketed by c is the same as the L2in the moiety bracketed by d. In an embodiment, L2in the moiety bracketed by c is not the same as L2in the moiety bracketed by d. In an embodiment, the L2in the moieties bracketed by b, c and d is the same, for example when the linker moiety is a serinol-derived linker moiety.

[0531] Serinol derived linker moieties may be based on serinol in any stereochemistry i.e. derived from L-serine isomer, D-serine isomer, a racemic serine or other combination of isomers. In a preferred aspect of the invention, the serinol-GalNAc moiety (SerGN) has the following stereochemistry:

[0532]

[0533] (S)-Serinol building blocks

[0534] i.e., is based on an (S)-serinol-amidite or (S)-serinol succinate solid supported building block derived from L-serine isomer.

[0535] In a preferred aspect, the first strand of the nucleic acid is a compound of formula (VIII) and the second strand of the nucleic acid is a compound of formula (IX), wherein:

[0536] b is 0;

[0537] c and d are 1,

[0538] n is 0,

[0539] Z1and Z2are respectively the first and second strand of the nucleic acid,

[0540] Y is S,

[0541] R1is H, and

[0542] L is -(CH2)4-C(O)-, wherein the terminal C(O) of L is attached to the N atom of the linker (ie not a possible N atom of a targeting ligand). In another preferred aspect, the first strand of the nucleic acid is a compound of formula (V) and the second strand of the nucleic acid is a compound of formula (VI), wherein:

[0543] b is 0,

[0544] c and d are 1,

[0545] n is 0,

[0546] Z1and Z2are respectively the first and second strand of the nucleic acid,

[0547] Y is S,

[0548] L1is of formula (VII), wherein:

[0549] W1is -CH2-O-(CH2)3-,

[0550] W3is -CH2-,

[0551] W5is absent,

[0552] V is CH,

[0553] X is NH, and

[0554] L is -(CH2)4-C(O)- wherein the terminal C(O) of L is attached to the N atom of X in formula (VII).

[0555] In another preferred aspect, the first strand of the nucleic acid is a compound of formula (V) and the second strand of the nucleic acid is a compound of formula (VI), wherein:

[0556] b is 0,

[0557] c and d are 1,

[0558] n is 0,

[0559] Z1and Z2are respectively the first and second strand of the nucleic acid,

[0560] Y is S,

[0561] L1is of formula (VII), wherein:

[0562] W1, W3and W5are absent,

[0563] I

[0564] V

[0565]

[0566] is I I,

[0567] X is absent, and

[0568] L is -(CH2)4-C(O)-NH-(CH2)5-C(O)-, wherein the terminal C(O) of L is attached to the N atom of V in formula (VII).

[0569] In one aspect, the nucleic acid is conjugated to a triantennary ligand with the following structure:

[0570]

[0571] wherein the nucleic acid is conjugated to the triantennary ligand via the phosphate group of the ligand to the

[0572] a) 3' position of the ribose sugar of the terminal nucleotide of the second (sense) strand of Z (which is also referred to as strand “B” in Tables 5a, 5b, 5c), or

[0573] b) 5' position of the ribose sugar of the terminal nucleotide of the second (sense) strand of Z (which is also referred to as strand “B” in Tables 5a, 5b, 5c), or

[0574] c) 3' position of the ribose sugar of the terminal nucleotide of the first (antisense) strand of Z (which is also referred to as strand “A” in Tables 5a, 5b).

[0575] In one aspect of the nucleic acid, the cells that are targeted by the nucleic acid with a ligand are hepatocytes.

[0576] In any one of the above ligands where GalNAc is present, the GalNAc may be substituted for any other targeting ligand, such as those mentioned herein, in particular mannose, galactose, glucose, glucosamine and fucose.

[0577] In one aspect, the nucleic acid is conjugated to a heterologous moiety that comprises a lipid, and more preferably, a cholesterol.

[0578] In one aspect, the double-stranded nucleic acid for inhibiting expression of INHBE is one of the duplexes shown in Table 5c, which may be referred to by their Duplex ID number.

[0579] In one aspect, the double-stranded nucleic acid for inhibiting expression of INHBE is selected from EU2001, EU2008, EU2009, EU2011, EU2013, EU2017, EU2018, EU2021, EU2023, EU2031, EU2032, EU2033, EU2034, or EU2035. In one aspect, the double stranded nucleic acid for inhibiting expression of INHBE is selected from Duplex ID Numbers EU 2009, EU2011, EU2018, EU2033 or EU2034.

[0580]

[0581] uses and methods

[0582] The present invention also provides compositions comprising a nucleic acid of the invention. The nucleic acids and compositions may be used as therapeutic or diagnostic agents, alone or in combination with other agents. For example, one or more nucleic acid(s) of the invention can be combined with a delivery vehicle (e.g., liposomes) and / or excipients, such as carriers, diluents. Other agents such as preservatives and stabilizers can also be added. Pharmaceutically acceptable salts or solvates of any of the nucleic acids of the invention are likewise within the scope of the present invention. Methods for the delivery of nucleic acids are known in the art and within the knowledge of the person skilled in the art.

[0583] Compositions disclosed herein are particularly pharmaceutical compositions. Such compositions are suitable for administration to a subject.

[0584] In one aspect, the composition comprises a nucleic acid disclosed herein, or a pharmaceutically acceptable salt or solvate thereof, and a solvent (preferably water) and / or a delivery vehicle and / or a physiologically acceptable excipient and / or a carrier and / or a salt and / or a diluent and / or a buffer and / or a preservative.

[0585] Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral, rectal, nasal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, and transdermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Subcutaneous or transdermal modes of administration may be particularly suitable for the compounds described herein.

[0586] The prophylactically or therapeutically effective amount of a nucleic acid of the present invention will depend on the route of administration, the type of mammal being treated, and the physical characteristics of the specific mammal under consideration. These factors and their relationship to determining this amount are well known to skilled practitioners in the medical arts. This amount and the method of administration can be tailored to achieve optimal efficacy, and may depend on such factors as weight, diet, concurrent medication and other factors, well known to those skilled in the medical arts. The dosage sizes and dosing regimen most appropriate for human use may be guided by the results obtained by the present invention and may be confirmed in properly designed clinical trials. An effective dosage and treatment protocol may be determined by conventional means, starting with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Numerous factors may be taken into consideration by a clinician when determining an optimal dosage for a given subject. Such considerations are known to the skilled person.

[0587] Nucleic acids of the present invention, or salts thereof, may be formulated as pharmaceutical compositions prepared for storage or administration, which typically comprise a prophylactically or therapeutically effective amount of a nucleic acid of the invention, or a salt thereof, in a pharmaceutically acceptable carrier.

[0588] The nucleic acid or conjugated nucleic acid of the present invention can also be administered in combination with other therapeutic compounds, either administrated separately or simultaneously, e.g., as a combined unit dose. The invention also includes a composition comprising one or more nucleic acids according to the present invention in a physiologically / pharmaceutically acceptable excipient, such as a stabilizer, preservative, diluent, buffer, and the like.

[0589] In one aspect, the composition comprises a nucleic acid disclosed herein and a therapeutic agent selected from the group comprising an oligonucleotide, a small molecule, a monoclonal antibody, a polyclonal antibody and a peptide. Preferably, the therapeutic agent is an agent that targets, preferably inhibits the expression or the activity, of INHBE. Preferably, the therapeutic agent is one of the following: a) a peptide that inhibits the expression or activity of INHBE, b) an antibody that specifically binds under physiological conditions to INHBE, or one of their subunits or proteolytic cleavage products.

[0590] In some embodiments, the therapeutic agent is selected from the group consisting of 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase inhibitors / Statins - simvastatin, atorvastatin, rosuvastatin, pravastatin, pitavastatin, fluvastatin, lovastatin, 2-azetidinone -ezetimibe, bile acid sequestrants (cholestyramine, colestipol, colesevelam), proprotein convertase subtilisin / Kexin type 9 (PCSK9) inhibitors (alirocumab, evolocumab and Inclisiran), fibrates (fenofibrate, clofibrate and gemfibrozil), omega-3 fatty acid (Icosapent ethyl, Omega-3-acid ethyl esters, Omega-3-carboxylic acids, Omega-3-acid ethyl esters) and niacin / nicotinic acid. In certain embodiments, two or more nucleic acids of the invention with different sequences may be administered simultaneously or sequentially. In another aspect, the present invention provides a composition, e.g., a pharmaceutical composition, comprising one or a combination of different nucleic acids of the invention and at least one pharmaceutically acceptable carrier.

[0591] Dosage levels for the therapeutic agents and compositions of the invention can be determined by those skilled in the art by experimentation. In one aspect, a unit dose may contain between about 0.01 mg / kg and about 100 mg / kg body weight of nucleic acid or conjugated nucleic acid. Alternatively, the dose can be from 10 mg / kg to 25 mg / kg body weight, or 1 mg / kg to 10 mg / kg body weight, or 0.05 mg / kg to 5 mg / kg body weight, or 0.1 mg / kg to 5 mg / kg body weight, or 0.1 mg / kg to1 mg / kg body weight, or 0.1 mg / kg to 0.5 mg / kg body weight, or 0.5 mg / kg to 1 mg / kg body weight. Alternatively, the dose can be from about 0.5 mg / kg to about 10 mg / kg body weight, or about 0.6 mg / kg to about 8 mg / kg body weight, or about 0.7 mg / kg to about 7 mg / kg body weight, or about 0.8 mg / kg to about 6 mg / kg body weight, or about 0.9 mg / kg to about 5.5 mg / kg body weight, or about 1 mg / kg to about 5 mg / kg body weight, or about 1 mg / kg body weight, or about 3 mg / kg body weight, or about 5 mg / kg body weight, wherein “about” is a deviation of up to 30%, preferably up to 20%, more preferably up to 10%, yet more preferably up to 5% and most preferably 0% from the indicated value. Dosage levels may also be calculated via other parameters such as, e.g., body surface area.

[0592] A dose unit of these nucleic acids preferably comprises about 1 mg / kg to about 5 mg / kg body weight, or about 1 mg / kg to about 3 mg / kg body weight, or about 1 mg / kg body weight, or about 3 mg / kg body weight, or about 5 mg / kg body weight. The INHBE mRNA level in the liver and / or the INHBE protein level in the plasma or blood of a subject treated by a dose unit of the nucleic acid is preferably decreased at the time point of maximum effect by at least 30%, at least 40%, at least 50%, at least 60% or at least 70% as compared to a control that was not treatment with the nucleic acid or treated with a control nucleic acid under comparable conditions.

[0593] The dosage and frequency of administration may vary depending on whether the treatment is therapeutic or prophylactic (e.g., preventative), and may be adjusted during the course of treatment. In certain prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a relatively long period of time. Some subjects may continue to receive treatment over their lifetime. In certain therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient may be switched to a suitable prophylactic dosing regimen. Actual dosage levels of a nucleic acid of the invention alone or in combination with one or more other active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without causing deleterious side effects to the subject or patient. A selected dosage level will depend upon a variety of factors, such as pharmacokinetic factors, including the activity of the particular nucleic acid or composition employed, the route of administration, the time of administration, the rate of excretion of the particular nucleic acid being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the subject or patient being treated, and similar factors well known in the medical arts.

[0594] The pharmaceutical composition may be a sterile injectable aqueous suspension or solution, or in a lyophilized form.

[0595] The pharmaceutical compositions can be in unit dosage form. In such form, the composition is divided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparations, for example, packeted tablets, capsules, and powders in vials or ampoules. The unit dosage form can also be a capsule, cachet, or tablet itself, or it can be the appropriate number of any of these packaged forms. It may be provided in single dose injectable form, for example in the form of a pen. Compositions may be formulated for any suitable route and means of administration.

[0596] The therapeutic agents and pharmaceutical compositions of the present invention may be administered to a mammalian subject in a pharmaceutically effective dose. The mammal may be selected from a human, a non-human primate, a simian or prosimian, a dog, a cat, a horse, cattle, a pig, a goat, a sheep, a mouse, a rat, a hamster, a hedgehog and a guinea pig, or other species of relevance. On this basis, “INHBE” as used herein denotes nucleic acid or protein in any of the above-mentioned species, if expressed therein naturally or artificially, but preferably this wording denotes human nucleic acids or proteins.

[0597] Pharmaceutical compositions of the invention may be administered alone or in combination with one or more other therapeutic or diagnostic agents. A combination therapy may include a nucleic acid of the present invention combined with at least one other therapeutic agent selected based on the particular patient, disease or condition to be treated. Examples of other such agents include, inter alia, a therapeutically active small molecule or polypeptide, a single chain antibody, a classical antibody or fragment thereof, or a nucleic acid molecule which modulates gene expression of one or more additional genes, and similar modulating therapeutics which may complement or otherwise be beneficial in a therapeutic or prophylactic treatment regimen.

[0598] Pharmaceutical compositions are typically sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier may be a solvent or dispersion medium containing, for example, water, alcohol such as ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or any suitable mixtures. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by use of surfactants according to formulation chemistry well known in the art. In certain embodiments, isotonic agents, e.g., sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride may be desirable in the composition. Prolonged absorption of injectable compositions may be brought about by including in the composition an agent that delays absorption for example, monostearate salts and gelatine.

[0599] One aspect of the invention is a nucleic acid or a composition disclosed herein for use as a therapeutic agent. The nucleic acid or composition is preferably for use in the prophylaxis or treatment of a disease, disorder or syndrome.

[0600] The present invention provides a nucleic acid for use, alone or in combination with one or more additional therapeutic agents in a pharmaceutical composition, for treatment or prophylaxis of conditions, diseases and disorders responsive to inhibition of INHBE expression.

[0601] One aspect of the invention is the use of a nucleic acid or a composition as disclosed herein in the prophylaxis or treatment of a disease, disorder or syndrome.

[0602] Nucleic acids and pharmaceutical compositions of the invention may be used in the treatment of a variety of conditions, disorders or diseases. Treatment with a nucleic acid of the invention preferably leads to in vivo INHBE depletion, preferably in the liver and / or in blood. As such, nucleic acids of the invention, and compositions comprising them, will be useful in methods for treating a variety of pathological disorders in which inhibiting the expression of INHBE may be beneficial. The present invention provides methods for treating a disease, disorder or syndrome comprising the step of administering to a subject in need thereof a prophylactically or therapeutically effective amount of a nucleic acid of the invention.

[0603] The invention thus provides methods of prophylaxis or treatment of a disease, disorder or syndrome, the method comprising the step of administering to a subject (e.g., a patient) in need thereof a therapeutically effective amount of a nucleic acid or pharmaceutical composition comprising a nucleic acid of the invention.

[0604] The most desirable therapeutically effective amount is an amount that will produce a desired efficacy of a particular treatment selected by one of skill in the art for a given subject in need thereof. This amount will vary depending upon a variety of factors understood by the skilled worker, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through experimentation, namely by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly. See, e.g., Remington: The Science and Practice of Pharmacy 21st Ed., Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005.

[0605] In certain embodiments, nucleic acids and pharmaceutical compositions of the invention may be used to treat or prevent a disease, disorder or syndrome.

[0606] In certain embodiments, the present invention provides methods for prophylaxis or treatment of a disease, disorder or syndrome in a mammalian subject, such as a human, the method comprising the step of administering to a subject in need thereof a prophylactically or therapeutically effective amount of a nucleic acid as disclosed herein.

[0607] Administration of a "therapeutically effective dosage" of a nucleic acid of the invention may result in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.

[0608] Nucleic acids of the invention may be beneficial in treating or diagnosing a disease, disorder or syndrome that may be diagnosed or treated using the methods described herein. Treatment and diagnosis of other diseases, disorders or syndromes are also considered to fall within the scope of the present invention.

[0609] One aspect of the invention is a method of prophylaxis or treatment of a disease, disorder or syndrome comprising administering a pharmaceutically effective dose or amount a nucleic acid or a composition disclosed herein to an individual in need of treatment, preferably wherein the nucleic acid or composition is administered to the subject subcutaneously, intravenously or by oral, rectal, pulmonary, intramuscular or intraperitoneal administration. Preferably, it is administered subcutaneously.

[0610] The disease, disorder or syndrome is typically an INHBE-mediated disease, disorder or syndrome associated with aberrant activation and / or over-activation (hyper-activation) of INHBE and / or with over-expression or ectopic expression or localisation or accumulation of INHBE.

[0611] The INHBE-mediated disease, disorder or syndrome may comprise cardiovascular disease, T2D and cardiometabolic disease, NASH, obesity and dyslipidemia.

[0612] THE INHBE mediated disease, disorder or syndrome may comprise metabolic syndrome, type 2 diabetes, cardiovascular disease, hypertension, adipositas, abdominal obesity, dyslipidemia, metabolic dysfunction associated steatotic liver disease (MASLD), formally known as non-alcoholic fatty liver disease (NAFLD), including its advanced form, metabolic dysfunction associated steato-hepatitis MASH, formally known as NASH, alcoholic liver disease (ALD), liver cirrhosis and hepatocellular carcinoma (HCC), abdominal obesity, lipodystrophies.

[0613] A nucleic acid or compositions disclosed herein may be for use in a regimen comprising treatments once or twice weekly, every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks, every ten weeks, every eleven weeks, every twelve weeks, every three months, every four months, every five months, every six months or in regimens with varying dosing frequency such as combinations of the before-mentioned intervals. The nucleic acid or composition may be for use subcutaneously, intravenously or using any other application routes such as oral, rectal, pulmonary, or intraperitoneal. Preferably, it is for use subcutaneously. The level of inhibition is preferably measured in conditions that have been selected because they show the greatest effect of the nucleic acid on the target mRNA (here: INHBE mRNA) level in cells treated with the nucleic acid or the conjugated nucleic acid of the invention in vitro or in vivo. The level of inhibition may for example be measured after 24 hours, 48 hours, 1 week, 2 weeks, 4 weeks, 8 weeks or 12 weeks of treatment with a nucleic acid or with a conjugated nucleic acid of the invention at a concentration of between 0.03 nM – 10 μM, preferably 0.1 nM, 0.5 nM, 1 nM, 10 nM, 100 nM or 1000nM for in vitro testing, or with an amount of between 1 nmol – 100 μmol for testing in vivo samples from mouse or non-human primates, or with an amount of between 0.5 μmol – 100 μmol, preferably of between 8 μmol - 80 μmol for testing in vivo samples from human. These conditions may be different for different nucleic acids or conjugated nucleic acids of the invention. Examples of suitable conditions for determining levels of inhibition are described in the Example section below.

[0614] In cells and / or subjects treated with or receiving a nucleic acid or composition as disclosed herein, the INHBE expression may be inhibited compared to untreated cells and / or subjects by a range from 15% up to 100% but at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% or intermediate values. The level of inhibition may allow treatment of a disease associated with INHBE expression or overexpression, or may serve to further investigate the functions and physiological roles of the INHBE gene products. The level of inhibition is preferably measured in the liver or in the blood or in the kidneys, preferably in a liver sample (which may otherwise be referred to as a liver biopsy), of the subject treated with the nucleic acid or composition. In another preferred embodiment, the level of inhibition is measured in the blood.

[0615] The level of inhibition of INHBE expression may be measured by RT-qPCR. For example, the level of inhibition of INHBE expression may be measured in a liver biopsy sample, for example by RT-qPCR, for example in the liver biopsy of the subject treated with the nucleic acid or composition. The level of cholesterol and / or triglycerides may also be measured.

[0616] One aspect is the use of a nucleic acid or composition as disclosed herein in the manufacture of a medicament for treating a disease, disorder or syndromes, such as those as listed above or additional pathologies associated with elevated levels of INHBE, preferably in the blood, in the liver or in the kidneys, or additional therapeutic approaches where inhibition of INHBE expression is desired. A medicament is a pharmaceutical composition.

[0617] Each of the nucleic acids of the invention and pharmaceutically acceptable salts and solvates thereof constitutes an individual embodiment of the invention. Also included in the invention is a method of prophylaxis or treatment of a disease, disorder or syndrome, such as those listed above, comprising administration of a composition comprising a nucleic acid or composition as described herein, to an individual in need thereof. The nucleic acid or composition may, for example, be administered in a regimen comprising treatments twice every week, once every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, or every eight to twelve or more weeks or in regimens with varying dosing frequency such as combinations of the before-mentioned intervals. The nucleic acid or conjugated nucleic acid may be for use subcutaneously or intravenously or other application routes such as oral, rectal or intraperitoneal.

[0618] A nucleic acid of the invention may be administered by any appropriate administration pathway known in the art, including but not limited to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, vaginal, or transdermal (e.g., topical administration of a cream, gel or ointment, or by means of a transdermal patch). " Parenteral administration” is typically associated with injection at or in communication with the intended site of action, including infraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal administration.

[0619] The use of a chemical modification pattern of the nucleic acids confers nuclease stability in serum and makes for example subcutaneous application route feasible.

[0620] Solutions or suspensions used for intradermal or subcutaneous application typically include one or more of: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and / or tonicity adjusting agents such as, e.g., sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide, or buffers with citrate, phosphate, acetate and the like. Such preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0621] Sterile injectable solutions may be prepared by incorporating a nucleic acid in the required amount in an appropriate solvent with one or a combination of ingredients described above, as required, followed by sterilization microfiltration. Dispersions may be prepared by incorporating the active compound into a sterile vehicle that contains a dispersion medium and optionally other ingredients, such as those described above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient in addition to any additional desired ingredient from a sterile-filtered solution thereof.

[0622] When a prophylactically or therapeutically effective amount of a nucleic acid of the invention is administered by, e.g., intravenous, cutaneous or subcutaneous injection, the nucleic acid will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. Methods for preparing parenterally acceptable solutions, taking into consideration appropriate pH, isotonicity, stability, and the like, are within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection will contain, in addition to a nucleic acid, an isotonic vehicle such as sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection, or other vehicle as known in the art. A pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives well known to those of skill in the art.

[0623] The amount of nucleic acid which can be combined with a carrier material to produce a single dosage form will vary depending on a variety of factors, including the subject being treated, and the particular mode of administration. In general, it will be an amount of the composition that produces an appropriate therapeutic effect under the particular circumstances. Generally, out of one hundred percent, this amount will range from about 0.01% to about 99% of nucleic acid, from about 0.1% to about 70%, or from about 1% to about 30% of nucleic acid in combination with a pharmaceutically acceptable carrier.

[0624] The nucleic acid may be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0625] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a dose may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the particular circumstances of the therapeutic situation, on a case-by-case basis. It is especially advantageous to formulate parenteral compositions in dosage unit forms for ease of administration and uniformity of dosage when administered to the subject or patient. As used herein, a dosage unit form refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce a desired therapeutic effect. The specification for the dosage unit forms of the invention depends on the specific characteristics of the active compound and the particular therapeutic or prophylactic effect(s) to be achieved and the treatment and sensitivity of any individual patient.

[0626] The nucleic acid or composition of the present invention can be produced using routine methods in the art including chemical synthesis, such as solid phase chemical synthesis.

[0627] Nucleic acids or compositions of the invention may be administered with one or more of a variety of medical devices known in the art. For example, in one embodiment, a nucleic acid of the invention may be administered with a needleless hypodermic injection device. Examples of well-known implants and modules useful in the present invention are in the art, including e.g., implantable micro-infusion pumps for controlled rate delivery; devices for administering through the skin; infusion pumps for delivery at a precise infusion rate; variable flow implantable infusion devices for continuous drug delivery; and osmotic drug delivery systems. These and other such implants, delivery systems, and modules are known to those skilled in the art.

[0628] In certain embodiments, the nucleic acid or composition of the invention may be formulated to ensure a desired distribution in vivo. To target a therapeutic compound or composition of the invention to a particular in vivo location, they can be formulated, for example, in liposomes which may comprise one or more moieties that are selectively transported into specific cells or organs, thus enhancing targeted drug delivery.

[0629] The invention is characterized by high specificity at the molecular and tissue-directed delivery level. The sequences of the nucleic acids of the invention are highly specific for their target, meaning that they do not inhibit the expression of genes that they are not designed to target or only minimally inhibit the expression of genes that they are not designed to target and / or only inhibit the expression of a low number of genes that they are not designed to target. A further level of specificity is achieved when nucleic acids are linked to a ligand that is specifically recognised and internalised by a particular cell type. This is for example the case when a nucleic acid is linked to a ligand comprising GalNAc moieties, which are specifically recognised and internalised by hepatocytes. This leads to the nucleic acid inhibiting the expression of their target only in the cells that are targeted by the ligand to which they are linked. These two levels of specificity potentially confer a better safety profile than the currently available treatments. In certain embodiments, the present invention thus provides nucleic acids of the invention linked to a ligand comprising one or more GalNAc moieties, or comprising one or more other moieties that confer cell-type or tissue-specific internalisation of the nucleic acid thereby conferring additional specificity of target gene knockdown by RNA interference.

[0630] The nucleic acid as described herein may be formulated with a lipid in the form of a liposome. Such a formulation may be described in the art as a lipoplex. The composition with a lipid / liposome may be used to assist with delivery of the nucleic acid of the invention to the target cells. The lipid delivery system herein described may be used as an alternative to a conjugated ligand. The modifications herein described may be present when using the nucleic acid of the invention with a lipid delivery system or with a ligand conjugate delivery system. Such a lipoplex may comprise a lipid composition comprising:

[0631] i) a cationic lipid, or a pharmaceutically acceptable salt thereof;

[0632] ii) a steroid;

[0633] iii) a phosphatidylethanolamine phospholipid; and / or

[0634] iv) a PEGylated lipid.

[0635] The cationic lipid may be an amino cationic lipid.

[0636] The cationic lipid may have the formula (XII):

[0637]

[0638] (XII)

[0639] or a pharmaceutically acceptable salt thereof, wherein:

[0640] X represents O, S or NH;

[0641] R1and R2each independently represents a C4-C22 linear or branched alkyl chain or a C4-C22 linear or branched alkenyl chain with one or more double bonds, wherein the alkyl or alkenyl chain optionally contains an intervening ester, amide or disulfide;

[0642] when X represents S or NH, R3and R4each independently represent hydrogen, methyl, ethyl, a mono- or polyamine moiety, or R3and R4together form a heterocyclyl ring;

[0643] when X represents O, R3and R4each independently represent hydrogen, methyl, ethyl, a mono- or polyamine moiety, or R3and R4together form a heterocyclyl ring, or R3represents hydrogen and R4represents C(NH)(NH2).

[0644] The cationic lipid may have the formula (XIII):

[0645]

[0646] or a pharmaceutically acceptable salt thereof.

[0647] The cationic lipid may have the formula (XIV):

[0648]

[0649] or a pharmaceutically acceptable salt thereof.

[0650] The content of the cationic lipid component may be from about 55 mol% to about 65 mol% of the overall lipid content of the composition. In particular, the cationic lipid component is about 59 mol% of the overall lipid content of the composition.

[0651] The compositions can further comprise a steroid. The steroid may be cholesterol. The content of the steroid may be from about 26 mol% to about 35 mol% of the overall lipid content of the lipid composition. More particularly, the content of steroid may be about 30 mol% of the overall lipid content of the lipid composition.

[0652] The phosphatidylethanolamine phospholipid may be selected from the group consisting of 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1.2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1.2-Dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLoPE), 1 -Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), 1.2-Disqualeoyl-sn-glycero-3-phosphoethanolamine (DSQPE) and 1-Stearoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (SLPE). The content of the phospholipid may be about 10 mol% of the overall lipid content of the composition.

[0653] The PEGylated lipid may be selected from the group consisting of 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG) and C16-Ceramide-PEG. The content of the PEGylated lipid may be about 1 to 5 mol% of the overall lipid content of the composition. The content of the cationic lipid component in the composition may be from about 55 mol% to about 65 mol% of the overall lipid content of the lipid composition, preferably about 59 mol% of the overall lipid content of the lipid composition.

[0654] The composition may have a molar ratio of the components of i):ii):iii):iv) selected from 55:34:10:1; 56:33:10:1; 57:32:10:1; 58:31:10:1; 59:30:10:1; 60:29:10:1; 61:28:10:1; 62:27:10:1; 63:26:10:1; 64:25:10:1; and 65:24:10:1.

[0655]

[0656] a phosphatidylethanolamine phospholipid having the structure

[0657]

[0658] mPEG-2000-DMG Neutral liposome compositions may be formed from, for example, dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions may be formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes may be formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition may be formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and / or phosphatidylcholine and / or cholesterol.

[0659] A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells. DOTMA analogues can also be used to form liposomes.

[0660] Derivatives and analogues of lipids described herein may also be used to form liposomes.

[0661] A liposome containing a nucleic acid can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The nucleic acid preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the nucleic acid and condense around the nucleic acid to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of nucleic acid.

[0662] If necessary, a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also be adjusted to favour condensation.

[0663] Nucleic acid formulations of the present invention may include a surfactant. In one embodiment, the nucleic acid is formulated as an emulsion that includes a surfactant.

[0664] A surfactant that is not ionized is a non-ionic surfactant. Examples include non-ionic esters, such as ethylene glycol esters, propylene glycol esters, glyceryl esters etc., nonionic alkanolamides, and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers.

[0665] A surfactant that carries a negative charge when dissolved or dispersed in water is an anionic surfactant. Examples include carboxylates, such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates.

[0666] A surfactant that carries a positive charge when dissolved or dispersed in water is a cationic surfactant. Examples include quaternary ammonium salts and ethoxylated amines.

[0667] A surfactant that has the ability to carry either a positive or negative charge is an amphoteric surfactant. Examples include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

[0668] " Micelles" are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic. A micelle may be formed by mixing an aqueous solution of the nucleic acid, an alkali metal alkyl sulphate, and at least one micelle forming compound.

[0669] Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerol, polyglycerol, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof.

[0670] Phenol and / or m-cresol may be added to the mixed micellar composition to act as a stabiliser and preservative. An isotonic agent such as glycerine may as be added.

[0671] A nucleic acid preparation may be incorporated into a particle such as a microparticle. Microparticles can be produced by spray-drying, lyophilisation, evaporation, fluid bed drying, vacuum drying, or a combination of these methods. Definitions

[0672] As used herein, the terms “inhibit”, “down-regulate”, or “reduce” with respect to gene expression mean that the expression of the gene, or the level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits (e.g., mRNA), or the activity of one or more proteins or protein subunits, is reduced below that observed either in the absence of the nucleic acid or conjugated nucleic acid of the invention or as compared to that obtained with an siRNA molecule with no known homology to the human transcript (herein termed non-silencing control). Such control may be conjugated and modified in an analogous manner to the molecule of the invention and delivered into the target cell by the same route. The expression after treatment with the nucleic acid of the invention may be reduced to 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5% or 0% or to intermediate values, or less than that observed in the absence of the nucleic acid or conjugated nucleic acid. The expression may be measured in the cells to which the nucleic acid is applied. Alternatively, especially if the nucleic acid is administered to a subject, the level can be measured in a different group of cells or in a tissue or an organ or in a body fluid such as blood or plasma. The level of inhibition is preferably measured in conditions that have been selected because they show the greatest effect of the nucleic acid on the target mRNA level in cells treated with the nucleic acid in vitro. The level of inhibition may for example be measured after 24 hours or 48 hours of treatment with a nucleic acid at a concentration of between 0.038 nM – 10 μM, preferably 0.5 nM, 1 nM, 10 nM or 100 nM. These conditions may be different for different nucleic acid sequences or for different types of nucleic acids, such as for nucleic acids that are unmodified or modified or conjugated to a ligand or not. Examples of suitable conditions for determining levels of inhibition are described in the examples.

[0673] By nucleic acid it is meant a nucleic acid comprising two strands comprising nucleotides, that is able to interfere with gene expression. Inhibition may be complete or partial and results in down regulation of gene expression in a targeted manner. The nucleic acid comprises two separate polynucleotide strands; the first strand, which may also be a guide strand; and a second strand, which may also be a passenger strand. The first strand and the second strand may be part of the same polynucleotide molecule that is self-complementary which 'folds' back to form a double-stranded molecule. The nucleic acid may be an siRNA molecule.

[0674] The nucleic acid may comprise ribonucleotides, modified ribonucleotides, deoxynucleotides, deoxyribonucleotides, or nucleotide analogues non-nucleotides that are able to mimic nucleotides such that they may 'pair' with the corresponding base on the target sequence or complementary strand. The nucleic acid may further comprise a double-stranded nucleic acid portion or duplex region formed by all or a portion of the first strand (also known in the art as a guide strand) and all or a portion of the second strand (also known in the art as a passenger strand). The duplex region is defined as beginning with the first base pair formed between the first strand and the second strand and ending with the last base pair formed between the first strand and the second strand, inclusive.

[0675] By duplex region it is meant the region in two complementary or substantially complementary oligonucleotides that form base pairs with one another, either by Watson-Crick base pairing or any other manner that allows for a duplex between oligonucleotide strands that are complementary or substantially complementary. For example, an oligonucleotide strand having 21 nucleotide units can base pair with another oligonucleotide of 21 nucleotide units, yet only 19 nucleotides on each strand are complementary or substantially complementary, such that the “duplex region” consists of 19 base pairs. The remaining base pairs may exist as 5' and 3' overhangs, or as single-stranded regions. Further, within the duplex region, 100% complementarity is not required; substantial complementarity is allowable within a duplex region.

[0676] Substantial complementarity refers to complementarity between the strands such that they are capable of annealing under biological conditions. Techniques to empirically determine if two strands are capable of annealing under biological conditions are well known in the art. Alternatively, two strands can be synthesised and added together under biological conditions to determine if they anneal to one another. The portion of the first strand and second strand that forms at least one duplex region may be fully complementary and is at least partially complementary to each other. Depending on the length of a nucleic acid, a perfect match in terms of base complementarity between the first strand and the second strand is not necessarily required. However, the first and second strands must be able to hybridise under physiological conditions.

[0677] As used herein, the terms “non-pairing nucleotide analogue” means a nucleotide analogue which includes a non-base pairing moiety including but not limited to: 6 des amino adenosine (Nebularine), 4-Me-indole, 3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-Me ribo U, N3-Me riboT, N3-Me dC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, and N3-Me dC. In some embodiments the non-base pairing nucleotide analogue is a ribonucleotide. In other embodiments it is a deoxyribonucleotide. As used herein, the term, “terminal functional group” includes without limitation a halogen, alcohol, amine, carboxylic, ester, amide, aldehyde, ketone, and ether groups.

[0678] An “overhang” as used herein has its normal and customary meaning in the art, i.e. a singlestranded portion of a nucleic acid that extends beyond the terminal nucleotide of a complementary strand in a double-strand nucleic acid. The term “blunt end” includes doublestranded nucleic acid whereby both strands terminate at the same position, regardless of whether the terminal nucleotide(s) are base-paired. The terminal nucleotide of a first strand and a second strand at a blunt end may be base paired. The terminal nucleotide of a first strand and a second strand at a blunt end may not be paired. The terminal two nucleotides of a first strand and a second strand at a blunt end may be base-paired. The terminal two nucleotides of a first strand and a second strand at a blunt end may not be paired.

[0679] The term “serinol-derived linker moiety” means the linker moiety comprises the following structure:

[0680] HN.,

[0681]

[0682] An O atom of said structure typically links to an RNA strand and the N atom typically links to the targeting ligand.

[0683] The terms “patient,” “subject,” and “individual” may be used interchangeably and refer to either a human or a non-human animal. These terms include mammals such as humans, primates, livestock animals (e.g., bovines, porcines), companion animals (e.g., canines, felines) and rodents (e.g., mice and rats).

[0684] As used herein, “treating” or “treatment” and grammatical variants thereof refer to an approach for obtaining beneficial or desired clinical results. The term may refer to slowing the onset or rate of development of a condition, disorder or disease, reducing or alleviating symptoms associated with it, generating a complete or partial regression of the condition, or some combination of any of the above. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, reduction or alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. " Treatment" can also mean prolonging survival relative to expected survival time if not receiving treatment. A subject (e.g., a human) in need of treatment may thus be a subject already afflicted with the disease or disorder in question. The term “treatment” includes inhibition or reduction of an increase in severity of a pathological state or symptoms relative to the absence of treatment, and is not necessarily meant to imply complete cessation of the relevant disease, disorder or condition.

[0685] As used herein, the terms "prophylaxis" and grammatical variants thereof refer to an approach for inhibiting or preventing the development, progression, or time or rate of onset of a condition, disease or disorder, and may relate to pathology and / or symptoms. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, prevention, inhibition or slowing of symptoms, progression or development of a disease, whether detectable or undetectable. A subject (e.g., a human) in need of prophylaxis may thus be a subject not yet afflicted with the disease or disorder in question. The term “prophylaxis” includes slowing the onset of disease relative to the absence of treatment, and is not necessarily meant to imply permanent prevention of the relevant disease, disorder or condition. Thus “prophylaxis” of a condition may in certain contexts refer to reducing the risk of developing the condition, or preventing, inhibiting or delaying the development of symptoms associated with the condition. It will be understood that prophylaxis may be considered as treatment or therapy.

[0686] As used herein, an "effective amount," “prophylactically effective amount”, "therapeutically effective amount" or "effective dose" is an amount of a composition (e.g., a therapeutic composition or agent) that produces at least one desired therapeutic effect in a subject, such as preventing or treating a target condition or beneficially alleviating a symptom associated with the condition.

[0687] As used herein, the term “pharmaceutically acceptable salt” refers to a salt that is not harmful to a patient or subject to which the salt in question is administered. It may be a salt chosen, e.g., among acid addition salts and basic salts. Examples of acid addition salts include chloride salts, citrate salts and acetate salts. Examples of basic salts include salts wherein the cation is selected from alkali metal cations, such as sodium or potassium ions, alkaline earth metal cations, such as calcium or magnesium ions, as well as substituted ammonium ions, such as ions of the type N(R1)(R2)(R3)(R4)+, wherein R1, R2, R3and R4independently will typically designate hydrogen, optionally substituted C1 -6-alkyl groups or optionally substituted C2-6-alkenyl groups. Examples of relevant C1 -6-alkyl groups include methyl, ethyl, 1 -propyl and 2-propyl groups. Examples of C2-6-alkenyl groups of possible relevance include ethenyl, 1-propenyl and 2-propenyl. Other examples of pharmaceutically acceptable salts are described in “Remington’s Pharmaceutical Sciences”, 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and more recent editions thereof), in the “Encyclopaedia of Pharmaceutical Technology”, 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and in J. Pharm. Sci. 66: 2 (1977). A "pharmaceutically acceptable salt" retains qualitatively a desired biological activity of the parent compound without imparting any undesired effects relative to the compound. Examples of pharmaceutically acceptable salts include acid addition salts and base addition salts. Acid addition salts include salts derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphorous, phosphoric, sulfuric, hydrobromic, hydroiodic and the like, or from nontoxic organic acids such as aliphatic mono- and di-carboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include salts derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N, N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

[0688] The term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). For example, sterile saline and phosphate-buffered saline at slightly acidic or physiological pH may be used. Exemplary pH buffering agents include phosphate, citrate, acetate, tris / hydroxymethyl)aminomethane (TRIS), N-Tris(hydroxymethyl)methyl-3-aminopropanesulphonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine, which is a preferred buffer, arginine, lysine, or acetate or mixtures thereof. The term further encompasses any agents listed in the US Pharmacopeia for use in animals, including humans. A "pharmaceutically acceptable carrier" includes any and all physiologically acceptable, i.e., compatible, solvents, dispersion media, coatings, antimicrobial agents, isotonic and absorption delaying agents, and the like. In certain embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on selected route of administration, the nucleic acid may be coated in a material or materials intended to protect the compound from the action of acids and other natural inactivating conditions to which the nucleic acid may be exposed when administered to a subject by a particular route of administration.

[0689] The term “solvate” in the context of the present invention refers to a complex of defined stoichiometry formed between a solute ( / n casu, a nucleic acid compound or pharmaceutically acceptable salt thereof according to the invention) and a solvent. The solvent in this connection may, for example, be water or another pharmaceutically acceptable, typically small-molecular organic species, such as, but not limited to, acetic acid or lactic acid. When the solvent in question is water, such a solvate is normally referred to as a hydrate. The invention will now be described with reference to the following non-limiting Figures and Examples.

[0690] Brief description of the Figures

[0691] Figure 1. Inhibition of human INHBE mRNA level by ELI2009 and ELI2018 in a mouse model expressing human INHBE (hINHBE AAV mouse model), mean values + / - SD.

[0692] Figure 2. Inhibition of human INHBE mRNA in liver of hINHBE AAV mouse model by different siRNAs. Human INHBE AAV mice were dosed subcutaneously with 1 mg siRNA per kg body weight. Represented are group mean values + / - SD.

[0693] Figure 3. Inhibition of human INHBE mRNA expression by transfection of Hep3B cells with different siRNAs. Mean values + / - SD.

[0694] Figure 4. Dose dependent inhibition of human INHBE mRNA levels in human primary hepatocytes by receptor mediated uptake of different siRNA conjugates. Mean values + / - SD.

[0695] Figure 5. Inhibition of INHBE expression in cynomolgus monkeys. Animals were dosed with either 0.9% saline (vehicle) or 5 mg siRNA / kg body weight. INHBE mRNA levels were calculated relative to individual animal's base line expression levels set as 100%. Mean values + / - SD.

[0696] Figure 6. Inhibition of INHBE expression in liver of cynomolgus monkeys. All animals were dosed on day 0 with either vehicle or different dose levels of ELI2033. INHBE mRNA levels were measured in liver biopsy samples collected at designated time points. INHBE expression levels were normalized to PPIB expression levels and are depicted relative to individual animal's base line expression levels set as 100%. Mean values + / - SD.

[0697] Figure 7. Stability of different conjugates in tritosome stability assay.

[0698] Figure 8. Stability of different conjugates in tritosome stability assay conducted in the presence of 1 mM MgCI2.

[0699] Examples

[0700] Example 1

[0701] In vitro experiment conducted with Hep3B cells showing INHBE knockdown efficacy of tested siRNAs after transfection of 10 nM siRNA.

[0702] INHBE knockdown efficacy of siRNAs EU1001-EU1201 (Table 5b) was determined by transfection of 10 nM siRNA in Hep3B cells. The results are depicted in Table 3, below. At 10 nM, siRNA treatment reduced INHBE mRNA levels to minimum of 26% compared to untreated (Ut) cells. At 10 nM, the most potent siRNAs were, ELI1098, ELI1125, ELI1135, ELI1141, EU1142, EU1153, EU1155, and EU1174.

[0703] For transfection of Hep3B cells with siRNAs, cells were seeded at a density of 30,000 cells / well in 96-well tissue culture plates (TPP, Cat. 92096, Switzerland). T ransfection of siRNA was carried out with Lipofectamine RNAiMax (Invitrogen / Life Technologies, Cat. 13778-500, Germany) according to the manufacturer’s instructions, directly before seeding. The screen with INHBE-siRNAs was performed in triplicates at 10 nM, with luciferase siRNA (ELI1201) as non-targeting control. 24 hours post-treatment, cells were lysed and RNA isolated using Dynabeads mRNA DIRECT kit (Thermo Fisher Scientific, Cat. 61012, USA). RT-qPCR was performed using INHBE and PPIB specific primer probe sets and Takyon™ One-Step Low Rox Probe 5X MasterMix dTTP on the QuantStudio6 device from Applied Biosystems in single-plex 384 well format. Expression differences were calculated using the delta delta Ct method (Livak and Schmittgen, Methods, Volume 25, Issue 4, December 2001, pages 402-408). INHBE expression levels were normalized to the expression levels of the housekeeping gene PPIB. Results of INHBE expression levels after siRNA transfections are depicted as % remaining INHBE mRNA in comparison to INHBE mRNA levels in untreated cell set as 100% (Table 3).

[0704] Table 3: Results of single dose siRNAs testing on targeting human INHBE expression

[0705] % INHBE mRNA remaining at 10 nM

[0706] Duplex ID

[0707] Mean SD

[0708] EU1201 112 13

[0709] EU1001 60 2

[0710] EU1002 49 7

[0711] EU1003 42 4

[0712] EU1004 56 11

[0713] EU1005 43 4

[0714] EU1006 80 7

[0715] EU1007 46 5

[0716] EU1008 37 1

[0717] EU1009 37 7

[0718] EU1010 53 9

[0719] EU1011 90 2

[0720] EU1012 42 6

[0721] EU1013 52 6

[0722] EU1014 47 2

[0723] EU1015 89 14

[0724] EU1016 79 20

[0725] EU1017 49 3

[0726] EU1018 83 9

[0727] EU1019 56 9

[0728] EU1020 48 5

[0729] EU1021 81 15

[0730] EU1022 47 6

[0731] EU1023 98 13

[0732] EU1024 100 17

[0733]

[0734] EU1025 55 11 EU1026 57 7 EU1027 40 5 EU1028 58 5 EU1029 80 4 EU1030 68 18 EU1031 74 9 EU1032 59 7 EU1033 67 11 EU1034 69 2 EU1035 45 4 EU1036 64 3 EU1037 39 9 EU1038 65 15 EU1039 75 18 EU1040 65 5 EU1041 71 10 EU1042 88 6 EU1043 49 15 EU1044 61 17 EU1045 74 5 EU1046 73 9 EU1047 61 10 EU1048 40 5 EU1049 66 9 EU1050 85 10 EU1051 74 10 EU1052 48 1 EU1053 62 14 EU1054 60 12 EU1055 40 10 EU1056 46 3 EU1057 89 20 EU1058 64 5 EU1059 79 15 EU1060 86 15 EU1061 74 4 EU1062 63 11 EU1063 54 5 EU1064 69 4 EU1065 62 18 EU1066 61 13 EU1067 40 11 EU1068 52 8 EU1069 40 7 EU1070 65 14 EU1071 60 7 EU1072 91 13 EU1073 53 11 EU1074 69 4 EU1075 107 8 EU1076 80 15 EU1077 94 11 EU1078 98 14 EU1079 73 3 EU1080 87 13 EU1081 84 14 EU1082 52 18 EU1083 85 14 EU1084 48 11 EU1085 63 18 EU1086 57 10

[0735]

[0736] EU1087 94 14 EU1088 52 22 EU1089 76 10 EU1090 87 9 EU1091 45 2 EU1092 70 5 EU1093 80 18 EU1094 81 12 EU1095 51 3 EU1096 51 19 EU1097 40 5 EU1098 29 6 EU1099 88 22 EU1100 84 4 EU1101 52 7 EU1102 50 6 EU1103 40 8 EU1104 47 3 EU1105 49 8 EU1106 35 3 EU1107 67 6 EU1108 44 4 EU1109 60 18 EU1110 44 7 EU1111 72 13 EU1112 45 2 EU1113 69 6 EU1114 69 9 EU1115 45 12 EU1116 52 3 EU1117 36 1 EU1118 78 3 EU1119 33 8 EU1120 55 8 EU1121 53 4 EU1122 51 8 EU1123 49 1 EU1124 50 10 EU1125 34 10 EU1126 83 13 EU1127 53 12 EU1128 89 12 EU1129 65 10 EU1130 84 9 EU1131 119 6 EU1132 67 8 EU1133 98 24 EU1134 79 10 EU1135 33 9 EU1136 70 1 EU1137 44 5 EU1138 52 11 EU1139 54 9 EU1140 54 10 EU1141 34 13 EU1142 34 2 EU1143 42 10 EU1144 49 3 EU1145 72 1 EU1146 59 6 EU1147 49 6 EU1148 57 12

[0737]

[0738] EU1149 59 13 EU1150 53 4

[0739] EU1151 44 5

[0740] EU1152 39 2

[0741] EU1153 33 2

[0742] EU1154 46 6

[0743] EU1155 26 9

[0744] EU1156 86 16

[0745] EU1157 39 2

[0746] EU1158 48 7

[0747] EU1159 36 3

[0748] EU1160 75 23

[0749] EU1161 92 11

[0750] EU1162 72 9

[0751] EU1163 45 10

[0752] EU1164 65 7

[0753] EU1165 60 0

[0754] EU1166 50 12

[0755] EU1167 80 5

[0756] EU1168 60 19

[0757] EU1169 69 10

[0758] EU1170 53 14

[0759] EU1171 81 10

[0760] EU1172 51 10

[0761] EU1173 63 6

[0762] EU1174 30 0

[0763] EU1175 76 18

[0764] EU1176 65 17

[0765] EU1177 72 2

[0766] EU1178 76 8

[0767] EU1179 45 6

[0768] EU1180 82 17

[0769] EU1181 70 3

[0770] EU1182 35 5

[0771] EU1183 103 21

[0772] EU1184 50 4

[0773] EU1185 65 10

[0774] EU1186 49 4

[0775] EU1187 87 9

[0776] EU1188 43 10

[0777] EU1189 107 7

[0778] EU1190 36 2

[0779] EU1191 37 8

[0780] EU1192 63 5

[0781] EU1193 46 4

[0782] EU1194 107 2

[0783] EU1195 49 6

[0784] EU1196 72 6

[0785] EU1197 45 4

[0786] EU1198 45 7

[0787] EU1199 80 14

[0788]

[0789] EU1200 35 3

[0790] The identity of the single strands forming each of the siRNA duplexes as well as their sequences and modifications are shown in Tables 5a and 5b. Example 2

[0791] In vitro study in Hep3B cells showing INHBE knockdown efficacy after transfection of different siRNAs at dose levels of 100, 20, 4, 0.8, 0.16, and 0.032 nM siRNA.

[0792] INHBE knockdown efficacy of selected siRNAs (Table 6 and Table 5b) was determined after transfection of 100, 20, 4, 0.8, 0.16 and 0.032 nM siRNA in human Hep3B cells. The results are depicted in Table 6, below.

[0793] For transfection of Hep3B cells with siRNAs, cells were seeded at a density of 30,000 cells / well in 96-well tissue culture plates (TPP, Cat. 92096, Switzerland). T ransfection of siRNA was carried out with Lipofectamine RNAiMax (Invitrogen / Life Technologies, Cat. 13778-500, Germany) according to manufacturer’s instructions directly before seeding. The doseresponse screen was performed with INHBE siRNAs in triplicates at 100, 20, 4, 0.8, 0.16, or 0.032, respectively, with luciferase siRNA (ELI1201) as non-targeting siRNA control. 24 hours post-treatment, cells were lysed and RNA isolated using Dynabeads mRNA DIRECT kit (Thermo Fisher Scientific, Cat. 61012, USA). RT-qPCR was performed using INHBE and PPIB specific primer probe sets and Takyon™ One-Step Low Rox Probe 5X MasterMix dTTP on the QuantStudio6 device from Applied Biosystems in single-plex 384 well format. Expression differences were calculated using the delta delta Ct method. INHBE mRNA levels were normalized to the housekeeping gene PPIB. Results are expressed as % remaining INHBE mRNA in siRNA transfected cells and compared to expression levels of untreated control cells set as 100 (Table 6).

[0794] Table 6: Results of dose-response screening by transfection (100, 20, 4, 0.8, 0.16 or 0.032 nM) of selected siRNAs targeting INHBE.

[0795] The identity of the single strands forming each of the siRNA duplexes as well as their sequences and modifications are shown in Tables 5a and 5b.

[0796] % INHBE mRNA remaining

[0797] Duplex Cone. (nM)

[0798] Mean SD

[0799] EU1201 100 102 13

[0800] 100 43 5

[0801] 20 49 7

[0802] 4 62 2

[0803] EU1003

[0804] 0.8 102 17

[0805] 0.16 106 9

[0806] 0.032 101 2

[0807] 100 43 2

[0808] 20 54 16

[0809] 4 60 7

[0810] EU1005

[0811] 0.8 71 8

[0812] 0.16 92 12

[0813] 0.032 93 16

[0814] 100 35 4

[0815] EU1007

[0816]

[0817] 20 39 4 4 61 16 0.8 86 13 0.16 106 19 0.032 99 13 100 34 5 20 40 5 4 43 8 EU1008

[0818] 0.8 59 20 0.16 77 13 0.032 100 7 100 32 3 20 33 4 4 35 1 EU1009

[0819] 0.8 49 6 0.16 68 16 0.032 74 18 100 40 5 20 38 5 4 47 11 EU1012

[0820] 0.8 62 7 0.16 82 19 0.032 91 15 100 34 6 20 31 3 4 35 7 EU1027

[0821] 0.8 48 3 0.16 91 20 0.032 85 10 100 43 7 20 45 1 4 52 3 EU1035

[0822] 0.8 84 21 0.16 105 2 0.032 103 1 100 43 7 20 43 1 4 49 4 EU1037

[0823] 0.8 75 15 0.16 86 10 0.032 79 27 100 45 3 20 40 6 4 40 9 EU1048

[0824] 0.8 57 6 0.16 86 13 0.032 109 14 100 36 3 20 53 4 4 54 5 EU1052

[0825] 0.8 70 6 0.16 103 21 0.032 105 4 100 29 4 20 35 7 4 37 6 EU1055

[0826] 0.8 51 11 0.16 65 9 0.032 86 5 100 31 2 20 36 2 EU1056

[0827] 4 39 6

[0828]

[0829] 0.8 54 5 0.16 74 3 0.032 98 14 100 26 5 20 37 5 4 41 4 EU1067

[0830] 0.8 52 5 0.16 61 3 0.032 67 2 100 33 3 20 38 1 4 41 1 EU1069

[0831] 0.8 55 2 0.16 68 2 0.032 78 12 100 36 5 20 35 4 4 40 5 EU1084

[0832] 0.8 54 4 0.16 70 4 0.032 84 10 100 49 8 20 53 3 4 52 12 EU1091

[0833] 0.8 65 6 0.16 82 9 0.032 78 7 100 55 1 20 53 0 4 56 5 EU1097

[0834] 0.8 62 4 0.16 83 3 0.032 97 9 100 29 4 20 39 1 4 44 12 EU1098

[0835] 0.8 45 13 0.16 55 6 0.032 61 8 100 38 8 20 50 10 4 47 9 EU1103

[0836] 0.8 58 6 0.16 67 11 0.032 75 13 100 30 6 20 35 5 4 45 2 EU1106

[0837] 0.8 59 3 0.16 63 3 0.032 65 13 100 41 4 20 42 2 4 66 10 EU1108

[0838] 0.8 74 8 0.16 74 5 0.032 89 15 100 41 5 20 46 2 4 61 4 EU1110

[0839] 0.8 60 5 0.16 86 9

[0840]

[0841] 0.032 104 13 100 46 6 20 47 4 4 49 6 EU1117

[0842] 0.8 48 5 0.16 69 2 0.032 91 18 100 30 3 20 27 4 4 35 2 EU1119

[0843] 0.8 40 10 0.16 61 9 0.032 79 14 100 30 1 20 32 4 4 36 2 EU1125

[0844] 0.8 59 5 0.16 71 3 0.032 110 8 100 29 2 20 39 4 4 42 13 EU1135

[0845] 0.8 45 3 0.16 76 12 0.032 98 8 100 31 2 20 31 6 4 33 7 EU1141

[0846] 0.8 42 2 0.16 49 6 0.032 74 4 100 28 1 20 30 4 4 41 17 EU1142

[0847] 0.8 44 8 0.16 53 9 0.032 72 2 100 37 2 20 39 3 4 50 5 EU1152

[0848] 0.8 72 8 0.16 82 9 0.032 81 12 100 24 4 20 27 1 4 35 2 EU1153

[0849] 0.8 56 4 0.16 70 3 0.032 72 14 100 41 7 20 45 3 4 48 4 EU1154

[0850] 0.8 52 6 0.16 71 2 0.032 80 14 100 28 2 20 30 4 4 32 4 EU1155

[0851] 0.8 58 2 0.16 80 8 0.032 98 24 100 37 6 EU1157

[0852]

[0853] 20 37 4 4 46 10

[0854] 0.8 70 18

[0855] 0.16 108 20

[0856] 0.032 105 37

[0857] 100 36 5

[0858] 20 49 12

[0859] 4 48 5

[0860] EU1159

[0861] 0.8 70 7

[0862] 0.16 98 20

[0863] 0.032 104 9

[0864] 100 28 3

[0865] 20 28 5

[0866] 4 43 9

[0867] EU1174

[0868] 0.8 62 10

[0869] 0.16 85 20

[0870] 0.032 98 23

[0871] 100 41 5

[0872] 20 44 7

[0873] 4 54 3

[0874] EU1182

[0875] 0.8 77 1

[0876] 0.16 86 13

[0877] 0.032 84 19

[0878] 100 29 4

[0879] 20 28 2

[0880] 4 27 7

[0881] EU1190

[0882] 0.8 37 4

[0883] 0.16 55 0

[0884] 0.032 75 14

[0885] 100 46 6

[0886] 20 48 5

[0887] 4 56 5

[0888] EU1191

[0889] 0.8 71 11

[0890] 0.16 94 9

[0891] 0.032 93 18

[0892] 100 34 2

[0893] 20 38 3

[0894] 4 36 4

[0895] EU1200

[0896] 0.8 45 6

[0897] 0.16 63 3

[0898]

[0899] 0.032 83 9

[0900] Dose dependent inhibition of INHBE was observed for all selected INHBE siRNAs, while the non-targeting control siRNA, EU1202, did not affect INHBE expression (Table 6 and Table 5b).

[0901] Example 3 In vitro study in primary cynomolgus monkey hepatocytes showing INHBE knockdown efficacy by exposure to selected GalNAc-siRNA conjugates.

[0902] Expression of INHBE mRNA was assessed after incubation with the GalNAc-siRNA conjugates (further described in Table 5c) at 100 nM, 20 nM, 4 nM, and 0.8 nM and 0.16 nM and compared to INHBE mRNA expression levels in untreated cells.

[0903] The knockdown efficacy of the GalNAc-conjugated siRNAs targeting INHBE was evaluated in primary cynomolgus monkey hepatocytes (Supplier: Life Technologies, USA). Hepatocytes were seeded at 45,000 cells per well in collagen-coated 96-well plates (Life Technologies, Cat. A1142801, USA), while siRNA was added to the plating medium (Life Technologies, Cat. A1217601, CM3000, CM4000, USA), thus achieving final siRNA concentrations between 100 nM and 0.16 nM. Twenty-four hours post-treatment, cells were lysed and RNA isolated using Dynabeads mRNA DIRECT kit (Thermo Fisher Scientific, Cat. 61012, USA). RT-qPCR was performed using mRNA-specific primers and probes against INHBE and PPIB mRNA. Expression levels were calculated using the delta delta Ct method. INHBE expression was normalized to the housekeeping gene PPIB. Results are expressed as ratio of INHBE to PPIB mRNA relative to INHBE to PPIB ratio in untreated (Ut) cells set as 100 and can be found in Table 7 and Table 8. Dose dependent and significant reduction of INHBE expression was observed for several GalNAc siRNA molecules (EU2001 to 2023 and EU2025 to EU2030 and EU2031 to EU2035, but not with the non-targeting control siRNA (EU2036).

[0904] In Table 7 and Table 8, results are expressed as % remaining INHBE mRNA after siRNA exposure compared to INHBE expression levels in untreated (Ut) cells set as 100.

[0905] Table 7: Results of dose-response testing in primary cynomolgus monkey hepatocytes by GalNAc siRNA conjugates for inhibition of INHBE expression by receptor mediated uptake.

[0906] siRNA % INHBE mRNA

[0907] Duplex

[0908] concentration Mean SD

[0909] Ut - 100 1

[0910] 100 nM 38 6

[0911] 20 nM 38 5

[0912] EU2001 4 nM 52 3

[0913] 0.8 nM 68 1

[0914] 0.16 nM 88 11

[0915] 100 nM 44 5

[0916] 20 nM 54 6

[0917] EU2002 4 nM 59 8

[0918] 0.8 nM 80 7

[0919] 0.16 nM 85 10

[0920] 100 nM 75 4

[0921] 20 nM 74 10

[0922] EU2003 4 nM 81 4

[0923] 0.8 nM 104 7

[0924] 0.16 nM 91 2

[0925] 100 nM 61 4

[0926] 20 nM 60 4

[0927] EU2004 4 nM 77 5

[0928] 0.8 nM 80 15

[0929] 0.16 nM 99 3

[0930] 100 nM 53 5

[0931] EU2005 20 nM 49 5

[0932]

[0933] 4 nM 70 10 0.8 nM 81 8 0.16 nM 89 16 100 nM 51 7 20 nM 56 5 EU2006 4 nM 69 10

[0934] 0.8 nM 77 15 0.16 nM 91 11 100 nM 46 5 20 nM 50 4 EU2007 4 nM 60 5

[0935] 0.8 nM 90 13 0.16 nM 98 10 100 nM 39 5 20 nM 44 8 EU2008 4 nM 55 0

[0936] 0.8 nM 80 3 0.16 nM 93 7 100 nM 31 1 20 nM 34 3 EU2009 4 nM 43 7

[0937] 0.8 nM 60 7 0.16 nM 75 6 100 nM 42 4 20 nM 51 3 EU2010 4 nM 66 6

[0938] 0.8 nM 83 5 0.16 nM 99 2 100 nM 48 2 20 nM 48 4 EU2011 4 nM 60 1

[0939] 0.8 nM 80 5 0.16 nM 98 10 100 nM 42 3 20 nM 42 3 EU2012 4 nM 59 7

[0940] 0.8 nM 79 12 0.16 nM 86 13 100 nM 44 5 20 nM 51 3 EU2013 4 nM 65 3

[0941] 0.8 nM 68 10 0.16 nM 92 13 100 nM 48 3 20 nM 54 4 EU2014

[0942] 4 nM 74 0

[0943]

[0944] 0.8 nM 92 1 0.16 nM 87 7 100 nM 43 3 20 nM 52 1 EU2015 4 nM 63 2

[0945] 0.8 nM 74 1 0.16 nM 86 9 100 nM 48 7 20 nM 52 3 EU2016 4 nM 68 8

[0946] 0.8 nM 79 3 0.16 nM 88 2 100 nM 35 4 20 nM 37 5 EU2017 4 nM 56 7

[0947] 0.8 nM 77 4 0.16 nM 83 4 100 nM 38 5 20 nM 40 3 EU2018 4 nM 52 6

[0948] 0.8 nM 74 2 0.16 nM 84 7 100 nM 39 7 20 nM 48 1 EU2019 4 nM 68 5

[0949] 0.8 nM 82 13 0.16 nM 111 17 100 nM 48 3 20 nM 59 9 EU2020 4 nM 67 5

[0950] 0.8 nM 81 8 0.16 nM 90 5 100 nM 33 2 20 nM 42 5 EU2021 4 nM 52 4

[0951] 0.8 nM 66 4 0.16 nM 87 6 100 nM 43 3 20 nM 47 1 EU2022 4 nM 60 3

[0952] 0.8 nM 75 8 0.16 nM 95 11 100 nM 41 5 20 nM 49 1 EU2023 4 nM 68 7

[0953] 0.8 nM 82 10

[0954]

[0955] 0.16 nM 90 5 100 nM 92 6 20 nM 85 5 EU2024 4 nM 98 6

[0956] 0.8 nM 113 5 0.16 nM 115 7 100 nM 54 9 20 nM 56 7 EU2025 4 nM 63 7

[0957] 0.8 nM 78 10 0.16 nM 105 10 100 nM 52 5 20 nM 68 9 EU2026 4 nM 75 4

[0958] 0.8 nM 96 4 0.16 nM 102 3 100 nM 68 8 20 nM 93 3 EU2027 4 nM 91 5

[0959] 0.8 nM 89 4 0.16 nM 94 13 100 nM 41 5 20 nM 51 8 EU2028 4 nM 59 5

[0960] 0.8 nM 76 2 0.16 nM 93 6 100 nM 48 6 20 nM 52 10 EU2029 4 nM 71 5

[0961] 0.8 nM 80 8 0.16 nM 86 13 100 nM 51 2 20 nM 59 5 EU2030 4 nM 69 2

[0962] 0.8 nM 83 6 0.16 nM 100 4

[0963]

[0964] EU2036 100 nM 104 14 Table 8: Results of dose-response testing of GalNAc siRNA conjugates EU2031 to EU2035 in primary cynomolgus monkey hepatocytes by receptor mediated uptake.

[0965] % INHBE mRNA

[0966] Cone. remaining

[0967] Duplex

[0968] (nM)

[0969] Mean SD

[0970] Ut - 100 9

[0971] 100 nM 46 5

[0972] 20 nM 60 10

[0973] EU2031 4 nM 65 14

[0974] 0.8 nM 86 26

[0975] 0.16 nM 85 15

[0976] 100 nM 62 13

[0977] 20 nM 66 9

[0978] EU2032 4 nM 74 11

[0979] 0.8 nM 87 10

[0980] 0.16 nM 97 13

[0981] 100 nM 46 12

[0982] 20 nM 49 8

[0983] EU2033 4 nM 56 9

[0984] 0.8 nM 94 25

[0985] 0.16 nM 95 14

[0986] 100 nM 41 3

[0987] 20 nM 39 5

[0988] EU2034 4 nM 53 7

[0989] 0.8 nM 85 15

[0990] 0.16 nM 88 17

[0991] 100 nM 64 9

[0992] 20 nM 55 10

[0993] EU2035 4 nM 70 12

[0994] 0.8 nM 81 20

[0995] 0.16 nM 110 23

[0996]

[0997] EU2036 100 nM 96 9

[0998] Example 4

[0999] In vitro study in primary human hepatocytes showing INHBE knockdown efficacy by GalNAc-siRNA conjugates.

[1000] Expression of INHBE mRNA was assessed after incubation with the GalNAc-siRNA conjugates (further described in Table 5c) at concentrations of 100 nM, 20 nM, 4 nM, 0.8 nM, and 0.16 nM. no

[1001] To test the knockdown efficacy of the GalNAc-conjugated siRNAs targeting INHBE m primary human hepatocytes, 35,000 cells per well (Supplier: Life Technologies, USA) were added to siRNAs in plating medium (Life Technologies, Cat. A1217601, CM3000, CM4000, USA) for final concentrations between 100 nM and 0.16 nM in collagen-coated 96-well plates (Life Technologies, Cat. A1142801, USA). 24 hours post-treatment, cells were lysed and RNA isolated using Dynabeads mRNA DIRECT kit (Thermo Fisher Scientific, Cat. 61012, USA). RT-qPCR was performed using mRNA-specific primers and probes against INHBE and the house keeping gene PPIB. Expression differences were calculated using the delta delta Ct method and relative expression of INHBE normalized to the housekeeping gene PPIB were determined. Results are expressed as ratio of INHBE to PPIB mRNA relative to untreated (Ut) levels set as 100 and can be found in Table 10.

[1002] Dose-dependent knockdown of human INHBE mRNA was observed for all tested GalNAc conjugates (EU2001-EU2035), except the non-targeting control siRNA, EU2036.

[1003] Results are expressed as % remaining INHBE mRNA after siRNA exposure (Table 9 and Table 10).

[1004] Table 9: Results of dose-response testing of siRNAs targeting INHBE in primary human hepatocytes by receptor mediated uptake.

[1005] siRNA % INHBE mRNA

[1006] Duplex

[1007] concentration Mean SD

[1008] Ut 100 7

[1009] 100 nM 27 9

[1010] 20 nM 49 5

[1011] EU2001 4 nM 65 7

[1012] 0.8 nM 81 10

[1013] 0.16 nM 106 6

[1014] 100 nM 48 4

[1015] 20 nM 71 5

[1016] EU2002 4 nM 76 4

[1017] 0.8 nM 103 11

[1018] 0.16 nM 109 11

[1019] 100 nM 65 1

[1020] 20 nM 81 3

[1021] EU2003 4 nM 98 8

[1022] 0.8 nM 94 6

[1023] 0.16 nM 109 8

[1024] 100 nM 52 3

[1025] 20 nM 65 6

[1026] EU2004 4 nM 70 4

[1027] 0.8 nM 86 3

[1028]

[1029] 0.16 nM 84 8 Ill

[1030] 100 nM 57 5 20 nM 62 4 EU2005 4 nM 75 6

[1031] 0.8 nM 91 1 0.16 nM 90 2 100 nM 59 2 20 nM 68 3 EU2006 4 nM 80 9

[1032] 0.8 nM 85 4 0.16 nM 95 2 100 nM 43 1 20 nM 53 7 EU2007 4 nM 68 4

[1033] 0.8 nM 84 5 0.16 nM 91 2 100 nM 43 2 20 nM 56 4 EU2008 4 nM 71 4

[1034] 0.8 nM 85 2 0.16 nM 97 8 100 nM 29 1 20 nM 44 1 EU2009 4 nM 61 1

[1035] 0.8 nM 78 3 0.16 nM 91 5 100 nM 50 3 20 nM 62 3 EU2010 4 nM 74 3

[1036] 0.8 nM 87 2 0.16 nM 96 4 100 nM 41 2 20 nM 58 4 EU2011 4 nM 68 4

[1037] 0.8 nM 89 3 0.16 nM 98 7 100 nM 43 1 20 nM 60 3 EU2012 4 nM 67 1

[1038] 0.8 nM 77 6 0.16 nM 93 1 100 nM 50 4 20 nM 58 8 EU2013 4 nM 71 6

[1039] 0.8 nM 89 9 0.16 nM 106 6

[1040]

[1041] EU2014 100 nM 49 3 20 nM 58 3 4 nM 68 9 0.8 nM 84 8 0.16 nM 85 1 100 nM 46 2 20 nM 63 5 EU2015 4 nM 73 4

[1042] 0.8 nM 80 9 0.16 nM 88 5 100 nM 41 1 20 nM 58 6 EU2016 4 nM 68 2

[1043] 0.8 nM 81 1 0.16 nM 89 5 100 nM 38 3 20 nM 54 9 EU2017 4 nM 60 2

[1044] 0.8 nM 90 7 0.16 nM 90 3 100 nM 42 7 20 nM 43 6 EU2018 4 nM 60 8

[1045] 0.8 nM 79 13 0.16 nM 86 11 100 nM 48 2 20 nM 57 4 EU2019 4 nM 71 3

[1046] 0.8 nM 87 2 0.16 nM 91 9 100 nM 53 5 20 nM 68 1 EU2020 4 nM 85 16

[1047] 0.8 nM 92 4 0.16 nM 110 6 100 nM 46 3 20 nM 56 7 EU2021 4 nM 76 5

[1048] 0.8 nM 90 2 0.16 nM 94 12 100 nM 42 2 20 nM 60 10 EU2022 4 nM 63 3

[1049] 0.8 nM 96 2 0.16 nM 88 5 100 nM 46 2 EU2023

[1050]

[1051] 20 nM 67 6 4 nM 75 10 0.8 nM 103 6 0.16 nM 88 5 100 nM 88 3 20 nM 96 2 EU2024 4 nM 97 8

[1052] 0.8 nM 93 6 0.16 nM 103 10 100 nM 55 3 20 nM 67 4 EU2025 4 nM 79 8

[1053] 0.8 nM 82 11 0.16 nM 90 6 100 nM 79 1 20 nM 86 9 EU2026 4 nM 84 2

[1054] 0.8 nM 98 10 0.16 nM 96 18 100 nM 88 5 20 nM 87 5 EU2027 4 nM 96 11

[1055] 0.8 nM 95 5 0.16 nM 103 4 100 nM 47 1 20 nM 59 9 EU2028 4 nM 70 5

[1056] 0.8 nM 88 13 0.16 nM 93 10 100 nM 58 5 20 nM 63 9 EU2029 4 nM 77 11

[1057] 0.8 nM 88 6 0.16 nM 97 8 100 nM 64 8 20 nM 77 1 EU2030 4 nM 85 10

[1058] 0.8 nM 96 9 0.16 nM 95 9

[1059]

[1060] EU2036 100 nM 101 2 Table 10

[1061] Results of dose-response testing of siRNAs targeting INHBE in primary human hepatocytes by receptor mediated uptake.

[1062] Duplex Cone. (nM) Mean SD

[1063] Ut - 100 8

[1064] 100 nM 35 2

[1065] 20 nM 53 2

[1066] EU2031 4 nM 75 8

[1067] 0.8n M 93 5

[1068] 0.16n M 106 6

[1069] 100 nM 45 12

[1070] 20 nM 61 7

[1071] EU2032 4 nM 80 2

[1072] 0.8 nM 93 11

[1073] 0.16 nM 97 9

[1074] 100 nM 25 2

[1075] 20n M 40 5

[1076] EU2033 4 nM 53 4

[1077] 0.8 nM 72 9

[1078] 0.16 nM 85 6

[1079] 100 nM 45 3

[1080] 20 nM 55 4

[1081] EU2034 4nM 70 3

[1082] 0.8 nM 88 15

[1083] 0.16 nM 83 11

[1084] 100 nM 56 2

[1085] 20 nM 69 5

[1086] EU2035 4n M 79 9

[1087] 0.8 nM 91 10

[1088] 0.16 nM 100 7

[1089]

[1090] EU2036 100 nM 96 9

[1091] Example 5

[1092] In vitro study in human liver spheroids showing INHBE knockdown efficacy of selected GalNAc-siRNA conjugates.

[1093] Expression of INHBE mRNA was assessed after incubation of human spheroids with GalNAc-siRNA conjugates EU2001, EU2007, EU2008, EU2009. EU2011, EU2012, EU2017, EU2018, ELI2021, ELI2022 and the non-targeting control siRNA, ELI2036 (further described in Table 5c) at concentrations of 100 nM, 10 nM, and 1 nM. To test the knockdown efficacy of the GalNAc-conjugated siRNAs targeting INHBE in liver spheroids, primary human hepatocytes were cultured in 3D structure. 250 cells per microcavity were seeded to form spheroids in Elplasia plates (Corning, Cat. 4442, USA) and cultured in FCS-containing medium before treatment. On day 7, GalNAc-conjugated siRNAs targeting INHBE were added and overlayed by Matrigel (0.99 mg / ml, Corning, Cat. 354234, USA). FCS-free medium was changed every 2-3 days. Cells were lysed and RNA was isolated 7- and 14-days post-treatment using Dynabeads mRNA DIRECT kit (Thermo Fisher Scientific, Cat.

[1094] 61012, USA). RT-qPCR was performed using mRNA-specific primers and probes against INHBE and the housekeeper PPIB. Expression levels were calculated using the delta delta Ct method and relative expression of INHBE normalized to the housekeeping gene PPIB were determined. Results are expressed as ratio of INHBE to PPIB mRNA relative to untreated levels set as 100 and are shown in Table 11.

[1095] Dose-dependent knockdown of INHBE mRNA was stronger at the later time point (14 days post siRNA dosing) and observed for all tested GalNAc conjugates, except non-targeting control siRNA EU2036. At 100 nM dose level, the remaining INHBE mRNA levels of treated cells were in the range of 20% to 50% at 7 days post-treatment and in the range of 7% to 38% at 14 days post-treatment. At both time points, the strongest reduction of INHBE mRNA was observed with EU2009 followed by EU2022 and EU2018, which lowered INHBE mRNA levels down to 14% and 18% compared to INHBE mRNA levels in untreated cells at the 2-weeks time point post siRNA treatment.

[1096] Results are expressed as % remaining INHBE mRNA after siRNA exposure in Table 11.

[1097] Table 11: Results of dose-response testing in human liver spheroids (100, 10, or 1 nM) of siRNAs targeting INHBE at 7- or 14-days post siRNA treatment.

[1098] % remaining INHBE mRNA

[1099] siRNA

[1100] Duplex 7 days post dosing 14 days post dosing concentration

[1101] Mean SD Mean SD

[1102] Ut - 100 9 100 14

[1103] 100 nM 50 6 23 8

[1104] EU2001 10n M 114 36 67 22

[1105] 1n M 97 7 89 27 100 nM 37 7 14 15

[1106] EU2022 10n M 77 13 56 5

[1107] 1n M 94 6 82 12

[1108] 100 nM 33 9 24 4

[1109] EU2007 10n M 62 3 52 9

[1110] 1n M 84 10 76 5

[1111] 100 nM 37 6 26 8

[1112] EU2008 10n M 96 2 56 3

[1113]

[1114] 1n M 88 15 88 16 100 nM 20 2 7 2 EU2009 10n M 45 29 21 20

[1115] 1n M 97 36 85 14

[1116] 100 nM 30 4 27 6

[1117] EU2011 10n M 48 6 65 6

[1118] 1n M 83 3 108 40

[1119] 100 nM 39 8 34 6

[1120] EU2012 10n M 63 14 62 9

[1121] 1n M 70 31 77 23

[1122] 100 nM 32 3 14 2

[1123] EU2017 10n M 54 11 40 25

[1124] 1n M 89 1 95 46

[1125] 100 nM 31 8 18 10

[1126] EU2018 10n M 71 4 36 18

[1127] 1n M 87 18 75 13

[1128] 100 nM 43 6 38 13

[1129] EU2021 10n M 87 18 68 7

[1130] 1n M 85 2 79 13

[1131]

[1132] EU2036 100 nM 102 8 102 6

[1133] Example 6

[1134] In vivo studies demonstrating knockdown of human INHBE mRNA in murine liver tissue following AAV-mediated expression and single subcutaneous dosing of 1 or 3 mg / kg GalNAc-conjugated siRNA.

[1135] Recombinant AAV particles were purchased from VectorBuilder (Chicago, USA). In brief, a sequence encoding human INHBE including adjacent UTR regions (NM 031479.5) was cloned into a pAAV[Exp] vector downstream of an apoE / hAAT promoter. Serotype 8 AAVs were packaged in HEK293T cells and purified by PEG precipitation and CsCI gradient ultracentrifugation.

[1136] Animal experiments were performed at Experimental Pharmacology & Oncology Berlin-Buch GmbH (Berlin, Germany) according to ethical guidelines of the German Protection of Animals Act in its version of July 2013. AAVs were diluted to 4E11 GC / ml in phosphate buffered saline (PBS) followed by i.v. injection of male C57BL / 6 mice at 8-10 weeks of age. All animals were dosed at 2E12 GC / kg. A stable and extended expression of human INHBE was observed at 2 and 4 weeks after injection as determined in a previous experiment.

[1137] Two weeks post AAV dosing, mice were randomized into groups of 6 and then treated with a single subcutaneous dose of 1 or 3 mg / kg EU2009 or 1 or 3 mg / kg EU2018 (further described in Table 5c) dissolved in PBS, or with PBS only as vehicle control. The viability, body weight, and behaviour of the mice were monitored during the study without pathological findings. The study was terminated 2 weeks after siRNA dosing. All animals were fasted for four hours before termination. Liver samples were snap-frozen and stored at - 80°C until further analyses. In summary, total RNA was isolated using an InviTrap Spin Universal RNA Mini Kit (Invitek Molecular, Cat. 1060 100 300, Germany) as recommended by the manufacturer. To assess the integrity of isolated RNA, automated electrophoresis was performed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). 100 ng total RNA per reaction was used for RT-qPCR with amplicon sets specific for human INHBE and murine ApoB as a housekeeper gene. Expression differences were calculated using the AACt method and relative expression of hINHBE versus ApoB normalized to the PBS group. Furthermore, DNA was extracted from liver tissue samples using DNA Multi-Sample Ultra 2.0 Kit (Thermo Fisher, Cat. A36570). The viral load (AAV copy number) was determined by qPCR of viral DNA in DNA samples extracted from respective liver tissue samples, using amplicon sets specific for the WPRE region of the transferred expression cassette. hINHBE mRNA levels were normalized to the housekeeping gene, ApoB, and to the viral copy numbers detected in respective liver sample with PBS / vehicle group set as 1. Results from this study are presented in Figure 1. EU2009 and EU2018 show dose dependent inhibition of hINHBE mRNA in a human INHBE-expressing AAV mouse model.

[1138] In a separate study, female C57BL / 6 mice at 8-10 weeks of age were dosed intravenously with hINHBE AAV virus at 2E12 GC / kg, randomized into treatment groups (n=6) and two weeks later treated subcutaneously with 1 mg / kg EU2008, EU2009, EU2011, EU2013, EU2017, EU2021, EU2031, EU2032, EU2033 and EU2034 (detailed in Table 5c) or with the vehicle PBS. The viability, body weight, and behaviour of the mice were monitored during the study without pathological findings.

[1139] The study was terminated 2 weeks after siRNA dosing. All animals were fasted for four hours before termination and sample collection. Liver samples were snap-frozen, total RNA and DNA was isolated from liver samples as described above. hINHBE mRNA levels were normalized to the house keeping gene ApoB and to the viral load detected in respective liver sample with PBS group set as 1. Results from this study are presented in Figure 2. EU2008, EU2009, EU20011, EU2013, EU2017, EU2021, EU2031, EU2032, EU2033 and EU2034 lowered human INHBE expression in the liver of hINHBE AAV mouse model at a dose level of 1 mg / kg.

[1140] Example 7

[1141] Synthesis of (vp)-mU-phos was performed as described in Prakash, Nucleic Acids Res. 2015, 43(6), 2993-3011 and Haraszti, Nucleic Acids Res. 2017, 45(13), 7581-7592. Synthesis of the phosphoramidite derivatives of ST41 (ST41-phos) as well as ST23 (ST23-phos) can be performed as described in WO2017 / 174657. Synthesis of phosphorthioamidites was performed as described in Caruthers, J. Org. Chem. 1996, 61, 4272-4281.

[1142]

[1143] 8

[1144] Example compounds were synthesized according to methods described below and known to persons of skill in the art. Assembly of the oligonucleotide chain and linker building blocks was performed by solid phase synthesis applying phosphoramidite methodology.

[1145] Downstream cleavage, deprotection and purification were performed following standard procedures that are well known in the art. Oligonucleotide syntheses was performed on an AKTA oligopilot 10 using commercially available 2'0-Methyl RNA and 2Tluoro-2'Deoxy RNA base loaded CPG solid support and phosphoramidites (all standard protection, ChemGenes, LinkTech) were used.

[1146] Ancillary reagents were purchased from EMP Biotech and Biosolve. Synthesis was performed using a 0.1 M solution of the phosphoramidite in dry acetonitrile (<20 ppm H2O) and benzylthiotetrazole (BTT) was used as activator (0.3M in acetonitrile). Coupling time was 10 min. If phosphorthioamidites were used to introduce a phosphordithioate linkage (PS2) a repeated coupling wash cycle over 60 min was performed. A Cap / OX / Cap or Cap / Thio / Cap cycle was applied (Cap: Ac20 / NMI / Lutidine / Acetonitrile, Oxidizer: 0.05M l2in pyridine / H2O). Phosphorothioates and phosphordithioates were introduced using commercially available thiolation reagent 50mM EDITH in acetonitrile (Link technologies). DMT cleavage was achieved by treatment with 3% dichloroacetic acid in toluene. Upon completion of the programmed synthesis cycles a diethylamine (DEA) wash was performed. All oligonucleotides were synthesized in DMT-off mode.

[1147] Tri-antennary GalNAc clusters (ST23 / ST41) were introduced by successive coupling of the branching trebler amidite derivative (C4XLT-phos) followed by the GalNAc amidite (ST23-phos). Attachment of (vp)-mU moiety was achieved by use of (vp)-mU-phos in the last synthesis cycle. The (vp)-mU-phos does not provide a hydroxy group suitable for further synthesis elongation and therefore, does not possess an DMT-group. Hence coupling of (vp)-mU-phos results in synthesis termination.

[1148] For the removal of the methyl esters masking the vinylphosphonate, the CPG carrying the fully assembled oligonucleotide was dried under reduced pressure and transferred into a 20 ml PP syringe reactor for solid phase peptide synthesis equipped with a disc frit (Carl Roth GmbH). The CPG was then brought into contact with a solution of 250 pL TMSBr and 177 pL pyridine in CH2Cl2(0.5 ml / pmol solid support bound oligonucleotide) at room temperature and the reactor was sealed with a Luer cap. The reaction vessels were slightly agitated over a period of 2x15 min, the excess reagent discarded, and the residual CPG washed 2x with 10 ml acetonitrile. Further downstream processing did not alter from any other example compound.

[1149] The single strands were cleaved off the CPG by 40% aq. methylamine treatment (in presence of 20 mM DTT if phosphorodithioate linkages were present) in 90 min at RT. The resulting crude oligonucleotide was purified by ion exchange chromatography (Resource Q, 6 ml, GE Healthcare) on an AKTA Pure HPLC System using a sodium chloride gradient. Product containing fractions were pooled, desalted on a size exclusion column (Zetadex, EMP Biotech) and lyophilized until further use.

[1150] All final single-stranded products were analysed by AEX-HPLC to prove their purity. Identity of the respective single-stranded products was proved by LC-MS analysis.

[1151]

[1152] 9

[1153] Individual single strands were dissolved in a concentration of 60 OD / ml in H2O. Both individual oligonucleotide solutions were added together in a reaction vessel. For easier reaction monitoring a titration was performed. The first strand was added in 25% excess over the second strand as determined by UV-absorption at 260 nm. The reaction mixture was heated to 80°C for 5 min and then slowly cooled to RT. Double-strand formation was monitored by ion pairing reverse phase HPLC. From the UV-area of the residual single strand the needed amount of the second strand was calculated and added to the reaction mixture. The reaction was heated to 80°C again and slowly cooled to RT. This procedure was repeated until less than 10% of residual single strand was detected.

[1154]

[1155] Effect of the mismatch on INHBE knockdown efficacy

[1156] This example demonstrates the impact of unpaired mismatches of position 1 of the antisense strand on knock-down activity. The mismatch enhanced the knock-down activity.

[1157] An in vitro experiment was conducted with Hep3B cells showing INHBE knockdown efficacy of tested siRNAs after transfection of 10 nM, 1 nM, 0.1 nM and 0.01 nM siRNA.

[1158] INHBE knockdown efficacy of siRNAs EU1009, EU1097, EU1098 and EU1202, EU1202 and-EU1204 was determined by transfection of 10 nM, 1 nM, 0.1 nM and 0.01 nM siRNA in Hep3B cells. All molecules induced dose dependent reduction of human INHBE mRNA expression.

[1159] For transfection of Hep3B cells with siRNAs, cells were seeded at a density of 30,000 cells / well in 96-well tissue culture plates (TPP, Cat. 92096, Switzerland). T ransfection of siRNA was carried out with Lipofectamine RNAiMax (Invitrogen / Life Technologies, Cat. 13778-500, Germany) according to the manufacturer’s instructions, directly before seeding. The transfection with INHBE-siRNAs was performed in triplicates, with luciferase siRNA (ELI1201) as non-targeting control. 24 hours post-treatment, cells were lysed and RNA isolated using Dynabeads mRNA DIRECT kit (Thermo Fisher Scientific, Cat. 61012, USA). RT-qPCR was performed using INHBE and PPIB specific primer probe sets and Takyon™ One-Step Low Rox Probe 5X MasterMix dTTP on the QuantStudio6 device from Applied Biosystems in single-plex 384 well format. Expression differences were calculated using the delta delta Ct method. INHBE expression levels were normalized to the expression levels of the housekeeping gene PPIB. Results of INHBE expression levels after siRNA transfections are depicted as % remaining INHBE mRNA in comparison to INHBE mRNA levels in untreated cell set as 1. (Figure 3).

[1160] Example 11. Effect of siRNA molecules with LNA on the first strand on INHBE mRNA levels

[1161] In this example the impact of LNA in the first strand (penultimate position) was investigated. EU2038 contains LNA in penultimate position. EU2037 has the same base sequence without LNA. The data demonstrates that LNA enhanced KD activity.

[1162] Results of dose-response testing of siRNAs targeting INHBE in primary human hepatocytes by receptor mediated uptake. Expression of INHBE mRNA was assessed after incubation with the GalNAc-siRNA conjugates at 100 nM, 20 nM, 4 nM, 0.8 nM, and 0.16 nM. To test the knockdown efficacy of the GalNAc-conjugated siRNAs targeting INHBE in primary human hepatocytes, 35,000 cells per well (Supplier: Life Technologies, USA) were added to siRNAs in plating medium (Life Technologies, Cat. A1217601, CM3000, CM4000, USA) for final concentrations between 100 nM and 0.16 nM in collagen-coated 96-well plates (Life Technologies, Cat. A1142801, USA). 24 hours post-treatment, cells were lysed and RNA isolated using Dynabeads mRNA DIRECT kit (Thermo Fisher Scientific, Cat. 61012, USA). RT-qPCR was performed using mRNA-specific primers and probes against INHBE and the house keeping gene PPIB. Expression differences were calculated using the delta delta Ct method and relative expression of INHBE normalized to the housekeeping gene PPIB were determined. Results are expressed as ratio of INHBE to PPIB mRNA relative to untreated levels set as 1 and can be found in Figure 4A.

[1163] 12. Effect of the length of the duplex region on knock-down activity In this example the knock-down activity by molecules of different lengths is compared. EU2009 is a 19mer, containing vp on 5’ end with unpaired mismatch at P1 of the first strand. EU2042 and EU2043 are 23mer duplex variants, with vinylphosphonate at 5'end unpaired or with paired mismatch at the first position respectively. The tests were performed following the protocol of Example 11. The results demonstrate that 23mers (longer constructs) are less active than the 19mer molecule (Figure 4B).

[1164] Example 13. In vivo effect of different siRNA molecules on INHBE mRNA levels Results of in vivo target gene inhibition by siRNA conjugates in cynomolgus monkeys receiving a single dose of siRNA. After acclimatization, physical examinations, safety clinical chemistry profiling and training, non-naive female monkeys were selected for the study and randomized into 6 treatment groups (n=4). Two weeks prior to siRNA dosing, liver tissue samples were collected by needle biopsy for assessment of baseline INHBE mRNA levels. On day 0 animals were treated with either 0.9% saline (vehicle) or with ELI2009, ELI2033, ELI2011, ELI2013 or EU2018 by subcutaneous injection at the scapular / lateral cervical region in alert / non-sedated animals and monitored for 8 weeks follow up period. Additional liver biopsy samples were collected 2 weeks, 5 weeks and 8 weeks after siRNA dosing by ultrasound guided liver biopsy. For assessment of target gene expression total RNA was isolated, extracted and purified using MagMAX mirVana Total RNA Isolation Kit (ThermoFisher). Subsequently, one time RT-qPCR analysis was conducted for INHBE mRNA and for PPIB mRNA as house keeper. Each sample was analyzed in triplicate. Individual INHBE mRNA levels were calculated using the delta delta Ct method and normalized to expression levels of PPIB. Individual INHBE / PPIB expression levels at 2, 5 and 8 weeks were corrected to each individual animal's baseline (pre-dosing) values set as 100%. All tested molecules lowered INHBE mRNA levels in the liver for at least 8 weeks (see Figure 5) with EU2033 showing particular consistent and long-term effect.

[1165] 14. Dose-dependent and stable reduction in INHBE mRNA levels by EU2033.

[1166] Results of in vivo target gene inhibition by siRNA conjugates in cynomolgus monkeys receiving different dose levels of ELI2033. After acclimatization, physical examinations, safety clinical chemistry profiling and training, non-naive male monkeys were selected for the study and randomized into 5 treatment groups (n=4). Two weeks prior to siRNA dosing, liver tissue samples were collected by needle biopsy for assessment of baseline INHBE mRNA levels. On day 0 animals were treated with either 0.9% saline (vehicle) or a dose (dose increasing from dose 1 to 3) of EU2033 by subcutaneous injection at the scapular / lateral cervical region in alert / non-sedated animals. Animals were monitored for 12 weeks following the first dose. Additional liver biopsy samples were collected 4 weeks, 8 weeks and 12 weeks after first siRNA dosing by ultrasound guided liver biopsy. For assessment of target gene expression total RNA was isolated, extracted and purified using MagMAX mirVana Total RNA Isolation Kit (ThermoFisher). Subsequently, one time RT-qPCR analysis was conducted for INHBE mRNA and for PPIB mRNA as house keeper. Each sample was analyzed in triplicate. Individual INHBE mRNA levels were calculated using the delta delta Ct method and normalized to expression levels of PPIB. Individual INHBE / PPIB expression levels at 4, 8 and 12 weeks were corrected to each individual animal's baseline (pre-dosing) values set as 100%. Animals, that did not respond to the siRNA treatment were excluded. A dose dependent reduction of INHBE mRNA levels by ELI2033 was observed at the designated time points of liver biopsy collection and persisted for all three dose levels until the end of the observation period (day 28 to 84, see Figure 6).

[1167] Example 15. Tritosome stability of different siRNA molecules.

[1168] Here EU2033 was investigated for stability in tritosome assay. EU2033 is a molecule having LNA in position 18 of the first strand, and ELI2009 corresponds to the version of ELI2033 w / o LNA in the first strand. The tritosome assay demonstrated enhanced stability by EU2033 To assess stability, 5 pM siRNA conjugate was incubated with acidic rat liver tritosome extract (pH 5) at 37°C for 0, 24 and 96 h. After incubation, samples were separated on a 20% TBE polyacrylamide gels and visualized by CybrGold (Invitrogen) staining. The results are presented in Figure 7.

[1169] Example 16. Stability of siRNA constructs with different length

[1170] In this example sequences of different lengths were tested for stability in tritosome assay. EU2009 (19mer, blunt ended, with vinylphsphonate at 5’) was compared to longer RNA duplexes such as EU2044, EU2040 and EU2041 with vinylphosponate at 5' end of the antisense strand. The tritosome assay demonstrates enhanced stability by EU2009 compared to the other molecules.

[1171] To assess stability, 5 pM siRNA conjugate was incubated with acidic rat liver tritosome extract (pH 5) at 37°C for 0; 24 and 96 hours in the presence of 1 mM MgCI2. After incubation, samples were separated on a 20% TBE polyacrylamide gels and visualized by Ethidium Bromide staining. The results are presented in Figure 8.

[1172] Summary abbreviations table - Table 4

[1173] Abbreviation Meaning

[1174] mA, mU, mC, 2‘-O-Methyl RNA nucleotides

[1175] mG

[1176] 2’-OMe 2‘-O-Methyl modification

[1177] fA, fU, fC, fG 2’ deoxy-2‘-F RNA nucleotides

[1178] LNA

[1179] o

[1180] „ Basn

[1181] O _

[1182] i -. i

[1183] O ' 0

[1184] Locked nucleic acid nucleotides

[1185]

[1186] LA NH2

[1187] o 1 / \ JJ J ”

[1188] LT

[1189] O^o^X° 0^*6

[1190] LNArT

[1191] L5mC Nd?

[1192] 0^ t

[1193] „ N

[1194] ()XO

[1195] .. |

[1196] LNA-C

[1197] LG 0

[1198] 1 / 'll NH °x \tA A

[1199] 1 „0 T N NH2

[1200] cA'O

[1201] LNA-G

[1202] LU O

[1203] Ni l O

[1204] 1 oN J° io

[1205] o o

[1206] LNA-U

[1207] 2’-F 2’-fluoro modification (PS) phosphorothioate

[1208] (vp) Vinyl-(E)-phosphonate

[1209]

[1210] (vp)-mU 0

[1211] HQ, O fV

[1212] HO-P' <N^O

[1213] (Efl ^cU

[1214] 0 OMe

[1215] (vp)-mU-phos 0

[1216] / O-P' <NA0

[1217] (EfVoJ

[1218] 0 OMe

[1219] NC^0AN,P12

[1220] ivA, ivC, ivU, inverted RNA (3'-3’) nucleotides

[1221] ivG

[1222] ST23 OH OH

[1223] NHAc0

[1224] ST23-phos OAc. OAc I I

[1225] NHAc0 0

[1226] ST41 (or

[1227] C4XLT) AZ’X^C'' x

[1228] o

[1229] ST41-phos (or DMT.

[1230] 0 0

[1231] C4XLT-phos) DMT. J 1Ni / Pr22

[1232] 0 0 T\ / O. Q Q X / . CN

[1233] DMT. >

[1234] 0 0

[1235] Ser(GN) (when OH OH

[1236] UX^-0

[1237] at the end of a

[1238] NHAc T

[1239] chain, one of NH

[1240] the 0— is OH) z°xA / 0-..

[1241] [ST23 (ps)]3 OH

[1242] ST41 (ps) OxS

[1243] NHAc 0 0 >

[1244] OH I

[1245] o® I

[1246] , P. 0 RxNHAc 0 0 0 - © / A I S O OH 1

[1247] HO'U^o, 0 J /

[1248] H0^X--^>^^0 P. J

[1249] NHAc 0 0

[1250] [ST23]3 ST41

[1251]

[1252]

[1253] The abbreviations as shown in the above abbreviation table may be used herein. The list of abbreviations may not be exhaustive and further abbreviations and their meaning may be found throughout this document. Summary sequence tables

[1254] Table 5a - Unmodified sequences

[1255] Strand Name (*) Unmodified Sequence (5'->3') Seq ID no. (unmodified) EU1001-A UCAGUCUAGUUGCAGUUUC 1 EU1001-B GAAACUGCAACUAGACUGC 2 EU1002-A GUAUAAAUGCUUGUCUCCC 3 EU1002-B GGGAGACAAGCAUUUAUAC 4 EU1003-A UGACUCCUGUUUCUGGGGG 5 EU1003-B CCCCCAGAAACAGGAGUCA 6 EU1004-A CCAGGAUUUGCUGCUUGGC 7 EU1004-B GCCAAGCAGCAAAUCCUGG 8 EU1005-A GAAAGUGCCCAUUUGGGUC 9 EU1005-B GACCCAAAUGGGCACUUUC 10 EU1006-A CCAAGGAUUGUUGGCUUUG 11 EU1006-B CAAAGCCAACAAU CCU U GG 12 EU1007-A UCCGUCUUGACCACAUUGC 13 EU1007-B GCAAUGUGGUCAAGACGGA 14 EU1008-A AUCCGUCUUGACCACAUUG 15 EU1008-B CAAUGUGGUCAAGACGGAU 16 EU1009-A GGAAACUUCAUCUUGGUCU 17 EU1009-B AGACCAAGAUGAAGUUUCC 18 EU1010-A UUUGUGGACACCCCUGAAA 19 EU1010-B UUUCAGGGGUGUCCACAAA 20 EU1011-A ACCACAUUGCCAUUAUGAU 21 EU1011-B AUCAUAAUGGCAAUGUGGU 22 EU1012-A AGUAUAAAUGCUUGUCUCC 23 EU1012-B GGAGACAAGCAUUUAUACU 24 EU1013-A UACAUUGCCAUUAUGAUCC 25 EU1013-B GGAUCAUAAUGGCAAUGUA 26 EU1014-A GACAGUAGCAAAGCUGAUG 27 EU1014-B CAUCAGCUUUGCUACUGUC 28 EU1015-A ACACAUUCCAUUAACAAUG 29 EU1015-B CAUUGUUAAUGGAAUGUGU 30 EU1016-A CAGUAGCAAAGCUGAUGAC 31 EU1016-B GUCAUCAGCUUUGCUACUG 32 EU1017-A UUGAAGUGGAGUCUGUGAC 33 EU1017-B GUCACAGACUCCACUUCAG 34 EU1018-A CCAGACUUCUCACCCCUCA 35 EU1018-B UGAGGGGUGAGAAGUCUGG 36 EU1019-A AAAGCUGAUGACCUCCUCC 37 EU1019-B GGAGGAGGUCAUCAGCUUU 38 EU1020-A UUAGGCUGAAGUGGAGUCU 39 EU1020-B AGACUCCACUUCAGCCUAC 40 EU1021-A GACCACAUUGCCAUUAUGA 41 EU1021-B UCAUAAUGGCAAUGUGGUC 42 EU1022-A AUACAAUUUUAAGAUAAUG 43 EU1022-B CAUUAUCUUAAAAUUGUAU 44 EU1023-A GUAAUUCAGCUGGUACCCC 45 EU1023-B GGGGUACCAGCUGAAUUAC 46 EU1024-A UAUUCUGGGACGACUGGUC 47 EU1024-B GACCAGUCGUCCCAGAAUA 48 EU1025-A AUUGCCAGGUGGUUGUUGG 49 EU1025-B CCAACAACCACCU G G CAAU 50 EU1026-A UCAUAUUGCCAGGUGGUUG 51 EU1026-B CAACCACCUGGCAAUAUGA 52 EU1027-A AUCAGGAAUCUGAUGCCUC 53 EU1027-B GAGGCAUCAGAUUCCUGAU 54 EU1028-A CUGAAGACGGCAGAAUGGA 55 EU1028-B UCCAUUCUGCCGUCUUCAG 56

[1256]

[1257] EU1029-A CUCUUCACUCCAAAGCCCC 57 EU1029-B GGGGCUUUGGAGUGAAGAG 58 EU1030-A UGAUGUAAUCACAUGUCAC 59 EU1030-B GUGACAUGUGAUUACAUCA 60 EU1031-A GGAGGAUGAGUUAUUCUGG 61 EU1031-B CCAGAAUAACUCAUCCUCC 62 EU1032-A CACAUUGCCAUUAUGAUCC 63 EU1032-B GGAUCAUAAUGGCAAUGUG 64 EU1033-A GCAGUCUAGUUGCAGUUUC 65 EU1033-B GAAACUGCAACUAGACUGC 2 EU1034-A GGUUGUCCAGUAACUGUGC 66 EU1034-B GCACAG U U ACU GG ACAACC 67 EU1035-A GGAUCUUAAGCUCUAGGAA 68 EU1035-B UUCCUAGAGCUUAAGAUCC 69 EU1036-A GAGGACUUUCUCAUCUUGG 70 EU1036-B CCAAGAUGAGAAAGUCCUC 71 EU1037-A AAUUUUUCUCUGCCUUCCC 72 EU1037-B GGGAAGGCAGAGAAAAAUU 73 EU1038-A AUCUGGCACAUCCGUCUUG 74 EU1038-B CAAGACGGAUGUGCCAGAU 75 EU1039-A AAAACCAGGGAACUUCUUA 76 EU1039-B UAAGAAGUUCCCUGGUUUU 77 EU1040-A CU U CACUCCAAAGCCCCAG 78 EU1040-B CUGGGGCUUUGGAGUGAAG 79 EU1041-A UUCUGCUUGGGGUGCCAGU 80 EU1041-B ACUGGCACCCCAAGCAGAA 81 EU1042-A CAAAGCUGAUGACCUCCUC 82 EU1042-B GAGGAGGUCAUCAGCUUUG 83 EU1043-A UUAUAAAUGCUUGUCUCCC 84 EU1043-B GGGAGACAAGCAUUUAUAC 4 EU1044-A GGGAACUUCUUAGGCUUAG 85 EU1044-B CUAAGCCUAAGAAGUUCCC 86 EU1045-A UUGUUGGCUUUGAGGAGGC 87 EU1045-B GCCUCCUCAAAGCCAACAA 88 EU1046-A UUCUAGGGGUCUGCAGUCU 89 EU1046-B AGACUGCAGACCCCUAGAA 90 EU1047-A AUAUCUGGCACAUCCGUCU 91 EU1047-B AGACGGAUGUGCCAGAUAU 92 EU1048-A GUAGGCUGAAGUGGAGUCU 93 EU1048-B AGACUCCACUUCAGCCUAC 40 EU1049-A UGAAGUGGAGUCUGUGACA 94 EU1049-B UGUCACAGACUCCACUUCA 95 EU1050-A GUUCCUGGAAGUCUACGUA 96 EU1050-B UACGUAGACUUCCAGGAAC 97 EU1051-A UUGUAGGCUGAAGUGGAGU 98 EU1051-B ACUCCACUUCAGCCUACAG 99 EU1052-A AAACCAGGGAACUUCUUAG 100 EU1052-B CUAAGAAGUUCCCUGGUUU 101 EU1053-A GAUAAUGAGAAUUCAAAAG 102 EU1053-B CUUUUGAAUUCUCAUUAUC 103 EU1054-A CUAAGCAUCCUCCCUCAGC 104 EU1054-B GCUGAGGGAGGAUGCUUAG 105 EU1055-A AUUAAGAAAGUAUAAGCCA 106 EU1055-B UGGCUUAUACUUUCUUAAU 107 EU1056-A UUUCCUGACUCCUGUUUCU 108 EU1056-B AGAAACAGGAGUCAGGAAA 109 EU1057-A AAGUAUAAGCCAGGCGCGG 110 EU1057-B CCGCGCCUGGCUUAUACUU 111 EU1058-A GAAAAACCAGGGAACUUCU 112 EU1058-B AGAAGUUCCCUGGUUUUUC 113 EU1059-A CUCAUCUUGGGAGAGGCUA 114 EU1059-B UAGCCUCUCCCAAGAUGAG 115

[1258]

[1259] EU1060-A CAUUCCAUUAACAAUGAUG 116 EU1060-B CAUCAUUGUUAAUGGAAUG 117 EU1061-A AACCAGGGAACUUCUUAGG 118 EU1061-B CCUAAGAAGUUCCCUGGUU 119 EU1062-A AGGAUUUGCUGCUUGGCUA 120 EU1062-B UAGCCAAGCAGCAAAUCCU 121 EU1063-A AGAUGAUGUAAUCACAUGU 122 EU1063-B ACAUGUGAUUACAUCAUCU 123 EU1064-A UAUGAUCCAGGUAGAGGAG 124 EU1064-B CUCCUCUACCUGGAUCAUA 125 EU1065-A UAGUCUAGUUGCAGUUUCA 126 EU1065-B UGAAACUGCAACUAGACUG 127 EU1066-A AUCCAGGUAGAGGAGAGAG 128 EU1066-B CUCUCUCCUCUACCUGGAU 129 EU1067-A UAACACAUCAGCCAACCUG 130 EU1067-B CAGGUUGGCUGAUGUGUUG 131 EU1068-A AAGAUAAUGAGAAUUCAAA 132 EU1068-B UUUGAAUUCUCAUUAUCUU 133 EU1069-A AAUGCUUGUCUCCCAGUGG 134 EU1069-B CCACUGGGAGACAAGCAUU 135 EU1070-A UAUCUUAAGCUCUAGGAAG 136 EU1070-B CUUCCUAGAGCUUAAGAUC 137 EU1071-A AUUGGCUCGGAUCUUAAGC 138 EU1071-B GCUUAAGAUCCGAGCCAAU 139 EU1072-A GAUCUUAAGCUCUAGGAAG 140 EU1072-B CUUCCUAGAGCUUAAGAUC 137 EU1073-A UCUAGUUGCAGUUUCAGGA 141 EU1073-B UCCUGAAACUGCAACUAGA 142 EU1074-A CAACACAUCAGCCAACCUG 143 EU1074-B CAGGUUGGCUGAUGUGUUG 131 EU1075-A CUGCAACAUAAGGGGGUCG 144 EU1075-B CGACCCCCUUAUGUUGCAG 145 EU1076-A GGAUUUGCUGCUUGGCUAG 146 EU1076-B CUAGCCAAGCAGCAAAUCC 147 EU1077-A UGGAGGAUGAGUUAUUCUG 148 EU1077-B CAGAAUAACUCAUCCUCCA 149 EU1078-A GCUCGGAUCUUAAGCUCUA 150 EU1078-B UAGAGCUUAAGAUCCGAGC 151 EU1079-A CUGUAGGCUGAAGUGGAGU 152 EU1079-B ACUCCACUUCAGCCUACAG 99 EU1080-A UGAUUUGCUGCUUGGCUAG 153 EU1080-B CUAGCCAAGCAGCAAAUCC 147 EU1081-A UGAGGAUGAGUUAUUCUGG 154 EU1081-B CCAGAAUAACUCAUCCUCC 62 EU1082-A U UUCAGGACACCAGACU UC 155 EU1082-B GAAGUCUGGUGUCCUGAAA 156 EU1083-A CCCAGGUUGGUGAUGUGGU 157 EU1083-B ACCACAUCACCAACCUGGG 158 EU1084-A UAUAAAUGCUUGUCUCCCA 159 EU1084-B UGGGAGACAAGCAUUUAUA 160 EU1085-A UUGGAAGUCUACGUAAUGG 161 EU1085-B CCAUUACGUAGACUUCCAG 162 EU1086-A UCAGGAUUUGCUGCUUGGC 163 EU1086-B GCCAAGCAGCAAAUCCUGG 8 EU1087-A UUUCUCUGCCUUCCCUCCC 164 EU1087-B GGGAGGGAAGGCAGAGAAA 165 EU1088-A GAGUUAUUCUGGGACGACU 166 EU1088-B AGUCGUCCCAGAAUAACUC 167 EU1089-A UACCACAUUGCCAUUAUGA 168 EU1089-B UCAUAAUGGCAAUGUGGUC 42* EU1090-A GAAUGGAAAGAGGCAGCAA 169 EU1090-B UUGCUGCCUCUUUCCAUUC 170

[1260]

[1261] EU1091-A ACAAGAAAGUGCCCAUUUG 171 EU1091-B CAAAUGGGCACUUUCUUGU 172 EU1092-A UUGCUUACCCUGCUUCAAG 173 EU1092-B CUUGAAGCAGGGUAAGCAG 174 EU1093-A CCUUCUAGGGGUCUGCAGU 175 EU1093-B ACUGCAGACCCCUAGAAGG 176 EU1094-A UCUUAAGCUCUAGGAAGGG 177 EU1094-B CCCUUCCUAGAGCUUAAGA 178 EU1095-A CUCGGAUCUUAAGCUCUAG 179 EU1095-B CUAGAGCUUAAGAUCCGAG 180 EU1096-A UGAGAAUUCAAAAGGCAAA 181 EU1096-B UUUGCCUUUUGAAUUCUCA 182 EU1097-A GUCAAGUGAGUCAUAUUGC 183 EU1097-B GCAAUAUGACUCACUUGAC 184 EU1098-A AAAGACGGCAGAAUGGAAG 185 EU1098-B CUUCCAUUCUGCCGUCUUU 186 EU1099-A AAGCACACAU U CCAU U AAC 187 EU1099-B GUUAAUGGAAUGUGUGCUU 188 EU1100-A GGAAGUCUACGUAAUGGUC 189 EU1100-B GACCAUUACGUAGACUUCC 190 EU1101-A UAAAGUGCCCAUUUGGGUC 191 EU1101-B GACCCAAAUGGGCACUUUC 10 EU1102-A AUCUUAAGCUCUAGGAAGG 192 EU1102-B CCUUCCUAGAGCUUAAGAU 193 EU1103-A GAAAGUAUAAAUGCUUGUC 194 EU1103-B GACAAGCAUUUAUACUUUC 195 EU1104-A CUGGGAAACUUCAUCUUGG 196 EU1104-B CCAAGAUGAAGUUUCCCAG 197 EU1105-A AG ACAAG AAAG UGCCCAU U 198 EU1105-B AAUGGGCACUUUCUUGUCU 199 EU1106-A UAAGAAAGUAUAAGCCAGG 200 EU1106-B CCUGGCUUAUACUUUCUUA 201 EU1107-A GAGGAGUGGACAGGUGAAA 202 EU1107-B UUUCACCUGUCCACUCCUC 203 EU1108-A UCAAGUGAGUCAUAUUGCC 204 EU1108-B GGCAAUAUGACUCACUUGA 205 EU1109-A GCUUUGAGGAGGCUGAAGA 206 EU1109-B UCUUCAGCCUCCUCAAAGC 207 EU1110-A AAUUUUAAGAUAAUGAGAA 208 EU1110-B UUCUCAUUAUCUUAAAAUU 209 EU1111-A GAUUGUUGGCUUUGAGGAG 210 EU1111-B CUCCUCAAAGCCAACAAUC 211 EU1112-A GGAUUGUUGGCUUUGAGGA 212 EU1112-B U CCU CAAAG CCAACAAU CC 213 EU1113-A UAACAAUGAUGUCAGAAAG 214 EU1113-B CUUUCUGACAUCAUUGUUA 215 EU1114-A AAGCUGAUGACCUCCUCCC 216 EU1114-B GGGAGGAGGUCAUCAGCUU 217 EU1115-A GGAAUCUGAUGCCUCCAGU 218 EU1115-B ACUGGAGGCAUCAGAUUCC 219 EU1116-A AGAAAGUAUAAGCCAGGCG 220 EU1116-B CGCCUGGCUUAUACUUUCU 221 EU1117-A UUAUUAAGAAAGUAUAAGC 222 EU1117-B GCUUAUACUUUCUUAAUAA 223 EU1118-A UCUAUCUGCUUCCUCCUCC 224 EU1118-B GGAGGAGGAAGCAGAUAGA 225 EU1119-A UUUAAGAUAAUGAGAAUUC 226 EU1119-B GAAUUCUCAUUAUCUUAAA 227 EU1120-A GUAUUAUUAUGAAAAUAGC 228 EU1120-B GCUAUUUUCAUAAUAAUAC 229 EU1121-A AUUGUUGGCUUUGAGGAGG 230 EU1121-B CCUCCUCAAAGCCAACAAU 231

[1262]

[1263] EU1122-A UCUUGGUCUCUUCACUCCA 232 EU1122-B UGGAGUGAAGAGACCAAGA 233 EU1123-A AAUGAGAAUUCAAAAGGCA 234 EU1123-B UGCCUUUUGAAUUCUCAUU 235 EU1124-A AGUUAUUCUGGGACGACUG 236 EU1124-B CAGUCGUCCCAGAAUAACU 237 EU1125-A UACAGUAGCAAAGCUGAUG 238 EU1125-B CAUCAGCUUUGCUACUGUA 239 EU1126-A UGGCACAUCCGUCUUGACC 240 EU1126-B GGUCAAGACGGAUGUGCCA 241 EU1127-A U CU CAGACAAG AAAG UGCC 242 EU1127-B GGCACUUUCUUGUCUGAGA 243 EU1128-A CCAGUUCCUGGAAGUCUAC 244 EU1128-B GUAGACUUCCAGGAACUGG 245 EU1129-A GUCUUGACCACAUUGCCAU 246 EU1129-B AUGGCAAUGUGGUCAAGAC 247 EU1130-A CAGUCUAGUUGCAGUUUCA 248 EU1130-B UGAAACUGCAACUAGACUG 127 EU1131-A GUUAUUCUGGGACGACUGG 249 EU1131-B CCAGUCGUCCCAGAAUAAC 250 EU1132-A UUGCUGCUUGGCUAGCUCC 251 EU1132-B GGAGCUAGCCAAGCAGCAA 252 EU1133-A UUUGCUGCUUGGCUAGCUC 253 EU1133-B GAGCUAGCCAAGCAGCAAA 254 EU1134-A CUGCUUACCCUGCUUCAAG 255 EU1134-B CUUGAAGCAGGGUAAGCAG 174 EU1135-A AAGGAUUGUUGGCUUUGAG 256 EU1135-B CUCAAAGCCAACAAUCCUU 257 EU1136-A AUUCAAAAGGCAAAUAACA 258 EU1136-B UGUUAUUUGCCUUUUGAAU 259 EU1137-A GAAGACGGCAGAAUGGAAA 260 EU1137-B UUUCCAUUCUGCCGUCUUC 261 EU1138-A ACAAU U U U AAGAU AAU GAG 262 EU1138-B CUCAUUAUCUUAAAAUUGU 263 EU1139-A GUAGACCCCUUUAGAAGAA 264 EU1139-B UUCUUCUAAAGGGGUCUAC 265 EU1140-A AUCUUGGUCUCUUCACUCC 266 EU1140-B GGAGUGAAGAGACCAAGAU 267 EU1141-A AAGUAUAAAUGCUUGUCUC 268 EU1141-B GAGACAAGCAUUUAUACUU 269 EU1142-A AACUUCAUCUUGGUCUCUU 270 EU1142-B AAGAGACCAAGAUGAAGUU 271 EU1143-A UGAAAAACCAGGGAACUUC 272 EU1143-B GAAGUUCCCUGGUUUUUCC 273 EU1144-A AGUAGCAAAGCUGAUGACC 274 EU1144-B GGUCAUCAGCUUUGCUACU 275 EU1145-A GAUGUCAGAAAGAUGAUGU 276 EU1145-B ACAUCAUCUUUCUGACAUC 277 EU1146-A UCAUCUUGGUCUCUUCACU 278 EU1146-B AGUGAAGAGACCAAGAUGA 279 EU1147-A AGACGGCAGAAUGGAAAGA 280 EU1147-B UCUUUCCAUUCUGCCGUCU 281 EU1148-A GUCUCAGACAAGAAAGUGC 282 EU1148-B GCACUUUCUUGUCUGAGAC 283 EU1149-A UUUUCCUGACUCCUGUUUC 284 EU1149-B GAAACAGGAGUCAGGAAAA 285 EU1150-A AAUUCAGCUGGUACCCCUC 286 EU1150-B GAGGGGUACCAGCUGAAUU 287 EU1151-A UAGGAUUUGCUGCUUGGCU 288 EU1151-B AGCCAAGCAGCAAAUCCUG 289 EU1152-A UCAAAAGGCAAAUAACAUG 290 EU1152-B CAUGUUAUUUGCCUUUUGA 291

[1264]

[1265] EU1153-A UUAUGAUCCAGGUAGAGGA 292 EU1153-B UCCUCUACCUGGAUCAUAA 293 EU1154-A AUCCAGGAUUUGCUGCUUG 294 EU1154-B CAAGCAGCAAAU CCU GG AU 295 EU1155-A UCGGAUCUUAAGCUCUAGG 296 EU1155-B CCUAGAGCUUAAGAUCCGA 297 EU1156-A UAGUCUCCGGAGGGCUCUG 298 EU1156-B CAGAGCCCUCCGGAGACUA 299 EU1157-A CUGAAGUGGAGUCUGUGAC 300 EU1157-B GUCACAGACUCCACUUCAG 34 EU1158-A CAGGACACCAGACUUCUCA 301 EU1158-B UGAGAAGUCUGGUGUCCUG 302 EU1159-A CUGGAAGUCUACGUAAUGG 303 EU1159-B CCAUUACGUAGACUUCCAG 162 EU1160-A AAUCUGAUGCCUCCAGUCA 304 EU1160-B UGACUGGAGGCAUCAGAUU 305 EU1161-A UCCAGGUUGGUGAUGUGGU 306 EU1161-B ACCACAUCACCAACCUGGG 158 EU1162-A CAUUGGCUCGGAUCUUAAG 307 EU1162-B CUUAAGAUCCGAGCCAAUG 308 EU1163-A AAUGGAAAGAGGCAGCAAU 309 EU1163-B AUUGCUGCCUCUUUCCAUU 310 EU1164-A AGACUUCUCACCCCUCAAG 311 EU1164-B CUUGAGGGGUGAGAAGUCU 312 EU1165-A UGUUGUCCAGUAACUGUGC 313 EU1165-B GCACAG U U ACU GG ACAACC 67 EU1166-A AUGUAAUCACAUGUCACAC 314 EU1166-B GUGUGACAUGUGAUUACAU 315 EU1167-A GUCUAGUUGCAGUUUCAGG 316 EU1167-B CCUGAAACUGCAACUAGAC 317 EU1168-A UAGUUAUUCUGGGACGACU 318 EU1168-B AGUCGUCCCAGAAUAACUC 167 EU1169-A AUGAUCCAGGUAGAGGAGA 319 EU1169-B UCUCCUCUACCUGGAUCAU 320 EU1170-A CAGUAAUUCAGCUGGUACC 321 EU1170-B GGUACCAGCUGAAUUACUG 322 EU1171-A CGUCUUGACCACAUUGCCA 323 EU1171-B UGGCAAUGUGGUCAAGACG 324 EU1172-A UAAUUCAGCUGGUACCCCU 325 EU1172-B AGGGGUACCAGCUGAAUUA 326 EU1173-A GGAAAAACCAGGGAACUUC 327 EU1173-B GAAGUUCCCUGGUUUUUCC 273 EU1174-A ACAGUAGCAAAGCUGAUGA 328 EU1174-B UCAUCAGCUUUGCUACUGU 329 EU1175-A GACACCAGACUUCUCACCC 330 EU1175-B GGGUGAGAAGUCUGGUGUC 331 EU1176-A AUUAACAAUGAUGUCAGAA 332 EU1176-B UUCUGACAUCAUUGUUAAU 333 EU1177-A UAGGACACCAGACUUCUCA 334 EU1177-B UGAGAAGUCUGGUGUCCUG 302 EU1178-A CAAGAAAGUGCCCAUUUGG 335 EU1178-B CCAAAUGGGCACUUUCUUG 336 EU1179-A UGUGACAGUAGCAAAGCUG 337 EU1179-B CAGCUUUGCUACUGUCACA 338 EU1180-A AUUCCCUGGAGCCACACUC 339 EU1180-B GAGUGUGGCUCCAGGGAAU 340 EU1181-A CAUAUUGCCAGGUGGUUGU 341 EU1181-B ACAACCACCUGGCAAUAUG 342 EU1182-A UUACCCUGCUUCAAGCCUG 343 EU1182-B CAGGCUUGAAGCAGGGUAA 344 EU1183-A UUAUUCUGGGACGACUGGU 345 EU1183-B ACCAGUCGUCCCAGAAUAA 346

[1266]

[1267] EU1184-A GAAGUGGAGUCUGUGACAG 347 EU1184-B CUGUCACAGACUCCACUUC 348 EU1185-A UUUGAGGAGGCUGAAGACG 349 EU1185-B CGUCUUCAGCCUCCUCAAA 350 EU1186-A ACUUCAUCUUGGUCUCUUC 351 EU1186-B GAAGAGACCAAGAUGAAGU 352 EU1187-A CUUCUCACCCCUCAAGCCA 353 EU1187-B UGGCUUGAGGGGUGAGAAG 354 EU1188-A AGAAAGAUGAUGUAAUCAC 355 EU1188-B GUGAUUACAUCAUCUUUCU 356 EU1189-A CUGUAGUCUCCGGAGGGCU 357 EU1189-B AGCCCUCCGGAGACUACAG 358 EU1190-A UUCAUCUUGGUCUCUUCAC 359 EU1190-B G UGAAGAG ACCAAGAU GAA 360 EU1191-A CAGGAUUUGCUGCUUGGCU 361 EU1191-B AGCCAAGCAGCAAAUCCUG 289 EU1192-A AAGGCAAAUAACAUGUUAG 362 EU1192-B CUAACAUGUUAUUUGCCUU 363 EU1193-A AUUGCCAUUAUGAUCCAGG 364 EU1193-B CCUGGAUCAUAAUGGCAAU 365 EU1194-A GGGGUCAAGUGAGUCAUAU 366 EU1194-B AUAUGACUCACUUGACCCC 367 EU1195-A UGACUUUGUGGACACCCCU 368 EU1195-B AGGGGUGUCCACAAAGUCA 369 EU1196-A AGUAAUUCAGCUGGUACCC 370 EU1196-B GGGUACCAGCUGAAUUACU 371 EU1197-A GGACUUUCUCAUCUUGGGA 372 EU1197-B UCCCAAGAUGAGAAAGUCC 373 EU1198-A AACUUCUUAGGCUUAGUGC 374 EU1198-B GCACUAAGCCUAAGAAGUU 375 EU1199-A UGGCUCGGAUCUUAAGCUC 376 EU1199-B GAGCUUAAGAUCCGAGCCA 377 EU1200-A UUUGACUUUGUGGACACCC 378 EU1200-B GGGUGUCCACAAAGUCAAA 379 EU1201-A UCGAAGUAUUCCGCGUACG 380 EU1201-B CGUACGCGGAAUACUUCGA 381 EU1202-A UGAAACUUCAUCUUGGUCU 382 EU1203-A UGUAUAAAUGCUUGUCUCC 383 EU1204-A UUCAGGAAUCUGAUGCCUC 384 EU1205-A UAUGCUUGUCUCCCAGUGG 385 EU1206-A UCAAGAAAGUGCCCAUUUG 386 EU1207-A UUCAAGUGAGUCAUAUUGC 387 EU1208-A UAAGACGGCAGAAUGGAAG 388 EU1209-A UAAAGUAUAAAUGCUUGUC 389 EU1210-A UAGGAUUGUUGGCUUUGAG 390 EU1211-A UAGUAUAAAUGCUUGUCUC 391 EU1212-A UACUUCAUCUUGGUCUCUU 392 EU1213-A UCAGUAGCAAAGCUGAUGA 393 EU1214-A AACAGUAGCAAAGCUGAUG 394 EU1215A UAAGACGGCAGAAUGGAAA 838 EU1215B UUUCCAUUCUGCCGUCUUU 839 EU1216A UAAGACGGCAGAAUGGAAGGAUU 840 EU1216B UCCUUCCAUUCUGCCGUCUUA 841 EU1217B UCCUUCCAUUCUGCCGUCUUU 842 EU1218B AAUCCUUCCAUUCUGCCGUCUUA 843 EU1219B AAUCCUUCCAUUCUGCCGUCUUU 844 EU1220A UAAGACGGCAGAAUGGAAAGAGG 845

[1268]

[1269] EU1220B UCUUUCCAUUCUGCCGUCUUC 846 he duplexes listed in Table 5b have various modifications as shown, with reference to Table 4 for an explanation of the abbreviations used. Where ppropriate, the sequence ID No. of the equivalent unmodified strand from Table 5a is also indicated.

[1270] able 5b - Duplex IDs and Modified Sequences

[1271] unmodified equivalent Duplex ID Strand Name (*) Modified Sequence (5'-^3') Seq ID No. SEQ ID No. EU1OO1 EU1001-A mU (ps) fC ( ps) mA fG mU fC mU fA mG fU mU fG mC fA mG fU mU (ps) fU ( ps) mC 395 1 EU1OO1 EU1001-B mG (ps) mA (ps) mA mA mC mU fG fC fA mA mC mU mA mG mA mC mU (ps) mG (ps) mC 396 2 EU1OO2 EU1002-A mG (ps) fU (ps) mA fU mA fA mA fU mG fC mU fU mG fU mC fU mC (ps) fC (ps) mC 397 3 EU1OO2 EU1002-B mG (ps) mG (ps) mG mA mG mA fC fA fA mG mC mA mU mU mU mA mU (ps) mA (ps) mC 398 4 EU1OO3 EU1003-A mU (ps) fG (ps) mA fC mU fC mC fU mG fU mU fU mC fU mG fG mG (ps) fG (ps) mG 399 5 EU1OO3 EU1003-B mC (ps) mC (ps) mC mC mC mA fG fA fA mA mC mA mG mG mA mG mU (ps) mC (ps) mA 400 6 EU1004 EU1004-A mC (ps) fC (ps) mA fG mG fA mU fU mU fG mC fU mG fC mU fU mG (ps) fG (ps) mC 401 7 EU1004 EU1004-B mG (ps) mC (ps) mC mA mA mG fC fA fG mC mA mA mA mU mC mC mU (ps) mG (ps) mG 402 8 EU1OO5 EU1005-A mG (ps) fA (ps) mA fA mG fU mG fC mC fC mA fU mU fU mG fG mG (ps) fU (ps) mC 403 9 EU1OO5 EU1005-B mG (ps) mA (ps) mC mC mC mA fA fA fU mG mG mG mC mA mC mU mU (ps) mU (ps) mC 404 10 EU1006 EU1006-A mC (ps) fC (ps) mA fA mG fG mA fU mU fG mU fU mG fG mC fU mU (ps) fU (ps) mG 405 11 EU1006 EU1006-B mC (ps) mA (ps) mA mA mG mC fC fA fA mC mA mA mU mC mC mU mU (ps) mG (ps) mG 406 12 EU1007 EU1007-A mU (ps) fC (ps) mC fG mU fC mU fU mG fA mC fC mA fC mA fU mU (ps) fG (ps) mC 407 13 EU1007 EU1007-B mG (ps) mC (ps) mA mA mU mG fU fG fG mU mC mA mA mG mA mC mG (ps) mG (ps) mA 408 14 EU1008 EU1008-A mA (ps) fU (ps) mC fC mG fU mC fU mU fG mA fC mC fA mC fA mU (ps) fU (ps) mG 409 15 EU1008 EU1008-B mC (ps) mA (ps) mA mU mG mU fG fG fU mC mA mA mG mA mC mG mG (ps) mA (ps) mU 410 16 EU1OO9 EU1009-A mG (ps) fG (ps) mA fA mA fC mU fU mC fA mU fC mU fU mG fG mU (ps) fC (ps) mU 411 17 EU1OO9 EU1009-B mA (ps) mG (ps) mA mC mC mA fA fG fA mU mG mA mA mG mU mU mU (ps) mC (ps) mC 412 18 EU1O1O EU1010-A mU (ps) fU (ps) mU fG mU fG mG fA mC fA mC fC mC fC mU fG mA (ps) fA (ps) mA 413 19 EU1O1O EU1010-B mU (ps) mU (ps) mU mC mA mG fG fG fG mU mG mU mC mC mA mC mA (ps) mA (ps) mA 414 20 EU1O11 EU1011-A mA (ps) fC (ps) mC fA mC fA mU fU mG fC mC fA mU fU mA fU mG (ps) fA (ps) mU 415 21 EU1O11 EU1011-B mA (ps) mU (ps) mC mA mU mA fA fU fG mG mC mA mA mU mG mU mG (ps) mG (ps) mU 416 22 EU1O12 EU1012-A mA (ps) fG (ps) mU fA mU fA mA fA mU fG mC fU mU fG mU fC mU (ps) fC (ps) mC 417 23 EU1O12 EU1012-B mG (ps) mG (ps) mA mG mA mC fA fA fG mC mA mU mU mU mA mU mA (ps) mC (ps) mU 418 24 EU1O13 EU1013-A mU (ps) fA (ps) mC fA mU fU mG fC mC fA mU fU mA fU mG fA mU (ps) fC (ps) mC 419 25 EU1O13 EU1013-B mG (ps) mG (ps) mA mU mC mA fU fA fA mU mG mG mC mA mA mU mG (ps) mU (ps) mA 420 26 EU1014 EU1014-A mG (ps) fA (ps) mC fA mG fU mA fG mC fA mA fA mG fC mU fG mA (ps) fU (ps) mG 421 27 EU1014 EU1014-B mC (ps) mA (ps) mU mC mA mG fC fU fU mU mG mC mU mA mC mU mG (ps) mU (ps) mC 422 28 EU1O15 EU1015-A mA (ps) fC (ps) mA fC mA fU mU fC mC fA mU fU mA fA mC fA mA (ps) fU (ps) mG 423 29 EU1O15 EU1015-B mC (ps) mA (ps) mU mU mG mU fU fA fA mU mG mG mA mA mU mG mU (ps) mG (ps) mU 424 30

[1272]

[1273] EU1016 EU1016-A mC (ps) fA (ps) mG fU mA fG mC fA mA fA mG fC mU fG mA fU mG (ps) fA (ps) mC 425 31 EU1016 EU1016-B mG ( ps) mil (ps) mC mA mil mC fA fG fC mil mil mil mG mC mil mA mC ( ps) mil ( ps) mG 426 32 EU1017 EU1017-A mil (ps) fll (ps) mG fA mA fG mil fG mG fA mG fll mC fll mG fll mG (ps) fA (ps) mC 427 33 EU1017 EU1017-B mG (ps) mil (ps) mC mA mC mA fG fA fC mil mC mC mA mC mil mil mC (ps) mA (ps) mG 428 34 EU1018 EU1018-A mC (ps) fC (ps) mA fG mA fC mil fll mC fll mC fA mC fC mC fC mil (ps) fC (ps) mA 429 35 EU1018 EU1018-B mil (ps) mG (ps) mA mG mG mG fG fll fG mA mG mA mA mG mil mC mil (ps) mG (ps) mG 430 36 EU1019 EU1019-A mA (ps) fA (ps) mA fG mC fll mG fA mil fG mA fC mC fll mC fC mil (ps) fC (ps) mC 431 37 EU1019 EU1019-B mG (ps) mG (ps) mA mG mG mA fG fG fll mC mA mil mC mA mG mC mil (ps) mil (ps) mil 432 38 EU1020 EU1020-A mil (ps) fll (ps) mA fG mG fC mil fG mA fA mG fll mG fG mA fG mil (ps) fC (ps) mil 433 39 EU1020 EU1020-B mA (ps) mG (ps) mA mC mil mC fC fA fC mil mil mC mA mG mC mC mil (ps) mA (ps) mC 434 40 EU1021 EU1021-A mG (ps) fA (ps) mC fC mA fC mA fll mil fG mC fC mA fll mil fA mil (ps) fG (ps) mA 435 41 EU1021 EU1021-B mil (ps) mC (ps) mA mil mA mA fll fG fG mC mA mA mil mG mil mG mG (ps) mil (ps) mC 436 42 EU1022 EU1022-A mA (ps) fll (ps) mA fC mA fA mil fll mil fll mA fA mG fA mil fA mA (ps) fll (ps) mG 437 43 EU1022 EU1022-B mC (ps) mA (ps) mil mil mA mil fC fll fll mA mA mA mA mil mil mG mil (ps) mA (ps) mil 438 44 EU1023 EU1023-A mG (ps) fll (ps) mA fA mil fll mC fA mG fC mil fG mG fll mA fC mC (ps) fC (ps) mC 439 45 EU1023 EU1023-B mG (ps) mG (ps) mG mG mil mA fC fC fA mG mC mil mG mA mA mil mil (ps) mA (ps) mC 440 46 EU1024 EU1024-A mil (ps) fA (ps) mil fll mC fll mG fG mG fA mC fG mA fC mil fG mG (ps) fll (ps) mC 441 47 EU1024 EU1024-B mG (ps) mA (ps) mC mC mA mG fll fC fG mil mC mC mC mA mG mA mA (ps) mil (ps) mA 442 48 EU1025 EU1025-A mA (ps) fll (ps) mil fG mC fC mA fG mG fll mG fG mil fll mG fll mil (ps) fG (ps) mG 443 49 EU1025 EU1025-B mC (ps) mC (ps) mA mA mC mA fA fC fC mA mC mC mil mG mG mC mA (ps) mA (ps) mil 444 50 EU1026 EU1026-A mil (ps) fC (ps) mA fll mA fll mil fG mC fC mA fG mG fll mG fG mil (ps) fll (ps) mG 445 51 EU1026 EU1026-B mC (ps) mA (ps) mA mC mC mA fC fC fll mG mG mC mA mA mil mA mil (ps) mG (ps) mA 446 52 EU1027 EU1027-A mA (ps) fll (ps) mC fA mG fG mA fA mil fC mil fG mA fll mG fC mC (ps) fll (ps) mC 447 53 EU1027 EU1027-B mG (ps) mA (ps) mG mG mC mA fll fC fA mG mA mil mil mC mC mil mG (ps) mA (ps) mil 448 54 EU1028 EU1028-A mC (ps) fll (ps) mG fA mA fG mA fC mG fG mC fA mG fA mA fll mG (ps) fG (ps) mA 449 55 EU1028 EU1028-B mil (ps) mC (ps) mC mA mil mil fC fll fG mC mC mG mil mC mil mil mC (ps) mA (ps) mG 450 56 EU1029 EU1029-A mC (ps) fll (ps) mC fll mil fC mA fC mil fC mC fA mA fA mG fC mC (ps) fC (ps) mC 451 57 EU1029 EU1029-B mG (ps) mG (ps) mG mG mC mil fll fll fG mG mA mG mil mG mA mA mG (ps) mA (ps) mG 452 58 EU1030 EU1030-A mil (ps) fG (ps) mA fll mG fll mA fA mil fC mA fC mA fll mG fll mC (ps) fA (ps) mC 453 59 EU1030 EU1030-B mG (ps) mil (ps) mG mA mC mA fll fG fll mG mA mil mil mA mC mA mil (ps) mC (ps) mA 454 60 EU1031 EU1031-A mG (ps) fG (ps) mA fG mG fA mil fG mA fG mil fll mA fll mil fC mil (ps) fG (ps) mG 455 61 EU1031 EU1031-B mC (ps) mC (ps) mA mG mA mA fll fA fA mC mil mC mA mil mC mC mil (ps) mC (ps) mC 456 62 EU1032 EU1032-A mC (ps) fA (ps) mC fA mil fll mG fC mC fA mil fll mA fll mG fA mil (ps) fC (ps) mC 457 63 EU1032 EU1032-B mG (ps) mG (ps) mA mil mC mA fll fA fA mil mG mG mC mA mA mil mG (ps) mil (ps) mG 458 64 EU1033 EU1033-A mG (ps) fC (ps) mA fG mil fC mil fA mG fll mil fG mC fA mG fll mil (ps) fll (ps) mC 459 65 EU1033 EU1001-B mG (ps) mA (ps) mA mA mC mil fG fC fA mA mC mil mA mG mA mC mil (ps) mG (ps) mC 396 2 EU1034 EU1034-A mG (ps) fG (ps) mil fll mG fll mC fC mA fG mil fA mA fC mil fG mil (ps) fG (ps) mC 460 66 EU1034 EU1034-B mG (ps) mC (ps) mA mC mA mG fll fll fA mC mil mG mG mA mC mA mA (ps) mC (ps) mC 461 67 EU1035 EU1O35-A mG (ps) fG (ps) mA fll mC fll mil fA mA fG mC fll mC fll mA fG mG (ps) fA (ps) mA 462 68

[1274]

[1275] EU1035 EU1O35-B mil (ps) mil (ps) mC mC mil mA fG fA fG mC mil mil mA mA mG mA mil (ps) mC (ps) mC 463 69 EU1036 EU1036-A mG ( ps) fA ( ps) mG fG mA fC mil fU mil fC mil fC mA fll mC fll mil ( ps) fG ( ps) mG 464 70 EU1036 EU1036-B mC (ps) mC (ps) mA mA mG mA fll fG fA mG mA mA mA mG mil mC mC (ps) mil (ps) mC 465 71 EU1037 EU1037-A mA (ps) fA (ps) mil fll mil fll mil fC mil fC mil fG mC fC mil fll mC (ps) fC (ps) mC 466 72 EU1037 EU1037-B mG (ps) mG (ps) mG mA mA mG fG fC fA mG mA mG mA mA mA mA mA (ps) mil (ps) mil 467 73 EU1038 EU1038-A mA (ps) fll (ps) mC fll mG fG mC fA mC fA mil fC mC fG mil fC mil (ps) fll (ps) mG 468 74 EU1038 EU1038-B mC (ps) mA (ps) mA mG mA mC fG fG fA mil mG mil mG mC mC mA mG (ps) mA (ps) mil 469 75 EU1039 EU1039-A mA (ps) fA (ps) mA fA mC fC mA fG mG fG mA fA mC fll mil fC mil (ps) fll (ps) mA 470 76 EU1039 EU1039-B mil (ps) mA (ps) mA mG mA mA fG fll fll mC mC mC mil mG mG mil mil (ps) mil (ps) mil 471 77 EU1040 EU1040-A mC (ps) fll (ps) mil fC mA fC mil fC mC fA mA fA mG fC mC fC mC (ps) fA (ps) mG 472 78 EU1040 EU1040-B mC (ps) mil (ps) mG mG mG mG fC fll fll mil mG mG mA mG mil mG mA (ps) mA (ps) mG 473 79 EU1041 EU1041-A mil (ps) fll (ps) mC fll mG fC mil fll mG fG mG fG mil fG mC fC mA (ps) fG (ps) mil 474 80 EU1041 EU1041-B mA (ps) mC (ps) mil mG mG mC fA fC fC mC mC mA mA mG mC mA mG (ps) mA (ps) mA 475 81 EU1042 EU1042-A mC (ps) fA (ps) mA fA mG fC mil fG mA fll mG fA mC fC mil fC mC (ps) fll (ps) mC 476 82 EU1042 EU1042-B mG (ps) mA (ps) mG mG mA mG fG fll fC mA mil mC mA mG mC mil mil (ps) mil (ps) mG 477 83 EU1043 EU1043-A mil (ps) fll (ps) mA fll mA fA mA fll mG fC mil fll mG fll mC fll mC (ps) fC (ps) mC 478 84 EU1043 EU1002-B mG (ps) mG (ps) mG mA mG mA fC fA fA mG mC mA mil mil mil mA mil (ps) mA (ps) mC 398 4 EU1044 EU1044-A mG (ps) fG (ps) mG fA mA fC mil fll mC fll mil fA mG fG mC fll mil (ps) fA (ps) mG 479 85 EU1044 EU1044-B mC (ps) mil (ps) mA mA mG mC fC fll fA mA mG mA mA mG mil mil mC (ps) mC (ps) mC 480 86 EU1045 EU1045-A mil (ps) fll (ps) mG fll mil fG mG fC mil fll mil fG mA fG mG fA mG (ps) fG (ps) mC 481 87 EU1045 EU1045-B mG (ps) mC (ps) mC mil mC mC fll fC fA mA mA mG mC mC mA mA mC (ps) mA (ps) mA 482 88 EU1046 EU1046-A mil (ps) fll (ps) mC fll mA fG mG fG mG fll mC fll mG fC mA fG mil (ps) fC (ps) mil 483 89 EU1046 EU1046-B mA (ps) mG (ps) mA mC mil mG fC fA fG mA mC mC mC mC mil mA mG (ps) mA (ps) mA 484 90 EU1047 EU1047-A mA (ps) fll (ps) mA fll mC fll mG fG mC fA mC fA mil fC mC fG mil (ps) fC (ps) mil 485 91 EU1047 EU1047-B mA (ps) mG (ps) mA mC mG mG fA fll fG mil mG mC mC mA mG mA mil (ps) mA (ps) mil 486 92 EU1048 EU1048-A mG (ps) fll (ps) mA fG mG fC mil fG mA fA mG fll mG fG mA fG mil (ps) fC (ps) mil 487 93 EU1048 EU1020-B mA (ps) mG (ps) mA mC mil mC fC fA fC mil mil mC mA mG mC mC mil (ps) mA (ps) mC 434 40 EU1049 EU1049-A mil (ps) fG (ps) mA fA mG fll mG fG mA fG mil fC mil fG mil fG mA (ps) fC (ps) mA 488 94 EU1049 EU1049-B mil (ps) mG (ps) mil mC mA mC fA fG fA mC mil mC mC mA mC mil mil (ps) mC (ps) mA 489 95 EU1050 EU1050-A mG (ps) fll (ps) mil fC mC fll mG fG mA fA mG fll mC fll mA fC mG (ps) fll (ps) mA 490 96 EU1050 EU1050-B mil (ps) mA (ps) mC mG mil mA fG fA fC mil mil mC mC mA mG mG mA (ps) mA (ps) mC 491 97 EU1051 EU1051-A mil (ps) fll (ps) mG fll mA fG mG fC mil fG mA fA mG fll mG fG mA (ps) fG (ps) mil 492 98 EU1051 EU1051-B mA (ps) mC (ps) mil mC mC mA fC fll fll mC mA mG mC mC mil mA mC (ps) mA (ps) mG 493 99 EU1052 EU1052-A mA (ps) fA (ps) mA fC mC fA mG fG mG fA mA fC mil fll mC fll mil (ps) fA (ps) mG 494 100 EU1052 EU1052-B mC (ps) mil (ps) mA mA mG mA fA fG fll mil mC mC mC mil mG mG mil (ps) mil (ps) mil 495 101 EU1053 EU1053-A mG (ps) fA (ps) mil fA mA fll mG fA mG fA mA fll mil fC mA fA mA (ps) fA (ps) mG 496 102 EU1053 EU1053-B mC (ps) mil (ps) mil mil mil mG fA fA fll mil mC mil mC mA mil mil mA (ps) mil (ps) mC 497 103 EU1054 EU1054-A mC (ps) fll (ps) mA fA mG fC mA fll mC fC mil fC mC fC mil fC mA (ps) fG (ps) mC 498 104 EU1054 EU1054-B mG (ps) mC (ps) mil mG mA mG fG fG fA mG mG mA mil mG mC mil mil (ps) mA (ps) mG 499 105

[1276]

[1277] EU1055 EU1055-A mA (ps) fll (ps) mil fA mA fG mA fA mA fG mil fA mil fA mA fG mC (ps) fC (ps) mA 500 106 EU1055 EU1055-B mil (ps) mG ( ps) mG mC mil mil fA fll fA mC mil mil mil mC mil mil mA ( ps) mA (ps) mil 501 107 EU1056 EU1056-A mil (ps) fll (ps) mil fC mC fll mG fA mC fll mC fC mil fG mil fll mil (ps) fC (ps) mil 502 108 EU1056 EU1056-B mA (ps) mG (ps) mA mA mA mC fA fG fG mA mG mil mC mA mG mG mA (ps) mA (ps) mA 503 109 EU1057 EU1057-A mA (ps) fA (ps) mG fll mA fll mA fA mG fC mC fA mG fG mC fG mC (ps) fG (ps) mG 504 110 EU1057 EU1057-B mC (ps) mC (ps) mG mC mG mC fC fll fG mG mC mil mil mA mil mA mC (ps) mil (ps) mil 505 111 EU1058 EU1058-A mG (ps) fA (ps) mA fA mA fA mC fC mA fG mG fG mA fA mC fll mil (ps) fC (ps) mil 506 112 EU1058 EU1058-B mA (ps) mG (ps) mA mA mG mil fll fC fC mC mil mG mG mil mil mil mil (ps) mil (ps) mC 507 113 EU1059 EU1059-A mC (ps) fll (ps) mC fA mil fC mil fll mG fG mG fA mG fA mG fG mC (ps) fll (ps) mA 508 114 EU1059 EU1059-B mil (ps) mA (ps) mG mC mC mil fC fll fC mC mC mA mA mG mA mil mG (ps) mA (ps) mG 509 115 EU1060 EU1060-A mC (ps) fA (ps) mil fll mC fC mA fll mil fA mA fC mA fA mil fG mA (ps) fll (ps) mG 510 116 EU1060 EU1060-B mC (ps) mA (ps) mil mC mA mil fll fG fll mil mA mA mil mG mG mA mA (ps) mil (ps) mG 511 117 EU1061 EU1061-A mA (ps) fA (ps) mC fC mA fG mG fG mA fA mC fll mil fC mil fll mA (ps) fG (ps) mG 512 118 EU1061 EU1061-B mC (ps) mC (ps) mil mA mA mG fA fA fG mil mil mC mC mC mil mG mG (ps) mil (ps) mil 513 119 EU1062 EU1062-A mA (ps) fG (ps) mG fA mil fll mil fG mC fll mG fC mil fll mG fG mC (ps) fll (ps) mA 514 120 EU1062 EU1062-B mil (ps) mA (ps) mG mC mC mA fA fG fC mA mG mC mA mA mA mil mC (ps) mC (ps) mil 515 121 EU1063 EU1063-A mA (ps) fG (ps) mA fll mG fA mil fG mil fA mA fll mC fA mC fA mil (ps) fG (ps) mil 516 122 EU1063 EU1063-B mA (ps) mC (ps) mA mil mG mil fG fA fll mil mA mC mA mil mC mA mil (ps) mC (ps) mil 517 123 EU1064 EU1064-A mil (ps) fA (ps) mil fG mA fll mC fC mA fG mG fll mA fG mA fG mG (ps) fA (ps) mG 518 124 EU1064 EU1064-B mC (ps) mil (ps) mC mC mil mC fll fA fC mC mil mG mG mA mil mC mA (ps) mil (ps) mA 519 125 EU1065 EU1065-A mil (ps) fA (ps) mG fll mC fll mA fG mil fll mG fC mA fG mil fll mil (ps) fC (ps) mA 520 126 EU1065 EU1065-B mil (ps) mG (ps) mA mA mA mC fll fG fC mA mA mC mil mA mG mA mC (ps) mil (ps) mG 521 127 EU1066 EU1066-A mA (ps) fll (ps) mC fC mA fG mG fll mA fG mA fG mG fA mG fA mG (ps) fA (ps) mG 522 128 EU1066 EU1066-B mC (ps) mil (ps) mC mil mC mil fC fC fll mC mil mA mC mC mil mG mG (ps) mA (ps) mil 523 129 EU1067 EU1067-A mil (ps) fA (ps) mA fC mA fC mA fll mC fA mG fC mC fA mA fC mC (ps) fll (ps) mG 524 130 EU1067 EU1067-B mC (ps) mA (ps) mG mG mil mil fG fG fC mil mG mA mil mG mil mG mil (ps) mil (ps) mG 525 131 EU1068 EU1068-A mA (ps) fA (ps) mG fA mil fA mA fll mG fA mG fA mA fll mil fC mA (ps) fA (ps) mA 526 132 EU1068 EU1068-B mil (ps) mil (ps) mil mG mA mA fll fll fC mil mC mA mil mil mA mil mC (ps) mil (ps) mil 527 133 EU1069 EU1069-A mA (ps) fA (ps) mil fG mC fll mil fG mil fC mil fC mC fC mA fG mil (ps) fG (ps) mG 528 134 EU1069 EU1069-B mC (ps) mC (ps) mA mC mil mG fG fG fA mG mA mC mA mA mG mC mA (ps) mil (ps) mil 529 135 EU1070 EU1070-A mil (ps) fA (ps) mil fC mil fll mA fA mG fC mil fC mil fA mG fG mA (ps) fA (ps) mG 530 136 EU1070 EU1070-B mC (ps) mil (ps) mil mC mC mil fA fG fA mG mC mil mil mA mA mG mA (ps) mil (ps) mC 531 137 EU1071 EU1071-A mA (ps) fll (ps) mil fG mG fC mil fC mG fG mA fll mC fll mil fA mA (ps) fG (ps) mC 532 138 EU1071 EU1071-B mG (ps) mC (ps) mil mil mA mA fG fA fll mC mC mG mA mG mC mC mA (ps) mA (ps) mil 533 139 EU1072 EU1072-A mG (ps) fA (ps) mil fC mil fll mA fA mG fC mil fC mil fA mG fG mA (ps) fA (ps) mG 534 140 EU1072 EU1070-B mC (ps) mil (ps) mil mC mC mil fA fG fA mG mC mil mil mA mA mG mA (ps) mil (ps) mC 531 137 EU1073 EU1073-A mil (ps) fC (ps) mil fA mG fll mil fG mC fA mG fll mil fll mC fA mG (ps) fG (ps) mA 535 141 EU1073 EU1073-B mil (ps) mC (ps) mC mil mG mA fA fA fC mil mG mC mA mA mC mil mA (ps) mG (ps) mA 536 142 EU1074 EU1074-A mC (ps) fA (ps) mA fC mA fC mA fll mC fA mG fC mC fA mA fC mC (ps) fll (ps) mG 537 143

[1278]

[1279] EU1074 EU1067-B mC (ps) mA (ps) mG mG mil mil fG fG fC mil mG mA mil mG mil mG mil (ps) mil (ps) mG 525 131 EU1075 EU1075-A mC (ps) fU (ps) mG fC mA fA mC fA mil fA mA fG mG fG mG fG mil (ps) fC ( ps) mG 538 144 EU1075 EU1075-B mC (ps) mG (ps) mA mC mC mC fC fC fU mil mA mil mG mil mil mG mC (ps) mA (ps) mG 539 145 EU1076 EU1076-A mG (ps) fG (ps) mA fU mil fU mG fC mil fG mC fU mil fG mG fC mil (ps) fA (ps) mG 540 146 EU1076 EU1076-B mC (ps) mil (ps) mA mG mC mC fA fA fG mC mA mG mC mA mA mA mil (ps) mC (ps) mC 541 147 EU1077 EU1077-A mil (ps) fG (ps) mG fA mG fG mA fU mG fA mG fU mil fA mil fU mC (ps) fU (ps) mG 542 148 EU1077 EU1077-B mC (ps) mA (ps) mG mA mA mil fA fA fC mil mC mA mil mC mC mil mC (ps) mC (ps) mA 543 149 EU1078 EU1078-A mG (ps) fC (ps) mil fC mG fG mA fU mC fU mil fA mA fG mC fU mC (ps) fU (ps) mA 544 150 EU1078 EU1078-B mil (ps) mA (ps) mG mA mG mC fU fU fA mA mG mA mil mC mC mG mA (ps) mG (ps) mC 545 151 EU1079 EU1079-A mC (ps) fU (ps) mG fU mA fG mG fC mil fG mA fA mG fU mG fG mA (ps) fG (ps) mil 546 152 EU1079 EU1051-B mA (ps) mC (ps) mil mC mC mA fC fU fU mC mA mG mC mC mil mA mC (ps) mA (ps) mG 493 99 EU1080 EU1080-A mil (ps) fG (ps) mA fU mil fU mG fC mil fG mC fU mil fG mG fC mil (ps) fA (ps) mG 547 153 EU1080 EU1076-B mC (ps) mil (ps) mA mG mC mC fA fA fG mC mA mG mC mA mA mA mil (ps) mC (ps) mC 541 147 EU1081 EU1081-A mil (ps) fG (ps) mA fG mG fA mil fG mA fG mil fU mA fU mil fC mil (ps) fG (ps) mG 548 154 EU1081 EU1031-B mC (ps) mC (ps) mA mG mA mA fU fA fA mC mil mC mA mil mC mC mil (ps) mC (ps) mC 456 62 EU1082 EU1082-A mil (ps) fU (ps) mil fC mA fG mG fA mC fA mC fC mA fG mA fC mil (ps) fU (ps) mC 549 155 EU1082 EU1082-B mG (ps) mA (ps) mA mG mil mC fU fG fG mil mG mil mC mC mil mG mA (ps) mA (ps) mA 550 156 EU1083 EU1083-A mC (ps) fC (ps) mC fA mG fG mil fU mG fG mil fG mA fU mG fU mG (ps) fG (ps) mil 551 157 EU1083 EU1083-B mA (ps) mC (ps) mC mA mC mA fU fC fA mC mC mA mA mC mC mil mG (ps) mG (ps) mG 552 158 EU1084 EU1084-A mil (ps) fA (ps) mil fA mA fA mil fG mC fU mil fG mil fC mil fC mC (ps) fC (ps) mA 553 159 EU1084 EU1084-B mil (ps) mG (ps) mG mG mA mG fA fC fA mA mG mC mA mil mil mil mA (ps) mil (ps) mA 554 160 EU1085 EU1085-A mil (ps) fU (ps) mG fG mA fA mG fU mC fU mA fC mG fU mA fA mil (ps) fG (ps) mG 555 161 EU1085 EU1085-B mC (ps) mC (ps) mA mil mil mA fC fG fU mA mG mA mC mil mil mC mC (ps) mA (ps) mG 556 162 EU1086 EU1086-A mil (ps) fC (ps) mA fG mG fA mil fU mil fG mC fU mG fC mil fU mG (ps) fG (ps) mC 557 163 EU1086 EU1004-B mG (ps) mC (ps) mC mA mA mG fC fA fG mC mA mA mA mil mC mC mil (ps) mG (ps) mG 402 8 EU1087 EU1087-A mil (ps) fU (ps) mil fC mil fC mil fG mC fC mil fU mC fC mC fU mC (ps) fC (ps) mC 558 164 EU1087 EU1087-B mG (ps) mG (ps) mG mA mG mG fG fA fA mG mG mC mA mG mA mG mA (ps) mA (ps) mA 559 165 EU1088 EU1088-A mG (ps) fA (ps) mG fU mil fA mil fU mC fU mG fG mG fA mC fG mA (ps) fC (ps) mil 560 166 EU1088 EU1088-B mA (ps) mG (ps) mil mC mG mil fC fC fC mA mG mA mA mil mA mA mC (ps) mil (ps) mC 561 167 EU1089 EU1089-A mil (ps) fA (ps) mC fC mA fC mA fU mil fG mC fC mA fU mil fA mil (ps) fG (ps) mA 562 168 EU1089 EU1021-B mil (ps) mC (ps) mA mil mA mA fU fG fG mC mA mA mil mG mil mG mG (ps) mil (ps) mC 436 42 EU1090 EU1090-A mG (ps) fA (ps) mA fU mG fG mA fA mA fG mA fG mG fC mA fG mC (ps) fA (ps) mA 563 169 EU1090 EU1090-B mil (ps) mil (ps) mG mC mil mG fC fC fU mC mil mil mil mC mC mA mil (ps) mil (ps) mC 564 170 EU1091 EU1091-A mA (ps) fC (ps) mA fA mG fA mA fA mG fU mG fC mC fC mA fU mil (ps) fU (ps) mG 565 171 EU1091 EU1091-B mC (ps) mA (ps) mA mA mil mG fG fG fC mA mC mil mil mil mC mil mil (ps) mG (ps) mil 566 172 EU1092 EU1092-A mil (ps) fU (ps) mG fC mil fU mA fC mC fC mil fG mC fU mil fC mA (ps) fA (ps) mG 567 173 EU1092 EU1092-B mC (ps) mil (ps) mil mG mA mA fG fC fA mG mG mG mil mA mA mG mC (ps) mA (ps) mG 568 174 EU1093 EU1093-A mC (ps) fC (ps) mil fU mC fU mA fG mG fG mG fU mC fU mG fC mA (ps) fG (ps) mil 569 175 EU1093 EU1093-B mA (ps) mC (ps) mil mG mC mA fG fA fC mC mC mC mil mA mG mA mA (ps) mG (ps) mG 570 176

[1280]

[1281] EU1094 EU1094-A mil (ps) fC (ps) mil fU mA fA mG fC mil fC mil fA mG fG mA fA mG (ps) fG (ps) mG 571 177 EU1094 EU1094-B mC (ps) mC (ps) mC mil mil mC fC fll fA mG mA mG mC mil mil mA mA (ps) mG ( ps) mA 572 178 EU1095 EU1O95-A mC (ps) fll (ps) mC fG mG fA mil fC mil fll mA fA mG fC mil fC mil (ps) fA (ps) mG 573 179 EU1095 EU1O95-B mC (ps) mil (ps) mA mG mA mG fC fll fll mA mA mG mA mil mC mC mG (ps) mA (ps) mG 574 180 EU1096 EU1096-A mil (ps) fG (ps) mA fG mA fA mil fll mC fA mA fA mA fG mG fC mA (ps) fA (ps) mA 575 181 EU1096 EU1096-B mil (ps) mil (ps) mil mG mC mC fll fll fll mil mG mA mA mil mil mC mil (ps) mC (ps) mA 576 182 EU1097 EU1097-A mG (ps) fll (ps) mC fA mA fG mil fG mA fG mil fC mA fll mA fll mil (ps) fG (ps) mC 577 183 EU1097 EU1097-B mG (ps) mC (ps) mA mA mil mA fll fG fA mC mil mC mA mC mil mil mG (ps) mA (ps) mC 578 184 EU1098 EU1098-A mA (ps) fA (ps) mA fG mA fC mG fG mC fA mG fA mA fll mG fG mA (ps) fA (ps) mG 579 185 EU1098 EU1098-B mC (ps) mil (ps) mil mC mC mA fll fll fC mil mG mC mC mG mil mC mil (ps) mil (ps) mil 580 186 EU1099 EU1O99-A mA (ps) fA (ps) mG fC mA fC mA fC mA fll mil fC mC fA mil fll mA (ps) fA (ps) mC 581 187 EU1099 EU1O99-B mG (ps) mil (ps) mil mA mA mil fG fG fA mA mil mG mil mG mil mG mC (ps) mil (ps) mil 582 188 EU1100 EU11OO-A mG (ps) fG (ps) mA fA mG fll mC fll mA fC mG fll mA fA mil fG mG (ps) fll (ps) mC 583 189 EU11OO EU1100-B mG (ps) mA (ps) mC mC mA mil fll fA fC mG mil mA mG mA mC mil mil (ps) mC (ps) mC 584 190 EU11O1 EU1101-A mil (ps) fA (ps) mA fA mG fll mG fC mC fC mA fll mil fll mG fG mG (ps) fll (ps) mC 585 191 EU11O1 EU1005-B mG (ps) mA (ps) mC mC mC mA fA fA fll mG mG mG mC mA mC mil mil (ps) mil (ps) mC 404 10 EU11O2 EU1102-A mA (ps) fll (ps) mC fll mil fA mA fG mC fll mC fll mA fG mG fA mA (ps) fG (ps) mG 586 192 EU11O2 EU1102-B mC (ps) mC (ps) mil mil mC mC fll fA fG mA mG mC mil mil mA mA mG (ps) mA (ps) mil 587 193 EU11O3 EU1103-A mG (ps) fA (ps) mA fA mG fll mA fll mA fA mA fll mG fC mil fll mG (ps) fll (ps) mC 588 194 EU11O3 EU1103-B mG (ps) mA (ps) mC mA mA mG fC fA fll mil mil mA mil mA mC mil mil (ps) mil (ps) mC 589 195 EU1104 EU1104-A mC (ps) fll (ps) mG fG mG fA mA fA mC fll mil fC mA fll mC fll mil (ps) fG (ps) mG 590 196 EU1104 EU1104-B mC (ps) mC (ps) mA mA mG mA fll fG fA mA mG mil mil mil mC mC mC (ps) mA (ps) mG 591 197 EU11O5 EU1105-A mA (ps) fG (ps) mA fC mA fA mG fA mA fA mG fll mG fC mC fC mA (ps) fll (ps) mil 592 198 EU11O5 EU1105-B mA (ps) mA (ps) mil mG mG mG fC fA fC mil mil mil mC mil mil mG mil (ps) mC (ps) mil 593 199 EU1106 EU1106-A mil (ps) fA (ps) mA fG mA fA mA fG mil fA mil fA mA fG mC fC mA (ps) fG (ps) mG 594 200 EU1106 EU1106-B mC (ps) mC (ps) mil mG mG mC fll fll fA mil mA mC mil mil mil mC mil (ps) mil (ps) mA 595 201 EU1107 EU1107-A mG (ps) fA (ps) mG fG mA fG mil fG mG fA mC fA mG fG mil fG mA (ps) fA (ps) mA 596 202 EU1107 EU1107-B mil (ps) mil (ps) mil mC mA mC fC fll fG mil mC mC mA mC mil mC mC (ps) mil (ps) mC 597 203 EU1108 EU1108-A mil (ps) fC (ps) mA fA mG fll mG fA mG fll mC fA mil fA mil fll mG (ps) fC (ps) mC 598 204 EU1108 EU1108-B mG (ps) mG (ps) mC mA mA mil fA fll fG mA mC mil mC mA mC mil mil (ps) mG (ps) mA 599 205 EU11O9 EU1109-A mG (ps) fC (ps) mil fll mil fG mA fG mG fA mG fG mC fll mG fA mA (ps) fG (ps) mA 600 206 EU11O9 EU1109-B mil (ps) mC (ps) mil mil mC mA fG fC fC mil mC mC mil mC mA mA mA (ps) mG (ps) mC 601 207 EU111O EU1110-A mA (ps) fA (ps) mil fll mil fll mA fA mG fA mil fA mA fll mG fA mG (ps) fA (ps) mA 602 208 EU111O EU1110-B mil (ps) mil (ps) mC mil mC mA fll fll fA mil mC mil mil mA mA mA mA (ps) mil (ps) mil 603 209 EU1111 EU1111-A mG (ps) fA (ps) mil fll mG fll mil fG mG fC mil fll mil fG mA fG mG (ps) fA (ps) mG 604 210 EU1111 EU1111-B mC (ps) mil (ps) mC mC mil mC fA fA fA mG mC mC mA mA mC mA mA (ps) mil (ps) mC 605 211 EU1112 EU1112-A mG (ps) fG (ps) mA fll mil fG mil fll mG fG mC fll mil fll mG fA mG (ps) fG (ps) mA 606 212 EU1112 EU1112-B mil (ps) mC (ps) mC mil mC mA fA fA fG mC mC mA mA mC mA mA mil (ps) mC (ps) mC 607 213 EU1113 EU1113-A mil (ps) fA (ps) mA fC mA fA mil fG mA fll mG fll mC fA mG fA mA (ps) fA (ps) mG 608 214

[1282]

[1283] EU1113 EU1113-B mC (ps) mil (ps) mil mil mC mil fG fA fC mA mil mC mA mil mil mG mil (ps) mil (ps) mA 609 215 EU1114 EU1114-A mA ( ps) fA ( ps) mG fC mil fG mA fU mG fA mC fC mil fC mC fll mC (ps) fC ( ps) mC 610 216 EU1114 EU1114-B mG (ps) mG (ps) mG mA mG mG fA fG fG mil mC mA mil mC mA mG mC (ps) mil (ps) mil 611 217 EU1115 EU1115-A mG (ps) fG (ps) mA fA mil fC mil fG mA fll mG fC mC fll mC fC mA (ps) fG (ps) mil 612 218 EU1115 EU1115-B mA (ps) mC (ps) mil mG mG mA fG fG fC mA mil mC mA mG mA mil mil (ps) mC (ps) mC 613 219 EU1116 EU1116-A mA (ps) fG (ps) mA fA mA fG mil fA mil fA mA fG mC fC mA fG mG (ps) fC (ps) mG 614 220 EU1116 EU1116-B mC (ps) mG (ps) mC mC mil mG fG fC fll mil mA mil mA mC mil mil mil (ps) mC (ps) mil 615 221 EU1117 EU1117-A mil (ps) fll (ps) mA fll mil fA mA fG mA fA mA fG mil fA mil fA mA (ps) fG (ps) mC 616 222 EU1117 EU1117-B mG (ps) mC (ps) mil mil mA mil fA fC fll mil mil mC mil mil mA mA mil (ps) mA (ps) mA 617 223 EU1118 EU1118-A mil (ps) fC (ps) mil fA mil fC mil fG mC fll mil fC mC fll mC fC mil (ps) fC (ps) mC 618 224 EU1118 EU1118-B mG (ps) mG (ps) mA mG mG mA fG fG fA mA mG mC mA mG mA mil mA (ps) mG (ps) mA 619 225 EU1119 EU1119-A mil (ps) fll (ps) mil fA mA fG mA fll mA fA mil fG mA fG mA fA mil (ps) fll (ps) mC 620 226 EU1119 EU1119-B mG (ps) mA (ps) mA mil mil mC fll fC fA mil mil mA mil mC mil mil mA (ps) mA (ps) mA 621 227 EU1120 EU1120-A mG (ps) fll (ps) mA fll mil fA mil fll mA fll mG fA mA fA mA fll mA (ps) fG (ps) mC 622 228 EU1120 EU1120-B mG (ps) mC (ps) mil mA mil mil fll fll fC mA mil mA mA mil mA mA mil (ps) mA (ps) mC 623 229 EU1121 EU1121-A mA (ps) fll (ps) mil fG mil fll mG fG mC fll mil fll mG fA mG fG mA (ps) fG (ps) mG 624 230 EU1121 EU1121-B mC (ps) mC (ps) mil mC mC mil fC fA fA mA mG mC mC mA mA mC mA (ps) mA (ps) mil 625 231 EU1122 EU1122-A mil (ps) fC (ps) mil fll mG fG mil fC mil fC mil fll mC fA mC fll mC (ps) fC (ps) mA 626 232 EU1122 EU1122-B mil (ps) mG (ps) mG mA mG mil fG fA fA mG mA mG mA mC mC mA mA (ps) mG (ps) mA 627 233 EU1123 EU1123-A mA (ps) fA (ps) mil fG mA fG mA fA mil fll mC fA mA fA mA fG mG (ps) fC (ps) mA 628 234 EU1123 EU1123-B mil (ps) mG (ps) mC mC mil mil fll fll fG mA mA mil mil mC mil mC mA (ps) mil (ps) mil 629 235 EU1124 EU1124-A mA (ps) fG (ps) mil fll mA fll mil fC mil fG mG fG mA fC mG fA mC (ps) fll (ps) mG 630 236 EU1124 EU1124-B mC (ps) mA (ps) mG mil mC mG fll fC fC mC mA mG mA mA mil mA mA (ps) mC (ps) mil 631 237 EU1125 EU1125-A mil (ps) fA (ps) mC fA mG fll mA fG mC fA mA fA mG fC mil fG mA (ps) fll (ps) mG 632 238 EU1125 EU1125-B mC (ps) mA (ps) mil mC mA mG fC fll fll mil mG mC mil mA mC mil mG (ps) mil (ps) mA 633 239 EU1126 EU1126-A mil (ps) fG (ps) mG fC mA fC mA fll mC fC mG fll mC fll mil fG mA (ps) fC (ps) mC 634 240 EU1126 EU1126-B mG (ps) mG (ps) mil mC mA mA fG fA fC mG mG mA mil mG mil mG mC (ps) mC (ps) mA 635 241 EU1127 EU1127-A mil (ps) fC (ps) mil fC mA fG mA fC mA fA mG fA mA fA mG fll mG (ps) fC (ps) mC 636 242 EU1127 EU1127-B mG (ps) mG (ps) mC mA mC mil fll fll fC mil mil mG mil mC mil mG mA (ps) mG (ps) mA 637 243 EU1128 EU1128-A mC (ps) fC (ps) mA fG mil fll mC fC mil fG mG fA mA fG mil fC mil (ps) fA (ps) mC 638 244 EU1128 EU1128-B mG (ps) mil (ps) mA mG mA mC fll fll fC mC mA mG mG mA mA mC mil (ps) mG (ps) mG 639 245 EU1129 EU1129-A mG (ps) fll (ps) mC fll mil fG mA fC mC fA mC fA mil fll mG fC mC (ps) fA (ps) mil 640 246 EU1129 EU1129-B mA (ps) mil (ps) mG mG mC mA fA fll fG mil mG mG mil mC mA mA mG (ps) mA (ps) mC 641 247 EU1130 EU1130-A mC (ps) fA (ps) mG fll mC fll mA fG mil fll mG fC mA fG mil fll mil (ps) fC (ps) mA 642 248 EU1130 EU1065-B mil (ps) mG (ps) mA mA mA mC fll fG fC mA mA mC mil mA mG mA mC (ps) mil (ps) mG 521 127 EU1131 EU1131-A mG (ps) fll (ps) mil fA mil fll mC fll mG fG mG fA mC fG mA fC mil (ps) fG (ps) mG 643 249 EU1131 EU1131-B mC (ps) mC (ps) mA mG mil mC fG fll fC mC mC mA mG mA mA mil mA (ps) mA (ps) mC 644 250 EU1132 EU1132-A mil (ps) fll (ps) mG fC mil fG mC fll mil fG mG fC mil fA mG fC mil (ps) fC (ps) mC 645 251 EU1132 EU1132-B mG (ps) mG (ps) mA mG mC mil fA fG fC mC mA mA mG mC mA mG mC (ps) mA (ps) mA 646 252

[1284]

[1285] EU1133 EU1133-A mil (ps) fll (ps) mil fG mC fll mG fC mil fll mG fG mC fll mA fG mC (ps) fll (ps) mC 647 253 EU1133 EU1133-B mG ( ps) mA (ps) mG mC mil mA fG fC fC mA mA mG mC mA mG mC mA (ps) mA ( ps) mA 648 254 EU1134 EU1134-A mC (ps) fU (ps) mG fC mil fll mA fC mC fC mil fG mC fll mil fC mA (ps) fA (ps) mG 649 255 EU1134 EU1092-B mC (ps) mil (ps) mil mG mA mA fG fC fA mG mG mG mil mA mA mG mC (ps) mA (ps) mG 568 174 EU1135 EU1135-A mA (ps) fA (ps) mG fG mA fll mil fG mil fll mG fG mC fll mil fll mG (ps) fA (ps) mG 650 256 EU1135 EU1135-B mC (ps) mil (ps) mC mA mA mA fG fC fC mA mA mC mA mA mil mC mC (ps) mil (ps) mil 651 257 EU1136 EU1136-A mA (ps) fll (ps) mil fC mA fA mA fA mG fG mC fA mA fA mil fA mA (ps) fC (ps) mA 652 258 EU1136 EU1136-B mil (ps) mG (ps) mil mil mA mil fll fll fG mC mC mil mil mil mil mG mA (ps) mA (ps) mil 653 259 EU1137 EU1137-A mG (ps) fA (ps) mA fG mA fC mG fG mC fA mG fA mA fll mG fG mA (ps) fA (ps) mA 654 260 EU1137 EU1137-B mil (ps) mil (ps) mil mC mC mA fll fll fC mil mG mC mC mG mil mC mil (ps) mil (ps) mC 655 261 EU1138 EU1138-A mA (ps) fC (ps) mA fA mil fll mil fll mA fA mG fA mil fA mA fll mG (ps) fA (ps) mG 656 262 EU1138 EU1138-B mC (ps) mil (ps) mC mA mil mil fA fll fC mil mil mA mA mA mA mil mil (ps) mG (ps) mil 657 263 EU1139 EU1139-A mG (ps) fll (ps) mA fG mA fC mC fC mC fll mil fll mA fG mA fA mG (ps) fA (ps) mA 658 264 EU1139 EU1139-B mil (ps) mil (ps) mC mil mil mC fll fA fA mA mG mG mG mG mil mC mil (ps) mA (ps) mC 659 265 EU1140 EU1140-A mA (ps) fll (ps) mC fll mil fG mG fll mC fll mC fll mil fC mA fC mil (ps) fC (ps) mC 660 266 EU1140 EU1140-B mG (ps) mG (ps) mA mG mil mG fA fA fG mA mG mA mC mC mA mA mG (ps) mA (ps) mil 661 267 EU1141 EU1141-A mA (ps) fA (ps) mG fll mA fll mA fA mA fll mG fC mil fll mG fll mC (ps) fll (ps) mC 662 268 EU1141 EU1141-B mG (ps) mA (ps) mG mA mC mA fA fG fC mA mil mil mil mA mil mA mC (ps) mil (ps) mil 663 269 EU1142 EU1142-A mA (ps) fA (ps) mC fll mil fC mA fll mC fll mil fG mG fll mC fll mC (ps) fll (ps) mil 664 270 EU1142 EU1142-B mA (ps) mA (ps) mG mA mG mA fC fC fA mA mG mA mil mG mA mA mG (ps) mil (ps) mil 665 271 EU1143 EU1143-A mil (ps) fG (ps) mA fA mA fA mA fC mC fA mG fG mG fA mA fC mil (ps) fll (ps) mC 666 272 EU1143 EU1143...

Claims

Claims1. A double-stranded nucleic acid for inhibiting expression of INHBE, wherein the nucleic acid comprises a first strand and a second strand, wherein the unmodified equivalent of the first strand sequence comprises a sequence of at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the first strand sequences of SEQ ID No. 388, 200, 226, 292, 296, 378, 382, or 387.

2. The nucleic acid of claim 1, wherein the unmodified equivalent of the second strand sequence comprises a sequence from any one of the corresponding second strand sequences of SEQ ID No. 186, 201, 227, 293, 297, 379, 18, or 184.

3. The nucleic acid of claim 1, wherein the first strand sequence comprises a sequence corresponding to nucleotides 2 to 18 from the 5’ end of any one of the first strand sequences with a given SEQ ID No. 388, 200, 226, 292, 296, 378, 382, or 387.

4. The nucleic acid of claim 3, wherein the second strand sequence comprises a sequence corresponding to nucleotides 2 to 18 from the 5’ end of the corresponding second strand sequence with a given SEQ ID No. 186, 201, 227, 293, 297, 379, 18, or 184.

5. The nucleic acid of any one of the preceding claims, wherein the first strand and the second strand form a duplex region of 17-25 nucleotides in length.

6. The nucleic acid of any one of the preceding claims, wherein the first strand and the second strand form a duplex region of 19 nucleotides in length.

7. The nucleic acid of any one of the preceding claims, wherein at least one nucleotide of the first and / or second strand is a modified nucleotide.

8. The nucleic acid of any one of the preceding claims, wherein at least nucleotides 2 and 14 of the first strand are modified by a first modification, the nucleotides being numbered consecutively starting with nucleotide number 1 of the duplex at the 5’ end of the first strand sequence, wherein said first modification is 2’-F.

9. The nucleic acid of any one of the preceding claims, wherein the first strand sequence with a given SEQ ID No has a terminal 5’ (E)-vinylphosphonate nucleotide at its 5’ end.

10. The nucleic acid of any one of the preceding claims comprising one or more LNA nucleotides.

11. The nucleic acid of any one of the preceding claims comprising an LNA nucleotide at the penultimate position of the double stranded region at the 3’ end of the first strand.

12. The nucleic acid of any one of the preceding claims, wherein the nucleic acid comprises a phosphorothioate linkage between the terminal two or three 3’ nucleotides and / or 5’ nucleotides of the first and / or the second strand.

13. The nucleic acid of any one of the preceding claims, wherein the linkages between the remaining nucleotides of the first strand and / or of the second strand are phosphodiester linkages.

14. The nucleic acid of any one of the preceding claims, wherein the first and the second strands are partially complementary, having a mismatch at position 1 at 5’ end of the nucleic acid.

15. The nucleic acid of any one of the preceding claims, wherein the nucleic acid is conjugated to a heterologous moiety.

16. The nucleic acid of claim 15, wherein the heterologous moiety comprises (i) one or more N-acetyl galactosamine (GalNAc) moieties or derivatives thereof, and (ii) a linker, wherein the linker conjugates the at least one GalNAc moiety or derivative thereof to the nucleic acid.

17. The nucleic acid of claim 16, wherein the heterologous moiety comprises, orOHwherein Z is the nucleic acid according to any one of claims 1-14.

18. A composition comprising a nucleic acid of any one of the preceding claims and a solvent and / or a delivery vehicle and / or a physiologically acceptable excipient and / or a carrier and / or a salt and / or a diluent and / or a buffer and / or a preservative and / or a further therapeutic agent selected from the group comprising an oligonucleotide, a small molecule, a monoclonal antibody, a polyclonal antibody and a peptide.

19. A nucleic acid of any one of claims 1 to 17 or a composition of claim 18 for use as a therapeutic agent.

20. A nucleic acid of any one of claims 1 to 17 or a composition of claim 18 for use in the prophylaxis or treatment of a disease, disorder or syndrome, wherein the disease, disorder or syndrome is an INHBE-mediated disease, disorder or syndrome.

Images