This application claims priority from GB 2111463.2 filed 09 August 2021 and GB 2209388.4 filed 27 June 2022, the contents and elements of which are herein incorporated by reference for all purposes.

Technical Field

The present disclosure relates to the fields of molecular biology, more specifically nucleic acid technology. The present disclosure also relates to methods of medical treatment and prophylaxis.

Background

Hypercholesterolemia is a condition that in most cases occurs as a consequence of a high-fat diet and inactivity, combined with genetic risk factors. High levels of cholesterol and in particular high levels of low- density lipoprotein (LDL) are well-recognised risk factors for the development of cardiovascular disease.

Hypercholesterolemia also occurs in an inherited form, as a consequence of genetic mutation to genes encoding proteins that influence the level of LDL in the blood (familial hypercholesterolemia). Certain mutations in the gene encoding the low-density lipoprotein receptor (LDLR) reduce or prevent LDLR- mediated removal of LDL-C from the blood. Similarly, mutations in the gene encoding apolipoprotein B (ApoB) can give rise to hypercholesterolemia if they reduce the ability of ApoB to facilitate binding of LDL particles to LDLR. Hypercholesterolemia can also occur as a consequence of gain-of-fanction mutations in the gene encoding the proprotein convertase subtilisin/kexin type 9 (PCSK9). Binding of PCSK9 to the LDLR directs it to be degraded, and so increased degradation of LDLR and consequent reduction in the uptake of LDL-C from the bloodstream can result in hypercholesterolemia.

Targeted inhibition of PCSK9 is a promising strategy for the treatment and prevention of hypercholesterolemia, and diseases/conditions associated with hypercholesterolemia. Fitzgerald at al., NEJM (2017) 376 (1): 41-51 and WO 2014/089313 A1 describe siRNA targeting PCSK9.

Summary

In a first aspect, the present disclosure provides an inhibitory nucleic acid for reducing gene and/or protein expression of PCSK9.

In some embodiments, the inhibitory nucleic acid comprises or encodes antisense nucleic acid targeting a nucleotide sequence comprising, or consisting of, one of SEQ ID NOs:9 to 213 or SEQ ID NO: 422 to 626.

In some embodiments, the inhibitory nucleic acid comprises or encodes antisense nucleic acid targeting a nucleotide sequence comprising, or consisting of, SEQ ID NO:12, 21, 44, 49, 53, 109, 195, 196, 197, 201, 202 or 206. In some embodiments, the inhibitory nucleic acid comprises or encodes antisense nucleic acid comprising or consisting of a nucleotide sequence having at least 75% sequence identity to one of SEQ ID NOs:214 to 418.

In some embodiments, the inhibitory nucleic add comprises or encodes antisense nudeic add comprising or consisting of a nucleotide sequence having at least 75% sequence identity to SEQ ID NO:217, 226, 249, 253, 258, 314, 400, 401, 402, 406, 407 or 411.

In some embodiments, the inhibitory nucleic acid comprises: (i) nucleic acid comprising the nucleotide sequence of one of SEQ ID NOs:214 to 418, or a nucleotide sequence having at least 75% sequence identity to one of SEQ ID NOs:214 to 418; and (ii) nudeic add comprising a nucleotide sequence having the reverse complement of the nudeotide sequence of (i), or having at least 75% sequence identity to the reverse complement of the nucleotide sequence of (i).

In some embodiments, the inhibitory nucleic acid comprises one or more modified nucleotides selected from: Z-O-methyluridine-S'-phosphate, 2 ^, -O-methyladenosine-3'-phosphate, 2'-O-methylguanosine-3'- phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-0-methyluridine-3'-phosphorothioate, 2'-O- methyladenosine-3'-phosphorothioate, 2'-0-methylguanosine-3'-phosphorothioate, 2'-O-methylcytidine- 3'-phosphorothioate, 2'-fluorouridine-3'-phosphate, 2'-fluoroadenosine-3'-phosphate, 2'-fluoroguanosine- 3-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluorocytidine-3'-phosphorothioate, 2'-fluoroguanosine-3'- phosphorothioate, 2'-fluoroadenosine-3'-phosphorothioate, and 2 ^, -fluorouridine-3'-phosphorothioate.

In some embodiments, the inhibitory nucleic acid comprises: (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) shown in any one of SEQ ID NOs:832 to 1071, 1236 to 1259, 1264 to 1287, 1292 to 1315, 1320 to 1343, 1378 to 1416, or 1429 to 1431; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) shown in any one of SEQ ID NOs:1072 to 1111, 1260 to 1263, 1288 to 1291, 1316 to 1319, 1344 to 1377, or 1417 to 1428.

In some embodiments, nudeic acid (i) comprises a 3’ overhang of 2 uracil residues (a 'UU* 3’ overhang).

In some embodiments, the inhibitory nucleic acid comprises: (i) nucleic acid comprising a nucleotide sequence (including the modifications thereto) indicated in column A of Table 1, and (II) nucleic acid comprising a nucleotide sequence (including the modifications thereto) indicated in column B of Table 1, wherein the sequences of columns A and B are selected from the same row of Table 1.

In some embodiments, the inhibitory nucleic acid comprises, or consists of: (i) nucleic acid comprising a nucleotide sequence (including the modifications thereto) indicated in column A of Table 2 (Example 2), and (ii) nucleic acid comprising a nucleotide sequence (including the modifications thereto) indicated in column B of Table 2, wherein the sequences of columns A and B are selected from the same row of Table 2. In some embodiments, the inhibitory nudeic acid comprises, or consists of (i) nucleic add comprising a nucleotide sequence (including the modifications thereto) indicated in column A of Table 3 (Example 5), and (ii) nudeic acid comprising a nucleotide sequence (including the modifications thereto) indicated in column B of Table 3, wherein the sequences of columns A and B are selected from the same row of Table 3.

In some embodiments, the inhibitory nucleic add comprises, or consists of (i) nucleic acid comprising a nudeotide sequence (including the modifications thereto) indicated in column A of Table 4 (Example 6), and (ii) nudeic acid comprising a nudeotide sequence (including the modifications thereto) indicated in column B of Table 4, wherein the sequences of columns A and B are selected from the same row of Table 4.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising a nucleotide sequence (including the modifications thereto) indicated in column A of Table 8 (Example 6), and (ii) nucleic acid comprising a nucleotide sequence (including the modifications thereto) indicated in column B of Table 8, wherein the sequences of columns A and B are selected from the same row of Table 8.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising a nudeotide sequence (including the modifications thereto) indicated in column A of Table 10 (Example 6), and (ii) nudeic acid comprising a nudeotide sequence (including the modifications thereto) indicated in column B of Table 10, wherein the sequences of columns A and B are selected from the same row of Table 10.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising a nucleotide sequence (including the modifications thereto) indicated in column A of Table 12 (Example 6), and (ii) nucleic add comprising a nucleotide sequence (including the modifications thereto) indicated in column B of Table 12, wherein the sequences of columns A and B are selected from the same row of Table 12.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic add comprising a nudeotide sequence (including the modifications thereto) indicated in column A of Table 14 (Example 6), and (ii) nudeic acid comprising a nucleotide sequence (including the modifications thereto) indicated in column B of Table 14, wherein the sequences of columns A and B are selected from the same row of Table 14.

In some embodiments, the inhibitory nudeic add comprises, or consists of (i) nucleic acid comprising a nucleotide sequence (including the modifications thereto) indicated in column A of Table 16 (Example 7), and (ii) nudeic acid comprising a nucleotide sequence (induding the modifications thereto) indicated in column B of Table 16, wherein the sequences of columns A and B are selected from the same row of Table 16. In some embodiments, the inhibitory nucleic acid may comprise or consist of at least one of (a) to (r) below:

(a) (I) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:905; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:1427;

(b) (I) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:920; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:1427;

(c) (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:922; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1426;

(d) (I) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:922; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1427;

(e) (I) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1246; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1428;

(f) (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1254; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1428;

(g) (I) nucleic add comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO: 1246; and (ii) nudeic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1419;

(h) (i) nucleic add comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO: 1246; and (ii) nudek; acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1420;

(I) (I) nudeic add comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO: 1246; and (ii) nudeic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1421;

(j) (i) nudeic add comprising the nudeotide sequence (induding the modifications thereto) of SEQ ID NO: 1246; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1422;

(k) (I) nucleic acid comprising the nucleotide sequence (induding the modifications thereto) of SEQ ID NO: 1246; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1423;

(l) (I) nudeic acid comprising the nucleotide sequence (induding the modifications thereto) of SEQ ID NO: 1246; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1424;

(m) (i) nucleic add comprising the nudeotide sequence (induding the modifications thereto) of SEQ ID NO: 1378; and (ii) nucleic acid comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO: 1425; (n) (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1378; and (II) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1427;

(o) (I) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1382; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1427;

(p) (I) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1383; and (II) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1425;

(q) (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1383; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1427; or

(r) (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1387; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1427.

In some embodiments, the inhibitory nucleic acid further comprises a moiety facilitating uptake of the inhibitory nucleic acid by hepatocytes.

In some embodiments, the inhibitory nucleic add is an siRNA

The present disclosure also provides a nucleic acid, optionally isolated, encoding an inhibitory nucleic acid according to the present disclosure.

The present disclosure also provides an expression vector, comprising a nucleic add according to the present disclosure.

The present disclosure also provides a composition comprising an inhibitory nucleic acid, nucleic acid or expression vector according to the present disclosure, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

The present disclosure also provides a cell comprising an inhibitory nucleic acid, a nucleic add, or an expression vector according to the present disdosure.

The present disclosure also provides an in vitro or in vivo method for reducing gene and/or protein expression of PCSK9 in a cell, comprising introducing an inhibitory nucleic acid, nucleic acid, or expression vector according to the present disclosure into a cell.

The present disclosure also provides a method of treatment, e.g. comprising reducing gene and/or protein expression of PCSK9 in a cell, comprising administering to a subject, e.g. into a cell of a subject, a therapeutically- or prophylactically-effective amount of an inhibitory nudeic acid, nudeic add, expression vector, or composition according to the present disclosure.

The present disdosure also provides the use of an inhibitory nudeic add, nucleic acid, expression vector, or composition according to the present disdosure, to reduce gene and/or protein expression of PCSK9 in vitro or in vivo.

The present disclosure also provides an inhibitory nucleic add, nucleic add, expression vector, or composition according to the present disclosure, for use in a method of medical treatment or prophylaxis.

The present disclosure also provides an inhibitory nucleic add, nudeic add, expression vector, or composition according to the present disdosure, for use in a method of treating or preventing a disease or condition selected from: dyslipidemia, hyperlipidemia, hypercholesterolemia, familial hypercholesterolemia, autosomal dominant hypercholesterolemia, atherosderosis, cardiovascular disease, atherosclerotic cardiovascular disease, coronary artery disease, angina, myocardial infarction, cardiac failure, peripheral vascular disease, peripheral arterial disease, hypertension, stroke, ischemic stroke, transient ischemic attack, congestive heart failure, steatosis, hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH).

The present disclosure also provides the use of an inhibitory nucleic acid, nucleic acid, expression vector, or composition according to the present disclosure, in the manufacture of a medicament for treating or preventing a disease or condition selected from: dyslipidemia, hyperlipidemia, hypercholesterolemia, familial hypercholesterolemia, autosomal dominant hypercholesterolemia, atherosclerosis, cardiovascular disease, atherosclerotic cardiovascular disease, coronary artery disease, angina, myocardial infarction, cardiac failure, peripheral vascular disease, peripheral arterial disease, hypertension, stroke, ischemic stroke, transient ischemic attack, congestive heart failure, steatosis, hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH).

The present disclosure also provides a method of treating or preventing a disease or condition in a subject comprising administering to a subject a therapeutically- or prophylactically-effective amount of an inhibitory nucleic add, nucleic acid, expression vector, or composition according to the present disdosure, wherein the disease or condition is selected from: dyslipidemia, hyperlipidemia, hypercholesterolemia, familial hypercholesterolemia, autosomal dominant hyperchdesterolemia, atherosclerosis, cardiovascular disease, atherosclerotic cardiovascular disease, coronary artery disease, angina, myocardial infarction, cardiac failure, peripheral vascular disease, peripheral arterial disease, hypertension, stroke, ischemic stroke, transient ischemic attack, congestive heart failure, steatosis, hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). Also provided is an inhibitory nucleic acid for reducing gene and/or protein expression of one or more target genetsyproteints), wherein the inhibitory nucleic acid comprises a nucleotide sequence having nucleotides comprising a pattern of modifications according to one of rows 1 to 70 of Table A.

Also provided is an inhibitory nucleic acid for reducing gene and/or protein expression of one or more target gene(s)/protein(s), wherein the inhibitory nucleic acid comprises (i) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to one of rows 1 to 24 or rows 29 to 70 of Table A; and (ii) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to one of rows 25 to 28 of Table A.

Also provided is an inhibitory nucleic acid comprising or consisting of: (i) nucleic acid comprising a nucleotide sequence having nucleotides comprising a pattern of modifications indicated in column 1 of Table B, and (ii) nucleic acid comprising a nucleotide sequence having nucleotides comprising a pattern of modifications indicated in column 2 of Table B, wherein the sequences of columns 1 and 2 are selected from the same row of Table B.

Inhibitory nucleic acids comprising modifications as described in Tables A and/or B may be used for any method of treatment or prevention provided herein.

Any inhibitory nucleic acid described herein may comprise at least one GalNAc monomer as provided herein, such as one or more GalNAc monomers) according to a compound of Formula 1, Formula 2 or Formula 3 as described herein.

Description

PCSK9

The present disclosure relates particularly to inhibition of gene and/or protein expression of proprotein convertase subtilisin/kexin type 9 (PCSK9).

The structure and function of PCSK9 is described in e.g. in Sobati et al., Adv Pharm Bull. (2020) 10(4): 502-511 and Urban et al., J Am Coll Cardiol. (2013) 62(16):1401-1408, both of which are hereby incorporated by reference in their entirety.

Alterative splicing of the mRNA transcribed from the human PCSK9 gene yields two isoforms: isoform 1 (UniProtKB: Q8NBP7-1, v3; SEQ ID NO:1), and isoform 2 (UniProtKB: Q8NBP7-2; SEQ ID NO:2) in which the amino acid sequence corresponding to positions 1 to 174 of SEQ ID NO:1 are replaced with the sequence 'MSPWK', the amino acid sequence corresponding to positions 333 to 365 of SEQ ID NO:1 are replaced with the sequence 'GRTSLVPPATAAPALCHRVGHHRLLPTWLALQP', and in which the amino acid sequence corresponding to positions 366 to 692 of SEQ ID NO:1 are missing. The 692 amino acid sequence of human PCSK9 isoform 1 comprises: an N-terminal signal peptide (positions 1 to 30 of SEQ ID NO:1, shown in SEQ ID NO:3), followed by a 122 amino acid prodomain (positions 31 to 152 of SEQ ID NO:1, shown in SEQ ID NO:4) the mature protein region (positions 153 to 692 of SEQ ID NO:1 , shown in SEQ ID NO:5), which comprises a catalytic domain (positions 153 to 451 of SEQ ID NO:1, shown in SEQ ID NO:6) and a C-terminal cysteine-histidine-rich domain (CHRD; positions 452 to 692 of SEQ ID NO:1 , shown in SEQ ID NO:7). The active site of the catalytic domain is formed by D186, H226 and S386, and the oxyanion hole at N317 (numbering relative to SEQ ID NO:1).

PCSK9 is expressed by hepatocytes, renal mesenchymal cells, intestinal ileum, and colon epithelial cells and telencephalon neurons in the embryonic brain. PCSK9 has been shown to have a central role in regulation of cholesterol homeostasis, by enhancing the endosomal and lysosomal degradation of hepatic low-density lipoprotein receptor (LDLR), resulting in increased serum concentrations of LDL-cholesterol (LDL-C). Gain-of-function mutations such as S127R and F216L are associated with autosomal dominant hypercholesterolemia, while loss-of-fonction mutations are linked to low plasma LDL-C levels and a reduced risk of cardiovascular disease.

PCSK9 is autocatalytically cleaved between Q152 and S153 in the endoplasmic reticulum to form the 14 kDa prodomain and 63 kDa mature protein. The prodomain remains closely-associated with the catalytic domain of the mature protein, blocking the substrate binding site. After the ER, PCSK9 either progresses through the extracellular or intracellular pathways. In the extracellular pathway, PCSK9 is secreted from the cell via the Golgi network, and binds to extracellular LDLR. PCSK9:LDLR complexes are inte alised by cells via clathrin-mediated endocytosis and trafficked to the endosomes, resulting in degradation of LDLR. In the intracellular pathway, PCSK9 is sorted to lysosomes together with LDLR, leading to degradation of LDLR.

PCSK9 has been shown to bind to LDLR via interaction between the catalytic domain of PCSK9 and the EGF-like repeat homology domain of LDLR. Studies have shown that inactivation of the catalytic domain of PCSK9 does not inhibit LDLR degradation, indicating that secreted PCSK9 acts as a chaperone for LDLR degradation, rather than as a catalytic enzyme.

Plasma LDL-C is mainly cleared through the LDL receptor (LDLR) pathway. The LDLR: LDL-C pathway and the role of PCSK9 in its regulation is described e.g. in Gu and Zhang J Biomed Res. (2015) 29(5): 356-361, which is hereby incorporated by reference in its entirety.

In this specification, reference to 'PCSK9' encompasses: human PCSK9 isoform 1, homologues of human PCSK9 isoform 1 (/.e. encoded by the genome of a non-human animal), and variants thereof. In some embodiments, PCSK9 according to the present disclosure comprises or consists of an amino acid sequence having at 70% or greater amino acid sequence identity, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1. A homologue of human PCSK9 isoform 1 may be from any animal. In some embodiments, a homologue of human PCSK9 isoform may be from a mammal. In some embodiments, the mammal may be a nonhuman mammal, e.g. a primate (e.g. a non-human primate, e.g. an animal of the genus Macaco (e.g. Macaca fascicularis, Macaca mulatta), e.g. a non-human hominid (e.g. Pan troglodytes)). In some embodiments, the mammal may be a rabbit, guinea pig, rat mouse or animal of the order Rodentia, cat dog, pig, sheep, goat, an animal of the order Bos (e.g. cattle), an animal of the family Equidae (e.g. horse) or donkey.

Homologues of human PCSK9 isoform 1 may optionally be characterised as having 70% or greater amino acid sequence identity, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino add sequence identity to the amino add sequence of SEQ ID NO:1. Variants of human PCSK9 isoform 1 may optionally be characterised as having 70% or greater amino add sequence identity, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater amino acid sequence identity to the amino acid sequence of SEQ ID NO:1. articles

Aspects and embodiments of the present disdosure relate to inhibitory nucleic acids. As used herein, an ‘inhibitory nucleic acid' refers to a nucleic add capable of reducing or preventing the gene and/or protein expression of one or more given target gene(s)/protein(s).

Inhibitory nucleic acids according to the present disclosure are suitable for reducing gene and/or protein expression of PCSK9. It will be appreciated that where an inhibitory nucleic acid is described as reducing gene expression of PCSK9, inhibition of expression of the gene encoding PCSK9 is intended. That is, reference herein to inhibition of gene expression of PCSK9 contemplates inhibition of expression of PCSK9.

In some embodiments, an inhibitory nucleic acid according to the present disclosure may: reduce/prevent expression of a gene encoding PCSK9; reduce/prevent transcription of nucleic acid encoding PCSK9 (e.g. from DNA encoding PCSK9 to RNA encoding PCSK9); reduce the level of RNA encoding PCSK9; increase degradation of RNA encoding PCSK9; reduce the level of PCSK9 protein; reduce/prevent normal splicing of pre-mRNA encoding PCSK9 reduce/prevent translation of mRNA encoding PCSK9; reduce the level of a function of PCSK9 (e.g. reduce the level of binding of PCSK9 to LDLR; reduce the level of PCSK9:LDLR complexes, reduce degradation of LDLR, increase the level of LDLR); reduce the level of total lipid (e.g. in the serum of a subject administered the inhibitory nucleic acid); reduce the level of total cholesterol (e.g. in the serum of a subject administered the inhibitory nucleic add); and/or reduce the level of LDL cholesterol (e.g. in the serum of a subject administered the inhibitory nudeic acid).

It will be appreciated that a given nucleic acid may display more than one of the properties recited in the preceding paragraph. A given nucleic acid may be evaluated for the properties recited in the preceding paragraph using suitable assays. The assays may be e.g. in vitro assays, optionally cell-based assays or cell-free assays. The assays may be e.g. in vivo assays, i.e. performed in non-human animals.

Where assays are cell-based assays, they may comprise treating cells with a nucleic add in order to determine whether the nudeic acid displays one or more of the recited properties. Assays may employ species labelled with detectable entities in order to facilitate their detection. Assays may comprise evaluating the recited properties following treatment of cells separately with a range of quantities/concentrations of a given nudeic add (e.g. a dilution series). It will be appredated that the cells are preferably cells that express PCSK9, e.g. liver cells (e.g. HepG2 cells or HuH7 cells).

Analysis of the results of such assays may comprise determining the concentration at which 50% of the maximal level of the relevant activity is attained. The concentration of nucleic acid at which 50% of the maximal level of the relevant activity is attained may be referred to as the 'half-maximal effective concentration’ of the nucleic acid in relation to the relevant activity, which may also be referred to as the 'ECso'. By way of illustration, the ECso of a given inhibitory nucleic acid for increasing degradation of RNA encoding PCSK9 may be the concentration at which 50% of the maximal level of degradation of RNA encoding PCSK9 is achieved.

Depending on the property, the ECso may also be referred to as the ‘half-maximal inhibitory concentration’ or ICso', this being the concentration of nucleic add at which 50% of the maximal level of inhibition of a given property is observed. By way of illustration, the ICso of a given inhibitory nucleic add for reducing expression of a gene encoding PCSK9 may be the concentration at which 50% of the maximal level of inhibition of expression of the gene is achieved.

Nucleic acids capable of redudng/preventing gene expression of PCSK9 and/or reducing/preventing transcription of nucleic acid encoding PCSK9 and/or reducing the level of RNA encoding PCSK9 and/or increasing degradation of RNA encoding PCSK9 may be identified using assays comprising detecting and/or quantifying the level of RNA encoding PCSK9. Such assays may comprise quantifying RNA encoding PCSK9 by RT-qPCR (a technique well known to the skilled person). The methods may employ primers and/or probes for the detection and/or quantification of RNA encoding PCSK9. Such assays may comprise introducing (e.g. by transfection) into cells that express PCSK9 in in vitro culture (i) a putative inhibitory nucleic acid, or (ii) a control nucleic acid (e.g. a nucleic add known not to influence the level of RNA encoding PCSK9), and subsequently (e.g. after an appropriate period of time, i.e. a period of time sufficient for a reduction in the level of gene expression of PCSK9/transcription of nudeic acid encoding PCSK9/level of RNA encoding PCSK9 or an increase in the level of degradation of RNA encoding PCSK9 to be observed) measuring the level of RNA encoding PCSK9 in cells according to (i) and (ii), and (iii) comparing the level of RNA encoding PCSK9 detected to determine whether the putative inhibitory nucleic acid reduces/prevents gene expression of PCSK9/transcription of nucleic acid encoding PCSK9, and/or reduces the level of RNA encoding PCSK9, and/or increases degradation of RNA encoding PCSK9.

In particular embodiments, a nucleic acid may be evaluated for its ability to reduce/prevent gene expression of PCSK9 as described in Example 1.1 herein.

Nucleic acids capable of reducing/preventing normal splicing of pre-mRNA encoding PCSK9 may be identified using assays comprising detecting and/or quantifying the level of RNA (e.g. mature mRNA) encoding one or more isoforms of PCSK9. Such assays may comprise quantifying RNA (e.g. mature mRNA) encoding one or more isoforms of PCSK9 by RT-qPCR. The methods may employ primers and/or probes for the detection and/or quantification of mature mRNA produced by canonical splicing of pre- mRNA transcribed from a gene encoding PCSK9, and/or primers and/or probes for the detection and/or quantification of mature mRNA produced by alternative splicing of pre-mRNA transcribed from a gene encoding PCSK9. Mature mRNA produced by canonical splicing of pre-mRNA transcribed from a gene encoding PCSK9 may be mature mRNA encoding the major isoform produced by expression of the gene encoding PCSK9. The major isoform may be the most commonly produced/detected isoform. For example, mature mRNA produced by canonical splicing of pre-mRNA transcribed from human PCSK9 may be mature mRNA encoding human PCSK9 isoform 1 (/.e. having the amino acid sequence shown in SEQ ID NO:1). Mature mRNA produced by alternative splicing of pre-mRNA transcribed from a gene encoding PCSK9 may be mature mRNA encoding an isoform other than the major isoform produced by expression of the gene encoding PCSK9. For example, mature mRNA produced by alternative splicing of pre-mRNA transcribed from human PCSK9 may be mature mRNA encoding an isoform of human PCSK9 other than isoform 1 (/.e. having an amino acid sequence non-identical to SEQ ID NO:1); e.g. mature mRNA encoding human PCSK9 isoform 2 (/.e. having an amino acid sequence non-identical to SEQ ID NO:2). Such assays may comprise introducing (e.g. by transfection) into cells that express PCSK9 in in vitro culture (i) a putative inhibitory nucleic acid, or (ii) a control nucleic acid (e.g. a nucleic acid known not to influence splicing of pre-mRNA encoding PCSK9), and subsequently (e.g. after an appropriate period of time, i.e. a period of time sufficient for an effect on splicing of pre-mRNA encoding PCSK9 to be observed) measuring the level of mature mRNA encoding one or more isoforms of PCSK9 in cells according to (i) and (ii), and (iii) comparing the level of mature mRNA encoding the isoform(s) to determine whether the putative inhibitory nucleic acid reduces/prevents normal splicing of pre-mRNA encoding PCSK9.

Nucleic acids capable of reducing the level of PCSK9 protein and/or reducing/preventing translation of mRNA encoding PCSK9 may be identified using assays comprising detecting the level of PCSK9 protein, e.g. using techniques well known to the skilled person, such as antibody/reporter-based methods (western blot, ELISA, immunohisto/cytochemistry etc ) The methods may employ antibodies specific for PCSK9. Such assays may comprise introducing (e.g. by transfection) into cells that express PCSK9 in in vitro culture (I) a putative inhibitory nucleic acid, or (ii) a control nucleic acid (e.g. a nucleic acid known not to influence the level of PCSK9 protein), and subsequently (e.g. after an appropriate period of time, i.e. a period of time sufficient for a reduction in the level of PCSK9 protein to be observed) measuring the level of PCSK9 protein in cells according to (i) and (ii), and (iii) comparing the level of PCSK9 protein detected to determine whether the putative inhibitory nucleic acid reduces the level of PCSK9 protein and/or reduces/prevents translation of mRNA encoding PCSK9.

In particular embodiments, a nucleic acid may be evaluated for its ability to reduce/prevent the level of PCSK9 protein as described in Example 1.1 herein.

Nucleic acids capable of redudng the level of a function of PCSK9 (e.g. a function of PCSK9 as described hereinabove) may be identified using assays comprising detecting the level of the relevant function. Such assays may comprise introdudng (e.g. by transfection) into cells that express PCSK9 in in vitro culture (I) a putative inhibitory nudeic add, or (ii) a control nucleic add (e.g. a nucleic acid known not to influence PCSK9 function), and subsequently (e.g. after an appropriate period of time, i.e. a period of time sufficient for a reduction in the level of a function of PCSK9 to be observed) measuring the level of a function of PCSK9 in cells according to (i) and (ii), and (iii) comparing the level of the function of PCSK9 detected to determine whether the putative inhibitory nucleic add reduces the level of a function of PCSK9.

Reference herein to 'a function of PCSK9* may refer to any functional property of, and/or activity mediated by, PCSK9 protein. In some embodiments, a function of PCSK9 may be selected from: binding of PCSK9 to LDLR, formation of PCSK9:LDLR complexes, and degradation of LDLR.

As used herein, reference to a 'PCSK9:LDLR complex' may refer to a non-covalent protein:protein complex comprising PCSK9 and LDLR, formed e.g. via electrostatic interaction (e.g. ionic bonding, hydrogen bonding) and/or Van der Waals forces.

Analysis of the level of binding of PCSK9 to LDLR and/or analysis of the level of PCSK9:LDLR complexes may be performed using assays comprising detecting interaction between PCSK9 and LDLR, e.g. using antibody/reporter-based methods. The level of binding of PCSK9 to LDLR and/or the level of PCSK9:LDLR complexes can be analysed e.g. using resonance energy transfer techniques (e.g. FRET, BRET), co-immunoprecipitation, ELISA, surface plasmon resonance, biolayer interferometry or methods comprising detection of PCSK9:LDLR complexes. Such assays may comprise introducing (e.g. by transfection) into cells that express PCSK9 and LDLR in in vitro culture (I) a putative inhibitory nucleic acid, or (ii) a control nucleic add (e.g. a nucleic acid known not to influence the level of binding of PCSK9 to LDLR and/or the level of PCSK9:LDLR complexes), and subsequently (e.g. after an appropriate period of time, i.e. a period of time sufficient for a reduction in the level of binding of PCSK9 to LDLR and/or the level of PCSK9:LDLR complexes to be observed) measuring the level of interaction between PCSK9 and LDLR and/or the level of PCSK9:LDLR complexes in cells according to (i) and (ii), and (iii) comparing the level detected to determine whether the putative inhibitory nucleic acid reduces the level of binding of PCSK9 to LDLR and/or the level of PCSK9:LDLR complexes.

Analysis of the level of degradation of LDLR may be performed e.g. using assays comprising detecting and/or quantifying LDLR, e.g. LDLR localised to the lysosome, using antibody/reporter-based methods. The methods may comprise detecting and quantifying LDLR. The methods may comprise detecting the presence of, quantifying, and/or determining the proportion of, LDLR localised to the lysosome, e.g. using antibody/reporter-based methods (western blot, ELISA, immunohisto/cytochemistry, etc.). The subcellular localisation of LDLR may be analysed e.g. by immunocytochemistry, or western blot of extracts prepared from different cellular fractions, and may employ organelle (e.g. lysosomal) markers and/or labelled proteins of known subcellular localisation. Methods may employ agents for the detection of the products of degradation of LDLR. Such assays may comprise introducing (e.g. by transfection) into cells that express PCSK9 and LDLR in in vitro culture (i) a putative inhibitory nucleic acid, or (ii) a control nucleic acid (e.g. a nucleic acid known not to influence the level of degradation of LDLR), and subsequently (e.g. after an appropriate period of time, i.e. a period of time sufficient for a reduction in the level of degradation of LDLR to be observed) measuring the level of degradation of LDLR in cells according to (i) and (ii), and (iii) comparing the level detected to determine whether the putative inhibitory nucleic acid reduces the level of degradation of LDLR.

Nucleic acids capable of reducing the level of total lipid/total cholesterol/LDL cholesterol (e.g. in the serum of a subject administered the nucleic acid), may be identified in in vivo assays comprising administering the nucleic acid to a subject, and subsequently (e.g. after an appropriate period of time, i.e. a period of time sufficient for a reduction in the level of total lipid/total cholesterol/LDL cholesterol to be observed) measuring the level of total lipid/total cholesterol/LDL cholesterol in the serum of the subject The level measured may be compared to the level observed in a comparable subject not administered the nucleic acid, or administered a control nucleic acid (e.g. a nucleic acid known not to influence the level of total lipid/total cholesterol/LDL cholesterol in the serum), to determine whether the nucleic acid reduces the level of total lipid/total cholesterol/LDL cholesterol. Such in vivo assays may be performed in a nonhuman animal model of hypercholesterolemia. Such assays may employ non-human animals expressing human PCSK9. For example, the assay may employ transgenic mice expressing human PCSK9, e.g. hPCSK9-KI mice described in Carreras et al. BMC Biology (2019) 17(1):4 (which is hereby incorporated by reference in its entirety).

In some embodiments, an inhibitory nucleic acid according to the present disclosure may be capable of reducing expression of a gene encoding PCSK9 to less than 1 times, e.g. one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level of expression observed in the absence of the inhibitory nucleic acid, or in the presence of the same quantity of a control nucleic acid known not to inhibit expression of the relevant gene, in a given assay. In some embodiments, an inhibitory nucleic acid according to the present disclosure may be capable of reducing expression of a gene encoding PCSK9 to less than 100%, e.g. one of ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5%, or ≤1% of the level of expression observed in the absence of the inhibitory nucleic acid, or in the presence of the same quantity of a control nucleic acid known not to inhibit expression of the relevant gene, in a given assay.

In some embodiments, an inhibitory nucleic acid according to the present disclosure may be capable of reducing the level of RNA encoding PCSK9 to less than 1 times, e.g. one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level observed in the absence of the inhibitory nucleic acid, or in the presence of the same quantity of a control nucleic acid known not to reduce the level of RNA encoding PCSK9, in a given assay. In some embodiments, an inhibitory nucleic acid according to the present disclosure may be capable of reducing the level of RNA encoding PCSK9 to less than 100%, e.g. one of ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5%, or≤l% of the level observed in the absence of the inhibitory nucleic acid, or in the presence of the same quantity of a control nucleic acid known not to reduce the level of RNA encoding PCSK9, in a given assay.

In some embodiments, an inhibitory nucleic acid according to the present disclosure may be capable of reducing the level of transcription of nucleic acid encoding PCSK9 to less than 1 times, e.g. one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level observed in the absence of the inhibitory nucleic acid, or in the presence of the same quantity of a control nucleic acid known not to reduce transcription of nucleic acid encoding PCSK9, in a given assay. In some embodiments, an inhibitory nucleic acid according to the present disclosure may be capable of reducing the level of transcription of nucleic acid encoding PCSK9 to less than 100%, e.g. one of ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5%, or ≤l% of the level observed in the absence of the inhibitory nucleic acid, or in the presence of the same quantity of a control nucleic acid known not to reduce transcription of nucleic acid encoding PCSK9, in a given assay.

In some embodiments, an inhibitory nucleic acid according to the present disclosure may be capable of reducing the level of PCSK9 protein to less than 1 times, e.g. one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level observed in the absence of the inhibitory nucleic acid, or in the presence of the same quantity of a control nucleic acid known not to reduce the level of PCSK9 protein, in a given assay. In some embodiments, an inhibitory nucleic acid according to the present disclosure may be capable of reducing the level of PCSK9 protein to less than 100%, e.g. one of ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5%, or ≤1% of the level observed in the absence of the inhibitory nucleic acid, or in the presence of the same quantity of a control nucleic acid known not to reduce the level of PCSK9 protein, in a given assay.

In some embodiments, an inhibitory nucleic acid according to the present disclosure may be capable of reducing the level of a function of PCSK9 to less than 1 times, e.g. one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level observed in the absence of the inhibitory nucleic acid, or in the presence of the same quantity of a control nucleic acid known not to reduce the level of the function of PCSK9, in a given assay. In some embodiments, an inhibitory nucleic add according to the present disdosure may be capable of redudng the level of a function of PCSK9 to less than 100%, e.g. one of ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5%, or ≤1% of the level observed in the absence of the inhibitory nucleic add, or in the presence of the same quantity of a control nucleic acid known not to reduce the level of the function of PCSK9, in a given assay.

In some embodiments, an inhibitory nudeic acid according to the present disdosure may be capable of redudng the level of binding of PCSK9 to LDLR and/or the level of PCSK9:LDLR complexes to less than 1 times, e.g. one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level observed in the absence of the inhibitory nudeic add, or in the presence of the same quantity of a control nucleic add known not to reduce the level of the relevant property, in a given assay. In some embodiments, an inhibitory nucleic acid according to the present disclosure may be capable of redudng the level of binding of PCSK9 to LDLR and/or the level of PCSK9:LDLR complexes to less than 100%, e.g. one of ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5%, or ≤1% of the level observed in the absence of the inhibitory nucleic add, or in the presence of the same quantity of a control nudeic acid known not to reduce the level of the relevant property, in a given assay.

In some embodiments, an inhibitory nucleic acid according to the present disclosure may be capable of reducing the level of degradation of LDLR to less than 1 times, e.g. one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level observed in the absence of the inhibitory nucleic acid, or in the presence of the same quantity of a control nucleic acid known not to reduce the level of degradation of LDLR, in a given assay. In some embodiments, an inhibitory nucleic acid according to the present disclosure may be capable of reducing the level of degradation of LDLR to less than 100%, e.g. one of ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5%, or ≤1% of the level observed in the absence of the inhibitory nucleic acid, or in the presence of the same quantity of a control nucleic acid known not to reduce the level of degradation of LDLR, in a given assay.

In some embodiments, an inhibitory nucleic acid according to the present disclosure may be capable of reducing normal splicing of pre-mRNA encoding PCSK9 to less than 1 times, e.g. one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level observed in the absence of the inhibitory nucleic acid, or in the presence of the same quantity of a control nucleic acid known not to reduce normal splicing of pre- mRNA encoding PCSK9, in a given assay. In some embodiments, an inhibitory nucleic acid according to the present disclosure may be capable of reducing the level of normal splicing of pre-mRNA encoding PCSK9 to less than 100%, e.g. one of ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5%, or≤l% of the level observed in the absence of the inhibitory nucleic acid, or in the presence of the same quantity of a control nucleic acid known not to reduce normal splicing of pre-mRNA encoding PCSK9, in a given assay.

In some embodiments, an inhibitory nucleic acid according to the present disclosure may be capable of reducing translation of mRNA encoding PCSK9 to less than 1 times, e.g. one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level observed in the absence of the inhibitory nucleic acid, or in the presence of the same quantity of a control nucleic acid known not to reduce translation of mRNA encoding PCSK9, in a given assay. In some embodiments, an inhibitory nucleic acid according to the present disclosure may be capable of reducing translation of mRNA encoding PCSK9 to less than 100%, e.g. one Of ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5%, or ≤1% of the level observed in the absence of the inhibitory nucleic acid, or in the presence of the same quantity of a control nucleic acid known not to reduce translation of mRNA encoding PCSK9, in a given assay.

In some embodiments, an inhibitory nucleic acid according to the present disclosure may be capable of reducing the level of total lipid/total cholesterol/LDL cholesterol in the serum to less than 1 times, e.g. one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level observed in the absence of treatment with the inhibitory nucleic acid, or following treatment with the same quantity of a control nucleic acid known not to reduce the level of total lipid/total cholesterol/LDL cholesterol in the serum, in a given assay. In some embodiments, an inhibitory nucleic acid according to the present disclosure may be capable of reducing the level of total lipid/total cholesterol/LDL cholesterol in the serum to less than 100%, e.g. one Of ≤99%, ≤95%, ≤90%, ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, ≤25%, ≤20%, ≤15%, ≤10%, ≤5%, or ≤1% of the level observed in the absence of treatment with the inhibitory nucleic acid, or following treatment with the same quantity of a control nucleic acid known not to reduce the level of total lipid/total cholesterol/LDL cholesterol in the serum, in a given assay.

Preferred levels of reduction in accordance with the preceding ten paragraphs are reduction to less than 0.5 times/≤50%, e.g. one of less than 0.4 times/s40%, less than 0.3 times/s30%, less than 0.2 times/s20%, less than 0.15 times/815%, or less than 0.1 times/sio%.

In some embodiments, an inhibitory nucleic acid according to the present disclosure may be capable of increasing degradation of RNA encoding PCSK9 to more than 1 times, e.g. one of 21.01 times, 21.02 times, 21.03 times, 21.04 times, 21.05 times, 21.1 times, 21.2 times, 21.3 times, 21.4 times, 21.5 times, 21.6 times, 21.7 times, 21.8 times, 21.9 times, 22 times, 23 times, 24 times, 25 times, 26 times, 27 times, 28 times, 29 times or 210 times the level observed in the absence of the inhibitory nucleic acid, or in the presence of the same quantity of a control nucleic acid known not to increase degradation of RNA encoding PCSK9, in a given assay.

In some embodiments, an inhibitory nucleic add according to the present disdosure may be capable of increasing the level of LDLR to more than 1 times, e.g. one of 21.01 times, 21.02 times, 21.03 times, 21.04 times, 21.05 times, 21.1 times, 21.2 times, 21.3 times, 21.4 times, 21.5 times, 21.6 times, 21.7 times, 21.8 times, 21.9 times, 22 times, 23 times, 24 times, 25 times, 26 times, 27 times, 28 times, 29 times or 210 times the level observed in the absence of the inhibitory nucleic acid, or in the presence of the same quantity of a control nucleic acid known not to increase the level of LDLR, in a given assay.

In some embodiments, an inhibitory nucleic acid according to the present disclosure prevents or silences expression of a gene encoding PCSK9. In some embodiments, an inhibitory nucleic acid according to the present disclosure prevents or silences expression of PCSK9 at the protein level. As used herein, expression of a given gene/protein may be considered to be 'prevented' or 'silenced' where the level of expression is reduced to less than 0.1 times/^10% of the level observed in the absence of the inhibitory nucleic acid, or in the presence of the same quantity of a control nucleic acid known not to be an inhibitor of expression of the relevant gene(s)/protein(s).

In preferred embodiments, an inhibitory nucleic acid (e.g. an siRNA) according to the present disclosure inhibits greater than 50%, e.g. one of 260%, 261%, 262%, 263%, 264%, 265%, 266%, 267%, 268%, 269%, 270%, 271%, 272%, 273%, 274%, 275%, 276%, 277%, 278%, 279%, 280%, 281%, 282%, 283%, 284%, 285%, 286%, 287%, 288%, 289%, 290%, 291%, 292%, 293%, 294%, 295%, 296%, 297%, 298%, 299% or 100% of the gene and/or protein expression of PCSK9 observed in the absence of the inhibitory nucleic acid, or in the presence of the same quantity of a control nucleic acid known not to inhibit gene and/or protein expression of PCSK9, in a given assay.

In preferred embodiments, an inhibitory nucleic add (e.g. an siRNA) according to the present disdosure inhibits greater than 50%, e.g. one of 260%, 261%, 262%, 263%, 264%, 265%, 266%, 267%, 268%, 269%, 270%, 271%, 272%, 273%, 274% 275% 276% 277% 278%, 279%, 280%, 281%, 282%, 283%, 284%, 285%, 286%, 287%, 288%, 289%, 290%, 291%, 292%, 293%, 294%, 295%, 296%, 297%, 298%, 299% or 100% of the gene expression of PCSK9 (e.g. as determined by qRT-PCR) observed in the absence of the inhibitory nucleic acid, or in the presence of the same quantity of a control nucleic acid known not to inhibit gene and/or protein expression of PCSK9, in a given assay (e.g. the assay described in Example 1 herein).

In preferred embodiments, an inhibitory nucleic acid (e.g. an siRNA) according to the present disclosure inhibits greater than 50%, e.g. one of 260%, 261%, 262%, 263%, 264%, 265%, 266%, 267%, 268%, 269%, 270%, 271%, 272%, 273%, 274%, 275%, 276%, 277%, 278%, 279%, 280%, 281%, 282%, 283%, 284%, 285%, 286%, 287%, 288%, 289%, 290%, 291%, 292%, 293%, 294%, 295%, 296%, 297%, 298%, 299% or 100% of the protein expression of PCSK9 (e.g. as determined by ELISA) observed in the absence of the inhibitory nucleic acid, or in the presence of the same quantity of a control nucleic acid known not to inhibit gene and/or protein expression of PCSK9, in a given assay (e.g. the assay described in Example 1 herein).

In some embodiments, an inhibitory nucleic acid (e.g. an siRNA) according to the present disclosure may inhibit gene and/or protein expression of PCSK9 with an ICso of ≤1 pM, e.g. one of ≤500 nM, ≤100 nM, ≤75 nM, ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM, ≤15 nM, ≤12.5 nM, ≤10 nM, ≤9 nM, ≤8 nM, ≤7 nM, ≤6 nM, ≤5 nM, ≤4 nM ≤3 nM, ≤2 nM, ≤1 nM, ≤900 pM, ≤800 pM, ≤700 pM, ≤600 pM, ≤500 pM, ≤400 pM, ≤300 pM, ≤200 pM, ≤100 pM, ≤50 pM, ≤40 pM, ≤30 pM, ≤20 pM, ≤10 pM or ≤1 pM.

In some embodiments an inhibitory nucleic acid according to the present disclosure (e.g. an siRNA) may inhibit gene expression of PCSK9 (e.g. as determined by qRT-PCR) with an ICso of ≤1 nM, ≤900 pM, ≤800 pM, ≤700 pM, ≤600 pM, ≤500 pM, ≤400 pM, ≤300 pM, ≤200 pM, ≤100 pM, ≤50 pM, ≤40 pM, ≤30 pM, ≤20 pM, ≤10 pM or ≤1 pM.

In some embodiments an inhibitory nucleic acid according to the present disclosure (e.g. an siRNA) may inhibit protein expression of PCSK9 (e.g. as determined by ELISA) with an ICso of ≤1 nM, ≤900 pM, ≤800 pM, ≤700 pM, ≤600 pM, ≤500 pM, ≤400 pM, ≤300 pM, ≤200 pM, ≤100 pM, ≤50 pM, ≤40 pM, ≤30 pM, ≤20 pM, ≤10 pM or ≤l pM.

Inclisiran is a synthetic siRNA directed against PCSK9, and is described e.g. in Fitzgerald et a/., NEJM (2017) 376 (1): 41-51 and WO 2014/089313 A1 (both of which are hereby incorporated by reference in their entirety). The target nucleotide sequence for inclisiran is shown in SEQ ID NO:419. The nucleotide sequence of the antisense nucleic add of indisiran (i.e. the nucleotide sequence of the guide strand) is shown in SEQ ID NO:420, and the nucleotide sequence of the sense strand (i.e. the nucleotide sequence of the passenger strand) is shown in SEQ ID NO:421.

An inhibitory nucleic acid according to the present disclosure is non-identical to an inhibitory nucleic acid disclosed in WO 2014/089313 A1. An inhibitory nucleic acid according to the present disclosure is nonidentical to inclisiran. In preferred embodiments the inhibitory nucleic acids of the present disclosure possess novel and/or improved properties compared to an inhibitory nucleic acid disclosed in WO 2014/089313 A1. In preferred embodiments, the inhibitory nucleic acids of the present disclosure possess novel and/or improved properties compared to inclisiran.

In some embodiments, an inhibitory nucleic acid according to the present disclosure: reduces expression of a gene encoding PCSK9 to a greater extent than an siRNA disclosed in WO 2014/089313 A1 (e.g. an siRNA comprising a guide strand having the nucleotide sequence of SEQ ID NO:420); reduces transcription of nucleic acid encoding PCSK9 (e.g. from DNA encoding PCSK9 to RNA encoding PCSK9) to a greater extent than siRNA disclosed in WO 2014/089313 A1 (e.g. an siRNA comprising a guide strand having the nucleotide sequence of SEQ ID NO:420); reduces the level of RNA encoding PCSK9 to a greater extent than siRNA disclosed in WO 2014/089313 A1 (e.g. an siRNA comprising a guide strand having the nucleotide sequence of SEQ ID NO:420); increases degradation of RNA encoding PCSK9 to a greater extent than siRNA disclosed in WO 2014/089313 A1 (e.g. an siRNA comprising a guide strand having the nucleotide sequence of SEQ ID NO:420); reduces the level of PCSK9 protein to a greater extent than siRNA disclosed in WO 2014/089313 A1 (e.g. an siRNA comprising a guide strand having the nucleotide sequence of SEQ ID NO:420); reduces normal splicing of pre-mRNA encoding PCSK9 to a greater extent than siRNA disclosed in WO 2014/089313 A1 (e.g. an siRNA comprising a guide strand having the nucleotide sequence of SEQ ID NO:420); reduces translation of mRNA encoding PCSK9 to a greater extent than siRNA disclosed in WO 2014/089313 A1 (e.g. an siRNA comprising a guide strand having the nucleotide sequence of SEQ ID NO:420); reduces the level of a function of PCSK9 (e.g. reduces the level of binding of PCSK9 to LDLR; reduces the level of PCSK9:LDLR complexes, reduces degradation of LDLR, increases the level of LDLR) to a greater extent than siRNA disclosed in WO 2014/089313 A1 (e.g. an siRNA comprising a guide strand having the nucleotide sequence of SEQ ID NO:420); reduces the level of total lipid (e.g. in the serum of a subject administered the inhibitory nucleic acid) to a greater extent than siRNA disclosed in WO 2014/089313 A1 (e.g. an siRNA comprising a guide strand having the nucleotide sequence of SEQ ID NO:420); reduces the level of total cholesterol (e.g. in the serum of a subject administered the inhibitory nucleic acid) to a greater extent than siRNA disclosed in WO 2014/089313 A1 (e.g. an siRNA comprising a guide strand having the nucleotide sequence of SEQ ID NO:420); and/or reduces the level of LDL cholesterol (e.g. in the serum of a subject administered the inhibitory nucleic acid) to a greater extent than siRNA disclosed in WO 2014/089313 A1 (e.g. an siRNA comprising a guide strand having the nucleotide sequence of SEQ ID NO:420). In some embodiments, an inhibitory nucleic acid according to the present disclosure reduces expression of a gene encoding PCSK9 to less than 1 times, e.g. one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level to which siRNA comprising a guide strand having the nucleotide sequence of SEQ ID NO:420 reduces expression of the relevant gene in a given assay, at a comparable concentration.

In some embodiments, an inhibitory nucleic acid according to the present disclosure reduces transcription of nucleic acid encoding PCSK9 to less than 1 times, e.g. one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level to which siRNA comprising a guide strand having the nucleotide sequence of SEQ ID NO:420 reduces transcription of the nucleic acid in a given assay, at a comparable concentration.

In some embodiments, an inhibitory nucleic acid according to the present disclosure reduces the level of RNA encoding PCSK9 to less than 1 times, e.g. one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level to which siRNA comprising a guide strand having the nucleotide sequence of SEQ ID NO:420 reduces the level of the RNA in a given assay, at a comparable concentration.

In some embodiments, an inhibitory nucleic acid according to the present disclosure increases degradation of RNA encoding PCSK9 to more than 1 times, e.g. one of 61.01 times, 61.02 times, 61.03 times, 61.04 times, 61.05 times, 61.1 times, 61.2 times, 61.3 times, 61.4 times, 61.5 times, 61.6 times, 61.7 times, 61.8 times, 61.9 times, 62 times, 63 times, 64 times, 65 times, 66 times, 67 times, 68 times, 69 times or 610 times the level to which siRNA comprising a guide strand having the nucleotide sequence of SEQ ID NO:420 increases degradation of the RNA in a given assay, at a comparable concentration.

In some embodiments, an inhibitory nucleic acid according to the present disclosure reduces the level of PCSK9 protein to less than 1 times, e.g. one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level to which siRNA comprising a guide strand having the nucleotide sequence of SEQ ID NO:420 reduces the level of PCSK9 protein in a given assay, at a comparable concentration.

In some embodiments, an inhibitory nucleic acid according to the present disclosure reduces normal splicing of pre-mRNA encoding PCSK9 to less than 1 times, e.g. one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level to which siRNA comprising a guide strand having the nucleotide sequence of SEQ ID NO:420 reduces the level of normal splicing of pre-mRNA encoding PCSK9 in a given assay, at a comparable concentration.

In some embodiments, an inhibitory nucleic acid according to the present disclosure reduces translation of mRNA encoding PCSK9 to less than 1 times, e.g. one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level to which siRNA comprising a guide strand having the nucleotide sequence of SEQ ID NO:420 reduces the level of translation of mRNA encoding PCSK9 in a given assay, at a comparable concentration.

In some embodiments, an inhibitory nucleic acid according to the present disclosure reduces the level of a function of PCSK9 to less than 1 times, e.g. one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level to which siRNA comprising a guide strand having the nucleotide sequence of SEQ ID NO:420 reduces the level of the relevant function in a given assay, at a comparable concentration.

In some embodiments, an inhibitory nucleic acid according to the present disclosure reduces the level of binding of PCSK9 to LDLR to less than 1 times, e.g. one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level to which siRNA comprising a guide strand having the nucleotide sequence of SEQ ID NO:420 reduces the level of binding of PCSK9 to LDLR in a given assay, at a comparable concentration.

In some embodiments, an inhibitory nucleic acid according to the present disclosure reduces the level of PCSK9:LDLR complexes to less than 1 times, e.g. one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level to which siRNA comprising a guide strand having the nucleotide sequence of SEQ ID NO:420 reduces the level of PCSK9:LDLR complexes in a given assay, at a comparable concentration.

In some embodiments, an inhibitory nucleic acid according to the present disclosure reduces the level of degradation of LDLR to less than 1 times, e.g. one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level to which siRNA comprising a guide strand having the nucleotide sequence of SEQ ID NO:420 reduces the level of degradation of LDLR in a given assay, at a comparable concentration. In some embodiments, an inhibitory nucleic acid according to the present disclosure reduces the level of total lipid/total cholesterol/LDL cholesterol in the serum to less than 1 times, e.g. one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level to which siRNA comprising a guide strand having the nucleotide sequence of SEQ ID NO:420 reduces the level of total lipid/total cholesterol/LDL cholesterol in the serum in a given assay, at a comparable concentration.

Inhibitory nucleic acids according to the present disclosure may comprise or consist of DNA and/or RNA. Inhibitory nucleic acids may be single-stranded (e.g. in the case of antisense oligonucleotides (e.g. gapmers)). Inhibitory nucleic acids may be double-stranded or may comprise double-stranded region(s) (e.g. in the case of siRNA, shRNA, etc.). Inhibitory nucleic acids may comprise both double-stranded and single-stranded regions (e.g. in the case of shRNA and pre-miRNA molecules, which are double-stranded in the stem region of the hairpin structure, and single-stranded in the loop region of the hairpin structure).

In some embodiments, an inhibitory nucleic acid according to the present disclosure may be an antisense nucleic acid as described herein. In some embodiments, an inhibitory nucleic acid may comprise an antisense nucleic acid as described herein. In some embodiments, an inhibitory nucleic acid may encode an antisense nucleic acid as described herein.

As used herein, an ‘antisense nucleic acid’ refers to a nucleic acid (e.g. DNA or RNA) that is complementary to at least a portion of a target nucleotide sequence (e.g. of RNA encoding PCSK9). Antisense nucleic acids according to the present disclosure are preferably single-stranded nucleic acids, and bind via complementary Watson-Crick base-pairing to a target nucleotide sequence. Complementary base-pairing may involve hydrogen bonding between complementary base pairs. Antisense nucleic acids may be provided as single-stranded molecules, as for example in the case of antisense oligonucleotides, or may be comprised in double-stranded molecular species, as for example in the case of siRNA, shRNA and pre-miRNA molecules.

Complementary base-pairing between the antisense nucleic acid and its target nucleotide sequence may be complete. In such embodiments the antisense nucleic acid comprises, or consists of, the reverse complement of its target nucleotide sequence, and complementary base-pairing occurs between each nucleotide of the target nucleotide sequence and complementary nucleotides in the antisense nucleic acid. Alternatively, complementary base-pairing between the antisense nucleic acid and its target nucleotide sequence may be incomplete/partial. In such embodiments complementary base-pairing occurs between some, but not all, nucleotides of the target nucleotide sequence and complementary nucleotides in the antisense nucleic acid.

Such binding between nucleic acids through complementary base pairing may be referred to as 'hybridisation'. Through binding to its target nucleotide sequence, an antisense nucleic acid may form a nucleic acid complex comprising (i) the antisense nucleic acid and (ii) a target nucleic acid comprising the target nucleotide sequence.

The nucleotide sequence of an antisense nucleic acid is sufficiently complementary to its target nudeotide sequence such that it binds or hybridises to the target nudeotide sequence. It will be appreciated that an antisense nudeic acid preferably has a high degree of sequence identity to the reverse complement of its target nudeotide sequence. In some embodiments, the antisense nudeic add comprises or consists of a nudeotide sequence having at least 75% sequence identity (e.g. one of at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to the reverse complement of its target nucleotide sequence.

In some embodiments, an antisense nudeic add according to the present disclosure comprises: a nucleotide sequence which is the reverse complement of its target nudeotide sequence, or a nudeotide sequence comprising 1 to 10 (e.g. one of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) substitutions relative to the reverse complement of its target nucleotide sequence.

In some embodiments, the target nucleotide sequence for an antisense nucleic acid according to the present disclosure comprises, or consists of, 5 to 100 nucleotides, e.g. one of 10 to 80, 12 to 50, or 15 to 30 nucleotides (e.g. 20 to 27, e.g. ~21). In some embodiments, the target nucleotide sequence for an antisense nucleic acid according to the present disclosure comprises or consists of DNA and/or RNA. In some embodiments, the target nucleotide sequence for an antisense nucleic acid according to the present disclosure comprises or consists of RNA.

In some embodiments, the antisense nucleic acid reduces/prevents transcription of nucleic acid comprising its target nucleotide sequence. In some embodiments, the antisense nucleic acid reduces/prevents association of factors required for normal transcription (e.g. enhancers, RNA polymerase) with nucleic acid comprising its target nucleotide sequence.

In some embodiments, the antisense nucleic add increases/potentiates degradation of nucleic acid comprising its target nudeotide sequence, e.g. through RNA interference. In some embodiments, the antisense nudeic acid reduces/prevents translation of nudeic acid comprising its target nucleotide sequence, e.g. through RNA interference or antisense degradation via RNase H.

RNA interference is described e.g. in Agrawal et al., Microbiol. Mol. Bio. Rev. (2003) 67(4): 657-685 and Hu et al., Sig. Transduc. Tar. Ther. (2020) 5(101), both of which are hereby incorporated by reference in their entirety. Briefly, double-stranded RNA molecules are recognised by the argonaute component of the RNA-induced silencing complex (RISC). The double-stranded RNAs are separated into single strands and integrated into an active RISC, by the RISC-Loading Complex (RLC). The RISC-integrated strands bind to their target RNA through complementary base pairing, and depending on the identity of the RISC- integrated RNA and degree of complementarity to the target RNA the RISC then either cleaves the target RNA resulting in its degradation, or otherwise blocks access of ribosomes thereby preventing its translation. RNAi based therapeutics have been approved for a number of indications (Kim, Chonnam Med J. (2020) 56(2): 87-93).

In some embodiments, the antisense nucleic acid reduces/prevents normal post-transcriptional processing (e.g. splicing and/or translation) of nucleic acid comprising its target nucleotide sequence. In some embodiments, the antisense nucleic acid reduces or alters splicing of pre-mRNA comprising its target nucleotide sequence to mature mRNA. In some embodiments, the antisense nucleic acid reduces translation of mRNA comprising its target nucleotide sequence to protein.

In some embodiments, the antisense nucleic acid reduces/prevents association of factors required for normal post-transcriptional processing (e.g. components of the spliceosome) with nucleic acid comprising its target nucleotide sequence. In such instances, the antisense nucleic may be referred to as a 'spliceswitching’ nucleic acid.

Splice-switching nucleic acids are reviewed e.g. in Haves and Hastings, Nucleic Acids Res. (2016) 44(14): 6549-6563, which is hereby incorporated by reference in its entirety. Splice-switching nucleic acids include e.g. splice-switching oligonucleotides (SSOs). They disrupt the normal splicing of target RNA transcripts by blocking the RNA: RNA base-pairing and/or protein: RNA binding interactions that occur between components of the splicing machinery and pre-mRNA. Splice-switching nucleic acids may be employed to alter the number/proportion of mature mRNA transcripts encoding PCSK9. Spliceswitching nucleic acids may be designed to target a specific region of the target transcript, e.g. to effect skipping of exon(s) of interest, e.g. exons encoding domains/regions of interest. SSOs often comprise alterations to oligonucleotide sugar-phosphate backbones in order to reduce/prevent RNAse H degradation, such as e.g. phosphorothioate linkages, phosphorodiamidate linkages such as phosphorodiamidate morpholino (PMOs), and may comprise e.g. peptide nucleic acids (PNAs), locked nucleic acids (LNAs), methoxyethyl nucleotide modifications, e.g. 2’0-methyl (2'OMe) and 2'-O- methoxyethyl (MOE) ribose modifications and/or S’-methylcytosine modifications.

In some embodiments, the antisense nucleic acid inhibits/reduces translation of nucleic acid comprising its target nucleotide sequence. In some embodiments, the antisense nucleic acid reduces/prevents association of factors required for translation (e.g. ribosomes) with nucleic acid comprising its target nucleotide sequence.

It will be appreciated that the target nucleotide sequence to which an antisense nucleic acid binds is a nucleotide sequence encoding a protein which it is desired to inhibit expression of. Accordingly, in aspects and embodiments of the present disclosure, the target nucleotide sequence for an antisense nucleic acid is a nucleotide sequence of a gene encoding PCSK9.

In some embodiments, the target nucleotide sequence is a nucleotide sequence of RNA encoded by a gene encoding PCSK9. In some embodiments the target nucleotide sequence is a nucleotide sequence of RNA encoding PCSK9. In some embodiments, the target nucleotide sequence comprises one or more nucleotides of an exon of RNA encoding PCSK9. In some embodiments, the target nucleotide sequence is a nucleotide sequence of an exon of RNA encoding PCSK9.

In some embodiments, the target nucleotide sequence is a nucleotide sequence of SEQ ID NO:8, which is the corresponding RNA sequence of NCBI Reference Sequence: NM_000186.4, which is a cDNA sequence encoding PCSK9. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 1 and 133 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 134 and 319 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 320 and 425 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 426 and 502 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 503 and 694 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 695 and 865 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 866 and 1039 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 1040 and 1234 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 1235 and 1411 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 1412 and 1594 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 1595 and 1771 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 1772 and 1948 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 1949 and 2131 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 2132 and 2311 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 2312 and 2488 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 2489 and 2671 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 2672 and 2857 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 2858 and 3031 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 3032 and 3208 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 3209 and 3385 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 3386 and 3568 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 3569 and 3962 (inclusive) of SEQ ID NO:8.

In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 3542 and 3563 (inclusive) of SEQ ID NO:8 In some embodiments the target nucleotide sequence is a nucleotide sequence from between positions 3542 and 3562 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 3543 and 3563 (inclusive) of SEQ ID NO:8.

In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 2102 and 2122 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 2879 and 2899 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence is a nucleotide sequence from between positions 3469 and 3489 (inclusive) of SEQ ID NO:8.

In some embodiments, the target nucleotide sequence does not comprise a nucleotide sequence from between positions 3529 and 3551 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence does not comprise a nucleotide from between positions 3529 and 3551 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence does not comprise a nucleotide sequence from between positions 3529 and 3541 (inclusive) of SEQ ID NO:8. In some embodiments, the target nucleotide sequence does not comprise a nucleotide from between positions 3529 and 3541 (inclusive) of SEQ ID NO:8.

In some embodiments, the target nucleotide sequence is non-identical to the nucleotide sequence of SEQ ID NO:419. In some embodiments, the target nucleotide sequence does not comprise or consist of the nucleotide sequence of SEQ ID NO:419.

In some embodiments, the target nucleotide sequence is, or comprises, the nucleotide sequence of one of SEQ ID NOs:9 to 213. In some embodiments, the target nucleotide sequence is, or comprises, the nucleotide sequence of one of SEQ ID NOs:422 to 626. In some embodiments, the target nucleotide sequence is, or comprises, the nucleotide sequence of SEQ ID NO:12, 21, 44, 49, 53, 109, 195, 196, 197, 201, 202 or 206.

In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g. one of at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to the reverse complement of one of SEQ ID NOs:9 to 213. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g. one of at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to the reverse complement of one of SEQ ID NOs:422 to 626. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g. one of at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to the reverse complement of SEQ ID NO: 12, 21, 44, 49, 53, 109, 195, 196, 197, 201 or 206. In some embodiments, the nucleotide sequence of the antisense nucleic acid is non-identical to the reverse complement of SEQ ID NO:419. In some embodiments, the nucleotide sequence of the antisense nucleic acid does not comprise or consist of the reverse complement of SEQ ID NO:419.

In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g. one of at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to one of SEQ ID NOs:214 to 418. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g. one of at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to one of SEQ ID NOs:627 to 831. In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g. one of at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to SEQ ID NO:217, 226, 249, 253, 258, 314, 400, 401, 402, 406, 407, or 411.

In some embodiments, the antisense nucleic acid comprises or consists of a sequence having at least 75% sequence identity (e.g. one of at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to one of SEQ ID NOs:832 to 1071, 1236 to 1259, 1264 to 1287, 1292 to 1315, 1320 to 1343, 1378 to 1416, or 1429 to 1431.

In some embodiments, the nucleotide sequence of the antisense nucleic acid is non-identical to SEQ ID NO:420. In some embodiments, the nucleotide sequence of the antisense nucleic acid does not comprise or consist of SEQ ID NO:420.

In some embodiments, an inhibitory nucleic acid is selected from: an siRNA, dsiRNA, miRNA, shRNA, pri- miRNA, pre-miRNA, saRNA, snoRNA, or antisense oligonucleotide (e.g. a gapmer), or a nucleic acid encoding the same. In some embodiments, an inhibitory nucleic acid is selected from: an siRNA, dsiRNA, miRNA, shRNA. In some embodiments, an inhibitory nucleic acid is an siRNA.

In some embodiments, an inhibitory nucleic acid may comprise an antisense nucleic acid described herein, e.g. as part of a larger nucleic acid species. For example, in some embodiments, an inhibitory nucleic acid may be an siRNA, dsiRNA, miRNA, shRNA, pri-miRNA, pre-miRNA, saRNA or snoRNA comprising an antisense nucleic acid described herein.

In some embodiments, an inhibitory nucleic acid is a small interfering RNA (siRNA). As used herein, 'siRNA' refers to a double-stranded RNA molecule having a length between 17 to 30 (e.g. 20 to 27, e.g. ~21) base pairs, which is capable of engaging the RNA interference (RNAi) pathway for the targeted degradation of target RNA. Double-stranded siRNA molecules may be formed as a nucleic acid complex of RNA strands having a high degree of complementarity In some embodiments, siRNA molecules comprise symmetric 3' overhangs, e.g. comprising one or two nucleotides (e.g. a 'UU' 3' overhang). In some embodiments, an antisense nucleic acid described herein (e.g. one of SEQ ID NO: 214 to 418) comprises a 'UU' 3' overhang. The strand of the double-stranded siRNA molecule having complementarity to a target nucleotide sequence (/.e. the antisense nucleic add) may be referred to as the 'guide' strand, and the other strand may be referred to as the 'passenger' strand or 'sense' strand. The structure and function of siRNAs is described e.g. in Kim and Rossi, Biotechniques. 2008 Apr; 44(5): 613-616.

In some embodiments, the guide strand of an siRNA according to the present disclosure may comprise or consist of an antisense nucleic acid according to an embodiment of an antisense nucleic acid described herein.

In some embodiments the passenger strand of an siRNA according to the present disdosure may comprise or consist of a sense nudeic acid according to an embodiment of a sense nudeic add described herein. In some embodiments the passenger strand of an siRNA according to the present disclosure may comprise or consist of a nucleic add comprising the nudeotide sequence (including the modifications thereto) shown in one of SEQ ID NOs: 9 to 213, 1072 to 1111, 1260 to 1263, 1288 to 1291, 1316 to 1319, 1344 to 1377, or 1417 to 1428.

In some embodiments, an inhibitory nucleic acid is a dicer small interfering RNA (dsiRNA). As used herein, 'dsiRNA' refers to a double-stranded RNA molecule having a length of ~27 base pairs, which is processed by Dicer to siRNA for RNAkmediated degradation of target RNA. DsiRNAs are described e.g. in Raja et al., Asian J Pharm Sci. (2019) 14(5): 497-510, which is hereby incorporated by reference in their entirety. DsiRNAs are optimised for Dicer processing and may have increased potency compared with 21-mer siRNAs (see e.g. Kim et al., Nat Biotechnol. (2005) 23(2):222-226), which may be related to the link between Dicer-mediated nuclease activity and RISC loading.

In some embodiments, an inhibitory nudeic acid is a micro RNA (miRNA), or a precursor thereof (e.g. a pri-miRNA or a pre-miRNA). miRNA molecules have a similar structure to siRNA molecules, but are encoded endogenously, and derived from processing of short hairpin RNA molecules. They are initially expressed as long primary transcripts (pri-miRNAs), which are processed within the nucleus into 60 to 70 nucleotide hairpins (pre-miRNAs), which are further processed in the cytoplasm into smaller spedes that interact with RISC and target mRNA. miRNAs comprise ‘seed sequences’ that are essential for binding to target mRNA. Seed sequences usually comprise six nudeotides and are situated at positions 2 to 7 at the miRNA 5' end.

In some embodiments, an inhibitory nucleic acid is a short hairpin RNA (shRNA). shRNA molecules comprise sequences of nucleotides having a high degree of complementarity that associate with one another through complementary base pairing to form the stem region of the hairpin. The sequences of nudeotides having a high degree of complementarity may be linked by one or more nudeotides that form the loop region of the hairpin. shRNA molecules may be processed (e.g. via catalytic cleavage by DICER) to form siRNA or miRNA molecules. shRNA molecules may have a length of between 35 to 100 (e.g. 40 to 70) nucleotides. The stem region of the hairpin may have a length between 17 to 30 (e.g. 20 to 27, e.g. ~21) base pairs. The stem region may comprise G-U pairings to stabilise the hairpin structure. siRNA dsiRNA, miRNAs and shRNAs for the targeted inhibition of gene and/or protein expression of PCSK9 may be identified/designed In accordance with principles and/or using tools well known to the skilled person. Parameters and tools for designing siRNA and shRNA molecules are described e.g. in Fakhr et al., Cancer Gene Therapy (2016) 23:73-82 (hereby incorporated by reference in its entirety). Software that may be used by the skilled person for the design of such molecules is summarised in Table 1 of Fakhr et al., Cancer Gene Therapy (2016) 23:73-82, and includes e.g. siRNA Wizard (InvivoGen). Details for making such molecules can be found in the websites of commercial vendors such as Ambion, Dharmacon, GenScript, Invitrogen and OligoEngine.

In some embodiments, an inhibitory nucleic acid is an antisense oligonucleotide (ASO). ASOs are singlestranded nucleic acid molecules comprising or consisting of an antisense nucleic acid to a target nucleotide sequence. An antisense oligonucleotide according to the present disclosure may comprise or consist of an antisense nucleic acid as described herein.

ASOs can modify expression of RNA molecules comprising their target nucleotide sequence by altering splicing, or by recruiting RNase H to degrade RNA comprising the target nucleotide sequence. RNase H recognises nucleic acid complex molecules formed when the ASO binds to RNA comprising its target nucleotide sequence. ASOs according to the present disclosure may comprise or consist of an antisense nucleic acid according to the present disclosure. ASOs may comprise 17 to 30 (e.g. 20 to 27, e.g. ~21) nucleotides in length. Many ASOs are designed as chimeras, comprising a mix of bases with different chemistries, or as gapmers, comprising a central DNA portion surrounded by "wings’ of modified nucleotides. ASOs are described in e.g. Scoles et al., Neurol Genet 2019 Apr; 5(2): e323. ASOs sometimes comprise alterations to the sugar-phosphate backbone in order to increase their stability and/or reduce/prevent RNAse H degradation, such as e.g. phosphorothioate linkages, phosphorod iamidate linkages such as phosphorodiamidate morpholino (PMOs), and may comprise e.g. peptide nucleic acids (PNAs), locked nucleic acids (LNAs), methoxyethyl nucleotide modifications, e.g. 2'O-methyl (2'OMe) and 2'-O-methoxyethyl (MOE) ribose modifications and/or 5'-methylcytosine modifications.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises: (I) nucleic acid comprising the nucleotide sequence of one of SEQ ID NOs:214 to 418, or a nucleotide sequence having at least 75% sequence identity (e.g. one of at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to one of SEQ ID NOs:214 to 418; and (ii) nucleic acid comprising a nucleotide sequence having the reverse complement of the nucleotide sequence of (i), or having at least 75% sequence identity (e.g. one of at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to the reverse complement of the nucleotide sequence of (i).

In some embodiments, an inhibitory nucleic add according to the present disclosure comprises: (i) nucleic acid comprising the nucleotide sequence of one of SEQ ID NOs:627 to 831 , or a nucleotide sequence having at least 75% sequence identity (e.g. one of at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to one of SEQ ID NOs:627 to 831; and (ii) nucleic add comprising a nucleotide sequence having the reverse complement of the nudeotide sequence of (i), or having at least 75% sequence identity (e.g. one of at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to the reverse complement of the nucleotide sequence of (i).

In some embodiments, an inhibitory nudeic acid according to the present disclosure comprises: (i) nudeic add comprising the nucleotide sequence of one of SEQ ID NOs: 832 to 1071 , 1236 to 1259, 1264 to 1287, 1292 to 1315, 1320 to 1343, 1378 to 1416, or 1429 to 1431, or a nucleotide sequence having at least 75% sequence identity (e.g. one of at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to one of SEQ ID NOs: 832 to 1071, 1236 to 1259, 1264 to 1287, 1292 to 1315, 1320 to 1343, 1378 to 1416, or 1429 to 1431; and (ii) nudeic acid comprising a nucleotide sequence having the reverse complement of the nucleotide sequence of (i), or having at least 75% sequence identity (e.g. one of at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to the reverse complement of the nucleotide sequence of (i).

In some embodiments in accordance with the preceding two paragraphs, the nucleotide sequence of (i) and the nudeotide sequence of (ii) may be provided on different nudeic acids (/.e. separate oligonucleotides). As such, the nucleic add of (i) and (ii) may be different nucleic acids. In such embodiments, the inhibitory nucleic add may comprise or consist of a nucleic acid duplex formed by complementary base pairing between the different nucleic adds comprising the nudeotide sequences of (i) and (ii).

Alternatively, in some embodiments the nucleotide sequence of (i) and the nucleotide sequence of (ii) may be provided on the same nucleic acid (/.e. a single oligonucleotide). That is, the nucleic acid of (i) and (ii) may be the same nucleic acid. In such embodiments, the nucleotide sequence of (i) and the nucleotide sequence of (ii) may be connected by one or more linker nucleotides. The inhibitory nucleic acid may comprise a nucleic acid duplex region formed by complementary base pairing between the nucleotide sequences of (i) and (ii), and the linker regions may form a single-stranded loop region.

Inhibitory nucleic acids according to the present disdosure may comprise chemically modified nudeotides, e.g. in which the phosphonate and/or ribose and/or base is/are chemically modified. Such modifications may influence the activity, specificity and/or stability of nucleic acid. One or more (e.g. one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or all) nucleotides of an inhibitory nucleic acid may comprise such chemical modification.

An inhibitory nucleic acid comprising chemically modified nucleotides according to the present disclosure may be capable of reducing or preventing the gene and/or protein expression of any one or more given target gene(s)/protein(s). In some cases, the target gene/protein is PCSK9. In some cases, the target gene/protein is a gene/protein other than PCSK9 (i.e. that is not PCSK9). The target/gene protein may be a gene/protein that is pathologically implicated in a disease or condition associated with or affecting the liver. For example, the target/gene protein may be a gene/protein that is pathologically implicated in a disease or condition selected from: dyslipidemia, hyperlipidemia, hypercholesterolemia, familial hypercholesterolemia, autosomal dominant hypercholesterolemia, atherosclerosis, cardiovascular disease, atherosclerotic cardiovascular disease, coronary artery disease, angina, myocardial infarction, cardiac failure, peripheral vascular disease, peripheral arterial disease, hypertension, stroke, ischemic stroke, transient ischemic attack, congestive heart failure, steatosis, hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH).

In some embodiments, the target gene/protein is C-reactive protein (CRP), interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF), Patatin-like phospholipase domain-containing 3 (PNPLA3), tetraspanin 4 (TSPAN4), peptidoglycan recognition protein 2 (PGLYRP2), thrombospondin 1 (THBS1), dual specific phosphatase-4 (DUSP4), or any gene/protein described in V\fang R et al., Ann Hepatol. 2016;15(2):190-9, Liu J et al., Arch Med Sci. 2020; 16(2): 374-385, or Li etal., Mol Med Rep. 2018;17(6):7708-7720, which are all hereby incorporated by reference in their entirety.

Modifications contemplated in accordance with inhibitory nucleic acids according to the present disclosure include those described in Hu eta/., Sig. Transduc. Tar. Ther. (2020) 5(101) (incorporated by reference hereinabove), in particular those shown in Figure 2 of Hu eta/., Sig. Transduc. Tar. Ther. (2020) 5(101). Further modifications contemplated in accordance with inhibitory nucleic acids according to the present disclosure include those described in Selvam eta/., Chem Biol Drug Des. (2017) 90(5): 665-678, which is hereby incorporated by reference in its entirety).

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises one or more nucleotides comprising a phosphonate modification. In some embodiments, the phosphonate modification(s) may be selected from: phosphorothioate (e.g. Rp isomer, Sp isomer), phosphorodithioate, methylphosphonate, methoxypropylphosphonate, 5'-(E)-vinylphosphonate, S'-methylphosphonate, (S)-5- C-methyl with phosphate, S’-phosphorothioate, and peptide nucleic acid. In some embodiments, an inhibitory nucleic acid comprises one or more nucleotides comprising phosphorothioate modification.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises one or more nucleotides comprising a ribose modification. In some embodiments, the ribose modification(s) may be selected from: Z-O-methyl, 2'-O-methoxyethyl 2' fluoro 2' deoxy-2’-fluoro, Z-methoxyethyl, Z-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, 2'-arabino-fluoro, 2'-O-benzyl, 2’-O-methyl-4-pyridine, locked nucleic acid, (S)-cEt-BNA, tricyclo-DNA, PMO, unlocked nucleic acid, hexitol nucleic acid and glycol nucleic acid. In some embodiments, an inhibitory nucleic acid comprises one or more nucleotides comprising 2'-O-methyl modification. In some embodiments, an inhibitory nucleic acid comprises one or more nucleotides comprising 2-fluoro modification.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises one or more nucleotides comprising a base modification. In some embodiments, the base modification(s) may be selected from: pseudouridine, 2-thiouridine, N 6'-methyladenosine, S’-methylcytidine, 5’-fluoro-2’- deoxyuridine, N-ethylpiperidine 7-EAA triazole-modified adenine, N-ethylpiperidine 6'-triazole-modified adenine, O'-phenylpyrrolo-cytosine, 2',4'-difluorotoluyl ribonucleoside and S'-nitroindole.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises: one or more nucleotides comprising phosphorothioate modification, one or more nucleotides comprising 2'-O- methyl modification, and one or more nucleotides comprising 2-fluoro modification.

In some embodiments, an inhibitory nucleic add according to the present disdosure comprises one or more modified nucleotides selected from: 2 ^, -O-methyluridine-3 ^, -phosphate, 2'-O-methyladenosine-3'- phosphate, 2 ^, -O-methylguanosine-3'-phosphate, 2 ^, -O-methylcytidine-3'-phosphate, 2'-O-methyluridine-3'- phosphorothioate, 2 ^, -0-methyladenosine-3'-phosphorothioate, 2'-0-methylguanosine-3'- phosphorothioate, 2'-0-methylcytidine-3 ^, -phosphorothioate, 2 ^, -fluorouridine-3'-phosphate, 2'- fluoroadenosine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'- fluorocytidine-3'-phosphorothioate, 2'-fluoroguanosine-3'-phosphorothioate, 2'-fluoroadenosine-3'- phosphorothioate, and 2'-fluorouridine-3'-phosphorothioate.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises a nucleotide sequence comprising 3 to 10 (e.g. one of 3, 4, 5, 6, 7, 8, 9 or 10) nucleotides comprising 2'- fluoro modification. In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises 4 to 15 (e.g. one of 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15) nucleotides comprising 2-fluoro modification. In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises a nucleotide sequence comprising 2 to 6 (e.g. one of 2, 3, 4, 5 or 6) nucleotides comprising phosphorothioate modification. In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises 5 to 20 (e.g. one of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) nucleotides comprising 2-O-methyl modification. In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises a nucleotide sequence comprising 2 to 6 (e.g. one of 2, 3, 4, 5 or 6) nucleotides comprising 2'-O-methyl and phosphorothioate modification. In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises a nucleotide sequence comprising 1 to 4 (e.g. one of 1 , 2, 3 or 4) nucleotides comprising 2-fluoro and phosphorothioate modification. In various aspects and embodiments, the present disclosure provides an inhibitory nucleic add comprising a pattern of modified nucleotides. The patterns of modified nudeotides disclosed herein are applicable irrespective of the identities of the individual nudeotides within a nucleic add sequence, i.e. the patterns according to the present disdosure can be applied to any inhibitory nucleic acid capable of reducing or preventing the gene and/or protein expression of any target gene(s)/protein(s).

In some embodiments, an inhibitory nucleic acid according to the present disclosure (i.e. an inhibitory nudek: acid that targets any gene/protein, optionally targeting PCSK9) comprises a (one or more) nucleotide sequence(s) having nudeotides comprising a pattern of modifications according to one of rows 1 to 70 of Table A below. In some embodiments, an inhibitory nucleic add according to the present disclosure comprises a (one or more) nucleotide sequence(s) having nucleotides comprising a pattern of modifications according to one of rows 1 to 24 or rows 29 to 70 of Table A below. In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises an antisense nucleic acid (e.g. a guide strand) having a (one or more) nudeotide sequence(s) having nucleotides comprising a pattern of modifications according to one of rows 1 to 24 or rows 29 to 70 of Table A below.

In some embodiments, an inhibitory nucleic acid according to the present disdosure comprises a (one or more) nucleotide sequence(s) having nudeotides comprising a pattern of modifications according to one of rows 25 to 28 of Table A below. In some embodiments, an inhibitory nucleic add according to the present disdosure comprises a sense nucleic acid (e.g. a sense strand) having (one or more) a nucleotide sequence(s) having nucleotides comprising a pattern of modifications according to one of rows 25 to 28 of Table A below.

In some embodiments, an inhibitory nucleic add according to the present disclosure comprises (i) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to one of rows 1 to 24 of Table A below; and (ii) a sense nucleic add (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to one of rows 25 to 28 of Table A below.

Table A: modification patters.

In the table above, mN = nucleotide comprising 2’-O-methyl modification, fN = nucleotide comprising 2'- deoxy-2'-fluoro modification, ps = phosphorothioate linkage. If not specified, there are phosphate linkages between two nucleotides.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (I) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to one of rows 1 to 24 or rows 29 to 70 of Table A; and/or (ii) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to one of rows 25 to 28 of the Table A, following the pairs of patterns in Table B below. Row 1 is the row labelled ’ssOOT.

In some embodiments, the inhibitory nucleic acid comprises, or consists of: (i) nucleic acid (e.g. antisense nucleic acid/guide strand) comprising a nucleotide sequence having nucleotides comprising a pattern of modifications indicated in column 1 of Table B, and/or (ii) nucleic acid (e.g. a sense strand) comprising a nucleotide sequence having nucleotides comprising a pattern of modifications indicated in column 2 of Table B, wherein the sequences of columns 1 and 2 are selected from the same row of Table B. The modification patterns are described in Table A above.

In some embodiments, an inhibitory nucleic acid according to the present disdosure comprises (i) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nudeotides comprising a pattern of modifications according to row 1 of Table A; and/or (Ii) a sense nudeic add (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 26 of Table A.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (I) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 2 of Table A; and/or (ii) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 26 of Table A.

In some embodiments, an inhibitory nucleic add according to the present disdosure comprises (I) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 3 of Table A; and/or (ii) a sense nudeic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 26 of Table A.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (i) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 4 of Table A; and/or (ii) a sense nucleic add (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 26 of Table A.

In some embodiments, an inhibitory nudeic acid according to the present disdosure comprises (i) an antisense nucleic add (e.g. a guide uence having nucleotides comprising a pattern of modifications according to row 5 of Table A; and/or (ii) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 26 of Table A.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (i) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 6 of Table A; and/or (ii) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 26 of Table A.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (i) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 7 of Table A; and/or (ii) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a patte of modifications according to row 26 of Table A.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (i) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 8 of Table A; and/or (ii) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a patter of modifications according to row 26 of Table A

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (i) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 9 of Table A; and/or (ii) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a patte of modifications according to row 26 of Table A.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (i) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 10 of Table A; and/or (ii) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a patter of modifications according to row 26 of Table A.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (i) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a patter of modifications according to row 11 of Table A; and/or (ii) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 26 of Table A. In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (I) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 12 of Table A; and/or (ii) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 26 of Table A.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (i) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 13 of Table A; and/or (ii) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 26 of Table A.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (i) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 14 of Table A; and/or (ii) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 26 of Table A.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (i) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 15 of Table A; and/or (ii) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 26 of Table A.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (I) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 16 of Table A; and/or (ii) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 26 of Table A.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (i) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 17 of Table A; and/or (ii) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 26 of Table A.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (i) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 18 of Table A; and/or (ii) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 26 of Table A. In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (i) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 19 of Table A; and/or (ii) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 26 of Table A.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (I) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 20 of Table A; and/or (ii) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 26 of Table A.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (i) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 21 of Table A; and/or (ii) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 26 of Table A.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (I) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 22 of Table A; and/or (ii) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 26 of Table A.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (i) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 23 of Table A; and/or (II) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 26 of Table A.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (i) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 24 of Table A; and/or (ii) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 26 of Table A.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (i) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 1 of Table A; and/or (II) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 25 of Table A.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (i) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 1 of Table A; and/or (ii) a sense nucleic add (e.g. a sense strand) having a nucleotide sequence having nudeotides comprising a pattern of modifications according to row 27 of Table A.

In some embodiments, an inhibitory nudeic add according to the present disdosure comprises (i) an antisense nucleic add (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 1 of Table A; and/or (II) a sense nudeic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 28 of Table A.

In some embodiments, an inhibitory nucleic acid according to the present disdosure comprises (i) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nudeotides comprising a pattern of modifications according to row 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 of Table A; and/or (ii) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nudeotides comprising a pattern of modifications according to row 26 of Table A.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises (i) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 of Table A; and/or (II) a sense nucleic acid (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 27 of Table A.

In some embodiments, an inhibitory nudeic add according to the present disclosure comprises (i) an antisense nucleic acid (e.g. a guide strand) having a nucleotide sequence having nudeotides comprising a patter of modifications according to row 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 of Table A; and/or (II) a sense nucleic add (e.g. a sense strand) having a nucleotide sequence having nucleotides comprising a pattern of modifications according to row 28 of Table A

Modified nucleic adds according to the present disclosure may have advantageous properties, including one or more of increased potency, increased bioavailability, decreased toxicity, and decreased off-target effects, increased stability e.g. against nudease degradation, improved affinities, expanded chemical functionality, and/or improved targeting e.g. to organ or tissue of interest, e.g. compared to an unmodified nucleic add. In embodiments wherein inhibitory nucleic acids comprise nucleotides comprising chemical modification as described herein, the nucleotide sequence is nevertheless evaluated for the purposes of sequence comparison in accordance with the present disclosure as if the equivalent unmodified nucleotide were instead present

Nucleic acids comprising nucleotide(s) comprising a modified phosphonate group are evaluated for the purposes of nucleotide sequence comparison as if nucleotide(s) comprising a modified phosphonate group instead comprise the equivalent unmodified phosphonate group. Nucleic acids comprising nucleotide(s) comprising a modified ribose group are evaluated for the purposes of nucleotide sequence comparison as if nucleotide(s) comprising a modified ribose group instead comprise the equivalent unmodified ribose group. Nucleic acids comprising nucleotide(s) comprising a modified base group are evaluated for the purposes of nucleotide sequence comparison as if nucleotide(s) comprising a modified base group instead comprise the equivalent unmodified base group.

By way of illustration, nucleic acids comprising nucleotides comprising pseudouridine, 2-thiouridine and/or 5’-fluoro-2’-deoxyuridine are evaluated for the purposes of nucleotide sequence comparison as if nucleotides comprising uridine were instead present at their respective positions. By way of illustration, nucleic acids comprising nucleotides comprising N6'-methyladenosine, N-ethylpiperidine 7-EAA triazole- modified adenine and/or N-ethylpiperidine e'-triazole-modified adenine are evaluated for the purposes of nucleotide sequence comparison as if nucleotides comprising adenine were instead present at their respective positions. By way of illustration, nucleic acids comprising nucleotides comprising 5’- methylcytidine and/or e'-phenylpyrrolo-cytosine are evaluated for the purposes of nucleotide sequence comparison as if nucleotides comprising cytosine were instead present at their respective positions.

In some embodiments, an inhibitory nucleic acid according to the present disclosure comprises nucleic acid comprising the nucleotide sequence (including the modifications thereto) shown in one of SEQ ID NOs:832 to 1071.

In some embodiments, an inhibitory nucleic acid comprises nucleic acid comprising the nucleotide sequence (including the modifications thereto) shown in one of SEQ ID NOs:832 to 855. In some embodiments, an inhibitory nucleic acid comprises nucleic acid comprising the nucleotide sequence (including the modifications thereto) shown in one of SEQ ID NOs:856 to 879. In some embodiments, an inhibitory nucleic acid comprises nucleic acid comprising the nucleotide sequence (including the modifications thereto) shown in one of SEQ ID NOs:880 to 903. In some embodiments, an inhibitory nucleic acid comprises nucleic acid comprising the nucleotide sequence (including the modifications thereto) shown in one of SEQ ID NOs:904 to 927. In some embodiments, an inhibitory nucleic acid comprises nucleic acid comprising the nucleotide sequence (including the modifications thereto) shown in one of SEQ ID NOs:928 to 951. In some embodiments, an inhibitory nucleic acid comprises nucleic acid comprising the nucleotide sequence (including the modifications thereto) shown in one of SEQ ID NOs:952 to 975. In some embodiments an inhibitory nucleic acid comprises nucleic acid comprising the nucleotide sequence (including the modifications thereto) shown in one of SEQ ID NOs:976 to 999. In some embodiments, an inhibitory nucleic acid comprises nucleic acid comprising the nudeotide sequence (induding the modifications thereto) shown in one of SEQ ID NOs: 1000 to 1023. In some embodiments, an inhibitory nudeic acid comprises nucleic acid comprising the nudeotide sequence (induding the modifications thereto) shown in one of SEQ ID NOs:1024 to 1047. In some embodiments, an inhibitory nucleic add comprises nucleic add comprising the nucleotide sequence (induding the modifications thereto) shown in one of SEQ ID NOs:1048 to 1071.

In some embodiments, an inhibitory nudeic add according to the present disclosure comprises: (i) nucleic acid comprising the nudeotide sequence (including the modifications thereto) shown in one of SEQ ID NOs:832 to 1071; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) shown in one of SEQ ID NOs: 1072 to 1111.

In some embodiments, an inhibitory nucleic acid comprises, or consists of: (i) nucleic add comprising the nudeotide sequence (including the modifications thereto) shown in one of SEQ ID NOs:832 to 855; and (ii) nucleic add comprising the nudeotide sequence (induding the modifications thereto) shown in one of SEQ ID NOs:1072 to 1075. In some embodiments, an inhibitory nudeic acid comprises, or consists of: (i) nucleic add comprising the nudeotide sequence (including the modifications thereto) shown in one of SEQ ID NOs:856 to 879; and (ii) nucleic acid comprising the nudeotide sequence (induding the modifications thereto) shown in one of SEQ ID NOs: 1076 to 1079. In some embodiments, an inhibitory nucleic add comprises, or consists of: (i) nudeic acid comprising the nucleotide sequence (induding the modifications thereto) shown in one of SEQ ID NOs:880 to 903; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) shown in one of SEQ ID NOs: 1080 to 1083.. In some embodiments, an inhibitory nucleic add comprises, or consists of: (i) nucleic add comprising the nucleotide sequence (including the modifications thereto) shown in one of SEQ ID NOs: 904 to 927; and (ii) nucleic add comprising the nucleotide sequence (induding the modifications thereto) shown in one of SEQ ID NOs:1084 to 1087. In some embodiments, an inhibitory nucleic acid comprises, or consists of: (i) nucleic acid comprising the nudeotide sequence (including the modifications thereto) shown in one of SEQ ID NOs:928 to 951; and (ii) nucleic acid comprising the nudeotide sequence (induding the modifications thereto) shown in one of SEQ ID NOs:1088 to 1091. In some embodiments, an inhibitory nucleic acid comprises, or consists of: (i) nucleic acid comprising the nucleotide sequence (induding the modifications thereto) shown in one of SEQ ID NOs: 952 to 975; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) shown in one of SEQ ID NOs: 1092 to 1095. In some embodiments, an inhibitory nudeic acid comprises, or consists of: (i) nucleic add comprising the nudeotide sequence (including the modifications thereto) shown in one of SEQ ID NOs: 976 to 999; and (ii) nucleic acid comprising the nudeotide sequence (induding the modifications thereto) shown in one of SEQ ID NOs: 1096 to 1099. In some embodiments, an inhibitory nucleic acid comprises, or consists of: (i) nudeic add comprising the nucleotide sequence (including the modifications thereto) shown in one of SEQ ID NOs: 1000 to 1023; and (ii) nudeic acid comprising the nudeotide sequence (including the modifications thereto) shown in one of SEQ ID NOs:1100 to 1103, 1417 or 1418. In some embodiments, an inhibitory nucleic acid comprises, or consists of: (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) shown in one of SEQ ID NOs: 1024 to 1047; and (ii) nucleic add comprising the nucleotide sequence (induding the modifications thereto) shown in one of SEQ ID NOs: 1104 to 1107. In some embodiments, an inhibitory nucleic add comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) shown in one of SEQ ID NOs: 1048 to 1071; and (ii) nudeic acid comprising the nucleotide sequence (including the modifications thereto) shown in one of SEQ ID NOs:1108 to 1111.

In some embodiments, an inhibitory nucleic add comprises, or consists of (i) nucleic acid comprising a nudeotide sequence (induding the modifications thereto) indicated in column A of Table 1 (below), and (ii) nucleic acid comprising a nudeotide sequence (including the modifications thereto) indicated in column B of Table 1 , wherein the sequences of columns A and B are selected from the same row of Table 1.

By way of illustration, where the nucleotide sequences of (i) and (ii) are selected from row 1 of Table 1 , the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:832; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:1072. By way of further illustration, the inhibitory nucleic add designated ‘CG-100002’ described in Example 2 herein is formed of (i) an oligonucleotide having the nucleotide sequence (including the modifications thereto) of SEQ ID NO:878, and (ii) an oligonucleotide having the nudeotide sequence (induding the modifications thereto) of SEQ ID NO: 1077; this embodiment corresponds to row 53 of Table 1.

Table i:

Column A Column B

T SEQ ID NO:832 SEQ ID NO:1072 2 SEQ ID NO:832 SEQ ID NO:1073 3 SEQ ID NO:832 SEQ ID NO:1074 4 SEQ ID NO:832 SEQ ID NO:1075 5 SEQ ID NO:833 SEQ ID NO: 1073 6 SEQ ID NO:834 SEQ ID NO: 1073 -7 SEQ ID NO:835 SEQ ID NO: 1073 8 SEQ ID NO:836 SEQ ID NO: 1073 9 SEQ ID NO:837 SEQ ID NO: 1073 10 SEQ ID NO:838 SEQ ID NO: 1073 11 SEQ ID NO:839 SEQ ID NO: 1073 12 SEQ ID NO:840 SEQ ID NO: 1073 13 SEQ ID NO:841 SEQ ID NO: 1073

SEQ ID NO:842 SEQ ID NO: 1073

15 SEQ ID NO:843 SEQ ID NO: 1073

16 SEQ ID NO:844 SEQ ID NO: 1073

17 SEQ ID NO:845 SEQ ID NO: 1073

18 SEQ ID NO:846 SEQ ID NO: 1073

19 SEQ ID NO:847 SEQ ID NO: 1073

20 SEQ ID NO:848 SEQ ID NO: 1073

21 SEQ ID NO:849 SEQ ID NO: 1073

22 SEQ ID NO:850 SEQ ID NO: 1073

23 SEQ ID NO:851 SEQ ID NO: 1073

24 SEQ ID NO:852 SEQ ID NO: 1073

25 SEQ ID NO:853 SEQ ID NO: 1073

26 SEQ ID NO:854 SEQ ID NO: 1073

27 SEQ ID NO:855 SEQ ID NO: 1073

28 EQ ID NO: 1076

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:878; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1077. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:873; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1077. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the m difi ti th t ) f SEQ ID NO:872; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1077. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:858; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1077. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:867; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1077. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:868; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1077. In some embodiments, the inhibitory nucleic add comprises, or consists of (i) nucleic add comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO:866; and (ii) nudeic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1077. In some embodiments, the inhibitory nudeic add comprises, or consists of (i) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:859; and (ii) nucleic acid comprising the nudeotide sequence (induding the modifications thereto) of SEQ ID NO: 1077. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nudeic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:857; and (ii) nucleic add comprising the nudeotide sequence (induding the modifications thereto) of SEQ ID NO: 1077. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:861; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1077. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic add comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO:856; and (ii) nucleic acid comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO: 1077. In some embodiments, the inhibitory nudeic acid comprises, or consists of (i) nudeic add comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO:856; and (ii) nudeic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1078. In some embodiments, the inhibitory nudeic add comprises, or consists of (i) nudeic acid comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO:874; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1077. In some embodiments, the inhibitory nudeic add comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:856; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1079. In some embodiments, the inhibitory nudeic add comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:860; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1077. In some embodiments, the inhibitory nudeic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:863; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1077. In some embodiments, the inhibitory nucleic acid comprises or consists of (i) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:864; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1077. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:877; and (II) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1077. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:879; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1077. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:875; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1077. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:869; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1077. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:876; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1077. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:871; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1077. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:865; and (ii) nucleic add comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO: 1077. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nudeic add comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO:870; and (ii) nucleic add comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO: 1077. In some embodiments, the inhibitory nudeic acid comprises, or consists of (i) nudek: add comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO:850; and (ii) nucleic acid comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO: 1073. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nudeic acid comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO:849; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1073. In some embodiments, the inhibitory nudeic acid comprises, or consists of (i) nudeic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:843; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1073. In some embodiments, the inhibitory nudeic acid comprises, or consists of (i) nudeic acid comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO:854; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1073. In some embodiments, the inhibitory nudeic acid comprises, or consists of (i) nudeic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:844; and (ii) nucleic acid comprising the nucleotide sequence (induding the modifications thereto) of SEQ ID NO: 1073. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:851; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1073. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:842; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1073. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:845; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1073. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence of SEQ ID NO:226; and (ii) nucleic add comprising the nucleotide sequence of SEQ ID NO:21. In some embodiments, the inhibitory nucleic add comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:852; and (ii) nudeic add comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO:

1073. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nudeic acid comprising the nucleotide sequence (induding the modifications thereto) of SEQ ID NO:846; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1073. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nudeic acid comprising the nucleotide sequence (induding the modifications thereto) of SEQ ID NO:855; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1073. In some embodiments, the inhibitory nucleic add comprises, or consists of (i) nudeic acid comprising the nudeotide sequence (induding the modifications thereto) of SEQ ID NO:853; and (ii) nucleic acid comprising the nucleotide sequence (induding the modifications thereto) of SEQ ID NO: 1073. In some embodiments, the inhibitory nucleic add comprises, or consists of (I) nudeic acid comprising the nudeotide sequence (induding the modifications thereto) of SEQ ID NO:833; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1073. In some embodiments, the inhibitory nucleic acid comprises, or consists of (I) nudeic acid comprising the nucleotide sequence (induding the modifications thereto) of SEQ ID NO:845; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1073. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nudeic acid comprising the nucleotide sequence (induding the modifications thereto) of SEQ ID NO:834; and (ii) nucleic add comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO: 1073. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:835; and (ii) nudeic acid comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO:

1073. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO:832; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:

1074. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic add comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO:832; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1073. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:837; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1073. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:841; and (II) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1073. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:832; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1075. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:832; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:

1072. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:836; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:

1073. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:840; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1073. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:838; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1073. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:839; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1073. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence of SEQ ID NO:258; and (ii) nucleic acid comprising the nucleotide sequence of SEQ ID NO:53. In some embodiments, the inhibitory nucleic acid comprises, or consists of (I) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:880; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1082. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:882; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1081. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:881; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1081. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:890; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1081. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:880; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1083. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:902; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1081 . In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:896; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1081 . In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:897; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1081 . In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:883; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1081. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:892; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1081. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:891; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1081. In some embodiments, the inhibitory nucleic acid comprises, or consists of (I) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:880; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1081. In some embodiments, the inhibitory nucleic acid comprises, or consists of (I) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:884; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1081 . In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:888; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1081 . In some embodiments, the inhibitory nucleic acid comprises, or consists of (I) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:886; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1081. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:887; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1081. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:901; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1081. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:889; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1081. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:894; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1081. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:903; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1081. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:893; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1081. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:900; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1081. In some embodiments, the inhibitory nucleic add comprises, or consists of (i) nudeic acid comprising the nucleotide sequence (induding the modifications thereto) of SEQ ID NO:889; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1081. In some embodiments, the inhibitory nucleic add comprises, or consists of (i) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:895; and (ii) nudeic acid comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO: 1081. In some embodiments, the inhibitory nudeic acid comprises, or consists of (i) nucleic acid comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO:401; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 196. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 954; and (ii) nudeic add comprising the nudeotide sequence (induding the modifications thereto) of SEQ ID NO: 1093. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 969; and (ii) nudeic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1093. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:952; and (ii) nucleic acid comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO:1451. In some embodiments, the inhibitory nucleic add comprises, or consists of (i) nudeic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:964; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1093. In some embodiments, the inhibitory nudeic add comprises, or consists of (i) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:952; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1093. In some embodiments, the inhibitory nucleic add comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:953; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1093. In some embodiments, the inhibitory nucleic add comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:968; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1093. In some embodiments, the inhibitory nucleic acid comprises or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:970; and (II) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1093. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:962; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1093. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:974; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1093. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:972; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1093. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:963; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1093. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:957; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1093. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:955; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1093. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:961; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1093. In some embodiments, the inhibitory nucleic acid comprises, or consists of. (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:971; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1093. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:952; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:1452. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:967; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1093. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:965; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1093. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:952; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:1092. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:975; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1093. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:973; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1093. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:966; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1093. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:956; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1093. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:958; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1093. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:959; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1093. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:960; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1093. In some embodiments, the inhibitory nucleic add comprises, or consists of (i) nucleic add comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO:938; and (ii) nucleic add comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO: 1089. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nudeic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:945; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1089. In some embodiments, the inhibitory nudeic acid comprises, or consists of (i) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:930; and (ii) nucleic acid comprising the nucleotide sequence (induding the modifications thereto) of SEQ ID NO: 1089. In some embodiments, the inhibitory nudeic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:940; and (ii) nucleic add comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO: 1089. In some embodiments, the inhibitory nucleic add comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:944; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1089. In some embodiments, the inhibitory nudeic acid comprises, or consists of (i) nudeic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:946; and (ii) nucleic acid comprising the nucleotide sequence (induding the modifications thereto) of SEQ ID NO: 1089. In some embodiments, the inhibitory nudeic add comprises, or consists of (i) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:929; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1089. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:933; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1089. In some embodiments, the inhibitory nucleic acid comprises, or consists of. (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:928; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1089. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:931; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1089. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:949; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1089. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:928; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1090. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:950; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1089. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:939; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1089. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:948; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1089. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:928; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:1091. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:937; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1089. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:943; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1089. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:942; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1089. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:402; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:197. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:941; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1089. In some embodiments, the inhibitory nucleic acid comprises or consists of (I) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:928; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1088. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:951; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1089. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:947; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1089. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic add comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO: 1055; and (ii) nudeic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1089. In some embodiments, the inhibitory nudeic add comprises, or consists of (i) nudeic add comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO:932; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1089. In some embodiments, the inhibitory nucleic add comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (induding the modifications thereto) of SEQ ID NO:935; and (ii) nucleic acid comprising the nudeotide sequence (induding the modifications thereto) of SEQ ID NO: 1089. In some embodiments, the inhibitory nucleic add comprises, or consists of (i) nudeic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:936; and (ii) nucleic acid comprising the nudeotide sequence (induding the modifications thereto) of SEQ ID NO: 1089. In some embodiments, the inhibitory nucleic add comprises, or consists of (i) nudeic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:916; and (ii) nucleic acid comprising the nudeotide sequence (induding the modifications thereto) of SEQ ID NO: 1085. In some embodiments, the inhibitory nucleic add comprises, or consists of (i) nudeic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:921; and (ii) nucleic add comprising the nudeotide sequence (induding the modifications thereto) of SEQ ID NO: 1085. In some embodiments, the inhibitory nucleic add comprises, or consists of. (i) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:920; and (ii) nucleic add comprising the nudeotide sequence (induding the modifications thereto) of SEQ ID NO: 1085. In some embodiments, the inhibitory nucleic add comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:922; and (ii) nucleic add comprising the nucleotide sequence (induding the modifications thereto) of SEQ ID NO: 1085. In some embodiments, the inhibitory nucleic add comprises, or consists of (i) nucleic acid comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO:914; and (ii) nucleic acid comprising the nucleotide sequence (induding the modifications thereto) of SEQ ID NO: 1085. In some embodiments, the inhibitory nudeic add comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:915; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1085. In some embodiments, the inhibitory nucleic acid comprises, or consists of. (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:905; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1085. In some embodiments, the inhibitory nucleic acid comprises, or consists oft (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:907; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1085. In some embodiments, the inhibitory nucleic acid comprises, or consists oft (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:926; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1085. In some embodiments, the inhibitory nucleic acid comprises, or consists of: (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:904; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1087. In some embodiments, the inhibitory nucleic acid comprises, or consists oft (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:904; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1085. In some embodiments, the inhibitory nucleic acid comprises, or consists oft (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:919; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1085. In some embodiments, the inhibitory nucleic acid comprises, or consists oft (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:925; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1085. In some embodiments, the inhibitory nucleic acid comprises, or consists oft (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:906; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1085. In some embodiments, the inhibitory nucleic acid comprises, or consists oft (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:904; and (ii) nucleic add comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO: 1086. In some embodiments, the inhibitory nucleic acid comprises, or consists oft (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:406; and (ii) nucleic add comprising the nudeotide sequence (induding the modifications thereto) of SEQ ID NO:201. In some embodiments, the inhibitory nucleic acid comprises, or consists oft (i) nudeic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:909; and (ii) nucleic acid comprising the nucleotide sequence (induding the modifications thereto) of SEQ ID NO: 1085. In some embodiments, the inhibitory nucleic acid comprises, or consists oft (i) nudeic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:918; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1085. In some embodiments, the inhibitory nucleic add comprises, or consists oft (i) nudeic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:923; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1085. In some embodiments, the inhibitory nucleic add comprises, or consists oft (i) nudeic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:924; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1085. In some embodiments, the inhibitory nudeic acid comprises, or consists oft (i) nudeic acid comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO:913; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1085. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic add comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO:927; and (ii) nucleic add comprising the nucleotide sequence (induding the modifications thereto) of SEQ ID NO: 1085. In some embodiments, the inhibitory nudeic acid comprises, or consists of (i) nudeic add comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO:904; and (ii) nucleic add comprising the nudeotide sequence (induding the modifications thereto) of SEQ ID NO: 1084.

In some embodiments, the Inhibitory nudeic acid comprises, or consists of (i) nucleic acid comprising a nucleotide sequence (including the modifications thereto) indicated in column A of Table 2 (Example 2), and (ii) nudeic add comprising a nudeotide sequence (induding the modifications thereto) indicated in column B of Table 2, wherein the sequences of columns A and B are selected from the same row of Table 2.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic add comprising a nucleotide sequence (including the modifications thereto) indicated in column A of Table 3 (Example 5), and (ii) nudeic acid comprising a nudeotide sequence (induding the modifications thereto) indicated in column B of Table 3, wherein the sequences of columns A and B are selected from the same row of Table 3.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of any one of SEQ ID NO:1116 to 1119; and (ii) nucleic add comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO:1120 to 1123.

In some embodiments, the inhibitory nucleic add comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1124 to 1147; and (ii) nudeic add comprising the nucleotide sequence (induding the modifications thereto) of SEQ ID NO: 1148 to 1151.

In some embodiments, the inhibitory nucleic add comprises, or consists of (i) nucleic acid comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO:1152 to 1175; and (ii) nudeic acid comprising the nudeotide sequence (induding the modifications thereto) of SEQ ID NO: 1176 to 1179.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1180 to 1203; and (ii) nudeic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1204 to 1207. In some embodiments, the inhibitory nucleic add comprises, or consists of (i) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:1208 to 1231; and (ii) nudeic acid comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO: 1232 to 1235.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nudeic acid comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO: 407 or 1236 to 1259; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 202, 1260 to 1263, 1363 to 1377, 1419 to 1424, or 1428.

In some embodiments, the inhibitory nucleic add comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1246; and (ii) nucleic add comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1428.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1254; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1428.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic add comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO: 406, 904 to 927, 1378 to 1416, or 1429 to 1431; and (ii) nucleic acid comprising the nudeotide sequence (induding the modifications thereto) of SEQ ID NO: 201, 1084 to 1087, 1348 to 1362, or 1425 to 1427. In some embodiments, nucleic add (ii) comprises the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1426. In some embodiments, nudeic add (ii) comprises the nudeotide sequence (induding the modifications thereto) of SEQ ID NO: 1427.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO:905; and (ii) nucleic acid comprising the nucleotide sequence (induding the modifications thereto) of SEQ ID NO:1427.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:920; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:1427.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:922; and (ii) nucleic add comprising the nudeotide sequence (induding the modifications thereto) of SEQ ID NO: 1426.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:922; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1427. In some embodiments, the inhibitory nucleic acid comprises, or consists of. (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1246; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1428.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (I) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1254; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1428.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1246; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1419.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (I) nucleic add comprising the nudeotide sequence (including the modifications thereto) of SEQ ID NO: 1246; and (ii) nudeic acid comprising the nucleotide sequence (induding the modifications thereto) of SEQ ID NO: 1420.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1246; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1421.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1246; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1422.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1246; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1423.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1246; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1424.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1378; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1425.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1378; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1427. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1382; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1427.

In some embodiments, the Inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1383; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1425.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1383; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1427.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1387; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 1427.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 406, 904 to 927, 1378 to 1416, or 1429 to 1431; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO:1085 or 1355.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 400, or 1264 to 1287; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 195, or 1288 to 1291.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 253, or 1292 to 1315; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 49 or 1316 to 1319.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 249, or 1320 to 1343; and (ii) nucleic acid comprising the nucleotide sequence (including the modifications thereto) of SEQ ID NO: 44 or 1344 to 1347.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising a nucleotide sequence (including the modifications thereto) indicated in column A of Table 4 (Example 6), and (ii) nucleic acid comprising a nucleotide sequence (including the modifications thereto) indicated in column B of Table 4, wherein the sequences of columns A and B are selected from the same row of Table 4. In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising a nucleotide sequence (including the modifications thereto) indicated in column A of Table 8 (Example 6), and (ii) nucleic acid comprising a nucleotide sequence (including the modifications thereto) indicated in column B of Table 8, wherein the sequences of columns A and B are selected from the same row of Table 8.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising a nucleotide sequence (including the modifications thereto) indicated in column A of Table 10 (Example 6), and (ii) nucleic acid comprising a nucleotide sequence (including the modifications thereto) indicated in column B of Table 10, wherein the sequences of columns A and B are selected from the same row of Table 10.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising a nucleotide sequence (including the modifications thereto) indicated in column A of Table 12 (Example 6), and (ii) nucleic acid comprising a nucleotide sequence (including the modifications thereto) indicated in column B of Table 12, wherein the sequences of columns A and B are selected from the same row of Table 12.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising a nucleotide sequence (including the modifications thereto) indicated in column A of Table 14 (Example 6), and (ii) nucleic acid comprising a nucleotide sequence (including the modifications thereto) indicated in column B of Table 14, wherein the sequences of columns A and B are selected from the same row of Table 14.

In some embodiments, the inhibitory nucleic acid comprises, or consists of (i) nucleic acid comprising a nucleotide sequence (including the modifications thereto) indicated in column A of Table 16 (Example 7), and (ii) nucleic acid comprising a nucleotide sequence (including the modifications thereto) indicated in column B of Table 16, wherein the sequences of columns A and B are selected from the same row of Table 16.

Inhibitory nucleic acids according to the present disclosure may be produced in accordance with techniques well known to the skilled person.

For example, inhibitory nucleic acids may be produced recombinantly by transcription of a nucleic acid sequence encoding the inhibitory nucleic acid. A nucleic acid encoding an inhibitory nucleic acid according to the present disclosure may e.g. be contained within an expression vector for expression of the inhibitory nucleic acid.

Transcription may be performed in cell-free transcription reactions using recombinant enzymes (e.g. RNA polymerase) fortranscription of the inhibitory nucleic acids Alternatively, production of an inhibitory nucleic acid according to the present disclosure may be performed in a cell comprising nucleic acid encoding the inhibitory nucleic acid, and may employ cellular enzymes (e.g. RNA polymerase) for transcription. Production of an inhibitory nucleic acid according to the present disclosure by expression within a cell may comprise transcription from a vector. Introduction of nucleic acid/vectors for the purposes of production of inhibitory nucleic acids according to the present disclosure may be performed in any of the ways known in the art (e.g. transfection, transduction, electroporation, etc.). Expression of an inhibitory nucleic acid can be regulated using a cell-specific promoter (e.g. a liver cell-specific promoter).

For example, an shRNA molecule according to the present disclosure may be produced within a cell by transcription from a vector encoding the shRNA. shRNAs may be produced within a cell by transfecting the cell with a vector encoding the shRNA sequence under control of an RNA polymerase promoter.

An siRNA molecule according to the present disclosure may be produced within a cell by transcription from a vector encoding shRNA encoding/comprising the siRNA, and subsequent processing of the shRNA molecule by cellular DICER to form the siRNA molecule.

Inhibitory nucleic acids may also be synthesised using standard solid or solution phase synthesis techniques which are well known in the art Solid phase synthesis may use phosphoramidite chemistry. Briefly, a solid supported nucleotide may be detritylated, then coupled with a suitably activated nucleoside phosphoramidite to form a phosphite triester linkage. Capping may then occur, followed by oxidation of the phosphite triester with an oxidant, typically iodine. The cycle may then be repeated to yield a polynucleotide.

The present disclosure provides nucleic acid comprising or encoding an inhibitory nucleic acid according to the present disclosure. In some embodiments, nucleic acid comprising or encoding an inhibitory nucleic acid comprises, or consists of, DNA and/or RNA.

The present disclosure also provides a vector comprising the nucleic acid comprising or encoding an inhibitory nucleic acid according to the present disclosure.

Nucleic acids and vectors according to the present disclosure may be provided in purified or isolated form, i.e. from other nucleic acid, or naturally-occurring biological material.

The nucleotide sequence of a nucleic acid comprising or encoding an inhibitory nucleic acid according to the present disclosure may be contained in a vector, e.g. an expression vector. A ‘vector' as used herein is a nucleic acid molecule used as a vehicle to transfer exogenous nucleic acid into a cell. The vector may be a vector for expression of the nucleic acid in the cell. Such vectors may include a promoter sequence operably linked to the nucleotide sequence encoding the sequence to be expressed. A vector may also include a termination codon and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express nucleic acid from a vector according to the present disclosure. The term ‘operably linked' may include the situation where a selected nucleic acid sequence and regulatory nucleic acid sequence (e.g. promoter and/or enhancer) are covalently linked in such a way as to place the expression of nucleic acid sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence is capable of affecting transcription of the nucleic acid sequence.

Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g. yeast artificial chromosomes).

In some embodiments, the vector may be a eukaryotic vector, e.g. a vector comprising the elements necessary for expression of nucleic acid from the vector in a eukaryotic cell. In some embodiments, the vector may be a mammalian vector, e.g. comprising a cytomegalovirus (CMV) or SV40 promoter to drive expression. In some embodiments, the vector comprises a cell- or tissue-specific promoter. In some embodiments, the vector comprises a liver cell-specific promoter.

The present disclosure also provides a plurality of inhibitory nucleic acids according to the present disclosure. The present disclosure also provides nucleic acids and vectors comprising or encoding a plurality of inhibitory nucleic acids according to the present disclosure.

Individual inhibitory nucleic acids of a plurality of inhibitory nucleic acids according to the present disclosure may be identical or non-identical. Similarly, in embodiments wherein a nucleic acid/vector comprising or encoding an inhibitory nucleic acid according to the present disclosure comprises/encodes more than one inhibitory nucleic acid according to the present disclosure, the inhibitory nucleic acids comprised/encoded by the nucleic acid/vector may be identical or non-identical.

In some embodiments, nucleic acids/vectors may encode one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 inhibitory nucleic acids according to the present disclosure. In some embodiments, nucleic acids/vectors may encode multiple (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) copies of a given inhibitory nucleic acid according to the present disclosure.

In some embodiments, a plurality of inhibitory nucleic acids according to the present disclosure may be a plurality of non-identical inhibitory nucleic acids. In some embodiments, a plurality of inhibitory nucleic acids may comprise one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nonidentical inhibitory nucleic acids. In some embodiments, nucleic acids/vectors may comprise/encode a plurality of non-identical inhibitory nucleic acids according to the present disclosure. The following two paragraphs further define pluralities of non-identical inhibitory nucleic adds in accordance with embodiments of pluralities of inhibitory nudeic adds according to the present disclosure, and also in accordance with embodiments of nucleic acids/vectors comprising/encoding a plurality of non- identical inhibitory nucleic acids according to the present disclosure.

In some embodiments, the non-identical inhibitory nucleic acids comprise or encode non-identical antisense nucleic adds. In such embodiments, the non-identical antisense nucleic adds may each independently conform to any embodiment of an antisense nucleic acid as described hereinabove.

In some embodiments, the non-identical inhibitory nucleic adds may comprise or encode antisense nudeic adds targeting non-identical target nudeotide sequences. In such embodiments, the non-identical target nucleotide sequences may each independently conform to any embodiment of a target nucleotide sequence for an antisense nucleic acid as described hereinabove.

The present disclosure also provides a cell comprising or expressing (i) an inhibitory nucleic acid according to the present disclosure, (ii) nudeic acid comprising or encoding an inhibitory nucleic acid according to the present disclosure, and/or (ill) a vector comprising nudeic acid comprising or encoding an inhibitory nudeic acid according to the present disdosure.

The cell may be a eukaryotic cell, e.g. a mammalian cell. The mammal may be a primate (rhesus, cynomolgous, non-human primate or human) or a non-human mammal (e.g. rabbit, guinea pig, rat, mouse or other rodent (including any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, e.g. dairy cows, or any animal in the order Bos), horse (including any animal in the order Equidae), donkey, and non-human primate). In preferred embodiments, the cell may be a human cell. In some embodiments, the cell may be a liver cell.

The present disdosure also provides a method for producing a cell comprising a nucleic add or vector according to the present disdosure, comprising introdudng a nudeic acid or vector according to the present disclosure into a cell. In some embodiments, introducing a nucleic acid or vector according to the present disclosure into a cell comprises transformation, transfection, electroporation or transduction (e.g. retroviral transduction).

The present disclosure also provides a method for producing an inhibitory nucleic acid according to the present disclosure or a nucleic acid comprising or encoding an inhibitory nucleic acid according to the present disclosure, comprising culturing a cell comprising nucleic acid comprising or encoding an inhibitory nucleic acid according to the present disclosure or a vector according to the present disclosure under conditions suitable for expression of the nucleic acid or vector by the cell. In some embodiments, the methods are performed in vitro. The present disclosure also provides compositions comprising nucleic acids (including inhibitory nucleic acids, nucleic acids comprising/encoding an inhibitory nucleic acid, expression vectors comprising/encoding such nucleic acids) or cells according to the present disclosure.

In therapeutic and prophylactic applications, the compositions of the present disclosure are preferably formulated as a medicament or pharmaceutical composition (suitable for clinical use). Such compositions may comprise the nucleic acid or cell together with one or more other pharmaceutically-acceptable ingredients well known to those skilled in the art Such ingredients include, but are not limited to, pharmaceutically-acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, preservatives, antioxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents.

The term ‘pharmaceutically-acceptable’ as used herein pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, adjuvant, excipient, etc. must also be 'acceptable' in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, adjuvants, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington’s Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.

Compositions according to the present disclosure may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active compound with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary.

The compositions may be prepared for topical, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intravitreal, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral, nasal or transdermal routes of administration which may include injection or infusion. Suitable formulations may comprise the selected agent in a sterile or isotonic medium. The formulation and mode of administration may be selected according to the agent to be administered, and disease to be treated/prevented.

The compositions of the present disclosure may be formulated in fluid, including gel, form. Fluid formulations may be formulated for administration by injection or infusion (e.g. via catheter) to a selected organ or region of the human or animal body. A further aspect of the present disclosure relates to a method of formulating or producing a medicament or pharmaceutical composition according to the present disclosure, the method comprising formulating a pharmaceutical composition or medicament by mixing an agent with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent Nucleic acids (including inhibitory nucleic acids, expression vectors), cells and compositions according to the present disclosure may be modified and/or be formulated to facilitate delivery to, and/or uptake by, a cell/tissue of interest, e.g. a liver cell (hepatocyte) or hepatic tissue.

Strategies for targeted delivery of such species are reviewed e.g. in Li eta/., Int J. Mol. Sd. (2015) 16: 19518-19536 and Fu eta/., Bioconjug Chem. (2014) 25(9): 1602-1608, which are hereby incorporated by reference in their entirety. In particular, nucleic acids according to the present disclosure may employ a delivery platform described in Hu eta/., Sig. Transduc. Tar. Ther. (2020) 5(101) (incorporated by reference hereinabove), or Tatiparti eta/. ‘siRNA Delivery Strategies: A Comprehensive Review of Recent Developments. ’ Ed. Thomas Nann. Nanomaterials 7.4 (2017): 77, and Lehto T eta/., Adv Drug Delhr Rev. 2016, 106(Pt A): 172-182, which are hereby incorporated by reference in their entirety.

In some embodiments, articles of the present disclosure may be encapsulated in a nanoparticle or a liposome. In some embodiments, articles of the present disclosure may be (covalently or non-covalently) associated with a cell-penetrating peptide (e.g. a protein transduction domain, trojan peptide, arginine-rich peptide, vectocell peptide), a cationic polymer, a cationic lipid or a viral carrier.

Nanoparticles may be organic, e.g. micelles, liposomes, proteins, solid-lipid particles, solid polymer particles, dendrimers, and polymer therapeutics. Nanoparticles may be inorganic, e.g. such as nanotubes or metal particles, optionally with organic molecules added. In some embodiments, a nanoparticle is a nanoparticle described in Chen eta/., Mol Ther Methods Clin Dev. (2016) 3:16023, which is hereby incorporated by reference in its entirety. In some embodiments, a nanoparticle is a RIGA, polypeptide, poly(P-amino ester), DOPE, p-cyclodextrin-containing polycation, linear PEI, PAMAM dendrimer, branched PEI, chitosan or polyphosophoester nanoparticle.

In some embodiments, a nucleic acid according to the present disclosure (e.g. an inhibitory nucleic acid, a nucleic acid comprising/encoding an inhibitory nucleic acid, or an expression vector) comprises modification to incorporate one or more moieties facilitating delivery to, and/or uptake by, a cell type or tissue of interest In some embodiments, a nucleic acid according to the present disclosure is linked (e.g. chemically conjugated to) one or more moieties facilitating delivery to, and/or uptake by, a cell type or tissue of interest

Modification to, and formulation of, nucleic acids to facilitate targeted delivery to cell types and/or tissues of interest is described e.g. in Lorenzer et el., J Control Release (2015) 203:1-15, which is hereby incorporated by reference in its entirety. The moiety facilitating delivery to, and/or uptake by, a cell type or tissue of interest may bind selectively to the target cell type/tissue of interest The moiety may facilitate traversal of the cell membrane of the target cell type and/or of cells of the tissue of interest The moiety may bind to a molecule expressed at the cell surface of the target cell type/tissue of interest The moiety may facilitate interalisation of the nucleic acid by the target cell type/tissue of interest (e.g. by endocytosis). Moieties facilitating delivery to, and/or uptake by, cell types or tissues of interest are described e.g. in Benizri et al., Bioconjug Chem. (2019) 30(2): 366-383, which is hereby incorporated by reference in its entirety. Such moieties include e.g. N-acetylgalactosamine (GalNAc), or-tocopherol, cell-penetrating peptide, nucleic add aptamer, antibody and antigen-binding fragments/derivatives thereof, cholesterol, squalene, polyethylene glycol (PEG), fatty acid (e.g. palmitic acid) and nudeolipid moieties.

In some embodiments, the moiety may e.g. be a peptide/polypeptide (e.g. an antibody, fragment or derivative thereof, peptide aptamer or cell-penetrating peptide) or nudeic acid (e.g. a nucleic acid aptamer) which binds to the target cell type/tissue of interest, e.g. via interaction with a molecule expressed at the cell surface of the target cell type/tissue of interest

In some embodiments, a nudeic acid according to the present disclosure comprises a moiety fadlitating delivery to, and/or uptake by, a liver cell (e.g. a hepatocyte) and/or hepatic tissue. In such embodiments, the moiety may facilitate traversal of the hepatocyte cell membrane. The moiety may bind to a molecule expressed at the cell surface of hepatocytes. In some embodiments, a molecule expressed at the cell surface of hepatocytes is an asialoglycoprotein receptor, e.g. ASGR1 or ASGR2. The moiety may fadlitate internalisation of a nudeic add by hepatocytes (e.g. by endocytosis).

In some embodiments, the moiety may e.g. be a peptide/polypeptide (e.g. an antibody, fragment or derivative thereof, peptide aptamer or cell-penetrating peptide) or nudeic add (e.g. a nucleic acid aptamer) which binds to a hepatocyte and/or hepatic tissue, e.g. via interaction with a molecule expressed at the cell surface of a hepatocyte (e.g. an asialoglycoprotein receptor, e.g. ASGR1 or ASGR2).

In some embodiments, the moiety is, or comprises, GalNAc. In some embodiments, a nucleic acid is conjugated to GalNAc. GalNAc interacts with asialoglycoprotein receptors expressed by hepatocytes. Nucleic acids conjugated to GalNAc are efficiently internalised by hepatic cells via receptor-mediated endocytosis following binding of GalNAc to ASGPR (see e.g. Nair et al, J. Am. Chem. Soc. (2014) 136(49): 16958-16961). In some embodiments, a nucleic add is conjugated to one or more (e.g. 1, 2, 3, 4 or more) GalNAc moieties. In some embodiments, one or more GalNAc moieties may be covalently associated to the 5* or 3' end of one or more strands of a nucleic acid. In some embodiments, a nucleic add is conjugated to a triantennary GalNAc (TriGai; triantennary N-acetylgalactosamine) carbohydrate moiety (such moieties are described e.g. in Nair et al, supra).

In some embodiments, the moiety is, or comprises, or-tocopherol (/.e. vitamin E). In some embodiments, a nucleic add is conjugated to or-tocopherol. Nudeic acid-a-tocopherol conjugates have been employed for targeted delivery of nucleic acids to the liver (see e.g. Nishina et al., Mol Ther. (2008) 16(4):734-740). In some embodiments, a nudeic acid is conjugated to one or more (e.g. 1 , 2, 3, 4 or more) or-tocopherol moieties. In some embodiments, one or more or-tocopherol moieties may be covalently assodated to the 5’ or 3* end of one or more strands of a nudeic add. In some embodiments, the sense (or passenger) strand of an Inhibitory nucleic acid described herein comprises the moiety, e.g. a GalNAc monomer described herein. In some embodiments, the sense (or passenger) strand of an inhibitory nucleic acid that comprises the moiety, e.g. triGai or a GalNAc monomer described herein, comprises SEQ ID NO: 1419, 1420, 1421, 1422, 1423, 1424, 1425, 1426, 1427 or 1428.

Nucleic acids according to the present disclosure (e.g. inhibitory nucleic acids according to the present disclosure) may comprise a GalNAc monomer as described below and in co-filed application PCT/EP2022/072271, which is incorporated herein by reference in its entirety. It will be appreciated that, dependent on the nature of Ri, the description below relates to both monomers suitable for oligonucleotide synthesis and GalNAc-oligonucleotide conjugates made using said GalNAc monomers, for example, GalNAc-bearing nucleic acids, e.g. siRNAs for reducing gene and/or protein expression of PCSK9.

The GalNAc monomer may be a compound of Formula 1, Formula 2, or Formula 3 or a pharmaceutically acceptable salt thereof; wherein

Ri is O-PN(Ci-4alkyl)2OCH2CH2CN, OH, a phosphoramidite linkage to an oligonucleotide, or a polystyrene bead or long chain alkylamine controlled-pore glass (LCAA-CPG) which is connected through a succinic, diglycolic or hydroquinone-O.O'diacetic acid linkage;

R2 is H or a protecting group; each Ra is independently Ci-«alkyl, O aloalkyl or H; R4 is H, OH, OCi-*alkyl or halogen;

L is -(W-Y)k-W-X-; k is 0 to 5; each W is independently L1 or 12; each L1 is (CH2)n, where n is independently 1 to 25; each L2 is CH2CH2(HetCH2CH2)m, where m independently is 1 to 24, and Het is independently a heteroatom;

X is a bond, Het, -CH2-, -CO-, ‘O-CH2-CO, *-(Het)CH2CEC-, or *-CH2C=C-, where * if present denotes the point of attachment to W; and each Y is independently CONZ, O-CH2-CONZ, NZCO, SO2NZ, O-CH2SO2NZ or NZSO2, where Z is H, Ci- xalky I or a protecting group; and wherein

GalNAc may be protected may be deprotected.

In some embodiments, X is a bond, Het, -CH2-, -CO-, *-(Het)CH2C=C-, or *-CH2C=C-, where * if present denotes the point of attachment to W; and each Y is independently CONZ, NZCO, SO2NZ or NZSO2, where Z is H, Ci-*alkyl or a protecting group

In some embodiments where the compound is a compound of Formula 1 , Formula 2, or Formula 3, L is a linker selected from -L1-X-, -L1-Y-L1-X-, -L1-Y-L2-X-, -L2-X-, -L2-Y-L1-X-, or -L2-Y-L2-X-;

L1 is (CH2)n, where n is 2 to 25;

L2 is CH2CH2(HetCH2CH2)m, where m is 1 to 12, and Het is a heteroatom;

X is a bond, Het, -CH2-, -CO-, *-(Het)CH2C=C-, or *-CH2C=C-; and

Y is CONZ, NZCO, SO2NZ or NZSO2, where Z is H, Ci-talkyl or a protecting group.

In some embodiments where the compound is a compound of Formula 1 , Formula 2, or Formula 3, L is a linker selected from -L1-X-, -L1-Y-L1-X-, -L1-Y-L2-X-, -L2-X-, -L2-Y-L1-X-, or -L2-Y-L2-X-;

L1 is (CH2)n, where n is 2 to 25;

L2 is CH2CH2(HetCH2CH2)m, where m is 1 to 12, and Het is a heteroatom;

X is a bond, Het, -CH2-, -CO-, *O-CH2-CO, *-(Het)CH2CEC-, or *-CH ₂ CEC-; and

Y is CONZ, O-CH2-CONZ, NZCO, SO2NZ, O-CH2SO2NZ or NZSO2, where Z is H, Ci-«alkyl or a protecting group.

In some embodiments, the compound is a compound of Formula 1 or a pharmaceutically acceptable salt thereof; wherein

R1 is O-PN(Ci^alkyl)2OCH2CH2CN, OH, a phosphoramidite linkage to an oligonucleotide or a polystyrene bead or long chain alkylamine controlled-pore glass (LCAA-CPG) which is connected through a succinic, diglycolic or hydroquinone-O.O'diacetic acid linkage;

R2 is H or a protecting group; each Ra is independently Ci-4alkyl, OCi-«alkyl, Ci-4haloalkyl, OCi-thaloalkyl or H;

R4 is H, OH, OCi-talkyl or halogen;

L is -(W-Y)k-W-X-; k is 0 to 5; each W is independently L1 or L2; each L1 is (CH2)n, where n is independently 1 to 25; each L2 is CH2CH2(HetCH2CH2)m, where m independently is 1 to 24, and Het is independently a heteroatom;

X is a bond, Het, -CH2- -CO- or ‘O-CH2-CO, for example a bond, Het, -CH2-, or -CO-; and each Y is independently CONZ, O-CH2-CONZ, NZCO, SO2NZ, O-CH2SO2NZ or NZSO2, for example, CONZ, NZCO, SO2NZ or NZSO2, where Z is H, Ci-*alkyl or a protecting group; and wherein GalNAc may be protected may be deprotected.

In some embodiments, the compound is a compound of Formula 2

Formula 2 or a pharmaceutically acceptable salt thereof; wherein

R1 is O-PN(Ci-*alkyl)2OCH2CH2CN, OH, a phosphoramidite linkage to an oligonucleotide, or a polystyrene bead or long chain alkylamine controlled-pore glass (LCAA-CPG) which is connected through a succinic, diglycolic or hydroquinone-O.O'diacetic acid linkage;

R2 is H or a protecting group; each Rs is independently Ci-talkyl, OCi-talkyl, CMhaloalkyl, OCi^haloalkyl or H;

R< is H, OH, OCi-«alkyl or halogen;

L is -(W-Y)irW-X-; k is 0 to 5; each W is independently L1 or L2; each L1 is (CH2)n, where n is independently 1 to 25; each L2 is CH2CH2(HetCH2CH2)m, where m independently is 1 to 24, and Het is independently a heteroatom;

X is a bond, Het, -CH2- -CO- or *O-CH2-CO, for example a bond, Het, -CH2-, or -CO-; and each Y is independently CONZ, O-CH2-CONZ, NZCO, SO2NZ, O-CH2SO2NZ or NZSO2, for example, CONZ, NZCO, SO2NZ or NZSO2, w cting group; and wherein GalNAc may be protected may be deprotected.

In some embodiments, the compound is a compound of Formula 3a or a pharmaceutically acceptable salt thereof; wherein

Ri is O-PN(C1-4alkyl)2OCH2CH2CN, OH, a phosphoramidite linkage to an oligonucleotide, or a polystyrene bead or long chain alkylamine controlled-pore glass (LCAA-CPG) which is connected through a succinic, diglycolic or hydroquinone-O.O’diacetic acid linkage;

R2 is H or a protecting group; each Ra is independently Cmalkyl, OCi^alkyl, Ci-*haloalkyl, OCi-thaloalkyl or H;

R* is H, OH, OCi-«alkyl or halogen;

L is -(W-Y)k-W-X-; k is 0 to 5; each W is independently L1 or L2; each L1 is (CH2)n, where n is independently 1 to 25; each L2 is CH2CH2(HetCH2CH2)m, where m independently is 1 to 24, and Het is independently a heteroatom;

X is a bond, Het, -CH2- -CO- or ‘O-CH2-CO, for example a bond, Het, -CH2-, or -CO-; and each Y is independently CONZ, O-CH2-CONZ, NZCO, SO2NZ, O-CH2SO2NZ or NZSO2, for example, CONZ, NZCO, SO2NZ or NZSO2, where Z is H, Ci-«alkyl or a protecting group; and wherein GalNAc may be protected may be deprotected.

In some embodiments where the compound is a compound of Formula 1 , Formula 2, or Formula 3a, L is a linker selected from -L1-X-, -L1-Y-L1-X-, -L1-Y-L2-X-, -L2-X-, -L2-Y-L1-X-, or -L2-Y-L2-X-;

L1 is (CH2)n, where n is 2 to 25;

L2 is CH2CH2(HetCH2CH2)m, where m is 1 to 12, and Het is a heteroatom;

X is a bond, Het, -CH2- -CO- or *O-CH2-CO, for example a bond, Het, -CH2-, or -CO-; and each Y is independently CONZ, O-CH2-CONZ, NZCO, SO2NZ, O-CH2SO2NZ or NZSO2, for example, CONZ, NZCO, SO2NZ or NZSO2, where Z is H, Ci-«alkyl or a protecting group.

It will be understood that references to compounds include, where appropriate, pharmaceutically acceptable salts, hydrates and solvates thereof.

GalNAc GalNAc (IUPAC name /V-acetylgalactosamine) is an amino sugar derivative of galactose. It is used as a targeting ligand in antisense and saRNA and siRNA hepatic therapies.

In the monomers and conjugates described herein, the GalNAc is attached via C1. The skilled person will appreciate that, as is conventional in sugar chemistry, a- and p-stereochemistries are possible. Both stereochemistries are envisaged. On some embodiments, the o-stereochemistry is used. In some embodiments, and as exemplified herein, the p-stereochemistry is used.

During the synthesis of oligonucleotides and oligonucleotides conjugates, the GalNAc may be protected, for example with acetyl groups. Accordingly, it will be appreciated that the term GalNAc as used herein refers both to the structure having free hydroxyls as shown and the structure in which these hydroxyls are protected with suitable protecting groups.

That is, GalNAc as written in the formulae described herein may, unless otherwise specified, be a moiety as shown below, where P is hydrogen or a protecting group (for example, acetyl).

Generally, in structures in which Ri is O-PN(Ci-4alkyl)2OCH2CH2CN, OH, or a polystyrene bead or LCAA- CPG, GalNAc is protected, such that the GalNAc is protected for the reaction to form an oligonucleotide or an oligonucleotide conjugate. For example, the GalNAc may be fully protected with acetyl groups. That is, each P may be a protecting group, for example acetyl.

In structures in which Ri is a phosphoramidite linkage to an oligonucleotide, the GalNAc may be protected (for example, immediately after synthesis) as described above or may be unprotected, as is normal for final products of this type. That is, each P may be hydrogen.

The Group Ri

Ri represents a chemical moiety for attaching the monomer to an oligonucleotide chain, a precursor group, or a point of attachment to an oligonucleotide chain. Phosphoramidite chemistry, as discussed herein, is preferred. Accordingly, in monomer units, Ri is suitably O-PN(Cmalkyl)2OCH2CH2CN, wherein said alkyl may be linear or branched For example, Ri may be

In some embodiments, Ri is OH, said free hydroxyl group being suitable for reaction with, for example, a chlorophosphoramidite such as 2-cyanoethyl /V./V-diisopropylchlorophosphoramidite.

In some embodiments, Ri is a phosphoramidite linkage to an oligonucleotide.

In some embodiments, Ri is a long chain alkylamine controlled-pore glass (LCAA-CPG) which is connected through a succinic, diglycolic or hydroquinone-O.O’diacetic add linkage.

Group Rs

Monomers disdosed herein may include hydroxyl groups which are protected during synthesis steps. It will be appreciated that the invention extends to both these free hydroxyls and protected forms thereof. Accordingly, Rs may be a protecting group or H.

In some cases, Rs is selected from Tr, MMTr, DMTr or TMTr protecting groups. Tr is trityl. MMTr is 4'- methoxytrityl. DMTr is dimethoxytrityl (IUPAC name bis-(4-methoxyphenyl)-phenylmethyl). TMTr is 4’, 4‘, 4'- trimethoxytrityl. They are protecting groups widely used for protection of the 5-hydroxy group in nucleosides, particularly in oligonucleotide synthesis. The skilled person will appreciate that other suitable protecting groups may be used.

Group Rs

Where present, each Rs may be H or a suitable substituent. Suitably, Rs is Cmalkyl, OCmalkyl, Ci-*haloalkyl, OCi-4haloalkyl or H.

In some embodiments, Rs is a methyl group, a methoxyl group or H. In some embodiments, Rs is H.

Monomers disclosed herein may include a substituent group which is commonly used in oligonucleotide monomers. This is denoted R*. Accordingly R4 may be a substiuent or H.

Suitable Rt substituents indude hydroxyl, OCi-olkyl (preferably OMe), and halogen (preferably F).

Linkers

The general formulae include linkers. Collectively, there are referred to herein as L. Linkers may be the same or different For example, different linkers may be preferred for different formulae described herein, and where more than one linker is present in a formula, those linkers may be the same or different Suitably when the compound is a compound of Formula 1 , Formula 2, or Formula 3,

L is -(W-Y)irW-X-; wherein k is 0 to 5; each W is independently L1 or 12; each L1 is (CH2)n, where n is 1 to 25; each 12 is CH2CH2(HetCH2CH2)m. where m is 1 to 24, and Het is a heteroatom;

X is a bond, a heteroatom (Het), -CH2-, -CO-, *O-CH2-CO, *-(Het)CH2C=C-, or *-CH2C=C-, where * if present denotes the point of attachment to W; and each Y is independently CONZ, O-CH2-CONZ, NZCO, SO2NZ, O-CH2SO2NZ or NZSO2, where Z is H, C1- *alky I or a protecting group.

In some embodiments, k is 0 to 5; each W is independently L1 or 12; each L1 is (CH2)n, where n is 1 to 25; each 12 is CH2CH2(HetCH2CH2)m, where m is 1 to 24, and Het is a heteroatom;

X is a bond, a heteroatom (Het), -CH2-, -CO-, *-(Het)CH2C=C-, or *-CH2C=C-, where * if present denotes the point of attachment to W; and each Y is independently CONZ, NZCO, SO2NZ or NZSO2, where Z is H, Ci-*alkyl or a protecting group.

In some embodiments, k is 0 to 2. In some embodiments, k is 0 or 1.

In some embodiments, L is -L1-X-, -L1-Y-L1-X-, -L1-Y-L2-X-, -L2-X-, -L2-Y-L1-X-, -L2-Y-L2-X-, or -L1-Y- L2-Y-L1-X-.

In some embodiments, L is -L1-X-, -L1-Y-L1-X-, -L1-Y-L2-X-, -L2-X-, -L2-Y-L1-X-, or -L2-Y-L2-X-; wherein each L1 is (CH2)n, where n is 2 to 25; each 12 is CH2CH2(HetCH2CH2)m. where m is 1 to 12, and Het is a heteroatom;

X is a bond, a heteroatom (Het), -CH2-, -CO-, ‘O-CH2-CO, *-(Het)CH2C=C-, or *-CH2C=C-; for example, a bond, a heteroatom (Het), -CH2-, -CO-, *-(Het)CH2C=C-, or *-CH2C=C-; and each Y is CONZ, O-CH2-CONZ, NZCO, SO2NZ, O-CH2SO2NZ or NZSO2, where Z is H, Cmalkyl or a protecting group; for example Y is CONZ, NZCO, SO2NZ or NZSO2.

In some embodiments, L is -L1-X-, -L1-Y-L1-X-, -L1-Y-L2-X-, -L2-X-, -L2-Y-L1-X-, or -L2-Y-L2-X-; each L1 is (CH2)n, where n is 2 to 10; each 12 is CH2CH2(OCH2CH2)m, where m is 1 to 5;

X is a bond, -CH2-, -CO-, *O-CH2-CO, -O-, *-OCH2C=C-, or *-CH2C=C-, for example, a bond, -CH2-, -CO-, -O-, *-OCH2CEC-, or ‘-CH2CEC-; and Y is CONZ or O-CH2-CONZ, for example O-CH2-CONZ, where Z is H, Cmalkyl or a protecting group.

In some embodiments, L is -L1-X-, -L1-Y-L1-X-, -L1-Y-L2-X-, -L2-X-, -L2-Y-L1-X-, or -L2-Y-L2-X-; each L1 is (CH2)n, where n is 2 to 10; each 12 is CH2CH2(OCH2CH2)m, where m is 1 to 5;

X is a bond, -CO-, ‘O-CH2-CO, -O-, *-OCH2C=C-, or *-CH2C=C-, for example, a bond, -CO-, -O-, *- OCH2CEC-, or *-CH2CEC-; and

Y is CONZ or O-CH2-CONZ, for example O-CH2-CONZ, where Z is H, Ci-*alkyl or a protecting group.

Suitably when the compound is a compound of Formula 1 , Formula 2, or Formula 3a, L is -(W-Y)k-W-X-; wherein k is 0 to 5; each W is independently L1 or L2; each L1 is (CH2)n, where n is 1 to 25; each 1.2 is CH2CH2(HetCH2CH2)m, where m is 1 to 24, and Het is a heteroatom;

X is a bond, a heteroatom (Het), -CH2-, *O-CH2-CO, or -CO-, for example, a bond, a heteroatom (Het), - CH2-, or -CO-; and each Y is independently CONZ, O-CH2-CONZ, NZCO, SO2NZ, O-CH2SO2NZ or NZSO2, for example, CONZ, NZCO, SO2NZ or NZSO2, where Z is H, Ci-olkyl or a protecting group.

In some embodiments, k is 0 to 2. In some embodiments, k is 0 or 1.

In some embodiments, L is -L1-X-, -L1-Y-L1-X-, -L1-Y-L2-X-, -L2-X-, -L2-Y-L1-X-, -L2-Y-L2-X-, or -L1-Y- L2-Y-L1-X-.

In some embodiments, L is -L1-X-, -L1-Y-L1-X-, -L1-Y-L2-X-, -L2-X-, -L2-Y-L1-X-, or -L2-Y-L2-X-; wherein each L1 is (CH2)n, where n is 2 to 25; each L2 is CH2CH2(HetCH2CH2)m, where m is 1 to 12, and Het is a heteroatom;

X is a bond, Het, -CH2-, ‘O-CH2-CO, or -CO-, for example, a bond, Het, -CH2-, or -CO-; and each Y is CONZ, O-CH2-CONZ, NZCO, SO2NZ, O-CH2SO2NZ or NZSO2, for example, CONZ, NZCO, SO2NZ or NZSO2, where Z is H, Ci-talkyl or a protecting group.

In some embodiments, L is -L1-X-, -L1-Y-L1-X-, -L1-Y-L2-X-, -L2-X-, -L2-Y-L1-X-, or -L2-Y-L2-X-; each L1 is (CH2)n, where n is 2 to 10; each L2 is CH2CH2(OCH2CH2)m, where m is 1 to 5;

X is a bond, -CO-/O-CH2-CO, or -O-, for example, a bond, -CO-, or -O-; and

Y is CONZ or O-CH2-CONZ, for example O-CH2-CONZ, where Z is H, Ci-*alky I or a protecting group. It will be understood that where the linker is attached to GalNAc, the arrangement is GalNAc-L-. It will be understood that where the linker is attached to R1, the arrangement is R1-L-. That is, the directionality of the linker should be read GalNAc-L1-X-, GalNAc-L1-Y-L1-X-, GalNAc-L1-Y-L2-X-, GalNAc-L2-X-, GalNAc-L2-Y-L1-X-, GalNAc-L2-Y-L2-X-, R1-H-X-, R1-LI-Y-LI-X-, Ri-L1-Y-L2-X-, Ri-L2-X-, Ri-L2-Y-L1- X-, R1-L2-Y-L2-X-, etc.

In some embodiments, a linker L is selected from -L1-X-, -L1-Y-L1-X-, and -L1-Y-L2-X-

L1

L1 is (CH2)n. That is, where present L1 is an alkylene chain. Optionally, the alkylene chain may be optionally substituted with one or more substituents selected from halogen (for example, F or Cl), Ci-4alkyl, Ci-<haloalkyl, OCi-«alkyl and OCi-ihaloalkyl. n is 1 to 25. That is, the alkylene chain comprises 1 to 25 carbon atoms. In some embodiments, n is 2 to 25. That is, the alkylene chain comprises 2 to 25 carbon atoms. In some embodiments, n is 2 to 10. That is, the alkylene chain comprises 2 to 10 carbon atoms. In some embodiments, n is 2; that is the alkylene chain is ethylene. In some embodiments, n is 3 to 10. In some embodiments, n is 8 to 10. In some embodiments, n is 3 to 6.

In some embodiments, n is 4 to 9. In some embodiments n is 4; that is, the alkylene chain is butylene. In some embodiments n is 5; that is, the alkylene chain is pentylene. In some embodiments n is 6; that is, the alkylene chain is hexylene. In some embodiments n is 7; that is, the alkylene chain is heptylene. In some embodiments n is 8; that is, the alkylene chain is octylene. In some embodiments n is 9; that is, the alkylene chain is nonylene.

12

1.2 is CH2CH2(HetCH2CH2>n; that is, where present 12 is a (HetCH2CH2) chain, m is 1 to 24, and Het is a heteroatom, such that the linker comprises 1 to 24 repeating (HetCH2CH2) units, in addition to leading ethylene. Additional valencies on a heteroatom, where present, may be occupied by H or Cmalkyl, preferably H. Exemplary heteroatoms include O, S and N (for example, NH).

In some embodiments, m is 1 to 12. In some embodiments, Het is an oxygen atom.

Where m is 1 and Het is an oxygen atom, L2 is -CH2CH2OCH2CH2-; where m is 2 and Het is an oxygen atom, L2 is -CH2CH2OCH2CH2OCH2CH2-, and so on. In some embodiments, m is 1, 2, 3 or 4. In some embodiments, m is 1, 2 or 3. In some embodiments, m is 1 or 2. In some embodiments, m is 2.

The X group

In some embodiments, X is -CO-. In other words, the linker is attached via an acyl group. In some embodiments the linker is -L1-CO-, for example pentanoyl (-CO(CH2)<-) or decanoyl (-CO(CH2)»-). In some embodiments, X is a bond. In other words, the linker may be attached to the leading CH2 of L1 or L2, as appropriate. For example, the linker may be -L1-, -L1-Y-L1-, -L1-Y-L2-, -L2-, -L2-Y-L1-, or -L2-Y-L2-. In some embodiments the linker is -L1-; for example ethylene.

In some embodiments, X is a heteroatom (Het). Additional valencies on a heteroatom, where present, may be occupied by H or Ci^alkyl, preferably H. Exemplary heteroatoms include O, S and N (for example, NH). In some preferred embodiments, X is -O-.

In some embodiments, X is *-(Het)CH2C=C-, where * is the point of attachment to W. Additional valencies on a heteroatom, where present, may be occupied by H or Ci-olkyl, preferably H. Exemplary heteroatoms include O, S and N (for example, NH). In some preferred embodiments, X is *-OCH2C=C-.

In some embodiments, X is *-CH2C=C-, where * is the point of attachment to W. In other words, the linker is attached via a propargylene group. In some embodiments the linker is -H-CH2CEC-.

In some embodiments, X is -GHz-.

In some embodiments, X is *O-CH2-CO. It will be appreciated that in units of formula L2-X, X is •O-CH2-CO may be appropriate as the synthetic route may include oxidising the terminal alcohol of a PEG chain.

77>e Y group

Each Y is independently CONZ, O-CH2-CONZ, NZCO, SO2NZ, O-CH2SO2NZ or NZSO2, where Z is H, Ci-«alkyl or a protecting group. In some embodiments, Y is CONZ or SO2NZ, where Z is H, Ci^alkyl (for example, methyl) or a protecting group.

Similarly, it will be appreciated that in units of formula L2-Y, Y is O-CH2-CONZ or O-CH2SO2NZ may be suitable as the synthetic route may include oxidising the terminal alcohol of a PEG chain.

In some embodiments where more than one Y is present, each Y is the same. In some embodiments, Y is CONZ; in other words, Y provides an amide linkage which may be optionally protected. Suitable nitrogen protecting groups are known in the art and include benzyl (Bn). In some embodiments, Z is H, Bn or Ci-talkyl.

In some embodiments, Y is CONH. That is, the linker is selected from -L1-CONH-L1-X-, -L1-CONH-L2-X-, -L2-CONH-L1-X-, and -L2-CONH-L2-X-; preferably -L1-CONH-L1-X- and -L1-CONH-L2-X-; more preferably -L1-CONH-L2-X-.

In one preferred embodiment, the linker is -(CH2)4-CONH-CH2CH2OCH2CH2-; that is the linker is -L1-Y-L2-X-, L1 is present and n is 4, Y is CONH, L2 is present and m is 1 , and X is a bond. In one preferred embodiment, the linker is -(CH2)s-CONH-(CH2)4-CO-; that is the linker is -L1-Y-L1-X-, where X is -CO-, Y is CONH, both L1 chains are present, and one n is 4 and the other n is 5.

In one preferred embodiment, the linker is -(CH2)4-CONH-CH2CH2-(OCH2CH2)2-CONH-(CH2)5-; that is the linker is -L1-Y-L2-Y-L1-X-, where X is a bond, each Y is CONH, the first L1 has n is 4, the second L1 has n is 5, and m is 2.

In one preferred embodiment, the linker is -(CH2)4-CONH-CH2CH2(OCH2CH2)-O-(CH2)C=C-; that is the linker is -L1-Y-L2-X-, L1 is present and n is 4, Y is CONH, L2 is present and m is 1 , and the X is *-O(CH ₂ )CEC-.

In some embodiments, the linker L is -L1-Y-L1-X- (such that k is 1 and both W are L1), where X is -CO-, Y is CONH, one n is 4 and the other n is 5. That is, the linker is -(CH2)4-CONH-(CH2)5-CO-. [Linker 1]

In some embodiments, the linker L is -L1-Y-L2-X- (such that k is 1, one W is L1 and the other W is 12), where n is 4, Y is CONH, m is 4, Het is O, and X is -CO-. That is, the linker is -(CH2)4-CONH- CH ₂ CH2(OCH ₂ CH2)4-CO-. [Unker 2]

In some embodiments, the linker L is -L1-X- (such that k is 0, and W is L1), where n is 9, and X is -CO-.

That is, the linker is -(CtoJe-CO-. [Linker 3]

In some embodiments, the linker L is -L1-Y-L2-X- (such that k is 1 , one W is L1 and the other W is L2), where n is 4, Y is CONH, m is 1, Het is O, and X is -O-CH2-CO. That is, the linker is -(CH2)4-CONH- CH2CH2(OCH2CH ₂ )-O-CH2-CO-. [Unker 4]

In some embodiments, the linker L is -L2-X- (such that k is 0, and W is 12), where m is 3, Het is O, and X is -O-CH2-CO-. That is, the linker is -CH2CH2(OCH ₂ CH ₂ )3-O-CH2-CO-. [UnkerS]

In some embodiments, the linker L is -L2-Y-L1-X- (such that k is 1, and one W is 12 and the other W is L1), where m is 1, Het is O, Y is O-CH2-CONH (that is, Z is H), n is 5, and X is -CO-. That is, the linker is -CH2CH ₂ (OCH2CH2)-O-CH2-CONH-(CH2)5-CO-. [Unker 6]

In some embodiments, the linker L is -(L1-Y)2-L1-X- (such that k is 2, and all three W are L1), where one n is 4, another n is 3 and the final n is 1 , one Y is CONH (that is, Z is H), and the other Y is NHCO (that is, Z is H), and X is -CH ₂ CEC-. That is, the linker is -(CH ₂ )4-CONH-(CH2)3-NHCO-CH2-CH2CEC-. [Unker 7]

In some embodiments, the linker L is -(L1-Y)2-L1-X- (such that k is 2, and all three W are L1), where one n is 4, another n is 3 and the final n is 4, one Y is CONH (that is, Z is H), and the other Y is NHCO (that is, Z is H), and X is a bond. That is, the linker is -(CH2)4-CONH-(CH2)3-NHCO-(CH2)4-. [Linker 7 - hydrogenated].

In some embodiments, the linker L is -L2-Y-L2-X- (such that k is 1, and both Ware 12), where one m is 1, and the other m is 1 , both Het are O, Y is O-CH2-CONH (that is, Z is H), and X is -OCH2CEC- (that is, Het is O). That is, the linker is -CH2CH ₂ (OCH2CH2)-O-CH2-CONH-CH2CH2(OCH2CH2)-OCH2CEC-. [UnkerS], In some embodiments, the linker L is -L2-Y-L2-X- (such that k is 1 , and both W are 12), where one m is 1 , and the other m is 2, both Het are O, Y is O-CH2-CONH (that is, Z is H), and X is -CH2-. That is, the linker is -CH2CH2(OCH2CH2)-O-CH2-CONH-CH2CH2(OCH2CH2)2-CH2-. [Linker 8 - hydrogenated]

In some embodiments, the linker L is -L2-Y-L1-Y-L1-X- (such that k is 2, and one W is L2 and the other two W are L1), where m is 1, one Y is O-CH2-CONH (that is, Z is H), and one Y is NHCO (that is, Z is H), one n is 3, and the other n is 1 , and X is -CH2CEC-. That is, the linker is -CH2CH2(OCH2CH2)-O-CH2- CONH-(CH2)3-NHCO-CH2-CH2CEC-. [Linker 9]

In some embodiments, the linker L is -L2-Y-L1-Y-L1-X- (such that k is 2, and one W is 12 and the other two W are L1), where m is 1 , one Y is O-CH2-CONH (that is, Z is H), and one Y is NHCO (that is, Z is H), one n is 3, and the other n is 4, and X is a bond. That is, the linker is -CH2CH2(OCH2CH2)-O-CH2-CONH- (CH ₂ )3-NHCO-{CH ₂ )4-. [Linker 9 - hydrogenated]

In some embodiments, the linker L is -L1-Y-L2-X- (such that k is 1 , and one W is L1 and the other W is 12), where n is 4, Y is CONH (that is, Z is H), m is 1 , and X is -OCH2CEC- (that is, Het is O). That is, the linker is -(CH ₂ )4-CONH-CH2CH2(OCH2CH2)-OCH2CEC-. [Linker 10]

In some embodiments, the linker L is -L1-Y-L2-X- (such that k is 1 , and one W is L1 and the other W is 12), where n is 4, Y is CONH (that is, Z is H), m is 2, and X is -CH2-. That is, the linker is -(CH2)4-CONH- CH ₂ CH ₂ (OCH ₂ CH ₂ ) ₂ -CH ₂ -. [Linker 10 - hydrogenated]

Formula 1

In some embodiments, the compound is a compound of Formula 1. Suitably, the two linkers are different and may be distinguished as L ^a and L ^b , as shown in Formula 1a. In some embodiments, the stereochemistry is as shown in Formula 1b.

Preferably, L ^a is a linker of formula -L1-X-, optionally wherein n is 2 and X is an oxygen atom. That is, L ^a is -(ethylene)O-, and the substituent is Ri-(ethylene)O-.

That is, the compound may be a compound of the following formula:

In some alternative embodiments, L ^a is L2, optionally where m is 1 or 2, preferably 1.

Preferably, L ^b is a linker of formula -L1-X-, optionally wherein n is 4 and X is -CO-. That is, L ^b is - (butylene)CO-.

That is, the compound may be a compound of the following formula:

In some embodiments, the invention provides a monomer of the following formula (GalNAc Monomer 1):

That is, Ri is O-PN(Pr)2OCH2CH2CN; R2 is DMTr; a first linker (L ^a ) is -L1-X-, where n = 2 and X is an oxygen atom; and the second linker (L ^b ) is -L1-X-, where X is -CO- and n is 4; and GalNAc is protected with acetyl groups (that is, P is Ac).

Formula 2

In some embodiments, the compound is a compound of Formula 2.

Formula 2

Preferably, each Rs is independently a methyl group, a methoxyl group or H. More preferably, Rs is H.

In some embodiments, the compound is a compound of Formula 2a or 2b:

Formula 2a Formula 2b

Preferably, the linker is -L1-X-, where X is -CO-. That is, the linker is attached to the amine of the core motif by an amide bond. In some embodiments, n is 8 to 10. In some embodiments n is 9; that is, the linker is decanoyl.

In some embodiments, the linker is -L1-Y-L1-X-, where X is -CO- and Y is CONZ. That is, the linker is attached to the amine of the core motif by an amide bond. In some embodiments, the L1 groups may have different values for n. In some embodiments, each n is independently 4 or 5. In some embodiments, the linker is -(CH ₂ ) ₅ -Y-(CH2)4-X-, for example, -(CH2)s-CONH-(CH2)4-CO-.

In some embodiments, the invention provides a monomer of the following formula (GalNAc Monomer 2):

That is, Ri is O-PN(/Pr)2OCH2CH2CN; R2 is DMTr; both Rs are H; the linker is -L1-X-, where X is -CO- and n is 9, and GalNAc is protected with acetyl groups (that is, P is Ac). In some embodiments, the invention provides a compound of Formula 2 in which Ri is a phosphoramidite linkage to an oligonucleotide, or a polystyrene bead or long chain alkylamine controlled-pore glass (LCAA-CPG) which is connected through a succinic, diglycolic or hydroquinone-O.O'diacetic acid linkage; R ₂ is OH, DMTr); both Ra are H; the linker L is -L1-Y-L1-X- (such that k is 1 and both Ware L1), where X is -CO-, Y is CONH, one n is 4 and the other n is 5 (that is, the linker is -(CH ₂ )4-CONH-(CH ₂ )5-CO-) [Linker 1], and GalNAc is protected with acetyl groups (that is P is Ac).

In some embodiments, the invention provides a monomer of the following formula (GalNAc Monomer 4):

That is, Ri is O-PN(Pr) ₂ OCH2CH ₂ CN; R ₂ is DMTr; both Ra are H; the linker is -L1-Y-L1-X-, where X is -CO-, Y is CONH, one n is 4 and the other n is 5 (that is, the linker is -(CH ₂ )s-CONH-(CH ₂ )4-CO-), and GalNAc is protected with acetyl groups (that is, P is Ac).

In some embodiments, the invention provides a compound of Formula 2 in which Ri is a phosphoramidite linkage to an oligonucleotide, or a polystyrene bead or long chain alkylamine controlled-pore glass (LCAA-CPG) which is connected through a succinic, diglycolic or hydroquinone-O.O'diacetic acid linkage; R ₂ is OH or DMTr, both Rs are H; the linker L is -L1-Y-L2-X- (such that k is 1 , one W is L1 and the other W is L2), where n is 4, Y is CONH, m is 4, Het is O, X is -CO- (that is, the linker is -(CH ₂ )4-CONH- CH ₂ CH ₂ (OCH ₂ CH ₂ )4-CO- [Linker 2ft, and GalNAc is protected with acetyl groups (that is P is Ac).

In some embodiments, the invention provides a compound of Formula 2 in which Ri is a phosphoramidite linkage to an oligonucleotide, or a polystyrene bead or long chain alkylamine controlled-pore glass (LCAA-CPG) which is connected through a succinic, diglycolic or hydroquinone-O.O'diacetic acid linkage; Rs is OH or DMTr; both Rs are H; the linker L is -L1-X- (such that k is 0, and W is L1), where n is 9, X is - CO- (that is, the linker is -(CH ₂ )g-CO- [Linker 3J), and GalNAc is protected with acetyl groups (that is P is Ac).

In some embodiments, the invention provides a compound of Formula 2 in which Ri is a phosphoramidite linkage to an oligonucleotide, or a polystyrene bead or long chain alkylamine controlled-pore glass (LCAA-CPG) which is connected through a succinic, diglycolic or hydroquinone-O.O'diacetic acid linkage;

R ₂ is OH or DMTr; both Rs are H; the linker L is -L1-Y-L2-X- (such that k is 1 , one W is L1 and the other W is 12), where n is 4, Y is CONH, m is 1 , Het is O, X is -O-CHr-CO (that is, the linker is -(CH ₂ )4-CONH- CH ₂ CH ₂ (OCH ₂ CH ₂ )-O-CH ₂ -CO- [Lin G d with acetyl groups (that is P is Ac). In some embodiments, the invention provides a compound of Formula 2 in which Ri is a phosphoramidite linkage to an oligonucleotide, or a polystyrene bead or long chain alkylamine controlled-pore glass (LCAA-CPG) which is connected through a succinic, diglycolic or hydroquinone-O.O’diacetic acid linkage; R ₂ is OH or DMTr; both Rs are H; the linker L is -L2-X- (such that k is 0, and W is L2), where m is 3, Het is O, X is O-CH ₂ -CO (that is, the linker is -CH ₂ CH ₂ (OCH ₂ CH ₂ ) ₃ -O-CH ₂ -CO- [Linkers]), and GalNAc is protected with acetyl groups (that is P is Ac).

In some embodiments, the invention provides a compound of Formula 2 in which Ri is a phosphoramidite linkage to an oligonucleotide, or a polystyrene bead or long chain alkylamine controlled-pore glass (LCAA-CPG) which is connected through a succinic, diglycolic or hydroquinone-O.O'diacetic acid linkage; R ₂ is OH or DMTr; both Rs are H; the linker L is -L2-Y-L1-X- (such that k is 1, and one W is L2 and the other W is L1), where m is 1, Het is O, Y is O-CHs-CONH (that is, Z is H), n is 5, X is -CO- (that is, the linker is -CH ₂ CH ₂ (OCH ₂ CH ₂ )-O-CH ₂ -CONH-(CH ₂ )5-CO- [Linker 6J), and GalNAc is protected with acetyl groups (that is P is Ac).

In some embodiments, the invention provides a compound of Formula 2 in which Ri is a phosphoramidite linkage to an oligonucleotide, or a polystyrene bead or long chain alkylamine controlled-pore glass (LCAA-CPG) which is connected through a succinic, diglycolic or hydroquinone-O.O’diacetic acid linkage; R ₂ is OH or DMTr; both Rs are H; the linker L is -(L1-Y)2-L1-X- (such that k is 2, and all three W is L1), where one n is 4, another n is 3 and the final n is 1 , one Y is CONH (that is, Z is H), and the other Y is NHCO (that is, Z is H), and X is -CHZCEC- (that is, the linker is -(CH^-CONH-fCH^s-NHCO-CHs- CH ₂ C=C- [Linker 7J), and GalNAc is protected with acetyl groups (that is P is Ac).

In some embodiments, the invention provides a compound of Formula 2 in which Ri is a phosphoramidite linkage to an oligonucleotide, or a polystyrene bead or long chain alkylamine controlled-pore glass (LCAA-CPG) which is connected through a succinic, diglycolic or hydroquinone-O.O'diacetic acid linkage; Rs is OH or DMTr; both Rs are H; the linker L is -(L1-Y)2-L1-X- (such that k is 2, and all three W are L1), where one n is 4, another n is 3 and the final n is 4, one Y is CONH (that is, Z is H), and the other Y is NHCO (that is, Z is H), and X is a bond (that is, the linker is -(CH ₂ )4-CONH-(CH ₂ )3-NHCO-(CH ₂ )4- [Linker 7 - hydrogenated]), and GalNAc is protected with acetyl groups (that is P is Ac).

In some embodiments, the invention provides a compound of Formula 2 in which Ri is a phosphoramidite linkage to an oligonucleotide, or a polystyrene bead or long chain alkylamine controlled-pore glass (LCAA-CPG) which is connected through a succinic, diglycolic or hydroquinone-O.O'diacetic acid linkage; Rs is OH or DMTr; both Rs are H; the linker L is -L2-Y-L2-X- (such that k is 1 , and both W are L2), where one m is 1 , and the other m is 1, both Het are O, Y is O-CHs-CONH (that is, Z is H), and X is -OCHSCEC- (that is, Het is O) (that is, the linker is -CH ₂ CH ₂ (OCH ₂ CH ₂ )-O-CH ₂ -CONH-CH ₂ CH ₂ (OCH ₂ CH ₂ )-OCH ₂ CEC- [Unker 8]), and GalNAc is protected with acetyl groups (that is P is Ac).

In some embodiments, the invention provides a compound of Formula 2 in which Ri is a phosphoramidite linkage to an oligonucleotide, or a polystyrene bead or long chain alkylamine controlled-pore glass (LCAA-CPG) which is connected through a succinic, diglycolic or hydroquinone-O.O'diacetic acid linkage; R ₂ is OH or DMTr; both Rs are H; th hat k is 1 , and both W are L2), where one m is 1 , and the other m is 2, both Het are O, Y is O-CH2-CONH (that is, Z is H), and X is -CH2- (that is, the linker is -CH2CH2(OCH2CH2)-O-CH2-CONH-CH2CH2(OCH2CH2)2-CH2- /L/n/rer8- hydrogenated), and GalNAc is protected with acetyl groups (that is P is Ac).

In some embodiments, the invention provides a compound of Formula 2 in which Ri is a phosphoramidite linkage to an oligonucleotide, or a polystyrene bead or long chain alkylamine controlled-pore glass (LCAA-CPG) which is connected through a succinic, diglycolic or hydroquinone-O.O'diacetic acid linkage; R ₂ is OH or DMTr; both Ra are H; the linker L is -L2-Y-L1-Y-L1-X- (such that k is 2, and one W is L2 and the other two W are L1), where m is 1 , one Y is O-CH2-CONH (that is, Z is H), and one Y is NHCO (that is, Z is H), one n is 3, and the other n is 1 , and X is -CH2CEC- (that is, the linker is -CHaCHafOCHsCHa)- O-CH2-CONH-(CH2)3-NHCO-CH2-CH2C=C- [Linker 9J), and GalNAc is protected with acetyl groups (that is P is Ac).

In some embodiments, the invention provides a compound of Formula 2 in which R1 is a phosphoramidite linkage to an oligonucleotide, or a polystyrene bead or long chain alkylamine controlled-pore glass (LCAA-CPG) which is connected through a succinic, diglycolic or hydroquinone-O.O’diacetic acid linkage;

R2 is OH or DMTr; both Ra are H; the linker L is -L2-Y-L1-Y-L1-X- (such that k is 2, and one W is L2 and the other two W are L1), where m is 1, one Y is O-CH2-CONH (that is, Z is H), and one Y is NHCO (that is, Z is H), one n is 3, and the other n is 4, and X is a bond (that is, the linker is -CH2CH2(OCH2CH2)-O- CH2-CONH-(CH2)3-NHCO-(CH2)4- [Linker 9 - hydrogenated), and GalNAc is protected with acetyl groups (that is P is Ac).

In some embodiments, the invention provides a compound of Formula 2 in which R1 is a phosphoramidite linkage to an oligonucleotide, or a polystyrene bead or long chain alkylamine controlled-pore glass (LCAA-CPG) which is connected through a succinic, diglycolic or hydroquinone-O.O’diacetic acid linkage;

R2 is OH or DMTr, both Ra are H; the linker L is -L1-Y-L2-X- (such that k is 1 , and one W is L1 and the other W is L2), where n is 4, Y is CONH (that is, Z is H), m is 1 , and X is -OCH2CEC- (that is, Het is O) (that is, the linker is -(CH ₂ )4-CONH-CH2CH2(OCH2CH2)-OCH2C=C- [Linker 10J), and GalNAc is protected with acetyl groups (that is P is Ac).

In some embodiments, the invention provides a compound of Formula 2 in which R1 is a phosphoramidite linkage to an oligonucleotide, or a polystyrene bead or long chain alkylamine controlled-pore glass (LCAA-CPG) which is connected through a succinic, diglycolic or hydroquinone-O.O'diacetic acid linkage;

R2 is OH or DMTr; both Rs are H; the linker L is -L1-Y-L2-X- (such that k is 1 , and one W is L1 and the other W is L2), where n is 4, Y is CONH (that is, Z is H), m is 2, and X is -CH2- (that is, the linker is - (CH2)4-CONH-CH2CH2(OCH2CH2)2-CH2-), and GalNAc is protected with acetyl groups (that is P is Ac).

Formula 3

In some embodiments, the compound is a compound of Formula 3.

Preferably, Rt is H, OH, OCi-olkyl or halogen. More preferably, R« is H.

Preferably, X is *-OCH2C=C-, where • denotes the point of attachment to W. In some embodiments, the linker is -L1-Y-L2-X-, such as -L1-Y-L2-OCH2C=C- (that is, X is *-OCH2C=C-). In some embodiments, Y is CONH. In some embodiments, n is 4, Y is CONH, and m is 1.

In some embodiments, the compound is a compound of Formula 3a

Preferably, X is an oxygen atom. In some embodiments, the linker is -L1-Y-L2-X-, such as -L1-Y-L2-O- (that is, X is an oxygen atom). In some embodiments, Y is CONH. In some embodiments, n is 4, Y is CONH, and m is 1.

In some embodiments, the linker is -(CH2)4-CONH-(CH2)5-CO- [Linker 1]

In some embodiments, the linker is -(CH2)4-CONH-CH2CH2(OCH2CH2)4-CO- /L/n*er2J

In some embodiments, the linker is -(CH2)9-CO- [Linker 3]

In some embodiments, the linker is -(CH2)4-CONH-CH2CH2(OCH2CH2)-O-CH2-CO- [Linker 4]

In some embodiments, the linker is -CH2CH2(OCH2CH2)3-O-CH2-CO- [Linkers]

In some embodiments, the linker is -CH2CH2(OCH ₂ CH ₂ )-O-CH2-CONH-(CH2)5-CO- [Linkers]

In some embodiments, the linker is -(CH ₂ )4-CONH-(CH2)3-NHCO-CH2-CH2CEC- [Linker 7]

In some embodiments, the linker is -(CH2>4-CONH-(CH2)3-NHCO-(CH2)4- [Linker 7 - hydrogenated]

In some embodiments, the linker is -CH2CH2(OCH2CH2)-O-CH2-CONH-CH2CH2(OCH2CH2)-OCH2C=C- [Linker 8] In some embodiments, the linker is -CH2CH2(OCH2CH2)-O-CH2-CONH-CH2CH2(OCH2CH2)z-CH2- [Unker 8 - hydrogenated]

In some embodiments, the linker Is -CHZCHZ(OCHZCHZ)-O-CHZ-CONH-(CHZ)3-NHCO-CH2<DHZCEC- [Linker 9]

In some embodiments, the linker is -CH2CH2(OCH2CH2)-O-CH2-CONH-(CH2)3-NHCO-(CH2)4- [Unker 9 - hydrogenated]

In some embodiments, the linker is -(CH2)4-C0NH-CH2CH2(0CH2CH2)-0CH2C=C- [Unker 10]

In some embodiments, the linker is -(CH2)4-C0NH-CH2CH2(0CH2CH2)z-CH2- [Unker 10- hydrogenated]

In some embodiments, the invention provides a monomer of the following formula (GalNAc Monomer 3):

That is in Formula 3, Ri is O-PN(/Pr)zOCH2CH2CN; R2 is DMTr; R* is H; the linker L is -L1-Y-L2-X-, where n is 4, Y is CONH, m is 1 and X is *-OCH2C=C-, and GalNAc is protected with acetyl groups (that is, P is Ac). That is in Formula 3a, Ri is O-PN(/Pr)zOCH2CH2CN; R2 is DMTr; R< is H; the linker L is -L1-Y-L2-X-, where n is 4, Y is CONH, m is 1 and X is an oxygen atom, and GalNAc is protected with acetyl groups (that is, P is Ac).

In some embodiments, the invention provides a monomer of the following formula (GalNAc Monomer 5):

That is in Formula 3, R1 is O-PN(/Pr)zOCH2CH2CN; R2 is DMTr; R< is H; the linker L is -L1-Y-L2-X-, where n is 4, Y is CONH, m is 2 and X is -CH2-, and GalNAc is protected with acetyl groups (that is, P is Ac). That is, 12 is -CH2CH2OCH2CH2OCH2CH2-. The term ‘monomer residue' refers to a monomer unit bound within the oligonucleotide chain at one or more positions. In the structure below, the wavy lines denote points of attachment within an oligonucleotide chain or a terminus of an oligonucleotide chain if appropriate.

Accordingly, an inhibitory nucleic acid according to the present disclosure may comprise at least one GalNAc monomer residue as described herein.

Suitably, an inhibitory nucleic acid (oligonucleotide) according to the present disclosure may comprise adjacent GalNAc monomer residues as described herein. In some embodiments, the inhibitory nucleic acid comprises preferably two, or more preferably three, adjacent monomer residues. Preferably, the two or more monomer residues will be multiple residues of the same monomer. It will be appreciated that said monomer residue(s) may be located at any point within the nucleotide chain. In some preferred embodiments, said monomer residue(s) are located at or near the 3’ end of the oligonucleotide. In some emboidments, said monomer residue(s) are located at or near the 5' end of the oligonucleotide.

In some embodiments, the disclosure provides an oligonucleotide (e.g. inhibitory nucleic acid) comprising at least one monomer residue of formula: optionally comprising three copies of said monomer residue; that is, three copies of a monomer of formula (A), (B) or (C). Suitably, said copies are successive. In other words, the oligonucleotide comprises three adjacent monomer residues, said monomer residues being selected from one of formula (A), (B) or (C). In some embodiments, X is O. In some embodiments, X is S. Oligonucleotides and siRNAs exemplified herein comprise three monomer residues of formula (A) wherein X is O, X is S and X is S, respectively. Other oligonucleotides and siRNAs exemplified herein comprise three monomer residues of formula (B) wherein X is O, X is S and X is S, respectively. Yet other oligonucleotides and siRNAs exemplified herein comprise three monomer residues of formula (C) wherein X is O, X is S and X is S, respectively. Said monomer residues) may be located at the 3‘ end of the oligonucleotide. Alternatively, said monomer residue(s) may be located at the 5’ end of the oligonucleotide or at another point in the chain.

In some embodiments, an inhibitory nucleic acid according to the present disclosure is selected from those drawn in Figure 13A, 13B or 13C. The waved line indicates a nucleotide chain. In some embodiments, an inhibitory nucleic acid (e.g. siRNA) according to the invention is selected from those drawn in Figure 14A, 14B or 14C. The waved lines indicate RNA strands. That is, an inhibitory nucleic acid according to the invention may comprise three successive monomers as shown in Figure 13 or Figure 14.

In some embodiments, the monomer is selected from: and, where the monomer contains an alkyne bond, the corresponding monomer in which that bond is fully hydrogenated. Conjugates of biomolecules may be produced utilising 'click chemistry’, as described e.g. in Nwe and Brechbiel Cancer Bother Radiopharm. (2009) 24(3):289-302 and Astakhova eta/., Mol Pharm. (2018) 15(8): 2892-2899, both of which are hereby incorporated by reference in their entirety. In some embodiments, conjugation may employ akyne-azide or thio-maleimide approaches. In some embodiments, a nucleic acid may be conjugated to a moiety facilitating delivery to, and/or uptake by, a cell type or tissue of interest e.g. at the 3' and/or 5' end of one or more strands of the nucleic acid.

Nucleic acids may be conjugated to one or more moieties facilitating delivery to, and/or uptake by, cell types or tissues of interest via a linker. In some embodiments, a linker may be or comprise a nucleotide sequence. The nucleotide sequence of a linker may comprise one or more modified nucleotides as described herein. tfjgj tactic

The inhibitory nucleic acids, nucleic acids, expression vectors, cells and compositions described herein find use in therapeutic and prophylactic methods. These articles are a viable alternative to antibodies with respect to effects on atherogenic lipoproteins.

The present disclosure provides an inhibitory nucleic acid, nucleic acid, expression vector, or composition described herein for use in a method of medical treatment or prophylaxis. Also provided is the use of an inhibitory nucleic acid, nucleic acid, expression vector, or composition described herein in the manufacture of a medicament for treating or preventing a disease or condition. Also provided is a method of treating or preventing a disease or condition, comprising administering to a subject a therapeutically or prophylactically effective amount of an inhibitory nucleic acid, nucleic acid, expression vector, or composition described herein.

The terms ‘disorder', ‘disease’ and 'condition' may be used interchangeably and refer to a pathological issue of a body part, organ or system which may be characterised by an identifiable group of signs or symptoms.

Therapeutic or prophylactic intervention in accordance with the present disclosure may be effective to reduce the development or progression of a disease/condition, alleviate the symptoms of a disease/condition or reduce the pathology of a disease/condition. The intervention may be effective to prevent progression of the disease/condition, e.g. to prevent worsening of, or to slow the rate of development of, the disease/condition. In some embodiments the intervention may lead to an improvement in the disease/condition, e.g. a reduction in the symptoms of the disease/condition or reduction in some other correlate of the severity/activity of the disease/condition. In some embodiments the intervention may prevent development of the disease/condition to a later stage (e.g. a more severe stage, or a chronic stage). The terms ‘develop’, ‘developing’, and ’development’, e.g. of a disorder, as used herein refer both to the onset of a disease as well as the progression, exacerbation or worsening of a disease state/correlate thereof.

It will be appreciated that the articles of the present disclosure (inhibitory nucleic acids, nucleic acids, expression vectors, cells and compositions described herein) may be used for the treatment/prevention of any disease/condition that would derive therapeutic or prophylactic benefit from a reduction in the level of gene and/or protein expression of PCSK9, and/or a reduction in the level of a function of PCSK9 (e.g. LDLR degradation).

The disease/condition to be treated/prevented in accordance with the present disclosure may be a disease/condition in which gene and/or protein expression of PCSK9 and/or a function of PCSK9 (e.g. LDLR degradation) is pathologically-implicated. For example, the disease/condition may be a disease/condition in which elevated gene and/or protein expression of PCSK9, and/or an increased level of the function of PCSK9 (e.g. LDLR degradation) is implicated in the pathology of the disease/condition.

The disease/condition may be characterised by an increased level of gene and/or protein expression of PCSK9, or an increased level of a function of PCSK9 (e.g. as compared to the level of expression or the relevant function in the absence of the disease/condition). The disease/condition may be a disease/condition in which an increased level of gene and/or protein expression of PCSK9, and/or an increased level of a function of PCSK9, is positively associated with the onset, development or progression of the disease/condition. The disease/condition may be a disease/condition in which an increased level of gene and/or protein expression of PCSK9, and/or an increased level of a function of PCSK9, is positively associated with the severity of one or more symptoms of the disease/condition. The disease/condition may be a disease/condition for which an increased level of gene and/or protein expression of PCSK9, and/or an increased level of a function of PCSK9, is a risk factor for the onset, development or progression of the disease/condition.

The increased level of gene and/or protein expression of PCSK9, and/or the increased level of a function of PCSK9, in accordance with the preceding paragraph may be in cells, tissue and/or an organ in which one or more symptoms of the disease/condition manifest. In some embodiments, the increased level of gene and/or protein expression of PCSK9, and/or the increased level of a function of PCSK9, may be in cells of the liver (e.g. hepatocytes), hepatic tissue and/or in the liver.

Therapeutic or prophylactic intervention in accordance with the present disclosure may achieve a reduction in the level of gene and/or protein expression of PCSK9, and/or a reduction in the level of a function of PCSK9 (/.e. in the treated subject). In some embodiments, the therapeutic/prophylactic intervention may achieve a reduction in the level of gene and/or protein expression of PCSK9, and/or a reduction in the level of a function of PCSK9, in cells, tissue and/or an organ in which one or more symptoms of the disease/condition manifest In some embodiments, the therapeutic/prophylactic intervention may achieve a reduction in the level of gene and/or protein expression of PCSK9, and/or a reduction in the level of a function of PCSK9, in cells of the liver (e.g. hepatocytes), hepatic tissue and/or in the liver.

The articles of the present disclosure also find use in the treatment/prevention of any disease/condition that would derive therapeutic or prophylactic benefit from one or more of: a reduction in the level of degradation of LDLR, an increase in the level of LDLR (e.g. in the serum); an increase in the level of an activity of LDLR; an increase in the level of LDLR:LCL-C complexes (e.g. in the serum); and/or a reduction in the level of LCL-C (e.g. free LCL-C; e.g. in the serum).

The disease/condition to be treated/prevented in accordance with the present disclosure may be a disease/condition in which LCL-C is pathologically-implicated. The disease/condition may be a disease/condition in which an elevated level of LCL-C (e.g. in the serum), a reduced level of LDLR gene/protein expression, and/or a reduced level of a function of LDLR is implicated in the pathology of the disease/condition.

Reference herein to 'a function of LDLR' may refer to any functional property of, and/or activity mediated by, LDLR protein. In some embodiments, a function of LDLR may be selected from: binding of LDLR to LDL-C, formation of LDLR: LDL-C complexes, and internalisation of LDLR: LDL-C complexes (e.g. by endocytosis).

The disease/condition may be characterised by an increased level of LDL-C (e.g. in the serum), a reduced level of LDLR gene/protein expression, a reduced level of a function of LDLR, and/or a reduced level of LDLR: LDL-C complexes, e.g. as compared to the level in the absence of the disease/condition. The disease/condition may be a disease/condition in which an increased level of LDL-C (e.g. in the serum), a reduced level of LDLR gene/protein expression, a reduced level of a function of LDLR, and/or a reduced level of LDLR: LDL-C complexes, is positively associated with the onset, development or progression of the disease/condition. The disease/condition may be a disease/condition in which an increased level of LDL-C (e.g. in the serum), a reduced level of LDLR gene/protein expression, a reduced level of a function of LDLR, and/or a reduced level of LDLR: LDL-C complexes, is positively associated with the severity of one or more symptoms of the disease/condition. The disease/condition may be a disease/condition for which an increased level of LDL-C (e.g. in the serum), a reduced level of LDLR gene/protein expression, a reduced level of a function of LDLR, and/or a reduced level of LDLR:LDL-C complexes, is a risk factor for the onset development or progression of the disease/condition.

The reduced level of LDLR gene/protein expression and/or reduced level of a function of LDLR in accordance with the preceding paragraph may be in cells, tissue and/or an organ in which one or more symptoms of the disease/condition manifest In some embodiments, the reduced level of LDLR gene/protein expression and/or reduced level of a function of LDLR may be in cells of the liver (e.g. hepatocytes), hepatic tissue and/or in the liver. Therapeutic or prophylactic intervention in accordance with the present disclosure may achieve a reduction in the level of LDL-C (e.g. in the serum), an increase in the level of LDLR protein (e.g. in the serum), an increase in the level of a function of LDLR, and/or an increase in the level of LDLR:LCL-C complexes (e.g. in the serum), i.e. in the treated subject. In some embodiments, the therapeutic/prophylactic intervention may achieve an increase in the level of LDLR protein (e.g. in the serum), an increase in the level of a function of LDLR, and/or an increase in the level of LDLR:LCL-C complexes in cells, tissue and/or an organ in which one or more symptoms of the disease/condition manifest. In some embodiments, the therapeutic/prophylactic intervention may achieve an increase in the level of LDLR protein (e.g. in the serum), an increase in the level of a function of LDLR, and/or an increase in the level of LDLR:LCL-C complexes in cells of the liver (e.g. hepatocytes), hepatic tissue and/or in the liver.

An inhibitory nucleic acid reducing expression of PCSK9 has been shown to be an effective treatment for reducing serum levels of LDL-C, and thus for the treatment and prevention of hypercholesterolemia and associated diseases (e.g. cardiovascular diseases). See e.g. Raal et al, N Engl J Med (2020) 382(16):1520-1530, Ray et al., N Engl J Med. (2020) 382(16): 1507-1519 and Stoekenbroek eta/., Future Cardiol. (2018) 14(6):433-442, all of which are hereby incorporated by reference in its entirety. The inhibitory nucleic acid inclisiran is approved in the EU for the treatment of dyslipidemias and hypercholesterolemia.

In some embodiments, the disease/condition to be treated/prevented according to the present invention is dyslipidemia and/or hypercholesterolemia. In some embodiments, the disease/condition is a disease/condition characterised by dyslipidemia and/or hypercholesterolemia. In some embodiments, the disease/condition is a disease/condition associated with dyslipidemia and/or hypercholesterolemia (e.g. a disease/condition for which dyslipidemia and/or hypercholesterolemia is a risk factor for the onset, development or progression of the disease/condition).

Dyslipidemia is defined as having blood lipid level that is too high or. too low. The present disclosure is particularly concerned with the treatment of hyperlipidemias, where the level of lipid or lipoprotein in the blood is elevated. Hyperlipidemias includes hypertriglyceridemia, hypercholesterolemia and combined hyperlipidemia (combination of hypertriglyceridemia and hypercholesterolemia). Hyperlipidemia is associated e.g. with atherosclerosis, hypertension and cardiovascular disease.

Hypercholesterolemia refers to a high level of cholesterol in the blood. In many cases, hypercholesterolemia arises as a consequence of a high-fat diet and inactive lifestyle in combination with genetic risk factors. Hypercholesterolemia may also occur as a consequence of genetic mutation (e.g. in the case of familial hypercholesterolemia), type 2 diabetes, hypothyroidism, renal disease, or as a side effect of treatment with certain drugs, e.g. corticosteroids.

Hypercholesterolemia is described e.g. in Bhatnagar et a/., BMJ (2008) 337:a993. The UK NHS defines hypercholesterolemia as blood total cholesterol level of 65 mmol/L or blood low-density lipoprotein (LDL) level of 63 mmol/L. The US NIH defines hypercholesterolemia as blood total cholesterol level of 2240 mg/dL. Hypercholesterolemia is a well-recognised risk factor for the development of cardiovascular disease, in particular cardiovascular disease arising as a consequence of atherosclerosis - see e.g. Nelson Prim Care. (2013) Mar; 40(1):195-211. Hypercholesterolemia has also been reported to lead to steatosis and non-alcoholic liver disease - see e.g. Arguello et el., Biochim Biophys Acta (2015) 1852(9): 1765-78.

In some embodiments, the disease/condition to be treated/prevented in accordance with the present disclosure is selected from: dyslipidemia, hyperlipidemia, hypercholesterolemia, familial hypercholesterolemia, autosomal dominant hypercholesterolemia, atherosclerosis, cardiovascular disease, atherosclerotic cardiovascular disease, coronary artery disease, angina, myocardial infarction, cardiac failure, peripheral vascular disease, peripheral arterial disease, hypertension, stroke, ischemic stroke, transient ischemic attack, congestive heart failure, steatosis, hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH).

In aspects and embodiments of the present disclosure, the disease/condition may be associated with a mutation. The mutation may give rise to the disease/condition (e.g. the mutation may be causal). The mutation may be positively associated with the onset development or progression of the disease/condition, and/or may be a risk factor for the onset development or progression of the disease/condition.

In aspects and embodiments of the present disclosure, the disease/condition may be associated with a mutation resulting in an increased level of gene and/or protein expression of PCSK9, and/or an increased level of a function of PCSK9. Herein, diseases/conditions which are 'associated with' mutations to a given gene are diseases/conditions which are caused or exacerbated by such mutations, or for which such mutations are a risk factor for the development or progression of the disease/condition. In some embodiments, the mutation may give rise to an increased level of gene and/or protein expression of PCSK9, and/or an increased level of a function of PCSK9 in cells comprising one or more copies of the mutant allele of the gene as compared to cells comprising two copies of (/.e. homozygous for) the nonmutated (wildtype) reference allele of the gene.

In aspects and embodiments of the present disclosure, the disease/condition may be associated with a mutation resulting in an increased level of LDL-C (e.g. in the serum), a reduced level of LDLR gene/protein expression, a reduced level of a function of LDLR, and/or a reduced level of LDLR: LDL-C complexes. In some embodiments, the mutation may give rise to a reduced level of LDLR gene/protein expression and/or a reduced level of a function of LDLR in cells comprising one or more copies of the mutant allele of the gene as compared to cells comprising two copies of (/.e. homozygous for) the nonmutated (wildtype) reference allele of the gene. In aspects and embodiments of the present disclosure, the disease/condition may be associated with a mutation in PCSK9, e.g. a mutation resulting in an increase in gene and/or protein expression of PCSK9, or resulting in an increase in the level of a function of PCSK9 (e.g. LDLR degradation).

In aspects and embodiments of the present disclosure, the disease/condition may be associated with a mutation in LDLR, e.g. a mutation resulting in a decrease in gene and/or protein expression of LDLR, or a reduced level of a function of LDLR (e.g. binding to LDL-C, formation of LDLR:LDL-C complexes).

In aspects and embodiments of the present disclosure, the disease/condition may be associated with a mutation in APOB, e.g. a mutation resulting in an increase in gene and/or protein expression of APOB (e.g. ApoB100), resulting in an increase in the level of a function of APOB (e.g. ApoB100), and/or resulting in an increase in the level of LDL-C (e.g. in the serum).

In some embodiments, a mutation according to the present disclosure may be a mutation giving rise to hypercholesterolemia. In some embodiments, a mutation giving rise to hypercholesterolemia may be a mutation in one or more of PCSK9, LDLR or APOB. Mutations in PCSK9 giving rise to hypercholesterolemia are described e.g. in Abifedel etel., Hum Mutat (2009) 30(4):520-9, which is hereby incorporated by reference in its entirety. Mutations in LDLR giving rise to hypercholesterolemia are described e.g. in Benito-Vicente etel., Int J. Mol. Sci. (2018) 19(6): 1676, which is hereby incorporated by reference in its entirety. Mutations in APOB giving rise to hypercholesterolemia are described e.g. in Elbbitar ef a/., Scientific Reports (2018) 8:1943, which is hereby incorporated by reference in its entirety (see in particular Figure 4 of Elbbitar et al.).

Administration of the articles of the present disclosure is preferably in a ‘therapeutically effective’ or ’prophylactically effective’ amount, this being sufficient to show therapeutic or prophylactic benefit to the subject. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease/condition and the particular article administered. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disease/disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington’s Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

Administration of the articles of the present disclosure may be topical, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intravitreal, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral, nasal or transdermal. Administration may be by injection or infusion.

In some aspects and embodiments in accordance with the present disclosure, inhibitory nucleic acids, nucleic acids, expression vectors, cells and compositions described herein may be administered to the liver, e.g. to one or more hepatocytes In some cases inhibitory nucleic acids, nucleic acids, expression vectors, cells and compositions described herein may be administered to the blood (/.e. intravenous/intra- arterial administration), subcutaneously or orally.

In some aspects and embodiments in accordance with the present disclosure there may be targeted delivery of articles of the present disclosure, i.e. wherein the concentration of the relevant agent in the subject is increased in some parts of the body relative to other parts of the body. In some embodiments, the methods comprise intravenous, intra-arterial, intramuscular or subcutaneous administration and wherein the relevant article is formulated in a targeted agent delivery system. Suitable targeted delivery systems include, for example, nanoparticles, liposomes, micelles, beads, polymers, metal particles, dendrimers, antibodies, aptamers, nanotubes or micro-sized silica rods. Such systems may comprise a magnetic element to direct the agent to the desired organ or tissue. Suitable nanocarriers and delivery systems will be apparent to one skilled in the art.

In some cases, the relevant agent is formulated for targeted delivery to specific cells, tissue(s) and/or organ(s). In some cases, the relevant agent is formulated for targeted delivery to cells of the liver (e.g. hepatocytes), hepatic tissue and/or the liver.

The particular mode and/or site of administration may be selected in accordance with the location where reduction of gene and/or protein expression of PCSK9 is required, e.g. cells of the liver (e.g. hepatocytes), hepatic tissue and/or the liver.

In some embodiments, therapeutic or prophylactic intervention according to the present disclosure may further comprise administering another agent for the treatment/prevention of the disease/condition.

Administration of an article of the present disclosure may be alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Simultaneous administration refers to administration with another therapeutic agent together, for example as a pharmaceutical composition containing both agents (combined preparation), or immediately after each other and optionally via the same route of administration (e.g. to the same tissue, artery, vein or other blood vessel). Sequential administration refers to administration of one agent followed after a given time interval by separate administration of another agent It is not required that the two agents are administered by the same route, although this is the case in some embodiments. The time interval may be any time interval.

Multiple doses of the articles of the present disclosure may be provided. One or more, or each, of the doses may be accompanied by simultaneous or sequential administration of another therapeutic agent

Multiple doses may be separated by a predetermined time interval, which may be selected to be one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1 , 2, 3, 4, 5, or 6 months. By way of example, doses may be given once every 7, 14, 21 or 28 days (plus or minus 3, 2, or 1 days). Articles of the present disclosure may be formulated in a sustained release delivery system, in order to release the inhibitory nucleic add, nucleic acid, expression vector or composition at a predetermined rate. Sustained release delivery systems may maintain a constant drug/therapeutic/prophylactic concentration for a specified period of time. In some embodiments, an inhibitory nucleic add, nucleic add, expression vector or composition described herein is formulated in a liposome, gel, implant, device, or drug-polymer conjugate e.g. hydrogel.

In accordance with various aspects of the present invention, methods are provided which are for, or which comprise (e.g. in the context of treatment/prevention of a disease/condition described herein), one or more of the following: reducing PCSK9 gene and/or protein expression; reducing the level of PCSK9 in the serum; reducing the level of total lipid in the serum; reducing the level of total cholesterol in the serum; and/or reducing the level of LDL cholesterol in the serum. Also provided are agents according to the present disclosure for use in such methods, and the use of agents according to the present disclosure in manufacture of compositions (e.g. medicaments) for use in such methods. It will be appreciated that the methods typically comprise administering an agent capable of inhibiting IL-11-mediated signalling to a subject.

Similarly, one or more of the following may be observed in a subject following therapeutic or prophylactic intervention in accordance with the present disclosure (e.g. compared to the level prior to intervention): reduced PCSK9 gene and/or protein expression; reduced level of PCSK9 in the serum; reduced the level of total lipid in the serum; reduced level of total cholesterol in the serum; and/or reduced level of LDL cholesterol in the serum.

In some embodiments, therapeutic/prophylactic intervention in accordance with the present disclosure may be described as being ‘associated with’ one or more of the effects described in the preceding paragraph. The skilled person is readily able to evaluate such properties using techniques that are routinely practiced in the art.

A subject in accordance with the various aspects of the present disclosure may be any animal or human. Therapeutic and prophylactic applications may be in human or animals (veterinary use). The subject to be treated with a therapeutic substance described herein may be a subject in need thereof. The subject is preferably mammalian, more preferably human. The subject may be a non-human mammal, e.g. mouse, rat, non-human primate, but is more preferably human. The subject may be male or female. The subject may be a patient.

A subject may have been diagnosed with a disease or condition described herein, may be suspected of having such a disease/condition, or may be at risk of developing/contracting such a disease/condition. In embodiments according to the present disclosure, a subject may be selected for treatment according to the methods based on characterisation for certain markers of such disease/condition. In some embodiments, a subject comprises a mutation according to any embodiment described hereinabove (that is, in some embodiments a subjects genome comprises one or more copies of an allele comprising a mutation according to any embodiment described hereinabove). In some embodiments, the subject comprises a mutation giving rise to a disease/condition described herein (e.g. hypercholesterolemia).

Kits

In some aspects of the present disclosure a kit of parts is provided. In some embodiments the kit may have at least one container having a predetermined quantity of an inhibitory nucleic add, nucleic add, expression vector, cell or composition described herein.

In some embodiments, the kit may comprise materials for produdng an inhibitory nucleic acid, nucleic add, expression vector, cell or composition described herein.

The kit may provide the Inhibitory nucleic acid, nucleic acid, expression vector, cell or composition together with instructions for administration to a patient in order to treat a specified disease/condition.

In some embodiments the kit may further comprise at least one container having a predetermined quantity of another therapeutic agent (e.g. as described herein). In such embodiments, the kit may also comprise a second medicament or pharmaceutical composition such that the two medicaments or pharmaceutical compositions may be administered simultaneously or separately such that they provide a combined treatment for the specific disease or condition.

Kits according to the present disclosure may include instructions for use, e.g. in the form of an instruction booklet or leaflet. The Instructions may include a protocol for performing any one or more of the methods described herein.

Sequence Identity

Pairwise and multiple sequence alignment for the purposes of determining percent identity between two or more amino add or nudeic add sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (SOding, J. 2005, Bioinformatics 21, 951-060), T-cotfee (Notredame eta/. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780) software. When using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used.

8 pj § CM § u

1 i ® o £

In the sequences of the table above, G = guanosine-3'-phosphate, C = cytidlne-3'-phosphate, U = uridlne- 3'-phosphate, T = thymidine-d'-phosphate, A = adenosine-3’-phosphate, dA = deoxyadenosjne-3'- phosphate, dT = deoxythymidine-S’-phosphate, mU = 2’-O-methyluridine-3'-pho8phate, mA = 2‘-O-

5 methyladenosine-S’-phosphate, mG = 2'-O-methylguanoslne-3'-phosphate, mC = 2*-O-methylcytjdlne-3*- phosphate, (mU)ps = 2'-O-methyluridine-3 ^, -phosphorothioate, (mA)ps = 2'-O-methyladenosine-3’- phosphorothioate, (mG)ps = 2'-0-methylguanosine-3'-phosphorothloate, (mC)ps = 2'-O-methylcytidine-3'- phosphorothioate, fU = 2'-fluorouridine-3’-phosphate, fA = 2’-fluoroadenosine-3’-phosphate ₁ fG = 2- fluoroguanosine-3’-phosphate, fC = 2'-fluorocytidine-3'-phosphate, (fC)ps = 2'-fluorocytidine-3'-

10 phosphorothloate, (fG)ps = 2‘-fluoroguanoslne-3 ^, -phosphorothloate, (fA)ps = 2’-fluoroadenoslne-3'- phosphorothioate, and (flJ)pa - 2’-fluorouridine-3 ^, -phosphorothioate.

SEQ ID NO:1419 to 1428 may comprise a TriGai (triantennary N-acetylgalactosamine) moiety or at least one GalNAc monomer as described herein.

15 ***

The Invention Includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

The section headings used herein are for organisational purposes only and are not to be construed as

20 limiting the subject matter described.

Aspects and embodiments of the present disclosure will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

131 Throughout this specification, including the claims which follow, unless the context requires otherwise, the word 'comprise,' and variations such as 'comprises' and 'comprising,' will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in the specification and the appended claims, the singular forms 'a,' 'an,' and "the' include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from 'about' one particular value, and/or to 'about' another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent ‘about,’ it will be understood that the particular value forms another embodiment

Where a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated.

Methods described herein may be performed in vitro or in vivo. In some embodiments, methods described herein are performed in vitro. The term ‘in vitro* is intended to encompass experiments with cells in culture whereas the term 'in vivo* is intended to encompass experiments with intact multi-cellular organisms.

Brief Description of the Figures

Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures.

Figures 1A and 1B. Bar charts showing knockdown of gene and protein expression of human PCSK9 in HuH7 cells by different dsiRNAs. HuH7 cells were transfected with the indicated dsiRNAs and analysed by qPCR for gene expression of PCSK9 as described in Example 1.1.

Figures 2A and 2B. Bar charts showing knockdown of gene and protein expression of human PCSK9 in HuH7 cells by different dsiRNAs. HuH7 cells were transfected with the indicated dsiRNAs and analysed by ELISA for protein expression of PCSK9 as described in Example 1.1.

Figure 3. Bar chart showing relative gene expression of PCSK9 in HepG2 cells after siRNA- mediated knockdown by different siRNAs. HepG2 cells were transfected with the indicated siRNAs or not transfected (NT), and analysed by qPCR gene as described in Example 1.2.

Figure 4A to 4F. Bar charts showing knockdown of protein expression of human PCSK9 in HuH7 cells by different siRNAs comprising chemical modifications. HuH7 cells were transfected with the indicated siRNA molecules at a final concentration of 20 nM (see Example 2), and analysed by ELISA for protein expression of PCSK9. Figures SA to 5F. Bar charts showing the chemically modified PCSK9 siRNA knockdown efficacy of human PCSK9 in HuH7 cells at 10nM and 1nM transfection concentrations. HuH7 cells were transfected with the indicated siRNA molecules at a final concentration of (SA) PCSK9-199 siRNAs at 10 nM, (SB) PCSK9-199 siRNAs at 1 nM, (SC) PCSK9-105 siRNAs at 1 nM, (5D) PCSK9-204 siRNAs at 1 nM, (5E) PCSK9-212 siRNAs at 1 nM, (5F) PCSK9-213 siRNAs at 1 nM (see Example 2), and analysed by ELISA for protein expression of PCSK9.

Figures 6A to 6D. Bar charts showing the knockdown efficacy test on chemically modified siRNAs targeting (6A) EGFP, (6B) HPRT, or (6C, 6D) mouse Gapdh (mmGapdh), respectively.

Figure 7. Heatmap showing the analysis of knockdown efficacy of 27 modification patterns across 9 different siRNA sequences against different targets in different species. 13 chemical modification patterns showed good knockdown efficacy across all the 9 siRNA sequences targeting PCSK9, HPRT, EGFP, or mouse Gapdh (mmGpadh) respectively.

Figures 8A to 8D. Bar charts showing the chemically modified PCSK9 siRNA knockdown efficacy of human PCSK9 in HuH7 cells at 1nM transfection concentrations. HuH7 cells were transfected with the indicated siRNA molecules at a final concentration of 1nM (8A) PCSK9-200, (8B) PCSK9-193, (8C) PCSK9-045 and (8D) PCSK9-040, and analysed by ELISA for protein expression of PCSK9.

Figure 9. Graph showing the in vivo efficacy on PCSK9 protein reduction and duration for the indicated siRNAs in hPCSK9-KI mice. siRNAs were administrated through subcutaneous injection at single dose of 10mg/kg and 0.5mg/kg. The hPCSK9 protein serum levels were determined by ELISA

Figure 10. Graph showing the in vivo efficacy on PCSK9 protein reduction and duration in hPCSK9- Kl mice for the indicated siRNAs at a single dose of 1mg/kg. MPCSK9 protein serum levels were determined by ELISA

Figure 11. Graph showing the in vivo efficacy on PCSK9 protein reduction and duration in hPCSK9- Kl mice for the indicated siRNAs derived from PCSK9-200 at a single dose of 1mg/kg. hPCSK9 protein serum levels were determined by ELISA.

Figure 12. Graph showing the in vivo efficacy on PCSK9 protein reduction and duration (fold change vs PBS) in hPCSK9-KI mice for the indicated siRNAs derived from PCSK9-199 at a single dose of 1mg/kg. hPCSK9 protein serum levels were determined by ELISA, hPCSK9 fold change to PBS group was calculated using level of PCSK9 from siRNA group divided by PCSK9 level from PBS group at each time point

Figures 13A to 13C. Schematic of an inhibitory nucleic acid according to the present disclosure conjugated with GalNAc monomers described herein The waved line indicates a nucleotide chain. Figures 14A to 14C. Schematic of a double-stranded inhibitory nucleic acid, e.g. siRNA, according to the present disclosure conjugated with GalNAc monomers described herein. The waved lines indicate nucleotide strands.

Examples

In the following Examples, the inventors describe the identification and characterisation of siRNA molecules for inhibiting gene and protein expression of PCSK9.

Example 1 : RNAt f PCSK9 expression siRNAs targeting different regions of mRNA encoding human PCSK9 were designed using a bioinformatics platform with an algorithmic score predicting the knockdown efficacy of the siRNA. The platform was also able to filter out stretches of the mRNA with minor allele frequency (MAE) < 0.01 , 0 mismatch with Macaca fascicularis, off-target score <0.1 in both human and Macaca fascicularis. The off- target score is a composite score which sums up the following: T is scored when the siRNA binds with 100% identity to an off-target gene, *0.T is scored when the siRNA binds with 1 mismatch to an off-target gene, *0.01 ’ is scored when the siRNA binds with 2 mismatches to an off-target gene.

The mRNA sequences of PCSK9 targeted by the siRNAs are shown in SEQ ID NOs:9 to 213. The nucleotide sequences of the antisense nucleic acids of the siRNAs (/.e. the guide strands) targeting SEQ ID NOs:9 to 213 are shown in SEQ ID NOs:214 to 418. The siRNAs were also converted to 27-mer dsiRNA molecules. The mRNA sequences of PCSK9 targeted by the dsiRNAs are shown in SEQ ID NOs:422 to 626. The nucleotide sequences of the antisense nucleic acids of the dsiRNAs targeting SEQ ID NOs:422 to 626 are shown in SEQ ID NOs:627 to 831. siRNA sequences having predicted knockdown efficacy scores greater than 0.7 were selected for further characterisation in assays in vitro, either in an siRNA format, or converted from siRNA to a 27-mer dsiRNA format

An siRNA targeting the same region of the mRNA sequence of PCSK9 as inclisiran (/.e. having the guide strand sequence shown in SEQ ID NO:420) was also obtained and characterised (referred to in the Figures as AD-57928). Three dsiRNA molecules not targeting sequences in the human genome were included as non-targeting (NT) negative controls.

Example 1.1 RNAi knockdown of PCSK9 expression using dsiRNA dsiRNAs were evaluated for their ability to inhibit gene expression of PCSK9, as follows.

Cells of the human hepatocyte carcinoma HuH7 cell line (Cellosaurus Accession: CVCL_0336) were maintained in culture in vitro. HuH7 cells were cultured with DMEM high glucose supplemented with 10%FBS, 1% sodium pyruvate and 1% PenStrep in a humidified incubator at 37°C and 5% CO2. 10,0000 HuH7 cells per well were reverse transfected in wells of 96-well plates with the different dsiRNAs (5 replicates for each siRNA) at a final concentration of 20 nM, using the transfection agent DharmaFECTS (Horizon), in accordance with the manufacturer’s instructions.

The transfected cells were maintained in culture as described above. The cells from three of the replicates were then harvested for analysis of the level of mRNA encoding PCSK9 at 48 hours posttransfection, and the cells from two of the replicates were harvested for analysis of the level of PCSK9 protein at 72 hours post-transfection.

The cells were washed with PBS, and followed with the process of Celkto-Ct assay for the quantification of the level of mRNA encoding PCSK9 using SingleShot Cell Lysis RT-qPCR kit (Bio-Rad), in accordance With the manufacturer’s instructions. The primers used for qPCR are shown in SEQ ID NOs:1112 to 1115. The level of PCSK9 knockdown was calculated by 2 ^A -ddct, normalizing to housekeeping gene GAPDH and further normalizing to non-targeting (NT) siRNA control. Fold change in the level of PCSK9 mRNA was calculated as: fold change = 2-C" ^m P ^|e i^^ ⁹ “■ ^QAPDH < ^PCSK9 CI-GATCH ct)

The results are shown in Figures 1A and 1B.

Total protein extracts were prepared from the cells, and the level of PCSK9 protein was quantified by ELISA using the PCSK9 ELISA kit from Abeam (Cat No. ab209884), in accordance with the manufacturer’s instructions.

The results are shown in Figures 2A and 2B. Several dsiRNAs were found to potently inhibit gene and protein expression of PCSK9, and to a greater extent than AD-57928.

Examole 1.2 Further f knockdown of PCSK9

The following dsiRNAs were evaluated in an independent human hepatocyte cell line HepG2 (Cellosaurus Accession: CVCL_0027): PCSK9-053, PCSK9-020, PCSK9-040, PCSK9-017, PCSK9-007, PCSK9-008, PCSK9-054, PCSK9-049, PCSK9-002, PCSK9-044, PCSK9-045, PCSK9-005, PCSK9-046, PCSK9-018, PCSK9-035. AD-57928 and Inclisiran were also evaluated in the experiment Two siRNA molecules not targeting any sequences in the human genome were included as non-targeting (NT) negative controls.

Cells of the human hepatocellular carcinoma HepG2 cell line were maintained in culture in vitro. HepG2 cells were cultured with DMEM high glucose supplemented with 10% FBS, 1% sodium pyruvate and 1% PenStrep with incubation at 37°C and 5% CO2.

120,000 HepG2 cells were seeded per well in wells of 24-well plates, for transfection the following day with the different dsiRNAs (4 replicates of transfection for each dsiRNA). dsiRNAs were transfected at a final concentration of 20 nM, using the transfection agent DharmaFECT3 (Horizon), in accordance with the manufacturer's instructions. The transfected cells were maintained in culture as described above for 48 hours. The cells from two of the replicates were then harvested for RNA isolation using RNA purification kit (Favorgen). 500ng of total RNA was used for cDNA synthesis using iScript™ Advanced cDNA Synthesis Kit (Bio-Rad) followed with qPCR using KAPA SYBR FAST (Merck). The analysis of the level of mRNA encoding PCSK9 was performed as described in Example 1.1.

The results are shown in Figure 3. Several of the siRNAs were found to inhibit gene expression of PCSK9 to a greater extent than inclisiran.

Example 2: Chemlcallv-modified siRNA-mediated knockdown of PCSK9 expression

The following siRNAs in Table 2, which showed good knockdown efficacy in Example 1, were synthesized comprising chemically-modified nucleotides and were evaluated for their ability to inhibit PCSK9 protein expression.

Two siRNA molecules not targeting any sequences in the human genome were included as non-targeting (NT) negative controls.

Briefly, 10,0000 HuH7 cells per well were reverse transfected in wells of 96-well plates with the different siRNAs (2 replicates for each siRNA) at a final concentration of 20 nM, using the DharmaFECTS (Horizon), in accordance with the manufacturer's instructions. The transfected cells were maintained in culture as described in Example 1.1 for 72 hours. Total protein extracts were prepared from the cells, and the level of PCSK9 protein was quantified by ELISA using the PCSK9 ELISA kit from Abeam (Cat No. ab209884), in accordance with the manufacturer's instructions. The results are shown in Figures 4A to 4F. Several of the siRNAs were found to potently inhibit protein expression of PCSK9. The siRNAs in the boxes potently inhibited PCSK9, knocking it down >70%. The chemical modification patterns used in the folly chemically modified siRNAs enabled very potent knockdown of PCSK9, with a greater knockdown than the unmodified siRNA nucleotide sequence and to a level even greater than that of inclisiran.

In further experiments, HuH7 cells were transfected with siRNAs as described immediately above, except that the siRNAs were provided at a final concentration of 10 nM or 1 nM. The results are shown in Figures 5A to 5F. siRNAs were identified that potently inhibited PCSK9, with some knocking it down >80% at 10nM, and >50% at 1nM. The chemical modification patterns used in the folly chemically modified siRNAs enabled very potent knockdown of PCSK9, better than the unmodified siRNA nucleotide sequence, and to a level even greater than that of inclisiran even at low concentration of 1nM.

In further experiments, siRNA molecules formed of (i) an oligonucleotide having a nucleotide sequence (including the modifications thereto) indicated in column A of Table 1 , and (ii) an oligonucleotide having a nucleotide sequence (including the modifications thereto) indicated in column B of Table 1, wherein the sequences of columns A and B are selected from the same row of Table 1 , are characterised for their ability to inhibit gene and protein expression of PCSK9.

HuH7 cells are transfected with the different siRNAs as described immediately above, and after 72 hours total protein extracts are prepared from the cells and the level of PCSK9 protein is quantified by ELISA as described above. RNA is also isolated from the cells as described in Example 1.2, and analysis of the level of mRNA encoding PCSK9 is performed as described in Example 1.1.

Several of the siRNAs are found to potently inhibit protein expression of PCSK9.

Examnle 3: Concentration titration of siRNAs targeting PCSK9 siRNA molecules described in Examples 1 and 2 are evaluated in order to determine the efficiency with which they knockdown expression of PCSK9 at different concentrations.

Briefly, HuH7 cells are transfected with the different siRNAs as described in Examples 1 and 2, at concentrations ranging from 10 nM to 0.1 nM (10 nM, 1 nM, 0.5 nM and 0.1 nM). At 72 hours posttransfection, total protein extracts are prepared from the cells and the level of PCSK9 protein is quantified by ELISA as described in Example 2.

Several of the siRNAs are found to inhibit protein expression of PCSK9 even at low concentration, with higher potency than Inclisiran at the same concentration.

Example 4: Knockdown of PCSK9 expression In ;

The efficacy of siRNAs targeting PCSK9 is evaluated in a human PCSK9 knock-in (hPCSK9-KI) mouse model of hypercholesterolemia. The endogenous mouse Pcsk9 gene is replaced with the cDNA encoding human PCSK9 via CRISPR/Cas-mediated recombination. The hPCSK9-KI mice are then fed with a high- fat diet in order to induce high serum levels of total cholesterol and LDL-cholesterol.

The hPCSK9-KI mouse fed with the high-fat diet is characterised by (/.e. relative to wildtype mice fed a normal chow diet) increased bodyweight (detectable at 8 weeks of age), and significantly elevated plasma levels of total cholesterol and LDL cholesterol (detectable at 8 and 16 weeks).

7 to 8 week old hPCSK9-KI mice are used in the experiments. Serum samples are collected after a 4- hour fasting, on days 14 and 7. siRNAs described in Example 1 , 2 or 3 that knockdown expression of PCSK9 are evaluated in the experiments.

On day 0, mice are injected subcutaneously with the selected siRNA at a dose of 0.5 mg/kg, 1 mg/kg, 2 mg/kg, or 10 mg/kg bodyweight (in PBS, total volume injected = 200 pl; 8 mice in each treatment group). Mice of a negative control group are injected subcutaneously with 200 pl PBS only.

Serum samples are collected from the mice in the various different treatment groups on days 1, 3, 7, 10, and 14 post-administration. This is followed by a weekly serum collection up to 8 weeks post-injection. The serum is analysed in order to determine the level of PCSK9 protein (/.e. by ELISA), and the levels of total cholesterol, LDL cholesterol, HDL cholesterol and total triglycerides (using the Agilent DxC 600 clinical analyser). hPCSK9-KI mice administered siRNAs that knockdown expression of PCSK9 are found to have lower levels of total cholesterol and LDL cholesterol compared to hPCSK9-KI mice of the control group administered PBS.

Example S: Chemical modifications Increase knockdown efficacy ofsiRNAs targeting EGFP, HPRT or mouse Gaodh (mmGaodhl siRNAs targeting EGFP, human and mus musculus HPRT, or mus musculus Gapdh (mmGapdh) comprising chemically-modified nucleotides were produced.

The siRNAs in Table 3 comprising chemically-modified nucleotides were evaluated for their ability to inhibit gene expression of EGFP, mm HPRT or mmGapdh, respectively.

Two siRNA molecules not targeting any sequences in the human or mouse genome were included as non-targeting (NT) negative controls.

A HepG2-EGFP cell line was generated using lentivirus carrying sequences coding enhance green fluorescent protein (EGFP) for evaluating the efficacy of siRNA knockdown. 20,000 HepG2-EGFP cells per well were reverse transfected in wells of 96-well plates with siRNAs targeting EGFP as described above. 10,000 mouse hepatocyte AML12 cells per well was used for testing knocking down efficacy of siRNAs for HRPT and mmGapdh. The results are shown in Figures 6A to 6D.

The knockdown efficiencies of 27 different chemical modification patterns which were used across nine siRNA sequences targeting different genes and species; on human PCSK9 (in example 2), EGFP, human/mouse HPRT or mouse Gapdh (mmGapdh), respectively, were analysed and shown in Figure 7.

Thirteen chemical modification patterns (underlined patterns in Figure 7) were able to efficiently improve the knockdown efficacy across different genes, species and siRNA sequences and can be broadly used as universal modifications to improve the knockdown efficacy of any siRNA sequences, irrespective of the specific sequence of the oligonucleotide. This was unexpected as it is well-known that a chemical modification pattern that works well for a particular sequence may not work well for another, different sequence.

Examole 6: siRNA PC8K9 knockdown and IC50 evaluation

Chemically modified siRNA as shown in Table 4 with working concentration of 10nM,1nM, 0.5nM, 0.25nM, 0.125nM, 0.0625nM, 0.03125nM, 0.015625nM, 0.003125nM, and 0.000625nM were transfected in HuH-7 cells using RNAimax. PCSK9 protein in the supernatant was determined by ELISA on 3 days post-transfection. IC50 values for selected duplex siRNAs are shown in Tables 5 and 6.

Figures 8A to 8D show knockdown efficiency of PCSK9 protein using chemically modified siRNAs targeting PCSK9.

Table 4. siRNA duplexes. CG-100390 to CG-100416 refer to 'PCSK9-200* siRNAs. CG-100417 to CG- 100443 refer to ‘PCSK9-199’ siRNAs. CG-100444 to GC-100470 refer to 'PCSK9-045' siRNAs. CG-

100471 to CG-100497 refer to 'PCSK9-040' siRNAs. e

PCSK9-199 derived siRNAs were generated to contain mismatches in the sense strand (SEQ ID NOs 1348-1362). siRNAs are shown in Table 8. Selected siRNAs were evaluated for IC50 values (Table 9).

Table 8. PCSK9-199 derived siRNAs strand. Os 11). PCSK9-199 derived siRNA sequences were generated with modified guide strands. siRNAs are shown in Table 12. Selected siRNAs were evaluated for IC50 values (Table 13).

Table 12. PCSK9-199 derived sequences.

Table 13. PCSK9-199 siRNA IC50 values

PCSK9-200 derived siRNAs were generated with DNA and position 11 mismatch and a triGai moiety attached to the sense strand. sRNA duplexes are shown in Table 14. Selected siRNAs were evaluated for IC50 values (Table 15).

Table 14. PCSK9-200 derived sequences with DNA and Position 11 mismatch and triGai on the sense strand.

Table 15. PCSK9-200 derived sequences IC50 values.

Examgle > In y/yo. evaluation. of siRNA sequences siRNA duplexes in Table 16 with a triGai (Triantennary N-acetylgalactosamine) moiety attached to their sense strand were tested for their ability to reduce PCSK9 protein production in vivo in hPCSK9-Knock-in mice.

7 to 8 week old hPCSK9-KI mice were used in the experiments. siRNAs were administered via subcutaneous injection at single dose of 10mg/kg, 1mg/kg or 0.5mg/kg. The hPCSK9 protein serum levels were determined by ELISA. The siRNA effects on PCSK9 protein reduction were determined up to 49 days post-injection.

Table 16. siRNA sequences tested for in vivo efficacy

Figure 9 shows that the indicated siRNAs were found to reduce human PCSK9 protein production in mice at 0.5mg/kg or 10mg/kg.

Figure 10 shows that the indicated siRNAs were found to reduce human PCSK9 protein production in mice at 1 mg/kg.

Figure 11 shows that the indicated siRNAs derived from PCSK9-200 were found to reduce human PCSK9 protein production in mice at 1mg/kg.

Figure 12 shows that the indicated siRNAs derived from PCSK9-199 were found to reduce human PCSK9 protein production in mice at 1mg/kg.

Example 8: In vivo evaluation of siRNA sequences in non-human primates siRNA duplexes with a GalNAc moiety attached to their sense strand are tested for their ability to reduce PCSK9 protein production in vivo in non-human primates (NHPs). siRNAs are administered via subcutaneous injection at single dose of 3 mg/kg. The PCSK9 protein serum levels are determined by ELISA. The siRNA effects on PCSK9 protein reduction are determined. siRNAs derived from PCSK9-199 and PCSK9-200 are found to reduce PCSK9 protein production in NHPs.