Nucleic acid for inhibiting expression of LPA in cells

Abstract

The present invention relates to products and compositions and their uses. In particular, the present invention relates to a nucleic acid product that interferes with or inhibits the expression of an LPA gene, preferably for use in the treatment, prevention of cardiovascular diseases such as coronary heart disease or aortic valve stenosis or stroke or any other condition, pathology or syndrome associated with an elevated level of Lp (a) particles, or reduce the risk of suffering from a cardiovascular disease such as coronary heart disease or aortic valve stenosis or stroke or any other condition, pathology or syndrome associated with an elevated level of Lp (a) particles.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201980074785.8, application date November 13, 2019, and invention name “Nucleic acid for inhibiting the expression of LPA in cells”. Technical Field

[0002] The present invention relates to products and compositions and their uses. In particular, the present invention relates to nucleic acid products that interfere with LPA gene expression or inhibit its expression. The therapeutic Lp(a) lowering therapy is used to prevent stroke, atherosclerosis, thrombosis and cardiovascular diseases such as coronary heart disease and aortic valve stenosis or any other condition, pathology or syndrome associated with elevated levels of Lp(a) particles, and to reduce the risk of suffering from stroke, atherosclerosis, thrombosis and cardiovascular diseases such as coronary heart disease and aortic valve stenosis or any other condition, pathology or syndrome associated with elevated levels of Lp(a) particles. Background Art

[0003] Double-stranded RNA (dsRNA) that has been shown to be able to complementarily bind to expressed mRNA can block gene expression through a mechanism that has been referred to as RNA interference (RNAi) (Fire et al., 1998, Nature. February 19, 1998; 391(6669): 806-11 and Elbashir et al., 2001, Nature. May 24, 2001; 411(6836): 494-8). Short dsRNAs guide gene-specific post-transcriptional silencing in many organisms including vertebrates and have become a tool that can be used to study gene function. RNAi is mediated by an RNA-induced silencing complex (RISC), which is a sequence-specific multicomponent nuclease that degrades messenger RNA homologous to a silencing trigger loaded into the RISC complex. Interfering RNAs (referred to herein as iRNAs) such as siRNAs, antisense RNAs, and microRNAs are oligonucleotides that inhibit the gene translation of proteins to prevent the formation of proteins by gene silencing, i.e., by degrading mRNA molecules. Gene silencing agents are becoming increasingly important for therapeutic applications in medicine.

[0004] According to Watts and Corey, Journal of Pathology (2012; Vol. 226, pp. 365-379), there are algorithms that can be used to design nucleic acid silencing triggers, but all of these algorithms have serious limitations. Various experimental methods may be used to identify powerful iRNAs because the algorithms do not consider factors such as the tertiary structure of the target mRNA or the involvement of RNA binding proteins. Therefore, it is a complex process to find a powerful nucleic acid silencing trigger with minimal off-target effects. For the drug development of these highly charged molecules, it is necessary that they can be economically synthesized, distributed in the target tissue, enter the cell, and function within acceptable toxicity limits.

[0005] Lp(a) particles are heterogeneous low-density lipoprotein particles expressed primarily in the liver (Witztum and Ginsberg, J Lipid Res. 2016 March; 57(3): 336-9). They consist of apolipoprotein(a) (Apo(a) or Lp(a) is encoded by the LPA gene) linked to LDL-like particles via the ApoB polypeptide. Genetically defined high serum levels of Lp(a) particles are not affected by diet and exercise and are associated with an increased risk of cardiovascular disease through associated atherosclerotic potential (Alonso et al., Journal of the American College of Cardiology Vol. 63, No. 19, 2014). According to diagnostics and preventive medicine, a patient's serum level of Lp(a) particles is a highly prevalent independent genetic risk factor for coronary heart disease and aortic stenosis (Saeedi and Frohlich Clinical Diabetes and Endocrinology (2016) 2: 7). There are no currently approved specific Lp(a) particle lowering therapies other than indirect standard general LDL lowering measures. Therefore, there is a current need for methods to effectively treat, prevent, and reduce the risk of conditions such as and associated with stroke, atherosclerosis, thrombosis, and cardiovascular diseases such as coronary heart disease, aortic stenosis, and other as yet unidentified related conditions, pathologies, or syndromes. The present invention addresses this unmet medical need. Summary of the invention

[0006] One aspect of the present invention relates to a nucleic acid for inhibiting the expression of LPA in a cell, the nucleic acid comprising at least one duplex region, the duplex region comprising at least a portion of a first strand and at least a portion of a second strand at least partially complementary to the first strand, wherein the first strand is at least partially complementary to at least a portion of RNA transcribed from the LPA gene, wherein the first strand comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43,

[0007] wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are modified with a 2' fluoro modification, and the nucleotides on the second strand corresponding to positions 11-13 of the first strand are modified with a 2' fluoro modification.

[0008] The present invention also provides a composition comprising a nucleic acid or a conjugated nucleic acid according to any aspect of the present invention and optionally a physiologically acceptable excipient.

[0009] One aspect relates to a nucleic acid capable of inhibiting the expression of LPA for use as a medicament.

[0010] Also provided is a nucleic acid or conjugated nucleic acid according to any aspect of the invention for use in treating a disease, disorder or syndrome and / or in the manufacture of a medicament for treating a disease, disorder or syndrome.

[0011] The invention provides a method for treating or preventing a disease, disorder or syndrome, the method comprising administering to an individual in need of treatment a composition comprising a nucleic acid or conjugated nucleic acid according to any aspect of the invention. The nucleic acid or conjugated nucleic acid can be administered to the subject subcutaneously, intravenously or using any other administration route such as oral, rectal or intraperitoneal administration.

[0012] Also included is a method of making a nucleic acid or conjugated nucleic acid of the invention.

[0013] The nucleic acid or composition comprising the nucleic acid or conjugated nucleic acid of the invention can be used to treat a disease, disorder or syndrome. Treatment can be for the prevention and reduction of the risk of stroke, atherosclerosis, thrombosis or cardiovascular disease such as coronary heart disease or aortic stenosis and any other disease or pathology associated with elevated levels of Lp(a)-containing particles. DETAILED DESCRIPTION

[0014] The present invention relates to a nucleic acid and a composition thereof, wherein the nucleic acid is double-stranded and is directed against the RNA transcript of the expression of LPA. These nucleic acids or conjugated nucleic acids can be used to treat and prevent a variety of diseases, disorders and syndromes in which reduced expression of LPA gene products is desirable.

[0015] One aspect relates to a nucleic acid for inhibiting the expression of LPA in a cell, the nucleic acid comprising at least one duplex region, the duplex region comprising at least a portion of a first strand and at least a portion of a second strand at least partially complementary to the first strand, wherein the first strand is at least partially complementary to at least a portion of RNA transcribed from the LPA gene, wherein the first strand comprises or preferably consists of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43,

[0016] wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are modified with a 2' fluoro modification, and the nucleotides on the second strand corresponding to positions 11-13 of the first strand are modified with a 2' fluoro modification.

[0017] One aspect relates to a nucleic acid for inhibiting the expression of LPA in a cell, the nucleic acid comprising at least one duplex region, the duplex region comprising a first strand and a second strand at least partially complementary to the first strand, wherein the first strand is at least partially complementary to at least a portion of RNA transcribed from the LPA gene, wherein the first strand comprises or preferably consists of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43,

[0018] wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are modified with a 2' fluoro modification, and the nucleotides on the second strand corresponding to positions 11-13 of the first strand are modified with a 2' fluoro modification.

[0019] Such nucleic acids can effectively reduce the expression of LPA in cells and are very stable. Under comparable conditions, the nucleic acids of the present invention are preferably capable of inhibiting the expression of LPA in cells to a similar (such as the same) or higher degree compared to the same nucleic acids with different modification patterns. More specifically, under comparable conditions, compared to the same nucleic acids with different modification patterns, the nucleic acids of the present invention are preferably capable of inhibiting the expression of LPA in cells by 80%, 90%, 100%, 105%, 110% or more percentages.

[0020] One aspect relates to a nucleic acid, wherein all nucleotides of the nucleic acid are modified at the 2' position of the sugar.

[0021] One aspect relates to a nucleic acid, wherein the nucleic acid is modified with alternating 2' O-methyl modifications and 2' fluoro modifications, preferably along the entire length of the first strand.

[0022] One aspect relates to a nucleic acid, wherein the remaining modifications of the second strand are naturally occurring modifications, preferably 2'O-methyl. In other words, the nucleotides on the second strand corresponding to positions 11-13 of the first strand are modified with 2'fluoro, and all other nucleotides of the second strand are modified with naturally occurring modifications, preferably 2'O-methyl.

[0023] The second strand may comprise the nucleotide sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42 or 44.

[0024] The nucleic acid may: a) be blunt ended at both ends; b) have an overhang at one end and a blunt end at the other end; or c) have an overhang at both ends. The nucleic acid is preferably blunt ended at both ends.

[0025] These nucleic acids have the advantages of being active in various species related to preclinical and clinical development and / or having few relevant off-target effects, as well as being stable in vivo and having a long duration of action. They also contain relatively few non-naturally occurring modified nucleotides, but are still able to effectively inhibit target genes for a long time. The specific modification pattern with few non-naturally occurring modified nucleotides (2'F modified nucleotides) also makes them easier to synthesize.

[0026] The nucleic acid may comprise: a first chain comprising or preferably consisting of the nucleotide sequence of SEQ ID NO: 9, and optionally, a second chain comprising or preferably consisting of the nucleotide sequence of SEQ ID NO: 10; or a first chain comprising or preferably consisting of the nucleotide sequence of SEQ ID NO: 5, and optionally, a second chain comprising or preferably consisting of the nucleotide sequence of SEQ ID NO: 6; or a first chain comprising or preferably consisting of the nucleotide sequence of SEQ ID NO: 1, and optionally, a second chain comprising or preferably consisting of the nucleotide sequence of SEQ ID NO: 2; or a first chain comprising or preferably consisting of the nucleotide sequence of SEQ ID NO: 3, and optionally, a second chain comprising or preferably consisting of the nucleotide sequence of SEQ ID NO: 4; or a first chain comprising or preferably consisting of the nucleotide sequence of SEQ ID NO: 7. NO:7, and optionally, a second chain comprising a nucleotide sequence of SEQ ID NO:8, or preferably consisting of a nucleotide sequence of SEQ ID NO:8; or a first chain comprising a nucleotide sequence of SEQ ID NO:11, or preferably consisting of a nucleotide sequence of SEQ ID NO:11, and optionally, a second chain comprising a nucleotide sequence of SEQ ID NO:12, or preferably consisting of a nucleotide sequence of SEQ ID NO:12; or a first chain comprising a nucleotide sequence of SEQ ID NO:13, or preferably consisting of a nucleotide sequence of SEQ ID NO:13, and optionally, a second chain comprising a nucleotide sequence of SEQ ID NO:14, or preferably consisting of a nucleotide sequence of SEQ ID NO:14; or a first chain comprising a nucleotide sequence of SEQ ID NO:15, or preferably consisting of a nucleotide sequence of SEQ ID NO:15, and optionally, a second chain comprising a nucleotide sequence of SEQ ID NO:16, or preferably consisting of a nucleotide sequence of SEQ ID NO:16; or a first chain comprising a nucleotide sequence of SEQ ID NO:17, or preferably consisting of a nucleotide sequence of SEQ ID NO:17, and optionally, a second chain comprising SEQ ID NO:17 NO: 18 or preferably a second chain consisting of the nucleotide sequence of SEQ ID NO: 18;or a first chain comprising, or preferably consisting of, the nucleotide sequence of SEQ ID NO: 19, and optionally, a second chain comprising, or preferably consisting of, the nucleotide sequence of SEQ ID NO: 20; or a first chain comprising, or preferably consisting of, the nucleotide sequence of SEQ ID NO: 21, and optionally, a second chain comprising, or preferably consisting of, the nucleotide sequence of SEQ ID NO: 22; or a first chain comprising, or preferably consisting of, the nucleotide sequence of SEQ ID NO: 23, and optionally, a second chain comprising, or preferably consisting of, the nucleotide sequence of SEQ ID NO: 24; or a first chain comprising, or preferably consisting of, the nucleotide sequence of SEQ ID NO: 25, and optionally, a second chain comprising, or preferably consisting of, the nucleotide sequence of SEQ ID NO: 26; or a first chain comprising, or preferably consisting of, the nucleotide sequence of SEQ ID NO: 27. NO:27, and optionally, a second chain comprising a nucleotide sequence of SEQ ID NO:28 or preferably consisting of a nucleotide sequence of SEQ ID NO:28; or a first chain comprising a nucleotide sequence of SEQ ID NO:29 or preferably consisting of a nucleotide sequence of SEQ ID NO:29, and optionally, a second chain comprising a nucleotide sequence of SEQ ID NO:30 or preferably consisting of a nucleotide sequence of SEQ ID NO:30; or a first chain comprising a nucleotide sequence of SEQ ID NO:31 or preferably consisting of a nucleotide sequence of SEQ ID NO:31, and optionally, a second chain comprising a nucleotide sequence of SEQ ID NO:32 or preferably consisting of a nucleotide sequence of SEQ ID NO:32; or a first chain comprising a nucleotide sequence of SEQ ID NO:33 or preferably consisting of a nucleotide sequence of SEQ ID NO:33, and optionally, a second chain comprising a nucleotide sequence of SEQ ID NO:34 or preferably consisting of a nucleotide sequence of SEQ ID NO:34; or a first chain comprising a nucleotide sequence of SEQ ID NO:35 or preferably consisting of a nucleotide sequence of SEQ ID NO:35, and optionally, a second chain comprising SEQ ID NO:35 NO:36 or preferably a second chain consisting of the nucleotide sequence of SEQ ID NO:36;or a first chain comprising or preferably consisting of a nucleotide sequence of SEQ ID NO:37, and optionally a second chain comprising or preferably consisting of a nucleotide sequence of SEQ ID NO:38; or a first chain comprising or preferably consisting of a nucleotide sequence of SEQ ID NO:39, and optionally a second chain comprising or preferably consisting of a nucleotide sequence of SEQ ID NO:40; or a first chain comprising or preferably consisting of a nucleotide sequence of SEQ ID NO:41, and optionally a second chain comprising or preferably consisting of a nucleotide sequence of SEQ ID NO:42; or a first chain comprising or preferably consisting of a nucleotide sequence of SEQ ID NO:43, and optionally a second chain comprising or preferably consisting of a nucleotide sequence of SEQ ID NO:44. ;

[0027] The nucleic acid, wherein the first strand comprises and preferably consists of the nucleotide sequence of SEQ ID NO: 9, and optionally, wherein the second strand comprises and preferably consists of the nucleotide sequence of SEQ ID NO: 10.

[0028] The LPA gene contains highly repetitive sequences. Therefore, a first strand of nucleic acid with a very similar sequence can have complete sequence complementarity with a very different target region of an mRNA.

[0029] One aspect is a nucleic acid for inhibiting the expression of LPA in a cell, wherein the nucleic acid comprises at least one duplex region, the duplex region comprising: a first strand; and a second strand, wherein the second strand is at least partially complementary to the first strand, wherein the first strand comprises a sequence having at least 15, preferably at least 16, more preferably at least 17, still more preferably at least 18, and most preferably at least 19 nucleotides of any one of the reference sequences SEQ ID NO: 9, 5, 1, 3, 7, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43, and wherein the number of single nucleotide mismatches and / or deletions and / or insertions in the first strand sequence relative to the portion having the reference sequence contained in the first strand sequence is at most three, preferably at most two, more preferably at most one, and most preferably zero. However, the sequence can be modified by many modifications that do not change the identity of the nucleotides. For example, modifications to the nucleic acid backbone do not change the identity of the nucleotides because the bases themselves remain the same as in the reference sequence.

[0030] In one aspect, the first strand of the nucleic acid comprises a sequence of at least 18 nucleotides having any one of the reference sequences, preferably having any one of the reference sequences SEQ ID NOs: 9 and 5, and wherein the number of single nucleotide mismatches and / or deletions and / or insertions in the first strand sequence relative to the portion having the reference sequence contained in the first strand sequence is at most one, and preferably is zero.

[0031] In one aspect, the first strand of the nucleic acid comprises a sequence of at least 19 nucleotides having any one of the reference sequences SEQ ID NOs: 9 and 5.

[0032] When the reference sequence comprising unmodified nucleotides or consisting of unmodified nucleotides is mentioned herein, this reference is not limited to sequences with unmodified nucleotides. The same mention also encompasses identical nucleotide sequences, wherein one, several (such as two, three, four, five, six, seven or more) (including all) nucleotides are modified by modifications such as 2'-OMe, 2'-F, ligands, joints, 3' end or 5' end modifications or any other modifications. It also refers to sequences in which two or more nucleotides are connected to each other by natural phosphodiester bonds or by any other bonds (such as phosphorothioate or phosphorodithioate bonds).

[0033] Double-stranded nucleic acid is such nucleic acid, wherein the first chain and the second chain hybridize to each other on at least a portion of their length and thus can form a duplex region under physiological conditions (such as at 37 ° C in PBS when the concentration of each chain is 1 μ M). The first chain and the second chain are preferably able to hybridize to each other and thus form a duplex region in a region of at least 15 nucleotides, preferably 16,17,18 or 19 nucleotides. This duplex region includes nucleotide base pairing between the two chains, preferably based on Watson-Crick (Watson-Crick) base pairing and / or wobble base pairing (such as GU base pairing). All nucleotides of the two chains in the duplex region do not have to base pair to form a duplex region. A certain number of mismatches, deletions or insertions between the nucleotide sequences of the two chains are acceptable. Unpaired nucleotides at the overhangs on either end of the first chain or the second chain or at either end of the double-stranded nucleic acid are also possible. The double-stranded nucleic acid is preferably a stable double-stranded nucleic acid under physiological conditions, and preferably has a melting temperature (Tm) of 45°C or higher, preferably 50°C or higher, and more preferably 55°C or higher, for example, in PBS at a concentration of 1 μM each, the first chain and the second chain are preferably able to form a duplex region (i.e., complementary to each other) over i) at least a portion of their length, preferably at least 15 nucleotides of both lengths; ii) over the entire length of the first chain; iii) over the entire length of the second chain; and / or iv) over the entire length of both the first chain and the second chain. Chains that are complementary to each other over a certain length mean that the chains can base pair with each other over the length by Watson-Crick or wobble base pairing. Each nucleotide of the length does not necessarily have to be able to base pair with its counterpart in the other chain over the entire given length, as long as a stable double-stranded nucleotide can be formed under physiological conditions. However, this is preferred.

[0034] A certain number of mismatches, deletions or insertions between the first (antisense) strand and the target sequence, or between the first and second (sense) strands, can be tolerated in the case of siRNAs, and in certain cases even have the potential to increase activity.

[0035] By nucleic acid it is meant a nucleic acid comprising two nucleotide-containing chains capable of interfering with gene expression. The inhibition may be complete or partial and results in downregulation of gene expression in a targeted manner. The nucleic acid comprises two separate polynucleotide chains; a first chain, which may also be a guide chain, and a second chain, which may also be a passenger chain. The first chain and the second chain may be part of the same polynucleotide molecule that is self-complementary, which 'folds' back to form a double-stranded molecule. The nucleic acid may be a siRNA molecule.

[0036] Nucleic acids may include ribonucleotides, modified ribonucleotides, deoxynucleotides, deoxyribonucleotides, or nucleotide analogs non-nucleotides that can mimic nucleotides so that they can 'pair' with corresponding bases on a target sequence or a complementary strand. Nucleic acids may also include a double-stranded nucleic acid portion or duplex region formed by all or a portion of a first strand (also referred to in the art as a guide strand) and all or a portion of a second strand (also referred to in the art as a passenger strand). A duplex region is defined as starting with the first base pair formed between the first strand and the second strand, and ending with the last base pair formed between the first strand and the second strand, including the first base pair and the last base pair.

[0037] With respect to duplex region, it means a region in two complementary or substantially complementary oligonucleotides that form base pairs with each other by Watson-Crick base pairing or any other means that allows a duplex to be achieved between complementary or substantially complementary oligonucleotide chains. For example, an oligonucleotide chain with 21 nucleotide units can be base paired with another oligonucleotide with 21 nucleotide units, but only 19 nucleotides on each chain are complementary or substantially complementary, so that the "duplex region" consists of 19 base pairs. The remaining base pairs can be in the form of 5' and 3' overhangs, or in the form of single-stranded regions. In addition, within the duplex region, 100% complementarity is not required; within the duplex region, substantially complementarity is permissible. Substantially complementarity refers to the complementarity between the chains that enables them to anneal under biological conditions. Techniques for empirically determining whether two chains can anneal under biological conditions are well known in the art. Alternatively, two chains can be synthesized and added together under biological conditions to determine whether they anneal to each other.

[0038] The portion of the first strand and the portion of the second strand forming at least one duplex region may be fully complementary, or at least partially complementary to each other.

[0039] Depending on the length of the nucleic acid, a perfect match in terms of base complementarity between the first and second strands is not necessarily required. However, the first and second strands must be able to hybridize under physiological conditions.

[0040] The complementarity between the first strand and the second strand in at least one duplex region may be complete because there are no nucleotide mismatches or extra / missing nucleotides in either strand. Alternatively, the complementarity may not be complete. The complementarity may be from about 70% to about 100%. More particularly, the complementarity may be at least 70%, 75%, 80%, 85%, 90% or 95% and intermediate values.

[0041] In the context of the present invention, "a portion of ..." as for example in the case of "a duplex region comprising at least a portion of the first strand" should be understood to mean that the duplex region comprises at least 10 nucleotides, preferably at least 12 nucleotides, more preferably at least 14 nucleotides, still more preferably at least 16 nucleotides, even more preferably at least 18 nucleotides, and most preferably all nucleotides of a given reference strand sequence. The portion of the reference sequence in the duplex region is at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95%, and most preferably 100% identical to the corresponding portion of the reference sequence. Alternatively, the number of single nucleotide mismatches relative to the portion of the reference sequence is at most three, preferably at most two, more preferably at most one, and most preferably zero.

[0042] The first strand and the second strand may each comprise a region of complementarity comprising at least 15 consecutive nucleotides that differ from any of the sequences listed in Table 1 by at most 3 nucleotides.

[0043] The use of nucleic acid of the present invention relates to forming a duplex region between all or part of the first strand and a part of the target nucleic acid. The portion of the target nucleic acid that forms the duplex region with the first strand is the target nucleic acid sequence or simply referred to as the target sequence, and the duplex region is defined as starting with the first base pair formed between the first strand and the target sequence, and ending with the last base pair formed between the first strand and the target sequence, including the first base pair and the last base pair. The duplex region formed between the first strand and the second strand does not need to be the same as the duplex region formed between the first strand and the target sequence. In other words, the second strand may have a sequence different from the target sequence; however, the first strand must be able to form a duplex structure with both the second strand and the target sequence at least under physiological conditions.

[0044] Complementarity between the first strand and the target sequence may be perfect (no nucleotide mismatches or extra / missing nucleotides in either nucleic acid).

[0045] The complementarity between the first strand and the target sequence may not be perfect. The complementarity may be about 70% to about 100%. More particularly, the complementarity may be at least 70%, 80%, 85%, 90% or 95% and intermediate values.

[0046] The identity between the first strand and the complementary sequence of the target sequence may be in the range of about 75% to about 100%. More particularly, the complementarity may be at least 75%, 80%, 85%, 90% or 95% and intermediate values, provided that the nucleic acid is capable of reducing or inhibiting the expression of LPA.

[0047] A nucleic acid having less than 100% complementarity between the first strand and the target sequence may be able to reduce the expression of LPA at the same level as a nucleic acid having complete complementarity between the first strand and the target sequence. Alternatively, it may be able to reduce the expression of LPA at a level that is 15%-100% of the reduction level achieved by a nucleic acid having complete complementarity.

[0048] In one aspect, the nucleic acid comprises a first nucleic acid strand and a second nucleic acid strand, wherein the first strand is capable of hybridizing under physiological conditions to a nucleic acid of sequence SEQ ID NO: 10, 6, 2, 4, 8, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, or 44;

[0049] wherein the second strand is capable of hybridizing with the first strand under physiological conditions to form a duplex region; and

[0050] wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are modified with a 2' fluoro modification, and the nucleotides on the second strand corresponding to positions 11-13 of the first strand are modified with a 2' fluoro modification.

[0051] A nucleic acid capable of hybridizing under physiological conditions is a nucleic acid capable of forming base pairs, preferably Watson-Crick or wobble base pairs, between at least a portion of the opposing nucleotides in the chain to form at least one duplex region. Such double-stranded nucleic acids are preferably stable double-stranded nucleic acids under physiological conditions (e.g., at 37° C. in PBS, at a concentration of 1 μM per chain), meaning that under such conditions, the two chains remain hybridized to each other. The Tm of the double-stranded nucleotide is preferably 45° C. or higher, preferably 50° C. or higher, and more preferably 55° C. or higher.

[0052] One aspect relates to a nucleic acid for inhibiting the expression of LPA, wherein the nucleic acid comprises a first sequence of at least 15, preferably at least 16, more preferably at least 17, more preferably at least 18, and most preferably all nucleotides that differ from any sequence in Table 1 by no more than 3 nucleotides, preferably no more than 2 nucleotides, more preferably no more than 1 nucleotide, and most preferably no more than any nucleotides, and the first sequence can hybridize with a target gene transcript (such as mRNA) under physiological conditions. Preferably, the nucleic acid further comprises a second sequence of at least 15, preferably at least 16, more preferably at least 17, more preferably at least 18, and most preferably all nucleotides that differ from any sequence in Table 1 by no more than 3 nucleotides, preferably no more than 2 nucleotides, more preferably no more than 1 nucleotide, and most preferably no more than any nucleotides, the second sequence can hybridize with the first sequence under physiological conditions, and preferably the nucleic acid is an siRNA capable of inhibiting LPA expression through the RNAi pathway.

[0053] The nucleic acids described herein may be capable of inhibiting the expression of LPA. The inhibition may be complete, i.e., 0% residual expression compared to the expression level of LPA in the absence of the nucleic acids of the present invention. The inhibition of LPA expression may be partial, i.e., it may be 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or an intermediate value of the expression of LPA in the absence of the nucleic acids of the present invention. The level of inhibition may be measured by comparing the treated sample with an untreated sample or with a sample treated with a control (e.g., an siRNA that does not target LPA). Inhibition may be measured by measuring LPA mRNA and / or protein levels or the level of a biomarker or indicator associated with the presence or activity of LPA. It may be measured in cells that may have been treated in vitro with the nucleic acids described herein. Alternatively or additionally, inhibition may be measured in cells (e.g., hepatocytes) or tissues (e.g., liver tissue) or organs (e.g., liver) or body fluids (e.g., blood, serum, lymph or any other body part) taken from a subject previously treated with a nucleic acid disclosed herein. Preferably, inhibition of LPA mRNA is determined by comparing the level of LPA mRNA measured in cells expressing LPA after treatment with a nucleic acid disclosed herein for 24 or 48 hours in vitro under ideal conditions (see the Examples for appropriate concentrations and conditions) with the level of LPA mRNA measured in the same cells that were not treated or mock treated or treated with a control nucleic acid.

[0054] As used herein, the terms "inhibit", "downregulate" or "reduce" with respect to gene expression means that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule (e.g., mRNA) encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits is reduced to below the expression or level or activity observed in the absence of a nucleic acid or conjugated nucleic acid of the invention or for an siRNA molecule that has no known homology to a human transcript (referred to herein as a non-silencing control). The control can be conjugated and modified in a similar manner to the molecules of the invention and delivered to the target cell by the same route; for example, expression can be reduced to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, or intermediate values ​​or less compared to expression observed in the absence of a nucleic acid or conjugated nucleic acid or in the presence of a non-silencing control.

[0055] The nucleic acid may comprise a first strand and a second strand each having a length of 19-25 nucleotides. The first strand and the second strand may have different lengths.

[0056] The length of the first strand and / or the second strand may each be 17-35, preferably 18-30, more preferably 19-25, and most preferably 19 nucleotides, and the length of at least one duplex region may be 10-25 nucleotides, preferably 18-23 nucleotides. The duplex may comprise two separate strands, or it may comprise a single strand comprising the first strand and the second strand.

[0057] The length of the first chain can be 17-25 nucleotides, preferably the length of the first chain can be 18-24 nucleotides, and the length of the first chain can be 18, 19, 20, 21, 22, 23 or 24 nucleotides. Most preferably, the length of the first chain is 19 nucleotides. The length of the second chain can be 17-25 nucleotides independently, preferably the length of the second chain can be 18-24 nucleotides, and the length of the second chain can be 18, 19, 20, 21, 22, 23 or 24 nucleotides. More preferably, the length of the second chain is 18 or 19 nucleotides, and most preferably, the length of the second chain is 18 nucleotides.

[0058] The nucleic acid may be 15-25 nucleotide pairs in length. The nucleic acid may be 17-23 nucleotide pairs in length. The nucleic acid may be 17-25 nucleotide pairs in length. The nucleic acid may be 23-24 nucleotide pairs in length. The nucleic acid may be 19-21 nucleotide pairs in length. The nucleic acid may be 21-23 nucleotide pairs in length.

[0059] The nucleic acid may comprise a duplex region consisting of 19-25 nucleotide base pairs. The duplex region may consist of 17, 18, 19, 20, 21, 22, 23, 24 or 25 base pairs that may be consecutive. Preferably, the duplex region consists of 19 base pairs.

[0060] Preferably, the nucleic acid mediates RNA interference.

[0061] In one embodiment, the nucleic acid for inhibiting the expression of LPA in a cell comprises at least one duplex region comprising a first strand and a second strand at least partially complementary to the first strand, wherein the first strand comprises a sequence of at least 15, preferably at least 16, more preferably at least 17, still more preferably at least 18, and most preferably at least 19 nucleotides having a sequence of at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95%, and most preferably 100% sequence identity to any one of the sequences of SEQ ID NO: 9, 5, 1, 3, 7, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43.

[0062] On the other hand, the nucleic acid or conjugated nucleic acid as described can reduce the expression of LPA by at least 15% compared to the expression observed in the absence of nucleic acid or conjugated nucleic acid. All preferred features of any of the previous aspects also apply to this aspect. In particular, the expression of LPA can be reduced to at least the following given % or less than the following given %: 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15% or less and intermediate values ​​compared to the expression of LPA observed in the absence of nucleic acid or conjugated nucleic acid or in the presence of a non-silencing control.

[0063] A nucleic acid may have an overhang at one end and a blunt end at the other end. A nucleic acid may have an overhang at both ends. A nucleic acid may be blunt-ended at both ends. A nucleic acid may have a 5' end at the first strand and a 3' end at the second strand, or a 3' end at the first strand and a 5' end at the second strand.

[0064] As used herein, "overhang" has its common and customary meaning in the art, i.e., a single-stranded portion of a nucleic acid that extends beyond the terminal nucleotides of the complementary strand in a double-stranded nucleic acid. The term "blunt end" includes double-stranded nucleic acids in which both strands terminate at the same position, regardless of whether one or more terminal nucleotides are base paired. The terminal nucleotides of the first strand and the second strand at the blunt end may be base paired. The terminal nucleotides of the first strand and the second strand at the blunt end may be unpaired. The first strand and the second strand may be base paired at both terminal nucleotides of the blunt end. The first strand and the second strand may be unpaired at both terminal nucleotides of the blunt end.

[0065] The nucleic acid may comprise an overhang at the 3' or 5' end. The nucleic acid may have a 3' overhang on the first strand. The nucleic acid may have a 3' overhang on the second strand. The nucleic acid may have a 5' overhang on the first strand. The nucleic acid may have a 5' overhang on the second strand. The nucleic acid may have an overhang at both the 5' and 3' ends of the first strand. The nucleic acid may have an overhang at both the 5' and 3' ends of the second strand. The nucleic acid may have a 5' overhang on the first strand and a 3' overhang on the second strand. The nucleic acid may have a 3' overhang on the first strand and a 5' overhang on the second strand. The nucleic acid may have a 3' overhang on the first strand and a 3' overhang on the second strand. The nucleic acid may have a 5' overhang on the first strand and a 5' overhang on the second strand.

[0066] The length of the overhang at the 3' end or 5' end of the second strand or the first strand may be selected from the group consisting of 1, 2, 3, 4 and 5 nucleotides. Optionally, the overhang may consist of 1 or 2 nucleotides which may or may not be modified.

[0067] Preferably, the nucleic acid is siRNA. siRNA is a short interfering RNA or short silencing RNA that can inhibit the expression of a target gene by RNA interference (RNAi) approach. Inhibition occurs by targeted degradation of the mRNA transcript of the target gene after transcription. siRNA forms a part of the RISC complex. The RISC complex specifically targets the target RNA by sequence complementarity of the first (antisense) strand and the target sequence.

[0068] Preferably, the nucleic acid mediates RNA interference (RNAi). The nucleic acid or at least the first strand of the nucleic acid is therefore preferably capable of being incorporated into the RISC complex. As a result, the nucleic acid or at least the first strand of the nucleic acid is therefore capable of guiding the RISC complex to a specific target RNA that is at least partially complementary to the nucleic acid or at least the first strand of the nucleic acid. The RISC complex then specifically cleaves this target RNA, and the result leads to inhibition of expression of the gene from which the RNA is derived.

[0069] Nucleic acid modification

[0070] Unmodified polynucleotides, particularly ribonucleotides, may tend to be degraded by cellular nucleases, and therefore, modified / modified nucleotides may be included in the nucleic acids of the present invention. The modifications may help stabilize the nucleic acids by making them more resistant to nucleases. This improved resistance allows the nucleic acids to be active in mediating RNA interference for a longer period of time, and is particularly desirable when the nucleic acids are to be used for treatment.

[0071] Modification of the nucleic acid of the present invention generally provides a powerful tool in overcoming potential limitations, including but not limited to the in vitro and in vivo stability and bioavailability inherent to natural RNA molecules. The nucleic acid of the present invention can be modified by chemical modification. The modified nucleic acid can also minimize the possibility of inducing interferon activity in humans. The modification can also enhance the functional delivery of nucleic acid to target cells. The modified nucleic acid of the present invention can include one or more chemically modified ribonucleotides of either or both of the first chain or the second chain. The ribonucleotide can include chemical modifications to bases, sugars or phosphate moieties. Ribonucleic acids can be modified by substitution or insertion with analogs of nucleic acids or bases.

[0072] Preferably, at least one nucleotide of the first and / or second strand of the nucleic acid is a modified nucleotide, preferably a non-naturally occurring nucleotide, such as preferably a 2'-F modified nucleotide.

[0073] One or more nucleotides of the nucleic acid of the present invention may be modified. The nucleic acid may comprise at least one modified nucleotide. The modified nucleotide may be in the first strand. The modified nucleotide may be in the second strand. The modified nucleotide may be in the duplex region. The modified nucleotide may be outside the duplex region, i.e., in the single-stranded region. The modified nucleotide may be on the first strand and may be outside the duplex region. The modified nucleotide may be on the second strand and may be outside the duplex region. The 3' terminal nucleotide of the first strand may be a modified nucleotide. The 3' terminal nucleotide of the second strand may be a modified nucleotide. The 5' terminal nucleotide of the first strand may be a modified nucleotide. The 5' terminal nucleotide of the second strand may be a modified nucleotide.

[0074] The nucleic acid of the invention may have 1 modified nucleotide, or the nucleic acid of the invention may have about 2-4 modified nucleotides, or the nucleic acid may have about 4-6 modified nucleotides, about 6-8 modified nucleotides, about 8-10 modified nucleotides, about 10-12 modified nucleotides, about 12-14 modified nucleotides, about 14-16 modified nucleotides, about 16-18 modified nucleotides, about 18-20 modified nucleotides, about 20-22 modified nucleotides, about 22-24 modified nucleotides, 24-26 modified nucleotides, or about 26-28 modified nucleotides, or all nucleotides may be modified. In each case, the nucleic acid comprising the modified nucleotide retains at least 50% of its activity compared to the same nucleic acid without the modified nucleotide, or vice versa. The nucleic acid may retain 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of its activity and intermediate values ​​compared to the same nucleic acid but without the modified nucleotides, or may have an activity that is greater than 100% of the activity of the same nucleic acid without the modified nucleotides.

[0075] The modified nucleotides may be purines or pyrimidines. At least half of the purines may be modified. At least half of the pyrimidines may be modified. All purines may be modified. All pyrimidines may be modified. The modified nucleotides may be selected from the group consisting of: 3' terminal deoxythymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2' modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides comprising non-natural bases, nucleotides comprising 5'-thiophosphate groups, nucleotides comprising 5' phosphates or 5' phosphate mimetics, and terminal nucleotides attached to cholesterol derivatives or dodecanoic acid bisdecylamide groups.

[0076] The nucleic acid may comprise nucleotides comprising modified nucleotides, wherein the base is selected from 2-aminoadenosine, 2,6-diaminopurine, inosine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g., 5-methylcytidine), 5-alkyluridine (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine), 6-azapyrimidine, 6-alkylpyrimidine (e.g., 6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxo sine), 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5′-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, β-D-galactosyl Q nucleoside, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, β-D-mannosyl Q nucleoside, uridine-5-oxyacetic acid and 2-thiocytidine.

[0077] Nucleic acid discussed herein includes unmodified RNA and modified RNA such as to improve efficacy, and polymers of nucleoside substitutes. Unmodified RNA refers to components of nucleic acid, i.e. sugar, base and phosphate moiety, and components existing in nature, such as components naturally present in human body, which are identical or substantially identical molecules. Modified nucleotides as used herein refer to components of nucleotides, i.e. sugar, base and phosphate moiety, which are different from those existing in nature, in which one or more of them. Although they are referred to as modified nucleotides, due to modification, they will certainly also include molecules that are not nucleotides, such as polynucleotide molecules replaced by non-ribose phosphate constructs that allow hybridization between chains, i.e. modified nucleotides simulate ribose phosphate backbones.

[0078] Many modifications present in nucleic acid described below will be repeated in polynucleotide molecules, such as modifications to bases or phosphate moieties, or non-connectivity O of phosphate moieties. In some cases, modification will be present in all possible positions / nucleotides in polynucleotides, but in many cases, it will not be so. Modification may be present only at 3' or 5' terminal positions, may be present only in terminal regions, such as at a certain position on terminal nucleotides or in the last 2, 3, 4, 5 or 10 nucleotides of a chain. Modification may be present in double-stranded regions, single-stranded regions or both. Modification may be present only in double-stranded regions of nucleic acid of the present invention, or may be present only in single-stranded regions of nucleic acid of the present invention. Phosphorothioate modification at non-connectivity O positions may be present only at one or both ends, may be present only in terminal regions, such as at a certain position on terminal nucleotides or in the last 2, 3, 4 or 5 nucleotides of a chain, or may be present in duplexes and / or in single-stranded regions, particularly at the ends. 5' end or 3' end may be phosphorylated.

[0079] The stability of the nucleic acids of the present invention can be increased by including specific bases in the overhangs, or by including modified nucleotides in the single-stranded overhangs, for example in the 5' or 3' overhangs, or in both. Purine nucleotides can be included in the overhangs. All or some of the bases in the 3' or 5' overhangs can be modified. Modifications can include the use of modifications at the 2'OH group of the ribose, the use of deoxyribonucleotides rather than ribonucleotides, and modifications in the phosphate groups, such as phosphorothioate modifications. The overhangs need not be homologous to the target sequence.

[0080] Nucleases can hydrolyze nucleic acid phosphodiester bonds. However, chemical modifications to nucleic acids can impart improved properties and can render oligoribonucleotides more stable to nucleases.

[0081] As used herein, modified nucleic acids may include one or more of the following:

[0082] (i) an alteration, e.g., a substitution, of one or both of the non-linking phosphate oxygens and / or of one or more of the linking phosphate oxygens (also referred to as linking even at the 5' and 3' ends of the nucleic acids of the invention);

[0083] (ii) a change, such as a substitution, to the composition of the ribose, such as the 2′ hydroxyl group on the ribose;

[0084] (iii) replacement of the phosphate moiety with a "dephospho" linker;

[0085] (iv) modification or substitution of naturally occurring bases;

[0086] (v) substitutions or modifications to the ribose-phosphate backbone;

[0087] (vi) Modification of the 3′ or 5′ end of the RNA, such as removal, modification or replacement of the terminal phosphate group, or conjugation of a moiety, such as a fluorescent labeling moiety, to the 3′ or 5′ end of the RNA.

[0088] The terms substitution, modification, alteration indicate a deviation from the naturally occurring molecule.

[0089] Specific modifications are discussed in more detail below.

[0090] The example of modified phosphate group includes phosphorothioate, phosphoroselenoate, borane phosphate, borane phosphate, hydrogen phosphonate, phosphoramidate, alkyl or aryl phosphonate and phosphotriester.In the case of dithiophosphate, two non-connective oxygens are replaced by sulfur.One, each or two non-connective oxygens in phosphate group can independently become any one of S, Se, B, C, H, N or OR (R is alkyl or aryl).

[0091] Phosphate linkers can also be modified by replacing the connecting oxygen with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylenephosphonates). The replacement can be at the terminal oxygen. It is possible to replace a non-connecting oxygen with a nitrogen.

[0092] Modified nucleotides may include modifications to the sugar group. The 2' hydroxyl (OH) group may be modified or replaced with a number of different "oxy" or "deoxy" substituents.

[0093] Examples of "oxy"-2' hydroxyl modifications include alkoxy or aryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG) O(CH2CH2O)nCH2CH2OR; "locked" nucleic acids (LNA), in which the 2' hydroxyl is linked to the 4' carbon of the same ribose, e.g., via a methylene bridge; O-amines (amine═NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino) and aminoalkoxy O(CH2)namines (e.g., amine═NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino).

[0094] "Deoxy" modifications include hydrogen, halogen, amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH2CH2NH)nCH2CH2-amine (amine = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino), -NHC(O)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), cyano; sulfhydryl; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl, and alkynyl groups that may be optionally substituted with, for example, an amino functional group. Other substituents in certain embodiments include 2'-methoxyethyl, 2'-OCH3, 2'-O-allyl, 2'-C-allyl, and 2'-fluoro.

[0095] The sugar group may also contain one or more carbons with the opposite stereochemical configuration compared to the configuration of the corresponding carbon in ribose. Thus, a modified nucleotide may contain a sugar such as arabinose.

[0096] Modified nucleotides may also include "abasic" sugars that lack a nucleobase at C-1'. These abasic sugars may also contain modifications at one or more of the constituent sugar atoms.

[0097] The 2' modification may be used in combination with one or more phosphate linker modifications (eg, phosphorothioate).

[0098] The phosphate groups can individually be replaced by non-phosphorus containing linkers.

[0099] Examples of moieties that can replace the phosphate group include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformal, methylal, oxime, methylene imino, methylene methyl imino, methylene hydrazono, methylene dimethyl hydrazono and methylene oxy methyl imino. In certain embodiments, replacement may include methylene carbonyl amino and methylene methyl imino.

[0100] The phosphate linker and ribose sugar can be replaced by nuclease-resistant nucleotides.

[0101] Examples include morpholino, cyclobutyl, pyrrolidine, and peptide nucleic acid (PNA) nucleoside surrogates. In certain embodiments, PNA surrogates may be used.

[0102] The 3' and 5' ends of the oligonucleotide may be modified. The modifications may be at the 3' end or the 5' end or both ends of the molecule. They may include modification or replacement of the entire terminal phosphate or of one or more of the atoms of the phosphate group. For example, the 3' and 5' ends of the oligonucleotide may be conjugated to other functional molecular entities, such as labeling moieties such as fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes), or protecting groups (e.g., based on sulfur, silicon, boron or esters). The functional molecular entity may be connected to the sugar via a phosphate group and / or a linker. The terminal atom of the linker may be connected to or replace the connecting atom of the phosphate group or the C-3' or C-5' O, N, S or C group of the sugar. Alternatively, the linker may be connected to or replace the terminal atom of a nucleotide substitute (e.g., PNA). These spacers or linkers may include, for example, -(CH2) n —, —(CH2) n N—,—(CH2) n O—、—(CH2) n S—,—(CH2CH2O) n CH2CH2O— (e.g. n=3 or 6), abasic sugars, amides, carboxyls, amines, oxyamines, oxyimines, thioethers, disulfides, thioureas, sulfonamides or morpholino groups, or biotin and fluorescein reagents. The 3′ end may be an —OH group.

[0103] Other examples of terminal modifications include dyes, intercalators (e.g., acridine), cross-linkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases, EDTA, lipophilic carriers (e.g., cholesterol, bile acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanol, palmityl acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate esters, amino groups, sulfhydryl groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled labels, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles).

[0104] Terminal modifications may be added for many reasons, including to modulate activity or to modulate resistance to degradation. Terminal modifications that may be used to modulate activity include modification of the 5' terminus with a phosphate or phosphate analog. The nucleic acids of the invention may be 5' phosphorylated on the first or second strand, or include a phosphoryl analog at the 5' terminus. 5'-phosphate modifications include those that are compatible with RISC-mediated gene silencing. Suitable modifications include: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylated or unmethylated) (7m-GO-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap (Appp) and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′- Monothiophosphates (phosphorothioates; (HO)2(S)P—O-5′); 5′-monodithiophosphates (phosphorodithioates; (HO)(HS)(S)P—O-5′), 5′-thiophosphates ((HO)2(O)P—S-5′); any additional combination of oxygen / sulfur substituted monophosphates, diphosphates, and triphosphates (e.g., 5′-α-thiotriphosphate, 5′-γ-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH 2 -), 5' vinyl phosphonate, 5'-alkyl ether phosphonate (R = alkyl ether = methoxymethyl (MeOCH2-), ethoxymethyl, etc., for example RP(OH)(O)-O-5'-).

[0105] Terminal modifications can also be used to monitor distribution, and in such cases, the group to be added can include a fluorophore, such as fluorescein or an Alexa dye. Terminal modifications can also be used to enhance uptake, and modifications that can be used for this include cholesterol. Terminal modifications can also be used to crosslink an RNA agent to another moiety.

[0106] Adenine, guanine, cytosine and uracil are the most common bases found in RNA. These bases can be modified or replaced to provide RNA with improved properties. For example, nuclease-resistant oligoribonucleotides can be prepared with these bases or with synthetic and natural nucleoside bases (e.g., inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanosine or tubercidine) and any of the above modifications. Alternatively, substituted or modified analogs of any of the above bases and "universal bases" can be used. Examples include 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-aminoallyl uracil, 8-halo, amino, thiol, sulfanyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines including 2-aminopropyladenine, 5-propynyl Uracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkylcytosine, 7-deazaadenine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3-carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N <4> -acetylcytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanine or O-alkylated bases.

[0107] As used herein, the term "non-pairing nucleotide analogue" means a nucleotide analogue that includes a non-base pairing portion, including but not limited to: 6-deaminoadenosine (adenosine), 4-Me-indole, 3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-Me ribose U, N3-Me ribose T, N3-Me dC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, N3-Me dC. In some embodiments, the non-base pairing nucleotide analogue is a ribonucleotide. In other embodiments, it is a deoxyribonucleotide.

[0108] As used herein, the term "terminal functional group" includes, but is not limited to, halogen, alcohol, amine, carboxylic acid, ester, amide, aldehyde, ketone, ether groups.

[0109] Certain moieties may be attached to the 5' terminus of the first or second strand. These moieties include abasic ribose moieties, abasic deoxyribose moieties, modifications to abasic ribose and abasic deoxyribose moieties, including 2'O alkyl modifications; inverted abasic ribose and abasic deoxyribose moieties and modifications thereof, C6-imino-Pi; mirror nucleotides, including L-DNA and L-RNA; 5'OMe nucleotides; and nucleotide analogs, including 4',5'-methylene nucleotides; 1-(β-D-erythrofuranosyl) nucleotides; 4'-thio nucleotides, carbocyclic nucleotides; 5'-amino-alkylphosphonucleotides; phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; α-nucleotide; threo-pentofuranosyl nucleotide; acyclic 3′,4′-bromonucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted abasic moiety; 1,4-butanediol phosphate; 5′-amino; and bridged or non-bridged methylphosphonate and 5′-thiol moieties.

[0110] The nucleic acids of the present invention may comprise abasic nucleotides. The term "abasic" as used herein refers to a moiety lacking a base or having other chemical groups at the 1' position instead of a base, such as a 3',3'-linked or 5',5'-linked deoxyabasic ribose derivative.

[0111] The nucleic acid may comprise one or more modified nucleotides on the second strand and / or the first strand.Alternating nucleotides may be modified to form modified nucleotides.

[0112] As used herein, alternation means occurring successively in a regular manner. In other words, alternation means repeating and occurring successively. For example, if one nucleotide is modified, then the next consecutive nucleotide is not modified, and then the consecutive nucleotide is modified, and so on. One nucleotide may be modified with a first modification, the next consecutive nucleotide may be modified with a second modification, and then the consecutive nucleotide is modified with the first modification, and so on, wherein the first modification and the second modification are different.

[0113] One or more of the odd-numbered nucleotides of the first strand of the nucleic acid of the present invention may be modified, wherein the first strand is numbered 5' to 3', and the 5'-most nucleotide is nucleotide No. 1 of the first strand. The term "odd numbering" as described herein means a number that is not divisible by two. Examples of odd numbering are 1, 3, 5, 7, 9, 11, etc. One or more of the even-numbered nucleotides of the first strand of the nucleic acid of the present invention may be modified, wherein the first strand is numbered 5' to 3'. The term "even numbering" as described herein means a number that is equally divisible by two. Examples of even numbering are 2, 4, 6, 8, 10, 12, 14, etc. One or more of the odd-numbered nucleotides of the second strand of the nucleic acid of the present invention may be modified, wherein the second strand is numbered 3' to 5', and the 3'-most nucleotide is nucleotide No. 1 of the second strand. One or more of the even-numbered nucleotides of the second strand of the nucleic acid of the present invention may be modified, wherein the second strand is numbered 3' to 5'.

[0114] One or more nucleotides on the first strand and / or the second strand may be modified to form a modified nucleotide. One or more of the odd-numbered nucleotides of the first strand may be modified. One or more of the even-numbered nucleotides of the first strand may be modified by at least a second modification, wherein the at least second modification is different from the modification on the one or more odd-numbered nucleotides. At least one of the one or more modified even-numbered nucleotides may be adjacent to at least one of the one or more modified odd-numbered nucleotides.

[0115] In the nucleic acid of the present invention, a plurality of odd-numbered nucleotides in the first strand may be modified. A plurality of even-numbered nucleotides in the first strand may be modified by a second modification. The first strand may include adjacent nucleotides modified by a common modification. The first strand may also include adjacent nucleotides modified by a second different modification.

[0116] One or more of the odd-numbered nucleotides of the second strand may be modified by a modification that is different from the modification of the odd-numbered nucleotides on the first strand, and / or one or more of the even-numbered nucleotides of the second strand may be modified by the same modification of the odd-numbered nucleotides of the first strand. At least one of the one or more modified even-numbered nucleotides of the second strand may be adjacent to one or more modified odd-numbered nucleotides. Multiple odd-numbered nucleotides of the second strand may be modified by a common modification, and / or multiple even-numbered nucleotides may be modified by the same modification present on the odd-numbered nucleotides of the first strand. Multiple odd-numbered nucleotides on the second strand may be modified by a second modification, wherein the second modification is different from the modification of the odd-numbered nucleotides of the first strand.

[0117] The second strand may comprise adjacent nucleotides modified by a common modification, which may be a second modification that is different from the modification of the odd-numbered nucleotides of the first strand.

[0118] In the nucleic acids of the invention, each of the odd-numbered nucleotides in the first strand and each of the even-numbered nucleotides in the second strand may be modified with a common modification, and each of the even-numbered nucleotides in the first strand may be modified with a second modification, and each of the odd-numbered nucleotides in the second strand may be modified with a second, different modification.

[0119] The nucleic acids of the invention may have a first strand of unmodified or differently modified nucleotides relative to a second strand, with modified nucleotides being displaced by at least one nucleotide.

[0120] One or more or each of the odd-numbered nucleotides in the first strand may be modified, and one or more or each of the even-numbered nucleotides in the second strand may be modified. One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification. One or more or each of the even-numbered nucleotides in the first strand may be modified, and one or more or each of the even-numbered nucleotides in the second strand may be modified. One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification. One or more or each of the odd-numbered nucleotides in the first strand may be modified, and one or more or each of the odd-numbered nucleotides in the second strand may be modified by a common modification. One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification. One or more or each of the even-numbered nucleotides in the first strand may be modified, and one or more or each of the odd-numbered nucleotides in the second strand may be modified by a common modification. One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification.

[0121] Nucleic acid of the present invention can comprise single-stranded or double-stranded constructs, and the construct comprises at least two regions with alternating modifications in one or both of the chains. These alternating regions can comprise up to about 12 nucleotides, but preferably comprise about 3 to about 10 nucleotides. The region of alternating nucleotides can be located at the end of one or both chains of nucleic acid of the present invention. Nucleic acid can comprise 4 to about 10 nucleotides with alternating nucleotides at each end (3' and 5'), and these regions can be separated by about 5 to about 12 continuous unmodified or modified nucleotides in different ways or in a common way.

[0122] The odd-numbered nucleotides of the first strand may be modified, and the even-numbered nucleotides may be modified with a second modification. The second strand may include adjacent nucleotides modified with a common modification, which may be the same as the modification of the odd-numbered nucleotides of the first strand. One or more nucleotides of the second strand may also be modified with a second modification. One or more nucleotides with the second modification may be adjacent to each other and adjacent to a nucleotide with the same modification as the modification of the odd-numbered nucleotides of the first strand. The first strand may also include a phosphorothioate bond between two nucleotides at the 3' end and at the 5' end. The second strand may include a phosphorothioate bond between two nucleotides at the 5' end. The second strand may also be conjugated to a ligand at the 5' end.

[0123] The nucleic acid of the present invention may comprise a first strand containing adjacent nucleotides modified with a common modification. One or more of the nucleotides may be adjacent to one or more nucleotides that may be modified with a second modification. One or more nucleotides with a second modification may be adjacent. The second strand may comprise adjacent nucleotides modified with a common modification, which may be the same as one of the modifications of the one or more nucleotides of the first strand. The one or more nucleotides of the second strand may also be modified with a second modification. One or more nucleotides with a second modification may be adjacent. The first strand may also comprise a phosphorothioate bond between two nucleotides at the 5' end and at the 3' end. The second strand may comprise a phosphorothioate bond between two nucleotides at the 3' end. The second strand may also be conjugated to a ligand at the 5' end.

[0124] Nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25 on the first strand may be modified by a modification from 5' to 3' on the first strand and from 3' to 5' on the second strand. Nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 on the first strand may be modified by a second modification. Nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 on the second strand may be modified by a modification. Nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 on the second strand may be modified by a second modification. For the nucleic acids of the present invention, the nucleotides are numbered from 5' to 3' on the first strand and from 3' to 5' on the second strand.

[0125] The nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 on the first strand may be modified by a modification. The nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 on the first strand may be modified by a second modification. The nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 on the second strand may be modified by a modification. The nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 on the second strand may be modified by a second modification.

[0126] Obviously, if the length of the first strand and / or the second strand is shorter than 25 nucleotides, such as 19 nucleotides in length, then none of the nucleotides numbered 20, 21, 22, 23, 24 and 25 are modified. The skilled person will appreciate that the above description applies accordingly to shorter strands.

[0127] One or more modified nucleotides on the first strand can be paired with a modified nucleotide with a common modification on the second strand. One or more modified nucleotides on the first strand can be paired with a modified nucleotide with different modifications on the second strand. One or more modified nucleotides on the first strand can be paired with an unmodified nucleotide on the second strand. One or more modified nucleotides on the second strand can be paired with an unmodified nucleotide on the first strand. In other words, the alternating nucleotides on the two strands can be aligned, such as (for example) all modifications in the alternating regions of the second strand are paired with the same modification in the first strand, or alternatively, the modification can be offset by one nucleotide, wherein the common modification in the alternating regions of one strand is paired with a different modification (i.e., the second or other modification) in the other strand. Another option is to have different modifications in each of the strands.

[0128] The modifications on the first strand may be shifted by one nucleotide relative to the modified nucleotides on the second strand so that nucleotides modified in a common manner do not pair with each other.

[0129] One or more modifications may each and individually be selected from the group consisting of: 3'-terminal deoxythymine, 2'-O-methyl, 2'-deoxy modification, 2'-amino modification, 2'-alkyl modification, morpholino modification, phosphoramidite modification, 5'-phosphorothioate group modification, 5' phosphate or 5' phosphate mimetic modification, and cholesterol derivative or dodecanoic acid bisdecylamide group modification, and / or the modified nucleotide may be any of a locked nucleotide, an abasic nucleotide or a nucleotide comprising a non-natural base.

[0130] At least one modification may be a 2'-O-methyl, and / or at least one modification may be a 2'-F. Other modifications as described herein may be present on the first strand and / or the second strand.

[0131] Throughout the invention description, "same or common modification" means the same modification made to any nucleotide, such as modification of A, G, C or U with a group such as methyl or fluoro. This is not intended to mean having the same additions on the same nucleotide. For example, 2'F-dU, 2'F-dA, 2'F-dC, 2'F-dG are all considered the same or common modification, as are 2'-OMe-rU, 2'-OMe-rA; 2'-OMe-rC; 2'-OMe-rG. The 2-'F modification is a different modification from the 2'-OMe modification.

[0132] Some representative modified nucleic acid sequences of the present invention are shown in the examples. These examples are intended to be representative and non-limiting.

[0133] Preferably, the nucleic acid may comprise a modification and a second or further modification, each and individually selected from the group comprising a 2'-O-methyl modification and a 2'-F modification. The nucleic acid may comprise a modification which is a 2'-O-methyl (2'-OMe) which may be a first modification, and a second modification which is a 2'-F. The nucleic acids of the invention may also include phosphorothioate modifications and / or deoxy modifications which may be present in or between the terminal 2 or 3 nucleotides at each or any end of each or both strands.

[0134] As another aspect, the present invention provides a nucleic acid for inhibiting the expression of LPA in a cell, the nucleic acid comprising a nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43, wherein the nucleotides of the first chain are modified by a first modification on an odd-numbered nucleotide and modified by a second modification on an even-numbered nucleotide, and the nucleotides of the second chain are modified by a third modification on an even-numbered nucleotide and modified by a fourth modification on an odd-numbered nucleotide, wherein at least the first modification is different from the second modification, and the third modification is different from the fourth modification. The third modification and the first modification may be the same or different, and the second modification and the fourth modification may be the same or different. The first modification and the second modification may be different from each other, and the third modification and the fourth modification may be different from each other.

[0135] The second strand may comprise a nucleotide sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 or 44. The nucleotides of the first strand may be modified by a first modification on an odd-numbered nucleotide and modified with a second modification on an even-numbered nucleotide, and the second strand may be modified with the second modification on an odd-numbered nucleotide and modified with the first modification on an even-numbered nucleotide. The first modification may be 2'OMe, and the second modification may be 2'F. The first strand may comprise a nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 9, and / or the second strand may comprise a nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 10. The modifications may be those set forth in Table 1.

[0136] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a 2'O-methyl modification, and the nucleotide on the second strand corresponding to position 13 of the first strand is not modified with a 2'O-methyl modification.

[0137] A nucleotide on the second strand "corresponding to" a position on the first strand is suitably the nucleotide that base pairs with that nucleotide on the first strand.

[0138] In one aspect, the nucleotide on the second strand corresponding to position 13 of the first strand is the nucleotide that forms a base pair with position 13 of the first strand.

[0139] In one aspect, the nucleotide on the second strand corresponding to position 11 of the first strand is the nucleotide that forms a base pair with position 11 of the first strand.

[0140] In one aspect, the nucleotide on the second strand corresponding to position 12 of the first strand is the nucleotide that forms a base pair with position 12 of the first strand.

[0141] This nomenclature can be applied to other positions of the second strand. For example, in a 19-mer nucleic acid that is double-stranded and has blunt ends, position 13 of the first strand will pair with position 7 of the second strand. Position 11 of the first strand will pair with position 9 of the second strand. This nomenclature can be applied to other positions of the second strand.

[0142] The nucleotide corresponding to position 13 of the first strand is suitably position 13 of the second strand starting from the first nucleotide of the double-stranded region and counting from the 3' of the second strand. Similarly, position 11 of the second strand is suitably the 11th nucleotide starting from the first nucleotide of the double-stranded region and counting from the 3' of the second strand. This nomenclature can be applied to other positions of the second strand.

[0143] In one aspect, in the case of partially complementary first and second strands, a nucleotide on the second strand that "corresponds to" a position on the first strand may not necessarily form a base pair (if that is where a mismatch exists), but the principles of nomenclature still apply.

[0144] It is preferred that the first and second strands are fully complementary across the duplex region (ignoring any overhang regions) and that no mismatches exist within the double-stranded region of the nucleic acid.

[0145] It is also preferred that:

[0146] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 of the first strand from the 5' end are not modified with a 2'O-methyl modification, and the nucleotide on the second strand corresponding to position 11 of the first strand is not modified with a 2'O-methyl modification.

[0147] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 of the first strand from the 5' end are not modified with a 2'O-methyl modification, and the nucleotides on the second strand corresponding to positions 11 and 13 of the first strand are not modified with a 2'O-methyl modification.

[0148] In one aspect, the nucleotide on the second strand corresponding to position 12 of the first strand is not modified with a 2' O-methyl modification.This limitation with respect to nucleic acids can be seen with any other limitations described herein.

[0149] Therefore, another aspect of the invention is a nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 of the first strand from the 5' end are not modified with a 2'O-methyl modification and the nucleotides on the second strand corresponding to positions 11-13 of the first strand are not modified with a 2'O-methyl modification.

[0150] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 of the first strand from the 5' end are not modified with a 2' O-methyl modification, and the nucleotides on the second strand corresponding to positions 11, or 13, or 11 and 13, or 11-13 of the first strand are modified with a 2' fluoro modification.

[0151] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 of the first strand from the 5' end are modified with a 2' fluoro modification, and the nucleotides on the second strand corresponding to positions 11, or 13, or 11 and 13, or 11-13 of the first strand are not modified with a 2' O-methyl modification.

[0152] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 of the first strand starting from the 5' end are modified with 2'fluoro modifications, and the nucleotides corresponding to positions 11, or 13, or 11 and 13, or preferably 11-13 of the first strand on the second strand are modified with 2'fluoro modifications. Preferably, in this embodiment, all even-numbered nucleotides of the first strand are modified with 2'fluoro, and all odd-numbered nucleotides of the first strand are modified with 2'O-methyl. In addition, the nucleotides on the second strand except those corresponding to positions 11, or 13, or 11 and 13, or preferably 11-13 of the first strand are modified with 2'O-methyl modifications. An advantage of this nucleic acid is that it contains relatively few non-naturally occurring modified nucleotides, but is still able to effectively inhibit the target gene for a long time. This nucleic acid is easier to synthesize than the corresponding nucleic acid with more non-naturally occurring (2'F modified) nucleotides.

[0153] A nucleic acid as disclosed herein, wherein greater than 50% of the nucleotides of the first strand and / or the second strand comprise a 2'O-methyl modification, such as greater than 55%, 60%, 65%, 70%, 75%, 80% or 85% or more of the first strand and / or the second strand comprise a 2'O-methyl modification, preferably measured as a percentage of the total nucleotides of both the first strand and the second strand.

[0154] A nucleic acid as disclosed herein, wherein greater than 50% of the nucleotides of the first and / or second strands comprise a naturally occurring RNA modification, such as wherein greater than 55%, 60%, 65%, 70%, 75%, 80% or 85% or more of the first and / or second strands comprise such a modification, preferably measured as a percentage of the total nucleotides of both the first and second strands. Suitable naturally occurring modifications include not only 2O' methyl groups, but also other 2' sugar modifications, particularly 2' H modifications that produce DNA nucleotides.

[0155] A nucleic acid as disclosed herein, comprising at most 20%, such as at most 15%, such as at most 10%, of nucleotides having a 2' modification other than a 2'O-methyl modification on the first strand and / or the second strand, preferably expressed as a percentage of the total nucleotides of both the first strand and the second strand.

[0156] A nucleic acid as disclosed herein comprising up to 20% (such as up to 15% or up to 10%) 2' fluoro modification on the first strand and / or the second strand, preferably expressed as a percentage of the total nucleotides of both strands.

[0157] A nucleic acid as disclosed herein, wherein all nucleotides are modified with a 2'O-methyl modification, with the exception of positions 2 and 14 of the first strand from the 5' end and the nucleotides on the second strand corresponding to positions 11, or 13, or 11 and 13, or preferably 11 to 13 of the first strand. Preferably, the nucleotides not modified with a 2'O-methyl are modified with a fluorine at the 2' position.

[0158] Preferred is a nucleic acid as disclosed herein, wherein all nucleotides of said nucleic acid are modified at the 2' position of the sugar.Preferably, the nucleotides are modified with a 2'-fluoro modification, wherein said modification is not a 2'O-methyl modification.

[0159] The nucleic acid of the present invention may comprise one or more nucleotides modified with 2'H at the 2' position, thus having DNA nucleotides within the nucleic acid. The nucleic acid of the present invention may comprise DNA nucleotides at positions 2 and / or 14 of the first strand, counting from the 5' end of the first strand. The nucleic acid may comprise DNA nucleotides on the second strand corresponding to positions 11, or 13, or 11 and 13, or 11-13 of the first strand.

[0160] In one aspect, there is at most one DNA per nucleic acid of the invention.

[0161] The nucleic acid of the invention may comprise one or more LNA nucleotides. The nucleic acid of the invention may comprise an LNA nucleotide at position 2 and / or 14 of the first strand, counted from the 5' end of the first strand. The nucleic acid may comprise an LNA on the second strand corresponding to positions 11, or 13, or 11 and 13, or 11-13 of the first strand.

[0162] In one aspect, the nucleic acid is modified on the first strand, preferably along the entire strand, with alternating 2'O-methyl modifications and 2'fluoro modifications, and positions 2 and 14 (starting from the 5' end) are modified with 2'fluoro. Preferably, the second strand is modified with a 2'fluoro modification at the nucleotides corresponding to positions 11, or 13, or 11 and 13, or preferably 11-13 of the first strand. Preferably, the second strand is modified with a 2'fluoro modification at the first position starting from the complementary (double-stranded) region, positions 11-13 counting from the 3' end, and the remaining modifications are naturally occurring modifications, preferably 2'O-methyl. At least in this case, the nucleic acid preferably has a blunt end at least at the end comprising the 5' end of the first strand.

[0163] The nucleotides of the second strand in positions corresponding to, for example, even-numbered nucleotides of the first strand are nucleotides of the second strand that base pair with the even-numbered nucleotides of the first strand.

[0164] In one aspect of the nucleic acid, the nucleotides in the position corresponding to nucleotide 11, or nucleotide 13, or nucleotides 11 and 13, or preferably nucleotides 11-13 of the second chain are modified by the fourth modification. Preferably, all nucleotides of the second chain except one or more nucleotides in the position corresponding to nucleotide 11, or nucleotide 13, or nucleotides 11 and 13, or preferably nucleotides 11-13 of the first chain are modified by the third modification. Preferably, in the same nucleic acid, nucleotides 2 and 14 of the first chain, or preferably all even-numbered nucleotides, are modified with the first modification. In addition or alternatively, the odd-numbered nucleotides of the first chain are modified with the second modification. The fourth modification is preferably different from the second modification, and is preferably different from the third modification, and the fourth modification is preferably the same as the first modification. The first modification and the fourth modification are preferably 2'-OMe modifications, and the second modification and the third modification are preferably 2'-F modifications. The nucleotides on the first chain are numbered continuously from the nucleotide number 1 at the 5' end of the first chain.

[0165] In one aspect of the nucleic acid, all even-numbered nucleotides of the first strand are modified by a first modification, all odd-numbered nucleotides of the first strand are modified by a second modification, all nucleotides of the second strand in positions corresponding to nucleotides 11-13 of the first strand are modified by a fourth modification, and all nucleotides of the second strand except for nucleotides corresponding to nucleotides 11-13 of the first strand are modified by a third modification, wherein the first modification and the fourth modification are 2'-F, and the second modification and the third modification are 2'-OMe. The nucleotides on the first strand are numbered consecutively starting from nucleotide number 1 at the 5' end of the first strand.

[0166] In one aspect of the nucleic acid, each nucleotide of the first strand and the second strand is a modified nucleotide.

[0167] One aspect is a double-stranded nucleic acid for inhibiting the expression of LPA, preferably in a cell, wherein the nucleic acid comprises a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 15 nucleotides, which differs from any one of the sequences of SEQ ID NO: 9, 5, 1, 3, 7, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43, preferably SEQ ID NO: 9 by no more than 3 nucleotides, wherein all even-numbered nucleotides of the first strand are modified by a first modification, all odd-numbered nucleotides of the first strand are modified by a second modification, all nucleotides of the second strand at positions corresponding to nucleotides 11-13 of the first strand are modified by a fourth modification, and all nucleotides of the second strand except for nucleotides corresponding to nucleotides 11-13 of the first strand are modified by a third modification, wherein the first modification and the fourth modification are 2'-F, and the second modification and the third modification are 2'-OMe.

[0168] One aspect is a nucleic acid that is a siRNA molecule, wherein the nucleotides at positions 2 and 14 of the first strand from the 5' end are not modified with a 2' O-methyl modification, and the nucleic acid comprises one or more or all of the following:

[0169] (i) an inverted nucleotide at the 3' terminus of the second strand, preferably using a 3'-3' linkage;

[0170] (ii) one or more phosphorodithioate bonds;

[0171] (iii) the second strand nucleotides corresponding to positions 11 or 13 of the first strand are not modified with a 2' O-methyl modification, preferably wherein one or both of these positions comprise a 2' fluoro modification;

[0172] (iv) the nucleic acid comprises at least 80% of all nucleotides having a 2'-O-methyl modification;

[0173] (v) The nucleic acid comprises at most 20% of nucleotides having a 2' fluoro modification.

[0174] The present invention also provides a nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand and the nucleotides at positions 7 and / or 9, or 7-9 from the 5' end of the second strand are modified with a 2' fluoro modification, and at least 90% of the remaining nucleotides are 2'-O methyl modified, or contain another naturally occurring 2' modification.

[0175] Specific preferred examples of blunt double-stranded 19-base nucleic acids having no overhangs are:

[0176] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a 2'O-methyl modification, and the nucleotide at position 7 from the 5' end of the second strand is not modified with a 2'O-methyl modification.

[0177] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a 2'O-methyl modification, and the nucleotide at position 9 from the 5' end of the second strand is not modified with a 2'O-methyl modification.

[0178] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a 2'O-methyl modification, and the nucleotides at positions 7 and 9 from the 5' end of the second strand are not modified with a 2'O-methyl modification.

[0179] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a 2'O-methyl modification, and the nucleotides at positions 7-9 from the 5' end of the second strand are not modified with a 2'O-methyl modification.

[0180] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a 2' O-methyl modification, and the nucleotides at positions 7 and / or 9, or 7-9 from the 5' end of the second strand are modified with a 2' fluoro modification.

[0181] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are modified with 2' fluoro modification, and the nucleotides at positions 7 and / or 9, or 7-9 from the 5' end of the second strand are not modified with 2' O-methyl modification.

[0182] A nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are modified with a 2' fluoro modification, and the nucleotides at positions 7 and / or 9, or 7-9 from the 5' end of the second strand are modified with a 2' fluoro modification.

[0183] A nucleic acid as disclosed herein, wherein greater than 50% of the nucleotides of the first strand and / or the second strand comprise a 2'O-methyl modification, such as greater than 55%, 60%, 65%, 70%, 75%, 80% or 85% or more of the first strand and / or the second strand comprise a 2'O-methyl modification, preferably measured as a percentage of the total nucleotides of both the first strand and the second strand.

[0184] A nucleic acid as disclosed herein, wherein greater than 50% of the nucleotides of the first and / or second strands comprise a naturally occurring RNA modification, such as wherein greater than 55%, 60%, 65%, 70%, 75%, 80% or 85% or more of the first and / or second strands comprise such a modification, preferably measured as a percentage of the total nucleotides of both the first and second strands. Suitable naturally occurring modifications include not only 2O' methyl groups, but also other 2' sugar modifications, particularly 2' H modifications that produce DNA nucleotides.

[0185] A nucleic acid as disclosed herein, comprising on the first strand and / or the second strand up to 20%, such as up to 15%, such as more than 10%, nucleotides with a 2' modification other than a 2'O-methyl modification, preferably expressed as a percentage of the total nucleotides of both the first strand and the second strand.

[0186] A nucleic acid as disclosed herein comprising up to 20% (such as up to 15% or up to 10%) 2' fluoro modification on the first strand and / or the second strand, preferably expressed as a percentage of the total nucleotides of both strands.

[0187] A nucleic acid as disclosed herein, wherein all nucleotides are modified with a 2'O-methyl modification, with the exception of the nucleotides at positions 2 and 14 from the 5' end of the first strand and at positions 7 and / or 9 from the 5' end of the second strand. Preferably, the nucleotides not modified with a 2'O-methyl are modified with a fluorine at the 2' position.

[0188] A nucleic acid as disclosed herein, wherein all nucleotides are modified with a 2'O-methyl modification, with the exception of the nucleotides at positions 2 and 14 from the 5' end of the first strand and at positions 7-9 from the 5' end of the second strand. Preferably, the nucleotides not modified with a 2'O-methyl are modified with a fluorine at the 2' position.

[0189] For a nucleic acid comprising a 20 base pair duplex region, the second strand preferably does not have a 2' O-methyl group at nucleotides 8 or 9 or 10 corresponding to positions 13, 12 and 11, respectively, of the first strand, counting from the 5' end of the duplex.

[0190] For a nucleic acid comprising a 21 base pair duplex region, the second strand preferably does not have a 2' O-methyl group at nucleotides 9 or 10 or 11 corresponding to positions 13, 12 and 11, respectively, of the first strand, counting from the 5' end of the duplex.

[0191] Nucleic acid of the present invention can comprise one or more phosphorothioate modifications on one or more of the ends of the first strand and / or the second strand. Optionally, each or any end of the first strand can comprise one or two or three phosphorothioate-modified nucleotides. Optionally, each or any end of the second strand can comprise one or two or three phosphorothioate-modified nucleotides.

[0192] In one embodiment, the first strand may include at least one phosphorothioate (ps) bond.

[0193] In one embodiment, the first strand may further comprise a phosphorothioate bond between the terminal two 3' nucleotides, or a phosphorothioate bond between the terminal three 3' nucleotides.

[0194] In one embodiment, the linkages between the other nucleotides in the first strand are phosphodiester bonds.

[0195] In one embodiment, the first strand may include more than one phosphorothioate bond.

[0196] In another embodiment, the second strand may comprise a phosphorothioate bond between the terminal two 3' nucleotides, or a phosphorothioate bond between the terminal three 3' nucleotides.

[0197] In another embodiment, the second strand may comprise a phosphorothioate bond between the terminal two 5' nucleotides, or a phosphorothioate bond between the terminal three 5' nucleotides.

[0198] In one aspect, the nucleic acid comprises one or more phosphorodithioate bonds, such as 1, 2, 3 or 4 phosphorodithioate bonds. Preferably, there are up to 4 phosphorodithioate bonds, one each at the 5' and 3' ends of the first and second strands.

[0199] Compared with the molecule that comprises thiophosphate at the same position, in nucleic acid of the present invention, use dithiophosphate bond to reduce the stereochemical variation of nucleic acid molecule colony.Thiophosphate bond introduces chiral center really, and is difficult to control which non-connected oxygen replaces sulphur.The use of dithiophosphate guarantees that there is no chiral center in the described key, and therefore reduces or eliminates any variation in nucleic acid molecule colony, and this depends on the number of used dithiophosphate and thiophosphate bond in nucleic acid molecule.

[0200] In one aspect, the nucleic acid comprises a phosphorothioate bond between each of the terminal three 3' nucleotides on the first strand and / or between each of the terminal three 5' nucleotides, and / or between each of the terminal three 3' nucleotides of the second strand and / or between each of the terminal three 5' nucleotides, when there is no phosphorodithioate bond at the terminus. The absence of a phosphorodithioate bond at the terminus refers to a bond between the terminal two nucleotides of the nucleic acid terminus in question, or preferably between the terminal three nucleotides, that is, a bond other than a phosphorodithioate bond.

[0201] The present invention also provides a nucleic acid according to any aspect of the present invention as described herein, wherein the first RNA strand has a terminal 5'(E)-vinylphosphonate nucleotide, and the terminal 5'(E)-vinylphosphonate nucleotide is connected to the second nucleotide in the first strand by a phosphodiester bond. The first strand may include more than 1 phosphodiester bond. In one embodiment, the first strand may include a phosphodiester bond between at least three terminal 5' nucleotides. In one embodiment, the first strand may include a phosphodiester bond between at least four terminal 5' nucleotides.

[0202] In one embodiment, the first chain may comprise formula (IV):

[0203] (vp)-N(po)[N(po)] n -(IV)

[0204] wherein '(vp)-' is 5'(E)-vinylphosphonate, 'N' is a nucleotide, 'po' is a phosphodiester bond, and n is 1 to (the total number of nucleotides in the first strand - 2), preferably, wherein n is 1 to (the total number of nucleotides in the first strand - 3), more preferably, wherein n is 1 to (the total number of nucleotides in the first strand - 4).

[0205] In one aspect, if the 5'-most nucleotide of the first strand is a nucleotide other than A or U, this nucleotide is replaced in the sequence by A or U. Preferably, if the 5'-most nucleotide of the first strand is a nucleotide other than U, this nucleotide is replaced in the sequence by U, and more preferably by U with a 5' vinylphosphonate.

[0206] Terminal 5' (E)-vinylphosphonate nucleotides are nucleotides in which the native phosphate group at the 5' terminus has been replaced with E-vinylphosphonate, wherein the bridging 5' oxygen atom of the terminal nucleotide of the 5' phosphorylated chain is replaced with a methynyl group (-CH=):

[0207]

[0208] 5'(E) vinylphosphonate is a 5' phosphate mimetic. A biomimetic is a molecule that can perform the same function as the original molecule being simulated and is structurally very similar to the original molecule. In the context of the present invention, 5'(E) vinylphosphonate mimics the function of a normal 5' phosphate, for example enabling efficient RISC loading. In addition, due to its slightly altered structure, 5'(E) vinylphosphonate can stabilize the 5' terminal nucleotide by protecting it from dephosphorylation by enzymes such as phosphatases.

[0209] In one embodiment, the terminal 5' (E)-vinylphosphonate nucleotide is an RNA nucleotide.

[0210] In one aspect, the nucleic acid:

[0211] (i) having a phosphorothioate bond between the terminal three 3' nucleotides and the terminal three 5' nucleotides of the first strand;

[0212] (ii) conjugated to a triantennary ligand at the 3' terminal nucleotide or the 5' terminal nucleotide of the second strand;

[0213] (iii) having phosphorothioate bonds between the terminal three nucleotides of the second strand at the end opposite to the one end conjugated to the triantennary ligand; and

[0214] (iv) all remaining bonds between nucleotides of the first and / or second strand are phosphodiester bonds.

[0215] In one aspect, the nucleic acid:

[0216] (i) having a terminal 5'(E)-vinylphosphonate nucleotide at the 5' end of the first strand;

[0217] (ii) having a phosphorothioate bond between the terminal three 3' nucleotides of the first and second strands and between the terminal three 5' nucleotides of the second strand; and

[0218] (iii) all remaining bonds between nucleotides of the first and / or second strand are phosphodiester bonds.

[0219] In one aspect, the nucleic acid, which is preferably a siRNA that preferably inhibits the expression of LPA by RNAi, comprises one or more or all of the following:

[0220] (i) modified nucleotides;

[0221] (ii) modified nucleotides other than 2'-OMe modified nucleotides at positions 2 and 14 from the 5' end of the first strand, preferably 2'-F modified nucleotides;

[0222] (iii) each of the odd-numbered nucleotides of the first strand, numbered from a starting point at the 5′ end of the first strand, is a 2′-OMe modified nucleotide;

[0223] (iv) each of the odd-numbered nucleotides of the first strand, numbered from a starting point at the 5′ end of the first strand, is a 2′-F modified nucleotide;

[0224] (v) the second strand nucleotide corresponding to position 11 or 13 of the first strand is not modified with a modification other than a 2'-OMe modification, preferably wherein one or both of these positions comprise a 2'-F modification;

[0225] (vi) an inverted nucleotide at the 3' end of the second strand, preferably using a 3'-3' linkage;

[0226] (vii) one or more phosphorothioate bonds;

[0227] (viii) one or more phosphorodithioate bonds; and / or

[0228] (ix) The first strand has a terminal 5'(E)-vinylphosphonate nucleotide at its 5' terminus, in which case the terminal 5'(E)-vinylphosphonate nucleotide is preferably a uridine and is preferably linked to the second nucleotide in the first strand by a phosphodiester bond.

[0229] The nucleic acids of the invention may include one or more inverted nucleotides, such as inverted thymidine or inverted adenine (see, eg, Takei et al., 2002. JBC 277(26):23800-06).

[0230] In one aspect, the nucleic acid of the invention comprises one or more inverted ribonucleotides, preferably inverted adenines, at the 3' end of the second strand using a 5'-5' linkage or a 3'-3' linkage, preferably using a 3'-3' linkage.

[0231] The nucleic acid of the present invention may comprise an inverted RNA nucleotide at one or more of the chain ends. The inverted nucleotide provides stability to the nucleic acid. Preferably, the nucleic acid comprises at least one inverted nucleotide at one or more of the 3' ends of at least one of the chains and / or at the 5' end of the second chain. More preferably, the nucleic acid comprises an inverted nucleotide at the 3' end of the second chain. Most preferably, the nucleic acid comprises an inverted RNA nucleotide at the 3' end of the second chain, and this nucleotide is preferably an inverted A. The inverted nucleotide is preferably not in the form of a protruding portion, but is present at the end of the chain in a manner relative to the corresponding nucleotide in the other chain. Nucleic acids with this modification are stable and easy to synthesize.

[0232] Ligand

[0233] The nucleic acids of the present invention may be conjugated to ligands. Efficient delivery of oligonucleotides, particularly double-stranded nucleic acids of the present invention, to cells in vivo is important and requires specific targeting as well as substantial protection from the extracellular environment, particularly serum proteins. One method of achieving specific targeting is to conjugate a ligand to the nucleic acid. The ligand helps target the nucleic acid to the desired target site. There is a need to conjugate a ligand suitable for a desired receptor molecule so that the conjugated molecule is taken up by the target cell through mechanisms such as different receptor-mediated endocytic pathways or functionally similar processes.

[0234] An example is the asialoglycoprotein receptor complex (ASGP-R) composed of multimers of membrane ASGR1 and ASGR2 receptors in varying ratios, which is highly abundant on hepatocytes and has a high affinity for the GalNAc moieties described herein. One of the first disclosures of using triantennary glycosides as conjugated ligands is in U.S. Pat. No. 5,885,968. Conjugates with three GalNAc ligands and containing phosphate groups are known and described in Dubber et al. (Bioconjug. Chem. 2003 Jan-Feb; 14(1): 239-46.). The ASGP-R complex has a 50-fold higher affinity for N-acetyl-D-galactosamine (GalNAc) than for D-Gal.

[0235] The asialoglycoprotein receptor complex (ASGP-R) that specifically recognizes the terminal β-galactosyl subunit of glycosylated proteins or other oligosaccharides (Weig el, PH et al., Biochim. Biophys. Acta. 2002 Sep 19; 1572(2-3): 341-63) can be used to deliver drugs to hepatocytes expressing the receptor complex in the liver by covalently coupling galactose or galactosamine to the drug substance (Ishibashi, S et al., J Biol. Chem. 1994 Nov 11; 269(45): 27803-6). In addition, the binding affinity can be significantly increased by the multivalent effect, which is achieved by duplication of the targeting moiety (Biessen EA et al., J Med Chem. 1995 Apr 28; 38(9): 1538-46.).

[0236] The ASGP-R complex is a mediator for achieving active uptake of glycoproteins containing terminal β-galactosyl groups into endosomes of cells. Therefore, the ASGPR is highly suitable for achieving targeted delivery of drug candidates such as nucleic acids conjugated to the ligand into receptor-expressing cells (Akinc et al., Mol Ther. 2010 July; 18(7): 1357-64).

[0237] More typically, the ligand may comprise a sugar selected to have affinity for at least one type of receptor on a target cell. In particular, the receptor is on the surface of a mammalian hepatocyte, such as the hepatic asialoglycoprotein receptor complex (ASGP-R) described previously.

[0238] The sugar may be selected from N-acetylgalactosamine, mannose, galactose, glucose, glucosamine and trehalose. The sugar may be N-acetylgalactosamine (GalNAc).

[0239] Thus, the ligands used in the present invention may comprise (i) one or more N-acetylgalactosamine (GalNAc) moieties and derivatives thereof, and (ii) a linker, wherein the linker conjugates the GalNAc moiety to a sequence as defined in any of the previous aspects. The linker may be a divalent or trivalent or tetravalent branched structure. The nucleotides may be modified as defined herein.

[0240] "GalNAc" refers to 2-(acetylamino)-2-deoxy-D-galactopyranose, commonly referred to as N-acetylgalactosamine in the literature. Reference to "GalNAc" or "N-acetylgalactosamine" includes both the β form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α form: 2-(acetylamino)-2-deoxy-α-D-galactopyranose. The β form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α form: 2-(acetylamino)-2-deoxy-α-D-galactopyranose are used interchangeably. Preferably, the compounds of the present invention contain the β form, i.e., 2-(acetylamino)-2-deoxy-β-D-galactopyranose.

[0241] Thus, the ligand may comprise GalNAc.

[0242] The ligand may include a compound of formula (I):

[0243] [SX 1 -PX 2 ]3-AX 3 -(I)

[0244] in:

[0245] S represents a sugar, wherein the sugar is N-acetylgalactosamine;

[0246] X 1 represents C3-C6 alkylene or (-CH2-CH2-O) m (-CH2)2-, where m is 1, 2 or 3;

[0247] P is phosphate or a modified phosphate (preferably phosphorothioate);

[0248] X2 is an alkylene group or a group of formula (-CH2) n -O-CH2-alkylene ether, wherein n=1-6;

[0249] A is the branching unit;

[0250] X 3 represents the bridge unit;

[0251] wherein the nucleic acid of the invention is conjugated to X via phosphate or modified phosphate (preferably via phosphorothioate) 3 .

[0252] In formula (I), the branching unit "A" is divided into three branches to accommodate three sugar ligands. The branching unit is covalently linked to the remaining tethered portion of the ligand and the nucleic acid. The branching unit may include a branched aliphatic group, the branched aliphatic group comprising a group selected from an alkyl group, an amide group, a disulfide group, a polyethylene glycol group, an ether group, a thioether group and a hydroxyamino group. The branching unit may include a group selected from an alkyl group and an ether group.

[0253] The branching unit A may have a structure selected from the following:

[0254]

[0255] Each of A 1 independently represent O, S, C═O or NH; and

[0256] Each n independently represents an integer from 1 to 20.

[0257] The branching unit may have a structure selected from the following:

[0258]

[0259] wherein each A1 independently represents O, S, C═O or NH; and

[0260] Each n independently represents an integer from 1 to 20.

[0261] The branching unit may have a structure selected from the following:

[0262]

[0263] wherein A1 is O, S, C=O or NH; and

[0264] Each n independently represents an integer from 1 to 20.

[0265] A branch unit can have the structure:

[0266]

[0267] A branch unit can have the structure:

[0268]

[0269] A branch unit can have the structure:

[0270]

[0271] Optionally, the branching units consist only of carbon atoms.

[0272] "X 3 The " portion is a bridging unit. The bridging unit is a linear chain and is covalently bound to the branch unit and the nucleic acid.

[0273] X 3 Can be selected from -C1-C 20 Alkylene-, -C2-C 20 Alkenylene-, formula -(C1-C 20 Alkylene)–O–(C1-C 20 Alkylene)-alkylene ether, -C(O)-C1-C 20 -C0-C4 alkylene-, -C0-C4 alkylene(Cy)C0-C4 alkylene-, wherein Cy represents a substituted or unsubstituted 5- or 6-membered cycloalkylene, arylene, heterocyclylene or heteroarylene ring, -C1-C4 alkylene-NHC(O)-C1-C4 alkylene-, -C1-C4 alkylene-C(O)NH-C1-C4 alkylene-, -C1-C4 alkylene-SC(O)-C1-C4 alkylene-, -C1-C4 alkylene-C(O)S-C1-C4 alkylene-, -C1-C4 alkylene-OC(O)-C1-C4 alkylene-, -C1-C4 alkylene-C(O)O-C1-C4 alkylene- and -C1-C6 alkylene-SS-C1-C6 alkylene-.

[0274] X 3 It can be expressed as -(C1-C 20 Alkylene)–O–(C1-C 20 Alkylene)-alkylene ether. 3 It can be expressed as -(C1-C 20 Alkylene)–O–(C4-C 20 Alkylene)-alkylene ether, wherein the (C4-C 20 X 3 Can be selected from the group consisting of: -CH2-O-C3H6-, -CH2-O-C4H8-, -CH2-O-C6H 12 - and -CH2-O-C8H 16 -, especially -CH2-O-C4H8-, -CH2-O-C6H 12- and -CH2-O-C8H 16 -, wherein in each case a -CH2- group is attached to A.

[0275] The ligand may include a compound of formula (II):

[0276] [SX 1 -PX 2 ]3-AX 3 -(II)

[0277] in:

[0278] S stands for sugar;

[0279] X 1 represents C3-C6 alkylene or (-CH2-CH2-O) m (-CH2)2-, where m is 1, 2 or 3;

[0280] P is phosphate or a modified phosphate (preferably phosphorothioate);

[0281] X 2 is C1-C8 alkylene;

[0282] A is a branching unit selected from the following:

[0283]

[0284] X 3 It is a bridge unit;

[0285] wherein the nucleic acid of the invention is conjugated to X via phosphate or modified phosphate (preferably via phosphorothioate) 3

[0286] The branch unit A may have the structure:

[0287]

[0288] The branch unit A may have the structure:

[0289] Where X 3 Attached to the nitrogen atom.

[0290] X 3 Can be C1-C 20 Preferably, X 3 Selected from the group consisting of: -C3H6-, -C4H8-, -C6H 12 -and-C8H 16 -, especially -C4H8-, -C6H 12 -and-C8H 16 -.

[0291] The ligand may include a compound of formula (III):

[0292] [SX 1 -PX 2 ]3-AX 3 -(III)

[0293] in:

[0294] S stands for sugar;

[0295] X 1 represents C3-C6 alkylene or (-CH2-CH2-O) m (-CH2)2-, where m is 1, 2 or 3;

[0296] P is phosphate or a modified phosphate (preferably phosphorothioate);

[0297] X 2 It is of the formula -C3H6-O-CH2-alkylene ether;

[0298] A is the branching unit;

[0299] X 3 is an alkylene ether having a formula selected from the group consisting of: -CH2-O-CH2-, -CH2-O-C2H4-, -CH2-O-C3H6-, -CH2-O-C4H8-, -CH2-O-C5H 10 -、-CH2-O-C6H 12 -、-CH2-O-C7H 14 - and -CH2-O-C8H 16 -, in which in each case a -CH2- group is attached to A,

[0300] And where X 3 Conjugation to the nucleic acids of the invention is via phosphate or modified phosphate, preferably via phosphorothioate.

[0301] The branching units may comprise carbon. Preferably, the branching units are carbon.

[0302] X 3 Can be selected from the group consisting of: -CH2-O-C4H8-, -CH2-O-C5H 10 -、-CH2-O-C6H 12 -、-CH2-O-C7H 14 - and -CH2-O-C8H 16 -. Preferably, X 3 Selected from the group consisting of: -CH2-O-C4H8-, -CH2-O-C6H12 - and -CH2-O-C8H 16 .

[0303] For any of the above aspects, when P represents a modified phosphate group, P may be represented by:

[0304]

[0305] where Y 1 and Y 2 Each independently represents =O, =S, -O -、 -OH, -SH, -BH 3、 -OCH2CO2, -OCH2CO2R x 、-OCH2C(S)OR x AND –OR x , wherein Rx represents a C1-C6 alkyl group, and wherein Indicates the connection to the rest of the compound.

[0306] In the case of modified phosphate, it means one or more replaced phosphate groups in non-connectivity oxygen. The example of modified phosphate group includes thiophosphate, selenophosphate, borane phosphate, borane phosphate, hydrogen phosphonate, phosphoramidate, alkyl or aryl phosphonate and phosphotriester. In the case of dithiophosphate, two non-connectivity oxygens are replaced by sulfur. One, each or two non-connectivity oxygens in phosphate group can independently become any one of S, Se, B, C, H, N or OR (R is alkyl or aryl).

[0307] Phosphates can also be modified by replacing the linking oxygen with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylenephosphonates). The replacement can be at the terminal oxygen. It is possible to replace a non-linking oxygen with nitrogen.

[0308] For example, Y 1 Can represent -OH, and Y 2 Can represent =O or =S; or

[0309] Y 1 Can represent -O-, and Y 2 Can represent =O or =S;

[0310] Y 1 Can represent =O, and Y 2 Can represent –CH3, -SH, -OR x or –BH3

[0311] Y 1 may represent =S, and Y2 may represent –CH 3 , ORx or –SH.

[0312] The skilled person will appreciate that in some cases, 1 With Y 2 There will be delocalization between them.

[0313] Preferably, the modified phosphate group is a thiophosphate group. The thiophosphate group includes dithiophosphates (i.e., wherein Y 1 represents = S, and Y 2 Stands for – S - ) and monothioate (i.e., wherein Y 1 Represents-O - , and Y 2 Represents = S, or Y 1 represents =O, and Y 2 Stands for – S - ). Preferably, P is a monothiophosphate. The inventors have found that conjugates having a phosphorothioate group replacing a phosphate group have improved potency and duration of action in vivo.

[0314] P can also be ethyl phosphate (ie, where Y 1 represents =O, and Y 2 represents OCH2CH3).

[0315] The sugar may be selected to have affinity for at least one type of receptor on the target cell. In particular, the receptor is on the surface of a mammalian hepatocyte, such as the hepatic asialoglycoprotein receptor complex (ASGP-R).

[0316] For any of the above aspects, the sugar may be selected from N-acetyl groups having one or more of galactosamine, mannose, galactose, glucose, glucosamine and fructose. Typically, the ligand to be used in the present invention may include N-acetylgalactosamine (GalNAc). Preferably, the compounds of the present invention may have 3 ligands, each of which will preferably include N-acetylgalactosamine.

[0317] "GalNAc" refers to 2-(acetylamino)-2-deoxy-D-galactopyranose, which is commonly referred to as N-acetylgalactosamine in the literature. Reference to "GalNAc" or "N-acetylgalactosamine" includes both the β form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α form: 2-(acetylamino)-2-deoxy-α-D-galactopyranose. In certain embodiments, the β form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α form: 2-(acetylamino)-2-deoxy-α-D-galactopyranose are used interchangeably. Preferably, the compounds of the present invention contain the β form, i.e., 2-(acetylamino)-2-deoxy-β-D-galactopyranose.

[0318]

[0319] 2-(Acetylamino)-2-deoxy-D-galactopyranose

[0320]

[0321] 2-(Acetylamino)-2-deoxy-β-D-galactopyranose

[0322]

[0323] 2-(Acetylamino)-2-deoxy-α-D-galactopyranose

[0324] For any of the compounds of formula (III) above, X 1 It can be (-CH2-CH2-O)(-CH2)2-. 1 It can be (-CH2-CH2-O)2(-CH2)2-. 1 It may be (-CH2-CH2-O)3(-CH2)2-. Preferably, X 1 is (-CH2-CH2-O)2(-CH2)2-. Or, X 1 represents a C3-C6 alkylene group. 1 It can be propylene. 1 It may be butylene. 1 X may be pentylene. 1 Preferably, the alkyl group is a straight chain alkylene group. In particular, X 1 It may be butylene.

[0325] For the compound of formula (III), X 2 It represents the formula -C3H6-O-CH2-alkylene ether, i.e. C3 alkoxymethylene or -CH2CH2CH2OCH2-.

[0326] The present invention provides a conjugated nucleic acid having one of the following structures:

[0327]

[0328]

[0329]

[0330]

[0331] wherein Z is a nucleic acid as defined herein before, and is preferably conjugated to the 5' end of the second strand of said nucleic acid.

[0332] Preferably, the conjugated nucleic acid has the following structure:

[0333]

[0334] wherein Z is a nucleic acid as defined herein before, and is preferably conjugated to the 5' end of the second strand.

[0335] The ligand of formula (I), (II) or (III) may be attached to the 3' end of the first (antisense) strand, and / or to either the 3' end and / or the 5' end of the second (sense) strand. The nucleic acid may contain more than one ligand of formula (I), (II) or (III). However, a single ligand of formula (I), (II) or (III) is preferred, since a single ligand is sufficient to achieve efficient targeting of the nucleic acid to a target cell. Preferably, in that case, at least the last two, preferably at least the last three, and more preferably at least the last four nucleotides of the nucleic acid termini to which the ligand is attached are linked by phosphodiester bonds.

[0336] Preferably, the 5' end of the first (antisense) strand is not linked to a ligand of formula (I), (II) or (III), since a ligand in this position could potentially interfere with the biological activity of the nucleic acid.

[0337] Nucleic acids having a single ligand of formula (I), (II) or (III) at the 5' end of the strand can be synthesized more easily and therefore more cheaply than the same nucleic acid having the same ligand at the 3' end. Thus, preferably, a single ligand having any of formula (I), (II) or (III) is covalently attached to (conjugated to) the 5' end of the second strand of the nucleic acid.

[0338] In one embodiment, the nucleic acid is conjugated to a ligand comprising a lipid, and more preferably to a ligand comprising cholesterol.

[0339] Alternatively, the nucleic acid of the invention may be conjugated to a ligand having the structure

[0340]

[0341] The conjugates of the invention may comprise any nucleic acid as disclosed herein conjugated to any one or more ligands as disclosed herein.

[0342] The present invention also relates to a conjugate for inhibiting the expression of an LPA gene in a cell, the conjugate comprising a nucleic acid portion containing a nucleic acid of any aspect of the present invention and at least one ligand portion, the nucleic acid portion comprising at least one duplex region, the duplex region comprising at least a portion of a first RNA chain and at least a portion of a second RNA chain at least partially complementary to the first chain, wherein the first chain is at least partially complementary to at least a portion of an RNA transcribed from the LPA gene, the at least one ligand portion comprising a linker portion, preferably a serinol-derived linker portion, and a targeting ligand for targeting cells in vivo and conjugated only to the 3' and / or 5' end of one or both RNA chains, wherein the 5' end of the first RNA chain is not conjugated, wherein:

[0343] (i) the second RNA chain is conjugated to the targeting ligand at the 5' end, and wherein (a) the second RNA chain is also conjugated to the targeting ligand at the 3' end, and the 3' end of the first RNA chain is not conjugated; or (b) the first RNA chain is conjugated to the targeting ligand at the 3' end, and the 3' end of the second RNA chain is not conjugated; or (c) both the second RNA chain and the first RNA chain are also conjugated to the targeting ligand at the 3' end; or

[0344] (ii) Both the second RNA strand and the first RNA strand are conjugated to the targeting ligand at the 3′ end, and the 5′ end of the second RNA strand is not conjugated.

[0345] The ligand may be monomeric or polymeric (eg, dimeric, trimeric, etc.).

[0346] Suitably, the ligand is monomeric and therefore contains a single targeting ligand moiety, for example a single GalNAc moiety.

[0347] Alternatively, the ligand may be a dimeric ligand, wherein the ligand moiety comprises two linker moieties, such as a serinol-derived linker moiety or a non-serinol linker moiety, each linked to a single targeting ligand moiety.

[0348] The ligand may be a trimeric ligand, wherein the ligand moiety comprises three linker moieties, such as a serinol-derived linker moiety or a non-serinol linker moiety, each linked to a single targeting ligand moiety.

[0349] Two or three serinol-derivatized linker moieties can be linked in series, for example as follows:

[0350]

[0351] wherein n is 1 or 2, and Y is S or O.

[0352] Preferably, the ligand is a monomer.

[0353] Suitably, the conjugated RNA strand is conjugated to the targeting ligand via a linker moiety comprising a further linker, wherein the further linker is or comprises a saturated, unbranched or branched C 1-15 An alkyl chain, wherein optionally one or more carbons (e.g. 1, 2 or 3 carbons, suitably 1 or 2 carbons, especially 1 carbon) are selected from O, N, S(O) p wherein p is 0, 1 or 2 (e.g. a CH2 group is replaced by O, or by NH, or by S, or by SO2, or a -CH3 group at the end of the chain or on a branch is replaced by OH or by NH2), wherein the chain is optionally substituted with one or more oxo groups (e.g. 1 to 3 such as 1 group).

[0354] Suitably, the linker moiety is a serinol-derivatized linker moiety.

[0355] The term "serinol-derived linker moiety" refers to a linker moiety comprising the following structure:

[0356]

[0357] The O atom of the structure is typically attached to the RNA strand, and the N atom is typically attached to the targeting ligand.

[0358] More preferably, the other linker comprises a saturated, unbranched C 1-15 An alkyl chain in which one or more carbons (for example 1, 2 or 3 carbons, suitably 1 or 2 carbons, especially 1 carbon) are replaced by an oxygen atom.

[0359] More suitably, the further linker comprises a PEG chain.

[0360] More preferably, the other linker comprises a saturated, unbranched C 1-15 Alkyl chain.

[0361] More preferably, the other linker comprises a saturated, unbranched C 1-6 Alkyl chain.

[0362] More suitably, the further linker comprises a saturated, unbranched C4 or C6 alkyl chain, such as a C4 alkyl chain.

[0363] In one embodiment of the invention, the first RNA strand is a compound of formula (X):

[0364]

[0365] where b is 0 or 1; and

[0366] The second RNA strand is a compound of formula (XI):

[0367]

[0368] in:

[0369] c and d are independently 0 or 1;

[0370] Z1 and Z2 are RNA moieties of the first RNA strand and the second RNA strand, respectively; Y is O or S;

[0371] n is 0, 1, 2 or 3; and

[0372] L1 is a linker attached to the ligand;

[0373] And where b+c+d is 2 or 3.

[0374] Suitably, the first RNA strand is of formula (XV)

[0375]

[0376] where b is 0 or 1; and

[0377] The second RNA strand is a compound of formula (XVI):

[0378]

[0379] Where c and d are independently 0 or 1;

[0380] in:

[0381] Z1 and Z2 are RNA portions of the first RNA strand and the second RNA strand, respectively;

[0382] Y is O or S;

[0383] R1 is H or methyl;

[0384] n is 0, 1, 2 or 3; and

[0385] In formula (XV) and (XVI), L is the same or different and is selected from the group consisting of:

[0386] -(CH2) r -C(O)-, wherein r = 2-12;

[0387] -(CH2-CH2-O) s -CH2-C(O)-, where s = 1-5;

[0388] -(CH2)t -CO-NH-(CH2) t -NH-C(O)-, wherein t is independently 1-5;

[0389] -(CH2) u -CO-NH-(CH2) u -C(O)-, wherein u is independently 1-5; and

[0390] -(CH2) v -NH-C(O)-, wherein v is 2-12; and

[0391] wherein if a terminal C(O) is present, it is attached to an NH group;

[0392] And where b+c+d is 2 or 3.

[0393] Suitably, the first RNA strand is a compound of formula (XII):

[0394]

[0395] where b is 0 or 1; and

[0396] The second RNA strand is a compound of formula (XIII):

[0397]

[0398] in:

[0399] c and d are independently 0 or 1;

[0400] Z1 and Z2 are RNA portions of the first RNA strand and the second RNA strand, respectively;

[0401] Y is O or S;

[0402] n is 0, 1, 2 or 3; and

[0403] In formulae (XII) and (XIII), L2 are the same or different, and in the bracketed parts by b, c and d, are the same or different and are selected from the group consisting of:

[0404] Or n is 0, and L2 is:

[0405]

[0406] And the terminal OH group is absent, so that the following parts are formed:

[0407]

[0408] in

[0409] F is saturated, branched, or unbranched (such as unbranched) C 1-8 Alkyl (e.g. C 1-6 alkyl) chain, wherein one of the carbon atoms is optionally replaced by an oxygen atom, provided that the oxygen atom is separated from another heteroatom (such as an O or N atom) by at least 2 carbon atoms;

[0410] In formula (XII) and (XIII), L is the same or different and is selected from the group consisting of:

[0411] -(CH2) r -C(O)-, wherein r = 2-12;

[0412] -(CH2-CH2-O) s -CH2-C(O)-, where s = 1-5;

[0413] -(CH2) t -CO-NH-(CH2) t -NH-C(O)-, wherein t is independently 1-5;

[0414] -(CH2) u -CO-NH-(CH2) u -C(O)-, wherein u is independently 1-5; and

[0415] -(CH2) v -NH-C(O)-, wherein v is 2-12; and

[0416] wherein if a terminal C(O) is present, it is attached to an NH group;

[0417] And where b+c+d is 2 or 3.

[0418] In any of the above formulae where GalNAc is present, the GalNAc may be replaced with any other targeting ligand such as those mentioned herein.

[0419] Suitably, b is 0, c is 1, and d is 1; b is 1, c is 0, and d is 1; b is 1, c is 1, and d is 0; or b is 1, c is 1, and d is 1.

[0420] More suitably, b is 0, c is 1, and d is 1; b is 1, c is 0, and d is 1; or b is 1, c is 1, and d is 1.

[0421] Most preferably, b is 0, c is 1, and d is 1.

[0422] In one embodiment, Y is O. In another embodiment, Y is S.

[0423] In one embodiment, R1 is H or methyl. In one embodiment, R1 is H. In another embodiment, R1 is methyl.

[0424] In one embodiment, n is 0, 1, 2 or 3. Suitably, n is 0.

[0425] In one embodiment, L is selected from the group consisting of:

[0426] -(CH2) r -C(O)-, wherein r = 2-12;

[0427] -(CH2-CH2-O) s -CH2-C(O)-, where s = 1-5;

[0428] -(CH2) t -CO-NH-(CH2) t -NH-C(O)-, wherein t is independently 1-5;

[0429] -(CH2) u -CO-NH-(CH2) u -C(O)-, wherein u is independently 1-5; and

[0430] -(CH2) v -NH-C(O)-, wherein v is 2-12;

[0431] The terminal C(O) is connected to the NH group.

[0432] Suitably, L is -(CH2) r -C(O)-, wherein r = 2-12. Suitably, r = 2-6. More suitably, r = 4 or 6, for example 4.

[0433] Suitably, L is:

[0434]

[0435] Example F portion includes (CH2) 1-6 , for example (CH2) 1-4 , such as CH2, (CH2)4, (CH2)5 or (CH2)6, or CH2O(CH2) 2-3 , for example CH2O(CH2)CH3.

[0436] Suitably, L2 is:

[0437]

[0438] Suitably, L2 is:

[0439]

[0440] Suitably, L2 is:

[0441]

[0442] Suitably, L2 is:

[0443]

[0444] Suitably, n is 0, and L2 is:

[0445]

[0446] And the terminal OH group is absent, so that the following parts are formed:

[0447]

[0448] wherein Y is as defined elsewhere herein.

[0449] In the part bracketed by b, c and d, L2 is usually the same. Between the parts bracketed by b, c and d, L2 can be the same or different. In one embodiment, the L2 in the part bracketed by c is the same as the L2 in the part bracketed by d. In one embodiment, the L2 in the part bracketed by c is not the same as the L2 in the part bracketed by d. In one embodiment, the L2 in the part bracketed by b, c and d is the same, for example, when the linker moiety is a serinol derived linker moiety.

[0450] The serinol-derived linker moiety can be based on serinol in any stereochemical form, i.e., derived from L-serine isomers, D-serine isomers, racemic serine or other combinations of isomers. In a preferred aspect of the invention, the serinol-GalNAc moiety (SerGN) has the following stereochemistry:

[0451]

[0452] That is, it is based on a solid-carrier building block of (S)-serinol-imide or (S)-serinol succinate derived from an L-serine isomer.

[0453] In one embodiment, the targeted cell is a hepatocyte.

[0454] One aspect is a nucleic acid for inhibiting the expression of LPA in a cell, the nucleic acid comprising at least one duplex region, the duplex region comprising at least a portion of a first strand and at least a portion of a second strand at least partially complementary to the first strand, wherein the first strand is at least partially complementary to at least a portion of an RNA transcribed from an LPA gene, wherein the first strand comprises a nucleotide sequence selected from the following sequences: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43, wherein the nucleic acid is conjugated to a ligand. The second strand may comprise a nucleotide sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 or 44. The nucleotides of the first strand and / or the second strand may be modified as described herein.

[0455] Preferably, the nucleic acid comprises SEQ ID NO: 5 or SEQ ID NO: 9 and SEQ ID NO: 6 or SEQ ID NO: 10 conjugated to a ligand of formula (I) (as described above), wherein the ligand is conjugated to the nucleic acid as described, and wherein the first strand is modified with 2'OMe modification on the odd-numbered nucleotides and 2'F on the even-numbered nucleotides, and the second strand is modified with 2'OMe on the even-numbered nucleotides and 2'F on the odd-numbered nucleotides.

[0456] Particularly preferred are nucleic acids wherein the first strand comprises or preferably consists of SEQ ID NO: 165 and the second strand optionally comprises or preferably consists of SEQ ID NO: 163. Such nucleic acids may further be conjugated to a ligand. Even more preferred are nucleic acids wherein the first strand comprises or preferably consists of SEQ ID NO: 165 and the second strand optionally comprises or preferably consists of SEQ ID NO: 164. Most preferred are siRNAs consisting of SEQ ID NO: 165 and SEQ ID NO: 164. One aspect of the invention is conjugate 21.

[0457] Compositions, uses and methods

[0458] The present invention also provides a pharmaceutical composition comprising a nucleic acid or conjugated nucleic acid of the present invention. The pharmaceutical composition can be used alone or in combination with other agents as a medicament or diagnostic agent. For example, one or more nucleic acid conjugates of the present invention can be combined with a delivery vehicle (e.g., liposome) and / or an excipient such as a carrier, a diluent. Other agents such as preservatives and stabilizers may also be added. Methods for delivering nucleic acids are known in the art and are within the knowledge of those skilled in the art.

[0459] The present invention also includes a pharmaceutical composition comprising one or more nucleic acids or conjugated nucleic acids of the present invention in a physiologically / pharmaceutically acceptable excipient such as a stabilizer, preservative, diluent, buffer, and the like.

[0460] The pharmaceutical composition may be a sterile injectable aqueous suspension or solution, or in lyophilized form, or attached to, absorbed or included in or into any other suitable galenic carrier material, such as pellets, tablets, capsules, nanoparticles, gels, tablets, beads or similar structures.

[0461] One aspect relates to a double-stranded nucleic acid capable of inhibiting the expression of LPA, preferably in a cell, for use as a medicament.

[0462] Another aspect of the invention relates to a nucleic acid or conjugated nucleic acid of the invention or a pharmaceutical composition comprising the nucleic acid or conjugated nucleic acid of the invention for use in treating a disease, disorder or syndrome, preferably a disease, disorder or syndrome associated with elevated levels of Lp(a)-containing particles. Treatment may be for the prevention of stroke, atherosclerosis, thrombosis or cardiovascular disease such as coronary heart disease or aortic stenosis and any other disease or pathology associated with elevated levels of Lp(a)-containing particles, and / or to reduce the risk of developing stroke, atherosclerosis, thrombosis or cardiovascular disease such as coronary heart disease or aortic stenosis and any other disease or pathology associated with elevated levels of Lp(a)-containing particles, and / or to treat stroke, atherosclerosis, thrombosis or cardiovascular disease such as coronary heart disease or aortic stenosis and any other disease or pathology associated with elevated levels of Lp(a)-containing particles. The treatment may be the prevention of, and / or the reduction of the risk of, and / or the treatment of, atherosclerotic cardiovascular disease, atherosclerotic cerebrovascular disease, hyperlipidemia and dyslipidemia, preferably wherein the disease is associated with elevated levels of Lp(a)-containing particles. The treatment can be preventing calcific aortic stenosis, ischemic stroke, coronary artery disease, peripheral arterial disease, abdominal aortic aneurysm, heart failure secondary to ischemic cardiomyopathy or familial hypercholesterolemia, and / or reducing the risk of developing calcific aortic stenosis, ischemic stroke, coronary artery disease, peripheral arterial disease, abdominal aortic aneurysm, heart failure secondary to ischemic cardiomyopathy or familial hypercholesterolemia, and / or treating calcific aortic stenosis, ischemic stroke, coronary artery disease, peripheral arterial disease, abdominal aortic aneurysm, heart failure secondary to ischemic cardiomyopathy or familial hypercholesterolemia, preferably wherein the disease is associated with elevated levels of Lp(a)-containing particles. Preferably, the treatment is the prevention of aortic stenosis (such as calcific aortic stenosis or familial hypercholesterolemia), and / or the reduction of the risk of developing aortic stenosis (such as calcific aortic stenosis or familial hypercholesterolemia), and / or the treatment of aortic stenosis (such as calcific aortic stenosis or familial hypercholesterolemia), preferably in each case when the disease or condition is associated with elevated levels of Lp(a)-containing particles. Ideal levels of Lp(a)-containing particles in serum are generally described as levels below 14 mg / dL. Elevated levels of Lp(a)-containing particles are at least 14, preferably at least 20, more preferably at least 30, more preferably at least 40 and most preferably at least 50 mg / dL of Lp(a)-containing particles in the subject's serum.

[0463] The present invention includes a pharmaceutical composition comprising one or more nucleic acids or conjugated nucleic acids of the present invention in a physiologically / pharmaceutically acceptable excipient such as a stabilizer, preservative, diluent, buffer, or the like.

[0464] Throughout this disclosure, the terms "Lp(a)-containing particles" and "Lp(a) particles" are used interchangeably.

[0465] The pharmaceutical compositions and agents of the present invention can be administered to mammalian subjects at a pharmaceutically effective dose. The mammal can be selected from humans, non-human primates, apes or monkeys, dogs, cats, horses, cattle, pigs, goats, sheep, mice, rats, hamsters, hedgehogs and guinea pigs, or other related species. On this basis, the term "LPA" or "LPA" as used herein refers to a nucleic acid or protein in any of the above-mentioned species, provided that the nucleic acid or protein is naturally or artificially expressed in the species, but preferably, this term refers to a human nucleic acid or protein.

[0466] The nucleic acids or conjugated nucleic acids of the present invention may also be administered in combination with other therapeutic compounds administered alone or simultaneously, for example, in a combined unit dosage form. Other therapeutic agents may be selected from the group consisting of oligonucleotides, small molecules, monoclonal antibodies, polyclonal antibodies, and peptides. It is also possible to carry out molecular conjugation with other biologically active molecular entities such as peptides, cellular ligands or artificial ligands or small molecules and macromolecules.

[0467] The dosage level of the medicament and pharmaceutical composition of the present invention can be determined by routine experiments by those skilled in the art. In one embodiment, the unit dose may contain a nucleic acid or conjugated nucleic acid between about 0.01 mg / kg body weight and about 100 mg / kg body weight. Alternatively, the dosage may be 10 mg / kg body weight to 25 mg / kg body weight, or 1 mg / kg body weight to 10 mg / kg body weight, or 0.05 mg / kg body weight to 5 mg / kg body weight, or 0.1 mg / kg body weight to 5 mg / kg body weight, or 0.1 mg / kg body weight to 1 mg / kg body weight, or 0.1 mg / kg body weight to 0.5 mg / kg body weight, or 0.5 mg / kg body weight to 1 mg / kg body weight. Alternatively, the dosage may be from about 0.5 mg / kg body weight to about 10 mg / kg body weight, or from about 0,6 mg / kg body weight to about 8 mg / kg body weight, or from about 0,7 mg / kg body weight to about 5 mg / kg body weight, or from about 0,8 mg / kg body weight to about 4 mg / kg body weight, or from about 0,9 mg / kg body weight to about 3,5 mg / kg body weight, or from about 1 mg / kg body weight to about 3 mg / kg body weight, or about 1 mg / kg body weight, or about 3 mg / kg body weight, wherein "about" is a deviation of up to 30%, preferably up to 20%, more preferably up to 10%, more preferably up to 5%, and most preferably 0% of the indicated value. The dosage level may also be calculated by other parameters such as, for example, body surface area.

[0468] The nucleic acids described herein may be capable of inhibiting the expression of LPA. The nucleic acids described herein may be capable of partially inhibiting the expression of LPA. The inhibition may be complete, i.e. 0% compared to the LPA expression level in the absence of the nucleic acids of the present invention. The inhibition of LPA expression may be partial, i.e. it may be 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or an intermediate value of the LPA expression in the absence of the nucleic acids of the present invention. When used in a subject such as a human patient, the inhibition may last for 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks or up to 3 months. The nucleic acid or conjugated nucleic acid of the present invention or the composition comprising the nucleic acid or conjugated nucleic acid can be used in a regimen comprising treatment once or twice a week, weekly, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, or every eight weeks, or in a regimen employing a combination of different dosing frequencies such as the intervals mentioned above. The nucleic acid can be used subcutaneously, intravenously or using any other route of administration such as oral, rectal or intraperitoneal, preferably subcutaneously.

[0469] In cells and / or subjects treated with or receiving nucleic acids or conjugated nucleic acids of the invention, LPA expression may be inhibited by 15% up to 100% but at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% or a range of intermediate values ​​compared to untreated cells and / or subjects. The level of inhibition may allow treatment of diseases associated with LPA expression or overexpression, or may be used to further explore the function and physiological role of LPA gene products.

[0470] Another aspect of the invention relates to a nucleic acid or conjugated nucleic acid of the invention for use in the manufacture of a medicament for treating a disease, disorder or syndrome such as those listed above or additional pathologies associated with elevated levels of Lp(a), or for use in additional therapeutic methods in which inhibition of LPA expression is desired.

[0471] Also included in the present invention is a method for treating or preventing diseases, disorders or syndromes such as those listed above, the method comprising administering to an individual in need of treatment a pharmaceutical composition comprising a nucleic acid or conjugated nucleic acid as described herein (to improve the pathology). Nucleic acid compositions may include a regimen for treatment twice a week, once a week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, or every eight weeks, or with a dosing interval more than once every eight weeks or with a regimen for administration using a combination of different dosing frequencies such as the intervals mentioned above. Nucleic acids or conjugated nucleic acids may be administered subcutaneously or intravenously or by other routes of application such as oral, rectal or intraperitoneal administration. Preferably, subcutaneous injection of nucleic acids or conjugated nucleic acids.

[0472] The nucleic acid or conjugated nucleic acid of the present invention can be produced using conventional methods in the art, including chemical synthesis, or expressing nucleic acid in vitro (e.g., by transcription) or in vivo. For example, solid phase chemical synthesis is used, or expression vectors based on nucleic acid are used, including viral derivatives or partially or completely synthetic expression systems. In one embodiment, expression vectors can be used to produce nucleic acids of the present invention in vitro, in intermediate host organisms or cell types, in intermediate or final organisms, or in desired target cells. The production methods of nucleic acids described herein (synthesis or enzymatic transcription) are known to those skilled in the art.

[0473] All features of the nucleic acids can be combined with all other aspects of the invention disclosed herein.

[0474] The present invention also relates to the unmodified sequences of all modified sequences disclosed herein.

[0475] The present invention will now be described with reference to the following non-limiting figures and examples.

[0476] Attached photos

[0477] Figure 1 Shown are the results of a screening of unconjugated siRNA molecules that inhibit LPA mRNA expression in human RT-4 cells.

[0478] Figure 2A and Figure 2B Shown is the dose response of non-conjugated LPA-targeting siRNA molecules on LPA mRNA expression in human RT-4 cells.

[0479] Figure 3 Shown is the inhibition of LPA mRNA expression in human and cynomolgus monkey primary hepatocytes by different doses of GalNAc-L1LPA-1038 conjugated siRNA molecules delivered via receptor-mediated uptake.

[0480] Figure 4 Shown are representative examples of LPA-mRNA knockdown in primary human hepatocytes by the indicated L6-conjugated GalNAc siRNAs delivered via receptor-mediated uptake.

[0481] Figure 5 Shown is the synthesis of A0268, which is a 3' mono-GalNAc conjugated single-stranded oligonucleotide and was the starting material in the synthesis of Conjugate 1 and Conjugate 3. (ps) indicates a phosphorothioate bond.

[0482] Figure 6 Shown is the synthesis of A0006, a 5' triantennary GalNAc conjugated single stranded oligonucleotide used to synthesize reference conjugate 4. (ps) indicates phosphorothioate linkage.

[0483] FIG. 7 illustrates an in vitro assay for TTR knockdown. In particular, FIG. 7A shows an in vitro assay for TTR knockdown achieved by reference conjugates (RC) 1 and 3 and an untreated control "UT"; FIG. 7B shows an in vitro assay for TTR knockdown achieved by reference conjugates (RC) 2 and 3 and an untreated control "UT"; and FIG. 7C shows an in vitro assay for TTR knockdown achieved by conjugates 1, 2, and 3 and by RC3 and an untreated control "UT". Reference conjugates 1 and 2 represent comparative conjugates. Reference conjugate 3 represents non-targeting GalNAc siRNA, and "untreated" ("UT") represents untreated cells. Both RC3 and UT are negative controls. mRNA levels are normalized to PtenII.

[0484] Figure 8 Shown is the time course of serum TTR in a cohort of n=4 c57BL / 6 mice 7, 14 and 27 days after subcutaneous treatment with 1 mg / kg-conjugates 1-3, reference conjugates (RC) 1, 2 and 4, and mock-treated (PBS) individuals.

[0485] Fig. 9 Oligonucleotide synthesis of 3' and 5' GalNAc conjugated oligonucleotide precursors such as compound X0385B-prec is shown.

[0486] Fig.10 It was shown that in primary cynomolgus monkey hepatocytes, LPA-targeted siRNA conjugated with two single GalNAc units at the 5' of the second strand achieved equivalent knockdown dose response compared to triantennary GalNAc units at the 5' of the second strand.

[0487] FIG11 illustrates an in vitro assay for TTR knockdown. In particular, Fig.11A In vitro determination of TTR knockdown achieved by conjugates 4, 5, 6 and 2 compared to "Luc" (reference conjugate 3) and untreated control "UT" is shown; Fig. 11B In vitro assays for TTR knockdown by conjugates 7 and 2 are shown compared to "Luc" (reference conjugate 3) and untreated control "UT". Luc or reference conjugate 3 (RC3) represents non-targeting GalNAc siRNA, and "untreated" ("UT") represents untreated cells. Both RC3 and UT are negative controls. mRNA levels were normalized to PtenII.

[0488] FIG12 illustrates an in vitro assay for TTR knockdown. In particular, Fig. 12A In vitro determination of TTR knockdown achieved by conjugates 8, 9, 10, 11 and 2 compared to "Luc" (reference conjugate 3) and untreated control "UT" is shown; Fig. 12B In vitro assays for TTR knockdown by conjugates 12 and 2 are shown compared to "Luc" (reference conjugate 3) and untreated control "UT". Luc or reference conjugate 3 represents non-targeting GalNAc siRNA, and "untreated" ("UT") represents untreated cells. Both RC3 and UT are negative controls. mRNA levels were normalized to PtenII.

[0489] Fig.13 In vitro determination of LPA mRNA knockdown achieved by conjugate 19 compared to control is illustrated. Ctr represents non-targeting GalNAc siRNA, and "untreated" ("UT") represents untreated cells. Both Ctr and UT are negative controls. mRNA levels were normalized to ACTB.

[0490] Fig.14Shown is the time course of Aldh2 liver mRNA levels in a group of n=6 c57BL / 6 mice 14, 28 and 42 days after subcutaneous treatment with 1 mg / kg-conjugate 15, reference conjugate (RC) 6, and mock-treated (PBS) individuals. mRNA levels were normalized to Pten.

[0491] Fig.15 Shown is the time course of Aldh2 liver mRNA levels in a group of n=6 c57BL / 6 mice 14, 28 and 42 days after subcutaneous treatment with 1 mg / kg-conjugate 16, reference conjugate (RC) 7, and mock-treated (PBS) individuals. mRNA levels were normalized to Pten.

[0492] Fig.16 Shown is the time course of Tmprss6 liver mRNA levels in a group of n=6 c57BL / 6 mice 14, 28 and 42 days after subcutaneous treatment with 1 mg / kg-conjugate 18, reference conjugate (RC) 8, and mock-treated (PBS) individuals. mRNA levels were normalized to Pten.

[0493] Fig.17 The serum stability of conjugates 4, 5, 6, 7 and 2 and untreated control (UT) over 3 days at 37°C is shown.

[0494] Fig.18 The serum stability of conjugates 8, 9, 10, 11, 12 and 2 and untreated control (UT) over 3 days at 37°C is shown.

[0495] Fig.19 Conjugate 21 was shown to reduce LPA mRNA in human primary hepatocytes.

[0496] Fig. 20 Conjugate 21 was shown to reduce LPA mRNA in primary cynomolgus monkey hepatocytes.

[0497] Fig.21 Conjugate 21 was shown to have no effect on the level of APOB gene expression.

[0498] Fig. 22 Conjugate 21 was shown to have no effect on the level of PLG gene expression.

[0499] Fig.23 Shown is the time course of serum Lp(a) inhibition over 29 days in cynomolgus monkeys with different doses of conjugate 21.

[0500] Fig.24 Shown is a dose response curve for conjugate 21 showing reduction in serum Lp(a) at day 29 in cynomolgus monkeys.

[0501] Example

[0502] The numbers mentioned in each example are specific to that example.

[0503] Example 1

[0504] A number of modified and conjugated siRNA molecules are shown here for use in functional examples.

[0505] LPA-1038 derivatives

[0506] GalNAc-LPA-1038-L1

[0507] First strand (SEQ ID NO: 119, based on SEQ ID NO 5)

[0508] OMeA-(ps)-FU-(ps)-OMeA-FA-OMeC-FU-OMeC-FU-OMeG-FU-OMeC-FC-OMeA-FU-OMeU-FA-OMeC-(ps)-FC-(ps)-OMeA 3'

[0509] Second strand (SEQ ID NO: 120, based on SEQ ID NO SEQ ID NO 6)

[0510] 5'[ST23(ps)]3 long triplet (ps)FU-OMeG-FG-OMeU-FA-OMeA-FU-OMeG-FG-OMeA-FC-OMeA-FG-OMeA-FG-OMeU-FU-(ps)-OMeA-(ps)-FU 3

[0511] GalNAc-LPA-1038-L6

[0512] First strand (SEQ ID NO: 121, based on SEQ ID NO 5)

[0513] OMeA-(ps)-FU-(ps)-OMeA-FA-OMeC-FU-OMeC-FU-OMeG-FU-OMeC-FC-OMeA-FU-OMeU-FA-OMeC-(ps)-FC-(ps)-OMeA 3'

[0514] Second strand (SEQ ID NO: 122, based on SEQ ID NO 6)

[0515] 5'[ST23(ps)]3ST43(ps)FU-OMeG-FG-OMeU-FA-OMeA-FU-OMeG-FG-OMeA-FC-OMeA-FG-OMeA-FG-OMeU-FU-(ps)-OMeA-(ps)-FU 3'

[0516] FN (N = A, C, G, U) represents 2' fluoro 2' deoxynucleoside

[0517] OMeN (N=A,C,G,U) represents 2'O methyl nucleoside

[0518] (ps) indicates phosphorothioate bond

[0519] ST23 and ST43 are as follows.

[0520] Other examples are LPA 1041 derivatives:

[0521] GalNAc-LPA-1041-L1

[0522] First strand (SEQ ID NO: 123, based on SEQ ID NO 9)

[0523] 5'OMeA-(ps)-FU-(ps)-OMeA-FA-OMeC-FU-OMeC-FU-OMeG-FU-OMeC-FC-OMeA-FU-OMeU-FA-OMeC-(ps)-FC-(ps)-OMeG 3'

[0524] Second strand (SEQ ID NO: 124, based on SEQ ID NO 10)

[0525] 5'[ST23(ps)]3 long triple son(ps)FC-OMeG-FG-OMeU-FA-OMeA-FU-OMeG-FG-OMeA-FC-OMeA-FG-OMeA-FG-OMeU-FU-(ps)-OMeA-(ps)-FU 3'

[0526] GalNAc-LPA-1041-L6

[0527] First strand (SEQ ID NO: 125, based on SEQ ID NO 9)

[0528] 5'OMeA-(ps)-FU-(ps)-OMeA-FA-OMeC-FU-OMeC-FU-OMeG-FU-OMeC-FC-OMeA-FU-OMeU-FA-OMeC-(ps)-FC-(ps)-OMeG 3'

[0529] Second strand (SEQ ID NO: 126, based on SEQ ID NO 10)

[0530] 5'[ST23(ps)]3ST43(ps)FC-OMeG-FG-OMeU-FA-OMeA-FU-OMeG-FG-OMeA-FC-OMeA-FG-OMeA-FG-OMeU-FU-(ps)-OMeA-(ps)-FU 3'

[0531] FN (N = A, C, G, U) represents 2' fluoro 2' deoxynucleoside

[0532] OMeN (N=A,C,G,U) represents 2'O methyl nucleoside

[0533] (ps) indicates phosphorothioate bond

[0534] All oligonucleotides were obtained from commercial oligonucleotide manufacturers (Biospring, Frankfurt, Germany, or RiboBio, Guangzhou, Guangdong, PRC) or synthesized on an AKTA oligopilot synthesizer (in-house) using standard phosphoramidite chemistry. Commercially available solid supports were used along with 2'O-methyl RNA phosphoramidites, 2' fluoro DNA phosphoramidites (all with standard protection), and commercially available long triplet phosphoramidites (Glen research). Synthesis was performed using 0.1 M phosphoramidites in anhydrous acetonitrile, and benzylthiotetrazolium (BTT) was used as an activator (0.3 M acetonitrile solution). All other reagents were commercially available standard reagents.

[0535] Conjugation of the corresponding GalNac synthons (eg ST23, ST41 or ST43) was achieved by coupling the corresponding phosphoramidite to the 5' end of the oligo under standard phosphoramidite coupling conditions. Phosphorothioates were introduced using standard commercially available thiolation reagents (EDITH, Link technologies).

[0536] The single strands were removed by cleavage from CPG using methylamine (40% aqueous solution), and the resulting crude oligonucleotides were purified by ion exchange chromatography (Resource Q, 6 mL, GE Healthcare) using a sodium chloride gradient on an AKTA Pure HPLC system. The fractions containing the product were combined, desalted on a size exclusion column (Zetadex, EMP Biotech), and lyophilized.

[0537] For annealing, equimolar amounts of the corresponding single strands were dissolved in water and heated to 80° C. for 5 minutes. After gradual cooling to room temperature, the resulting duplexes were lyophilized.

[0538] The sequences of the resulting nucleic acids (siRNAs) are set forth in Table 1 below.

[0539] Table 1 : Non-conjugated nucleic acid sequences tested for inhibition of LPA mRNA expression.

[0540] Indicates the sequence and the modification style applied

[0541]

[0542]

[0543]

[0544] Table 1

[0545] Table 1: Nucleotide modifications are depicted by the following numbers (column 4), 1 = 2'F-dU; 2 = 2'F-dA; 3 = 2'F-dC; 4 = 2'F-dG; 5 = 2'-OMe-rU; 6 = 2'-OMe-rA; 7 = 2'-OMe-rC; 8 = 2'-OMe-rG.

[0546] Table 2: Shown below are the sequences of the LPA, APOB, beta-Actin, and PTEN qPCR amplicons used to measure mRNA levels.

[0547]

[0548]

[0549] Table 2

[0550] Example 2

[0551] Screening of unconjugated siRNA molecules (Table 1) for inhibition of LPA mRNA expression in human RT-4 cells.

[0552] Lipofectamine transfection complexes were prepared in triplicate at a ratio of 1.5 μl RNAiMax (ThermoFisher) / 80 pmol of the indicated siRNA molecules. The complexes were diluted to the indicated concentrations of 2, 5 nM and 25 nM, respectively (values ​​are represented in pairs as light grey and dark grey bars). RT4 human bladder transitional cell papilloma cells endogenously expressing LPA were seeded in a 24-well format at a density of 125.000 cells per well on top of the transfection complexes previously plated at the indicated concentrations (reverse transfection). 24 hours after transfection, total RNA was isolated using the Spin Cell Mini Kit 250 (Stratec). LPA mRNA levels were determined by qRT-PCR relative to PPIB mRNA expression as a housekeeping transcript in the corresponding samples. Values ​​are normalized to the amount of LPA mRNA detected in untreated cells (intra-plate). Non-silencing siRNA compounds were transfected as additional controls. The mean and SD of the normalized triplicate values ​​are shown. The results are shown in Figure 1 middle.

[0553] Example 3

[0554] Dose response of unconjugated LPA-targeting siRNA compounds on LPA mRNA expression in human RT-4 cells.

[0555] RT4 human bladder transitional cell papilloma cells were reverse transfected as described above (Example 2) and treated with different non-conjugated siRNA compounds as labeled (Table 1) at the indicated concentrations (range 100 nM to 0.2 nM). Total RNA was isolated 24 hours after transfection using the Spin Cell Mini Kit 250 (Stratec). LPA mRNA levels were determined by qRT-PCR relative to PPIB mRNA expression as a housekeeping transcript in the corresponding samples. Values ​​were normalized to the amount of LPA mRNA detected in untreated cells. The bars represent the remaining LPA mRNA expression for each data point. The results are shown in Figure 2.

[0556] Example 4

[0557] Inhibition of LPA mRNA expression in human and cynomolgus monkey primary hepatocytes by different doses of GalNAc-L1 LPA-1038 conjugated siRNA molecules delivered via receptor-mediated uptake.

[0558] Primary hepatocytes (ThermoFisher) were plated on collagen-coated 96-well plates at a density of 45,000 cells per well (cynomolgus monkey) and 30,000 cells per well (human). GalNAc-L1-conjugated LPA-1038 was added immediately after plating at the indicated concentrations (nM). Total RNA was isolated 24 hours after siRNA treatment using the InviTrap RNA Cell HTS 96-well Kit (Stratec). LPA mRNA levels were determined by qRT-PCR relative to actin (cynomolgus monkey) or APOB (human) mRNA levels as housekeeping transcripts in the corresponding samples. Values ​​were normalized to LPA expression in untreated cells. The mean and SD of normalized triplicate values ​​of the remaining LPA mRNA levels are shown as black bars. Results are shown in Figure 3 middle.

[0559] Example 5

[0560] Knockdown of LPA-mRNA in human primary hepatocytes by different indicated L6-GalNAc-conjugated siRNAs after receptor-mediated delivery.

[0561] Primary human hepatocytes (ThermoFisher) were plated on collagen-coated 96-well plates at 30,000 cells per well (96-well format). GalNAc-L6 conjugated siRNAs including non-silencing controls were added immediately after cell plating at the two indicated concentrations. Total RNA was isolated 24 hours after siRNA treatment using the InviTrap RNA Cell HTS 96-well Kit (Stratec). LPA mRNA expression levels were determined by qRT-PCR relative to APOB mRNA as a housekeeping transcript. Values ​​are normalized to LPA mRNA expression in untreated cells, and the remaining LPA mRNA levels are shown in pairs as bars (100 nM black bars, 20 nM gray bars). The means and SD of normalized triplicate values ​​are shown in Figure 4 middle.

[0562] Example 6 - In vitro determination of TTR knockdown achieved with various TTR siRNA GalNAc conjugates

[0563] Murine primary hepatocytes were seeded into collagen pre-coated 96-well plates (Thermo Fisher Scientific, #A1142803) at a cell density of 30,000 cells per well and treated with siRNA conjugates at concentrations ranging from 10 nM to 0.0001 nM. 24 hours after treatment, cells were lysed and treated with siRNA according to the manufacturer's protocol. RNA was extracted using the RNA Cell HTS 96 Kit / C24 x 96 Preparation (Stratec #7061300400). Transcript levels of TTR and housekeeping mRNA (PtenII) were quantified by TaqMan analysis.

[0564] Compared to the negative control (see "UT" column and reference conjugate 3), 24 hours after treatment with the conjugates of the present invention, i.e., conjugates 1-3, the target gene expression in primary mouse hepatocytes showed that the target gene expression decreased with increasing doses of the conjugates, as shown in FIG7. This indicates that the first chain binds to the target gene, thereby reducing gene expression. FIG7 also shows the target gene expression level in the case of reference conjugates 1 and 2, which serve as comparative conjugates. As can be seen from the comparison between the data presented in FIG7A and FIG7C, and FIG7B and FIG7C, the conjugates of the present invention (conjugates 1-3) reduce target gene expression compared to reference conjugates 1 and 2. The most effective conjugate at 0.01 nM appears to be conjugate 2. The most effective conjugate at 0.1 nM, 0.5 nM, 1 nM, and 10 nM appears to be conjugate 3.

[0565] Example 7 - In vivo time course of serum TTR in mice

[0566] C57BL / 6 mice were treated subcutaneously with 1 mg / kg siRNA conjugates on day 0. Serum samples were obtained by orbital bleeding on days 7, 14, and 27 and stored at -20°C until analysis. Serum TTR quantification was performed using mouse prealbumin ELISA (ALPCO, 41-PALMS / Batch 22, 2008003B) according to the manufacturer's protocol (sample dilution 1:8000 or 1:800).

[0567] Results of the time course of serum TTR in a cohort of n=4 c57BL / 6 mice at 7, 14 and 27 days after subcutaneous treatment with 1 mg / kg conjugates 1-3, reference conjugates 1, 2 and 4, and mock-treated (PBS) individuals are shown in Figure 8 In. Figure 8 As indicated by the data in , the conjugates of the invention are particularly effective in reducing target gene expression compared to the negative control (PBS) and reference conjugates 1, 2, and particularly compared to reference conjugate 4. Conjugates 2 and 3 are also more effective than reference conjugates 1, 2, and 4. The most effective conjugate is conjugate 2. Therefore, it can be expected that the dosing level of conjugate 3 will be about 1 / 3 lower than that of reference conjugate 4 to achieve the same initial knockdown, and will also result in a longer duration of knockdown.

[0568] More specifically, in wild-type mice, compared to reference conjugate 4 at an equimolar dose, conjugate 2 resulted in a 1 / 3 reduction in target protein levels in serum on day 7, and a 1 / 4 reduction in target protein levels in serum on day 27. In addition, conjugate 2 resulted in an 85% reduction in target serum protein levels on day 27 after a single injection, compared to a 36% reduction achieved by an equimolar amount of reference conjugate 4.

[0569] Example 8

[0570] In primary cynomolgus monkey hepatocytes, LPA-targeted siRNA conjugated with two single GalNAc units at the 5' of the second strand achieved equivalent knockdown dose response compared to triantennary GalNAc units at the 5' of the second strand.

[0571] The siRNAs are modified with alternating 2'-OMe / 2'-F and each contains two phosphorothioate (PS) internucleotide linkages at their 5' and 3' terminal two internucleotide linkages. In conjugate 19, a serinol-GalNAc unit is each linked to the 5' and 3' of the second strand via a PS bond. In conjugate 20, there is a phosphodiester between the two terminal 5' nucleotides of the second strand, and a triantennary GalNAc linker is linked to this end via a PS bond.

[0572] The dose response of LPA knockdown in primary cynomolgus monkey hepatocytes was assessed 24 hours after treatment with 100, 20, 4, 0.8, 0.16, 0.032 and 0.006 nM siRNA. The reference control was construct 2 and the non-targeting control was named Cte. The transcript ct values ​​of each treatment group were normalized to the transcript ct values ​​of the housekeeping gene ACTB (Δct) and relative to untreated hepatocytes (named ut (ΔΔct)).

[0573] Data shown in Fig.10 middle.

[0574] Materials and methods:

[0575] siRNA

[0576]

[0577]

[0578] Legend

[0579]

[0580] Primers:

[0581]

[0582] General Approach

[0583] In vitro experiments

[0584] Primary mouse hepatocytes (Thermo Scientific: GIBCO batch: #MC798) were thawed and the cryopreservation medium was exchanged for Williams E medium supplemented with 5% FBS, 1 μM dexamethasone, 2 mM GlutaMax, 1% penicillin-streptomycin, 4 mg / ml human recombinant insulin, 15 mM Hepes. The cell density was adjusted to 250,000 cells per 1 ml. 100 μl of this cell suspension per well was seeded into a collagen pre-coated 96-well plate. The test article was pre-diluted in the same medium (5-fold concentrated) to obtain each concentration, and 25 μl of this pre-diluted siRNA or medium alone was added to the cells. The cells were cultured at 37°C and 5% CO2. 24 hours after treatment, the supernatant was discarded, and the cells were washed in cold PBS and 250 μl RNA lysis buffer S (Stratec) was added. After incubation at room temperature for 15 minutes, the plates were stored at -80°C until RNA isolation according to the manufacturer's protocol.

[0585] TaqMan Assays

[0586] For mTTR and PTEN multiplex TaqMan assays, 10 μl of isolated RNA from each treatment group was mixed with 10 μl of PCR master mix (TAKYON Low Rox) containing 600 nM mTTR primer, 400 nM ApoB primer, and 200 nM of each probe, as well as 0.5 units of Euroscript II RT polymerase and 0.2 units of RNase inhibitor. TaqMan assays were performed in 384-well plates with a 10-minute reverse transcription step at 48°C, a 3-minute initial denaturation at 95°C, and 40 cycles of 95°C for 10 seconds and 60°C for 1 minute. Primers contained two of BHQ1, FAM, and YY, one at each end of the sequence.

[0587] For TMPRSS6 and ApoB multiplex TaqMan analysis, 10 μl of isolated RNA from each treatment group was mixed with 10 μl of PCR master mix (TAKYON Low Rox) containing 800 nM TMPRSS6 primer, 100 nM ApoB primer and 200 nM of either probe, as well as 0.5 units of Euroscript II RT polymerase and 0.2 units of RNase inhibitor. TaqMan analysis was performed in 384-well plates with a 10-minute reverse transcription step at 48°C, a 3-minute initial denaturation at 95°C, and 40 cycles of 95°C for 10 seconds and 60°C for 1 minute.

[0588] In vivo experiments

[0589] To compare the in vivo efficacy of different siRNA conjugates, 1 mg / kg siRNA dissolved in PBS was administered subcutaneously in the scapular region of c57BL / 6 mice. Groups of n=6 were treated with siRNA targeting Aldh2 or Tmprss6 on day 1 and sacrificed at selected time points after treatment. Liver samples were snap frozen in liquid nitrogen and stored at -80°C until RNA was extracted using the InviTrap spin tissue RNA mini kit (stratec) according to the manufacturer's manual. Afterwards, transcript levels of Aldh2, Tmprss6, and Pten were quantified as described above.

[0590] Decontaminated lysosome stability assay

[0591] To probe the RNase stability in the endosomal / lysosomal compartment of hepatocytes in vitro, siRNA was incubated in Sprague Dawley rat liver decontaminated lysosomes (Tebu-Bio, catalog number: R0610.LT, batch: 1610405, pH: 7.4, 2.827 units / ml) for 0 hours, 4 hours, 24 hours or 72 hours. To simulate an acidified environment, the decontaminated lysosomes were mixed 1:10 with a low pH buffer (1.5 M acetic acid, 1.5 M sodium acetate, pH 4.75). Afterwards, 10 μl of siRNA (20 μM) was mixed with 30 μl of this acidified decontaminated lysosome and incubated at 37° C. for the indicated time. After incubation, RNA was isolated using a Clarity OTX Starter Kit-Cartridge (Phenomenex catalog number: KSO-8494) according to the manufacturer's protocol for biological fluids. Lyophilized RNA was reconstituted in 30 μl H2O, mixed with 4x loading buffer, and 5 μl was loaded onto 20% TBE-polyacrylamide gel electrophoresis (PAGE) for separation and semi-quantitative analysis. PAGE was run at 120 V for 2 hours, and RNA was visualized by ethidium bromide staining and subsequent digital imaging with a Biorad imaging system.

[0592] Example 9 - Synthesis of conjugates

[0593] Example compounds were synthesized according to the methods described below and methods known to those skilled in the art. Assembly of the oligonucleotide chain and the linker building blocks was performed by solid phase synthesis using the phosphoramidite method. GalNAc conjugation was achieved by forming a peptide bond between the GalNAc-carboxylic acid building block and a previously assembled and purified oligonucleotide to which the necessary number of amino-modified linker building blocks were attached.

[0594] Oligonucleotide synthesis, deprotection and purification followed standard procedures known in the art.

[0595] All oligonucleotides were synthesized on an AKTA oligopilot synthesizer using standard phosphoramidite chemistry. Commercially available solid supports and 2'O-methyl RNA phosphoramidites, 2'fluoro 2'deoxy RNA phosphoramidites (all standardly protected, ChemGenes, LinkTech) and the commercially available 3'-amino modifier TFA amino C-6lcaa CPG (Chemgenes), Fmoc-amino-DMT C-7CE phosphoramidite (GlyC3Am), 3'-amino modifier C-3lcaa CPG (C3Am), Fmoc-amino-DMT C-3CED phosphoramidite (C3Am) and TFA-amino C-6CED phosphoramidite (C6Am) (Chemgenes), 3'-amino modifier C7 CPG (C7Am) (Glen Research), non-nucleotide TFA amino phosphoramidite (Pip), non-nucleotide TFA amino solid support (PipAm) (AM Chemicals). Peracetylated galactosamine 8 is commercially available.

[0596] Auxiliary reagents were purchased from EMP Biotech. Synthesis was performed using a 0.1M solution of phosphoramidite in anhydrous acetonitrile, and benzylthio tetrazolium (BTT) was used as an activator (0.3M solution in acetonitrile). Coupling time was 15 minutes. Capping / oxidation / capping or capping / thio / capping cycles were applied (capping: Ac2O / NMI / dimethylpyridine / acetonitrile, oxidant: 0.1M I2 in pyridine / H2O). Phosphorothioates were introduced using standard commercially available thiolation reagents (EDITH, Link technologies). DMT cleavage was achieved by treatment with toluene containing 3% dichloroacetic acid. After completion of the programmed synthesis cycle, diethylamine (DEA) washing was performed. All oligonucleotides were synthesized in DMT removal mode.

[0597] The connection of serinol derived linker part is realized by using (S)-DMT-serinol (TFA)-succinate-lcaa-CPG 10 or (S)-DMT-serinol (TFA) phosphoramidite 7 (synthesis as described in document Hoevelmann et al. Chem. Sci., 2016, 7, 128-135). Three antennae GalNAc clusters (ST23 / C4XLT or ST23 / C6XLT) are introduced by successively coupling corresponding triple subamid derivatives (C4XLT-phos or C6XLT-phos) and GalNAc subamidite (ST23-phos).

[0598] Attachment of amino-modified moieties (non-serinol-derivatized linkers) was achieved by using the corresponding commercially available amino-modified building blocks CPG or amidite.

[0599] The single strand was removed from the CPG cleavage by treatment with 40% aqueous methylamine. The resulting crude oligonucleotide was purified on an AKTA Pure HPLC system using a sodium chloride gradient by ion exchange chromatography (Resource Q, 6 mL, GE Healthcare). The fractions containing the product were combined, desalted on a size exclusion column (Zetadex, EMP Biotech), and lyophilized.

[0600] The single strands were dissolved in H2O at a concentration of 60 OD / mL. Two single oligonucleotide solutions were added together in a reaction vessel. For easier reaction monitoring, titration was performed. The first strand was added in 25% excess compared to the second strand, as determined by UV absorption at 260 nm. The reaction mixture was heated to 80°C for 5 minutes and then slowly cooled to room temperature. Double-stranded formation was monitored by ion-pair reversed-phase HPLC. Based on the UV area of ​​the residual single strands, the required amount of the second strand was calculated and added to the reaction mixture. The reaction was heated to 80°C again and slowly cooled to room temperature. This procedure was repeated until less than 10% of the residual single strands were detected.

[0601] Synthesis of Compound 2-10

[0602] Compounds 2 to 5 and (S)-DMT-serinol (TFA)-phosphoramidite 7 were synthesized according to the method disclosed in the literature (Hoevelmann et al. Chem. Sci., 2016, 7, 128-135).

[0603] (S)-4-(3-(bis(4-methoxyphenyl)phenyl)methoxy)-2-(2,2,2-trifluoroacetamido)propoxy)-4-oxobutanoic acid (6).

[0604] To a pyridine solution of 5 was added succinic anhydride and DMAP in sequence. The resulting mixture was stirred at room temperature overnight. All starting material was consumed as judged by TLC. The reaction was concentrated. The crude material was chromatographed on silica gel using a gradient of 0% to 5% methanol in DCM (+1% triethylamine) to provide 1.33 g 6 (yield = 38%). m / z (ESI-): 588.2 (100%), (calcd. for C30H29F3NO8-: [MH] -588.6). 1H-NMR: (400MHz,CDCl3)δ[ppm]=7.94(d,1H,NH),7.39-7.36(m,2H,CH aryl),7.29-7.25(m,7H,CH aryl),6.82-6.79(m,4H,CH aryl),4.51–4.47(m,1H),4.31–4.24(m,2H),3.77(s,6H,2xDMTr-OMe),3.66-3.60(m,16H,HNEt3 + ),3.26-3.25(m,2H),2.97–2.81(m,20H,NEt3),2.50-2.41(4H,m),1.48–1.45(m,26H,HNEt3 + ),1.24-1.18(m,29H,NEt3).

[0605] (S)-DMT-serinol (TFA)-succinate-lcaa-CPG (10)

[0606] (S)-DMT-serinol (TFA)-succinate (159mg, 270umol) and HBTU (113mg, 299umol) are dissolved in CH in CN (10mL).Diisopropylethylamine (DIPEA, 94 μ L, 540umol) is added in solution, and the mixture is vortexed for 2 minutes, then natural amino-lcaa-CPG (500A, 3g, amine content: 136umol / g) is added.At room temperature on wrist-moving oscillator, gentle vibration suspension is 16 hours, then filtered, and washed with DCM and EtOH.The solid support is dried under vacuum for 2 hours.The unreacted amine on the support is capped by stirring at room temperature with acetic anhydride / lutidine / N-methylimidazole.The washing of support is repeated as above.The solid is dried under vacuum to produce solid support 10 (3g, 26umol / g loading).

[0607] GalNAc synthon(9)

[0608] The synthesis of GalNAc synthon 9 was performed as described in Nair et al. J. Am. Chem. Soc., 2014, 136(49), pp. 16958-16961 with 46% yield over two steps.

[0609] The representational data were matched with the publicly available data.

[0610] Oligonucleotide synthesis

[0611] All single-stranded oligonucleotides were prepared according to the Figure 5 and Figure 6 The reaction conditions described in were used for the synthesis and are summarized in Tables 3 and 4.

[0612] All final single-stranded products were analyzed by AEX-HPLC to demonstrate their purity. Purity was given as FLP% (full-length product%), which is the percentage of UV area under the designated product signal in the case of the UV trace of the AEX-HPLC analysis of the final product. The identity of the corresponding single-stranded product (non-modified, amino-modified precursor or GalNAc-conjugated oligonucleotide) was demonstrated by LC-MS analysis.

[0613] Table 3: Single-stranded unconjugated oligonucleotides

[0614]

[0615]

[0616] 5'1x NH2 means the position (5' end) and number (1xNH2) of free serinol-derivatized amino groups available for conjugation. For example, 1x3'NH2 on A0264 means there is a free amino group at the 3' end of chain A0264 that can react with GalNAc synthon 9. 3'5'1xNH2 means there is one serinol-derivatized free amino group at the 3' end and 5' end of the chain that can react with GalNAc linker 9.

[0617] Table 4: Single-stranded oligonucleotides with 5' and 3' modifications

[0618]

[0619]

[0620] Similarly, 3'5'1x NH2 refers to the position (3' and 5' ends) and number (1x NH2 each) of free amino groups available for conjugation. For example, 3'5'1xNH2 on A0561 means that there are 2 free amino groups (1 at the 3' end and 1 at the 5' end) that can react with GalNAc synthon 9 at the 3' end of chain A0561.

[0621] Synthesis of Certain Conjugates and Reference Conjugates 1-2

[0622] The conjugation of the GalNac synthon (9) was achieved by coupling with the serinol-amino functional group of the corresponding oligonucleotide chain 11 using a peptide coupling reagent. Therefore, the corresponding amino-modified precursor molecule 11 was dissolved in H2O (500OD / mL), and DMSO (DMSO / H2O, 2 / 1, v / v) and DIPEA (2.5% of the total volume) were added in sequence. In a separate reaction vessel, the pre-activation of the GalN(Ac4)-C4 acid (9) was carried out by reacting 2 equivalents (according to the amino functional group in the amino-modified precursor oligonucleotide 11) of the carboxylic acid component with 2 equivalents of HBTU in the presence of 8 equivalents of DIPEA in DMSO. After 2 minutes, the pre-activated compound 9 was added to the solution of the corresponding amino-modified precursor molecule. After 30 minutes, the reaction progress was monitored by LCMS or AEX-HPLC. After the conjugation reaction was completed, the crude product was precipitated by adding 10x iPrOH and 0.1x 2M NaCl and collected by centrifugation and decantation. To release the acetylated hydroxyl group in the GalNAc moiety, the resulting mass was dissolved in 40% MeNH2 (1 mL / 500 OD) and after 15 minutes at room temperature, diluted in H2O (1:10) and finally purified again by anion exchange and size exclusion chromatography and lyophilized to produce the final product 12 (Table 5).

[0623] Table 5: Single-stranded GalNAc-conjugated oligonucleotides

[0624]

[0625] Synthesis of Certain Conjugates of the Invention

[0626] The conjugation of the GalNac synthon (9) was achieved by coupling with the amino functional group of the corresponding oligonucleotide chain 14 using a peptide coupling reagent. Therefore, the corresponding amino-modified precursor molecule 14 was dissolved in H2O (500OD / mL), and DMSO (DMSO / H2O, 2 / 1, v / v) and DIPEA (2.5% of the total volume) were added in sequence. In a separate reaction vessel, the pre-activation of the GalN(Ac4)-C4 acid (9) was carried out by reacting 2 equivalents (according to the amino functional group in the amino-modified precursor oligonucleotide 14) of the carboxylic acid component with 2 equivalents of HBTU in the presence of 8 equivalents of DIPEA in DMSO. After 2 minutes, the pre-activated compound 9 was added to the solution of the corresponding amino-modified precursor molecule. After 30 minutes, the reaction progress was monitored by LCMS or AEX-HPLC. After the conjugation reaction was completed, the crude product was precipitated by adding 10x iPrOH and 0.1x 2M NaCl and collected by centrifugation and decantation. To release the acetylated hydroxyl group in the GalNAc moiety, the resulting mass was dissolved in 40% MeNH2 (1 mL / 500 OD) and after 15 minutes at room temperature, diluted in H2O (1:10) and finally purified again by anion exchange and size exclusion chromatography and lyophilized to produce the final product 15 (Table 6).

[0627] Table 6: Single-stranded GalNAc-conjugated oligonucleotides

[0628]

[0629]

[0630] Double strand formation

[0631] Double strand formation was performed according to the method described above.

[0632] Double-strand purity is given as % Double-strand, which is the percentage of the UV area under the designated product signal in the case of the UV trace of IP-RP-HPLC analysis (Table 7).

[0633] Table 7: Nucleic acid conjugates

[0634]

[0635]

[0636] sequence

[0637] Modified index for the following sequence:

[0638] f represents 2'fluoro 2'deoxyribonucleotide or 2'-fluororibonucleotide (the terms are interchangeable)

[0639] m represents 2'O methyl ribonucleotide

[0640] (ps) indicates phosphorothioate bond

[0641] FAM = 6-carboxyfluorescein

[0642] BHQ = Black Hole Quencher 1

[0643] YY=Yakima Yellow

[0644] definition

[0645] Ser(GN) is a GalNAc-C4 building block attached to a serinol-derived linker moiety:

[0646]

[0647] wherein O--- is a bond between an oxygen atom and, for example, H, a phosphodiester bond, or a phosphorothioate bond.

[0648] GN is:

[0649]

[0650] The C4XLT is:

[0651]

[0652] The C6XLT is:

[0653]

[0654] ST23 is:

[0655]

[0656] The synthesis of phosphoramidite derivatives of C4XLT (C4XLT-phos), phosphoramidite derivatives of C6XLT (C6XLT-phos) and phosphoramidite derivatives of ST23 (ST23-phos) can be carried out as described in WO2017 / 174657.

[0657] C4XLT-phos:

[0658]

[0659] C6XLT-phos:

[0660]

[0661] ST23-phos:

[0662]

[0663]

[0664]

[0665] Wherein G=H (before conjugation) or G=GN (after conjugation).

[0666] Conjugate 1

[0667] Antisense strand-STS16001AL33 (SEQ ID NO: 127)

[0668] 5′mU(ps)fU(ps)mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG(ps)fU(ps)mU(ps)Ser(GN)3′

[0669] Sense strand-STS16001BL20 (SEQ ID NO: 128)

[0670] 5'Ser(GN)(ps)fA mA fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA fU(ps)mA(ps)fA 3'

[0671] Conjugate 2

[0672] Antisense strand-STS16001A (SEQ ID NO: 129)

[0673] mU(ps)fU(ps)mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG(ps)fU(ps)mU

[0674] Sense strand-STS16001BV1L42 (SEQ ID NO: 130)

[0675] Ser(GN)(ps)fA(ps)mA(ps)fC mA fG mU fG mU fU mC fU mU fG mC fU mC fUmA fU(ps)mA(ps)fA(ps)Ser(GN)

[0676] Conjugate 3

[0677] Protein-STS16001AL33(SEQ ID NO:127)

[0678] 5′mU(ps)fU(ps)mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG(ps)fU(ps)mU(ps)Ser(GN)3′

[0679] SPECIFICATION-STS16001BV1L42(SEQ ID NO:130)

[0680] 5′Ser(GN)(ps)fA(ps)mA(ps)fC mA fG mU fG mU fU mC fU mU fG mC fU mC fUmA fU(ps)mA(ps)fA(ps)Ser(GN)3′

[0681] Conjugate 4

[0682] Protein-STS16001A(SEQ ID NO:129)

[0683] mU(ps)fU(ps)mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG(ps)fU(ps)mU

[0684] SPECIFICATION-STS16001BV1L75(SEQ ID NO:142)

[0685] 5'Ser(GN)fA(ps)mA(ps)fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mAfU(ps)mA(ps)fA Ser(GN)3

[0686] Conjugate 5

[0687] Protein-STS16001A(SEQ ID NO:129)

[0688] mU(ps)fU(ps)mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG(ps)fU(ps)mU

[0689] Sense strand - STS16001BV16L42 (SEQ ID NO:143)

[0690] 5’Ser(GN)(ps)fA mA fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA fUmA fA(ps)Ser(GN)3’

[0691] Conjugate 6

[0692] Antisense strand - STS16001A (SEQ ID NO:129)

[0693] mU(ps)fU(ps)mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG(ps)fU(ps)mU

[0694] Sense strand - STS16001BV20L75 (SEQ ID NO:144)

[0695] 5’Ser(GN)fA mA fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA fU mAfA Ser(GN)3’

[0696] Conjugate 7

[0697] Antisense strand - (SEQ ID NO:129)

[0698] mU(ps)fU(ps)mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG(ps)fU(ps)mU

[0699] Sense strand - STS16001BV1L94 (SEQ ID NO:145)

[0700] 5’Ser(GN)(ps)Ser(GN)(ps)fA(ps)mA(ps)fC mA fG mU fG mU fU mC fU mU fGmC fU mC fU mA fU(ps)mA(ps)fA(ps)Ser(GN)(ps)Ser(GN)3’

[0701] Conjugate 8

[0702] Antisense strand - STS16001A (SEQ ID NO:129)

[0703] 5’mU(ps)fU(ps)mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG(ps)fU(ps)mU 3’

[0704] Sense strand - STS16001V1BL96 (SEQ ID NO:146)

[0705] 5’C6Am(GN)(ps)fA(ps)mA(ps)fC mA fG mU fG mU fU mC fU mU fG mC fU mCfU mA fU(ps)mA(ps)fA(ps)C7Am(GN)3’

[0706] Conjugate 9

[0707] Antisense strand - STS16001A (SEQ ID NO:129)

[0708] 5’mU(ps)fU(ps)mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG(ps)fU(ps)mU 3’

[0709] Sense strand - STS16001V1BL97 (SEQ ID NO:147)

[0710] 5’GlyC3Am(GN)(ps)fA(ps)mA(ps)fC mA fG mU fG mU fU mC fU mU fG mC fUmC fU mA fU(ps)mA(ps)fA(ps)GlyC3Am(GN)3’

[0711] Conjugate 10

[0712] Antisense strand - STS16001A (SEQ ID NO:129)

[0713] 5’mU(ps)fU(ps)mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG(ps)fU(ps)mU 3’

[0714] Sense strand (SEQ ID NO: 148)

[0715] 5'PipAm(GN)(ps)fA(ps)mA(ps)fC mA fG mU fG mU fU mC fU mU fG mC fU mCfU mA fU(ps)mA(ps)fA(ps)PipAm(GN)3'

[0716] Conjugate 11

[0717] Antisense strand-STS16001A (SEQ ID NO: 129)

[0718] 5'mU(ps)fU(ps)mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG(ps)fU(ps)mU 3'

[0719] Sense strand-STS16001V1BL88 (SEQ ID NO: 149)

[0720] 5'C3Am(GN)(ps)fA(ps)mA(ps)fC mA fG mU fG mU fU mC fU mU fG mC fU mCfU mA fU(ps)mA(ps)fA(ps)C3Am(GN)3'

[0721] Conjugate 12

[0722] Antisense strand-STS16001A (SEQ ID NO: 129)

[0723] 5'mU(ps)fU(ps)mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG(ps)fU(ps)mU 3'

[0724] Sense strand-STS16001V1BL87 (SEQ ID NO: 150)

[0725] 5'C6Am(GN)(ps)fA(ps)mA(ps)fC mA fG mU fG mU fU mC fU mU fG mC fU mCfU mA fU(ps)mA(ps)fA(ps)GlyC3Am(GN)3'

[0726] Conjugate 15

[0727] Antisense strand (SEQ ID NO: 151)

[0728] mU(ps)fC(ps)mU fU mC fU mU fA mA fA mC fU mG fA mG fU mU(ps)fU(ps)mC

[0729] Sense strand (SEQ ID NO:152)

[0730] Ser(GN)(ps)fG(ps)mA(ps)fA mA fC mU fC mA fG mU fU mU fA mA fG mA fA(ps)mG(ps)fA(ps)Ser(GN)

[0731] Conjugate 16

[0732] Antisense strand (SEQ ID NO:153)

[0733] mA(ps)fU(ps)mG fU mA fG mC fC mG fA mG fG mA fU mC fU mU(ps)fC(ps)mU

[0734] Sense strand (SEQ ID NO:154)

[0735] Ser(GN)(ps)fA(ps)mG(ps)fA mA fG mA fU mC fC mU fC mG fG mC fU mA fC(ps)mA(ps)fU(ps)Ser(GN)

[0736] Conjugate 18

[0737] Antisense strand (SEQ ID NO:155)

[0738] mA(ps)fA(ps)mC fC mA fG mA fA mG fA mA fG mC fA mG fG mU(ps)fG(ps)mA

[0739] Sense strand (SEQ ID NO:156)

[0740] Ser(GN)(ps)fU(ps)mC(ps)fA mC fC mU fG mC fU mU fC mU fU mC fU mG fG(ps)mU(ps)fU(ps)Ser(GN)

[0741] Conjugate 19

[0742] Antisense strand (SEQ ID NO:135)

[0743] mA(ps)fU(ps)mA fA mC fU mC fU mG fU mC fC mA fU mU fA mC(ps)fC(ps)mG

[0744] Sense strand (SEQ ID NO:136)

[0745] Ser(GN)(ps)fC(ps)mG(ps)fG mU fA mA fU mG fG mA fC mA fG mA fG mU fU(ps)mA(ps)fU(ps)Ser(GN)

[0746] Reference conjugate 1

[0747] Antisense strand - STS16001A (SEQ ID NO:129)

[0748] mU(ps)fU(ps)mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG(ps)fU(ps)mU

[0749] Sense strand – STS16001BL20 (SEQ ID NO:128)

[0750] Ser(GN)(ps)fA mA fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA fU(ps)mA(ps)fA

[0751] Reference conjugate 2

[0752] Antisense strand - STS16001AL33 (SEQ ID NO:127)

[0753] mU(ps)fU(ps)mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG(ps)fU(ps)mU(ps)Ser(GN)

[0754] Sense strand - STS16001BV1 (SEQ ID NO:157)

[0755] fA(ps)mA(ps)fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA fU(ps)mA(ps)fA

[0756] Reference conjugate 3 – "Luc"

[0757] Antisense strand-STS18001A (A0130, SEQ ID NO: 132)

[0758] mU(ps)fC(ps)mG fA mA fG mU fA mU fU mC fC mG fC mG fU mA(ps)fC(ps)mG

[0759] Sense strand-STS18001BL4 (A0131, SEQ ID NO: 133)

[0760] [(ST23)(ps)]3C4XLT(ps)fC mG fU mA fC mG fC mG fG mA fA mU fA mC fU mUfC(ps)mG(ps)fA

[0761] Reference conjugate 4

[0762] Antisense strand-STS16001AL33 (SEQ ID NO: 127)

[0763] mU(ps)fU(ps)mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG(ps)fU(ps)mU

[0764] Sense strand-STS16001BL4 (SEQ ID NO: 134)

[0765] 5′[(ST23)(ps)]3C4XLT(ps)fA(ps)mA(ps)fC mA fG mU fG mU fU mC fU mU fGmC fU mC fU mA fU(ps)mA(ps)fA

[0766] Reference conjugate 5 – "Ctr"

[0767] Antisense strand (SEQ ID NO: 138)

[0768] mC(ps)fU(ps)mU fA mC fU mC fU mC fG mC fC mC fA mA fG mC(ps)fG(ps)mA

[0769] Sense strand (SEQ ID NO: 139)

[0770] [(ST23)(ps)]3(C6XLT)(ps)fU mC fG mC fU mU fG mG fG mC fG mA fG mA fGmU fA(ps)mA(ps)fG

[0771] Reference conjugate 6

[0772] Antisense strand (SEQ ID NO:151)

[0773] mU(ps)fC(ps)mU fU mC fU mU fA mA fA mC fU mG fA mG fU mU(ps)fU(ps)mC

[0774] Sense strand (SEQ ID NO:158)

[0775] [ST23(ps)]3ltrb(ps)fG mA fA mA fC mU fC mA fG mU fU mU fA mA fG mA fA(ps)mG(ps)fA

[0776] Reference conjugate 7

[0777] Antisense strand (SEQ ID NO:153)

[0778] mA(ps)fU(ps)mG fU mA fG mC fC mG fA mG fG mA fU mC fU mU(ps)fC(ps)mU

[0779] Sense strand (SEQ ID NO:159)

[0780] [ST23(ps)]3ltrb(ps)fA mG fA mA fG mA fU mC fC mU fC mG fG mC fU mA fC(ps)mA(ps)fU

[0781] Reference conjugate 8

[0782] Antisense strand (SEQ ID NO:160)

[0783] mU(ps)fA(ps)mC fC mA fG mA fA mG fA mA fG mC fA mG fG mU(ps)fG(ps)mA

[0784] Sense strand (SEQ ID NO:161)

[0785] [ST23(ps)]3ST41(ps)fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mG fG(ps)mU(ps)fA

[0786] Reference conjugate 9

[0787] Antisense strand (SEQ ID NO: 135)

[0788] mA(ps)fU(ps)mA fA mC fU mC fU mG fU mC fC mA fU mU fA mC(ps)fC(ps)mG

[0789] Sense strand (SEQ ID NO: 162)

[0790] [ST23(ps)]3C6XLT(ps)fC mG fG mU fA mA fU mG fG mA fC mA fG mA fG mUfU(ps)mA(ps)fU

[0791] Example 10 - In vitro determination of TTR knockdown achieved with various TTR siRNA GalNAc conjugates

[0792] Conjugates 4 to 7

[0793] The methods described above under "In vitro experiments" in the General Methods section were followed.

[0794] Compared to the negative control (see "UT" column and Luc [reference conjugate 3]), target gene expression in primary murine hepatocytes 24 hours after treatment with the conjugates of the invention, i.e., conjugates 4-7 at 0.01 nM, 0.1 nM, 0.5 nM, 1 nM, and 10 nM, showed that target gene expression decreased with increasing doses of the conjugates, as shown in Figure 11. This indicates that the first strand binds to the target gene, thereby reducing gene expression.

[0795] The in vitro data showed that where one or two serinol-derivatized linker moieties were provided at the 5' and 3' ends of the sense strand in conjugates 4-7, the number of phosphorothioate (PS) bonds between the terminal nucleotide and the linker and / or between the terminal three nucleotides in the sense strand could be varied while maintaining efficacy in reducing target gene expression.

[0796] Conjugates 8 to 12 and 19

[0797] The methods described above under "In vitro experiments" in the General Methods section were followed.

[0798] Compared to the negative control (see "UT" column and Luc [reference conjugate 3]), target gene expression in primary mouse hepatocytes 24 hours after treatment with the conjugates of the invention, i.e., conjugates 8-12 at 0.01 nM, 0.1 nM, 0.5 nM, 1 nM, and 10 nM, showed that target gene expression decreased with increasing doses of the conjugates, as shown in Figure 12. This indicates that the first strand binds to the target gene, thereby reducing gene expression. In particular, conjugates 8, 9, 10, and 11 appear to be comparable or better than conjugate 2, which was previously shown to be the most effective conjugate at 0.01 nM.

[0799] Conjugate 19 was also shown to reduce target gene expression compared to the negative control (see the "UT" column and is a non-targeting siRNA and is also referred to as the Ctr of reference conjugate 5), as shown in Fig.13 This indicates that the first strand binds to the target gene, thereby reducing gene expression.

[0800] The in vitro data of conjugates 8-12 and 19 show that many linkers conjugated to both ends of the sense strand are different in structure and effectively reduce target gene expression. Compared with "Luc" (for conjugates 8-12), which is reference conjugate 3, "Ctr" (for conjugate 19), which is reference conjugate 5, and untreated controls, conjugates 8-12 and 19 are more effective in reducing target gene expression.

[0801] Example 11 - In vivo time course of serum Ttr, Aldh2 and Tmprss6 in mice Conjugates 15 to 18

[0802] The methods described above under "In vivo experiments" in the General Methods section were followed.

[0803] Results of the time course of serum Aldh2 in a cohort of n=6 c57BL / 6 mice at 14, 28 and 42 days after subcutaneous treatment with 1 mg / kg conjugates 15 and 16, reference conjugates 6 and 7, and mock-treated (PBS) individuals are shown in Fig.14 and Fig.15 In. Fig.14 and Fig.15 As indicated by the data in , the conjugates of the present invention are particularly effective in reducing target gene expression compared to the negative control (PBS) and reference conjugates 6 and 7, respectively.

[0804] Results of the time course of serum Tmprss6 in a cohort of n=6 c57BL / 6 mice at 14, 28 and 42 days after subcutaneous treatment with 1 mg / kg conjugate 18, reference conjugate 8, and mock-treated (PBS) individuals are shown in Fig.16 In. Fig.16As indicated by the data in , the conjugates of the present invention are particularly effective in reducing target gene expression compared to the negative control (PBS) and the reference conjugate 8.

[0805] In summary, the in vivo data show that multiple example linkers conjugated to both ends of the second strand are effective in reducing target gene expression in vivo. Compared to the triantennary GalNAc linker control at the 5' end of the second strand (reference conjugates 6, 7 and 8), the positioning of the linker improves the in vivo efficacy of the conjugate.

[0806] Example 12 - Serum stability study

[0807] The method described above under "Decontaminated lysosomal stability assay" in the General Methods section was followed.

[0808] Fig.17 Results from serum stability studies for conjugates 2, 4, 5, 6, and 7 are shown. Fig.18 The serum stability of conjugates 2, 8, 9, 10, 11 and 12 is shown.

[0809] All conjugates of the invention tested were more stable in serum compared to the control.

[0810] All tested conjugates each contained one GalNAc linker unit at the 5' end of the second strand and another GalNAc linker unit at the 3' end of the second strand. Unless stated differently, siRNAs were modified with alternating 2'-OMe / 2'-F and each contained two phosphorothioate (PS) internucleotide linkages at their 5' and 3' terminal two internucleotide linkages.

[0811] In conjugate 4, the serinol-GalNAc unit is connected by a phosphodiester bond. In conjugate 5, the serinol-GalNAc unit is conjugated by PS, and all internucleotide linkages in the second chain are phosphodiester. In conjugate 6, the second chain does not contain PS. In conjugate 7, two serinol-GalNAc units are connected to each second chain end by a PS bond, and are connected to each other by a PS bond at the corresponding ends. In conjugate 8, the C6-amino modifier at the 5' end of the second chain and the C7-amino modifier at the 3' end of the second chain are applied to ligand connection. In conjugate 9, Gly-C3-amino modifier, in conjugate 10, piperidinyl-amino modifier, in conjugate 11, C3-amino modifier, and in conjugate 2, serinol-GalNAc unit, as a linker for achieving conjugation with the two ends of the second chain. In conjugate 2, PS is PS between the two terminal nucleotides and the nucleotide-serinol bond. In conjugate 12, a C6-amino modifier at the 5' end of the second strand and a GlyC3-amino modifier at the 3' end of the second strand were applied for ligand attachment. "ut" indicates an untreated sample to which other samples were normalized. "Luc" indicates a siRNA targeting luciferase (reference conjugate 3), which was used as a non-targeting control and did not reduce the target mRNA level.

[0812] The data show that where serinol-derivatized linker moieties are provided at the 5' and 3' ends of the sense strand, the number of phosphorothioate (PS) bonds between the terminal nucleotide and the linker and / or between the terminal three nucleotides in the sense strand can be varied while maintaining stability in serum.

[0813] Example 13

[0814] Primary human (Lot Hu1823) and cynomolgus monkey (Lot CY367) hepatocytes and culture media were from Life Technologies. Primary hepatocytes were thawed and plated in plating medium consisting of Williams' E medium (Life Technologies) supplemented with 5% fetal bovine serum, 1 μM dexamethasone in DMSO (final concentration of DMSO = 0.01%) and 3.6% v / v of Thawing / Plating Mixture-A (Thermo Fisher Scientific, CM3000) as described by the manufacturer.

[0815] Human primary hepatocytes were seeded at a density of 30,000 cells per well in collagen I-coated 96-well plates (Life Technologies). Cynomolgus monkey hepatocytes were seeded at a density of 45,000 cells per well. Conjugate 21 and control GalNAc-conjugated siRNA were serially diluted 5-fold in the concentration range of 0.006–100 nM and added immediately after plating in plating medium. The plates were then incubated at 37°C in a 5% CO2 atmosphere for 24 hours. Subsequently, the cells were lysed and RNA was isolated using the method described below.

[0816] Conjugate 21 sequence

[0817] Antisense strand-Conjugate 21 (SEQ ID NO: 165)

[0818] 5′mA(ps)fU(ps)mA fA mC fU mC fU mG fU mC fC mA fU mU fA mC(ps)fC(ps)mG 3′

[0819] Sense strand-STS16001BL20 (SEQ ID NO: 164)

[0820] 5′[ST23(ps)]3C6XLT(ps)mC mG mG mU mA mA fU fG fG mA mC mA mG mA mG mUmU(ps)mA(ps)mU 3′

[0821] Total RNA was extracted using the InviTrap HTS 96-well kit (Stratec Molecular GmbH, Berlin, Germany) according to the manufacturer's instructions, with the following changes to the protocol: After the final wash step, the plate was centrifuged at 6,000 rpm for 20 minutes. Subsequently, the RNA binding plate was placed on top of the elution plate and RNA was eluted by two rounds of adding 30 μl elution buffer and incubating at room temperature for 2 minutes, followed by centrifugation at 1,000 rpm for 1 minute. The final elution step was performed by centrifugation at 1700 g (4000 rpm) for 3 minutes, and the RNA was stored at -80°C.

[0822] 10 μl of RNA solution was used for gene expression analysis by reverse transcription quantitative polymerase chain reaction (RT-qPCR) with amplicon sets / sequences of LPA, ACTB (Eurogentec Deutschland GmbH, Cologne, Germany), APOB and PLG (BioTez GmbH, Berlin, Germany).

[0823] RT-qPCR reactions were performed using ABI StepOne Plus (Applied Biosystems, a division of Thermo Fisher Scientific, Massachusetts, USA) using a standard protocol for RT-PCR (48°C for 30 min, 95°C for 10 min, 40 cycles of 95°C for 15 sec, then 60°C for 1 min).

[0824] In primary hepatocytes, LPA and PLG analysis was performed in singleplex assays (primers: 300 nM, probe: 100 nM). APOB (200 nM) and ACTB (300 nM) were run in multiplex assays, with 100 nM of each probe added to the standard mix.

[0825] By using the comparative CT method (also known as 2 -ΔΔCt Data were calculated using the method of Livak and Schmittgen, 2001 and Schmittgen and Livak, 2008. Here, the amount of APOB, LPA or PLG mRNA normalized to the endogenous reference ACTB relative to the calibrator (untreated control) is given by

[0826] Fold change = 2 -ΔΔCt .

[0827] Unless otherwise stated, all values ​​presented in the examples are mean ± SD. IC was calculated using sigmoidal 4-parameter dose-response curves in GraphPadPrism 7. 50 value.

[0828] Fig.19 Conjugate 21 knocked down LPA mRNA via receptor-mediated uptake into primary human hepatocytes 24 hours after siRNA treatment (IC 50 =3.6 nM). LPA mRNA expression levels were normalized to ACTB and to cells treated with non-targeting siRNA control measured by RT-qPCR. Data are presented as mean ± SD of a single experiment. Fig. 20 Conjugate 21 knocked down LPA mRNA in primary cynomolgus monkey hepatocytes via receptor-mediated uptake 24 hours after siRNA treatment (IC 50 =0.7 nM). LPA mRNA expression levels were normalized to ACTB and to cells treated with non-targeting siRNA control measured by RT-qPCR. Data are presented as mean ± SD of a single experiment.

[0829] Fig.21Conjugate 21 knocks down APOB mRNA by receptor-mediated uptake into primary human hepatocytes 24 hours after siRNA treatment. APOB mRNA expression levels were normalized to ACTB and to cells treated with non-targeting siRNA control measured by RT-qPCR. Data are presented as mean ± SD of a single experiment.

[0830] Fig. 22 Shown is knockdown of PLG mRNA by conjugate 21 via receptor-mediated uptake into primary human hepatocytes 24 hours after siRNA treatment. PLG mRNA expression levels were normalized to ACTB and to cells treated with non-targeting siRNA control measured by RT-qPCR. Data are presented as mean ± SD of a single experiment.

[0831] Conjugate 21 achieved high levels of LPA mRNA inhibition without affecting the levels of APOB and PLG mRNA.

[0832] Embodiment 14

[0833] To test the efficacy of conjugate 21 in vivo, pharmacodynamic studies were performed using male cynomolgus monkeys. The animals were divided into 4 groups of 3 animals each. Before dosing, serum was prepared to establish the baseline Lp(a) level of each animal. On day 1, conjugate 21 was formulated in 0.9% saline, and each animal received a single dose of conjugate 21 at doses of 0.1, 0.3, 1.0, and 3.0 mg / kg.

[0834] Serial samples were collected over 29 days and Lp(a) levels were measured by ELISA (Mercodia, Catalog No. 10-1106-01 Lot: 27736; Uppsala, Sweden). All values ​​were normalized to the baseline value of each individual animal collected before dosing and presented as a percentage of the starting level ( Fig.23 ). Doses of 1.0 and 3.0 mg / kg showed significant reductions in serum Lp(a) levels (69.4% and 77.3% reductions after 29 days, respectively). Dose-dependent effects lead to ED 50 The dose is 0.56 mg / kg ( Fig.24 ). These data indicate that a significant and sustained reduction in serum Lp(a) can be achieved with a single dose of conjugate 21. Based on these data, infrequent dosing would be expected, no more than once every two months.

[0835] Claim of Invention

[0836] The following is the claim of the invention:

[0837] 1. A nucleic acid for inhibiting the expression of LPA in a cell, the nucleic acid comprising at least one duplex region, the duplex region comprising at least a portion of a first strand and at least a portion of a second strand at least partially complementary to the first strand, wherein the first strand is at least partially complementary to at least a portion of RNA transcribed from an LPA gene, wherein the first strand comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 9, 5, 1, 3, 7, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43.

[0838] 2. A nucleic acid as described in statement 1, wherein the first chain comprises the nucleotide sequence of SEQ ID NO:9, and optionally, wherein the second chain comprises the nucleotide sequence of SEQ ID NO:10; or wherein the first chain comprises the nucleotide sequence of SEQ ID NO:5, and optionally, wherein the second chain comprises the nucleotide sequence of SEQ ID NO:6.

[0839] 3. A nucleic acid for inhibiting the expression of LPA in a cell, the nucleic acid comprising at least one duplex region, the duplex region comprising at least a portion of a first strand and at least a portion of a second strand at least partially complementary to the first strand, wherein the first strand is at least partially complementary to at least a portion of RNA transcribed from an LPA gene, wherein the first strand comprises a nucleotide sequence selected from the following sequences: SEQ ID NO: 9 or 5.

[0840] 4. The nucleic acid of any of the preceding statements, wherein the first strand and / or the second strand are each 17-35 nucleotides in length.

[0841] 5. The nucleic acid of any of the preceding statements, wherein the at least one duplex region consists of 17-25, preferably 19-25, consecutive nucleotide base pairs.

[0842] 6. A nucleic acid as described in any of the preceding statements, wherein the nucleic acid:

[0843] a) are blunt-ended at both ends; or

[0844] b) has a protruding portion at one end and a blunt end at the other end; or

[0845] c) having overhangs at both ends.

[0846] 7. The nucleic acid of any of the preceding statements, wherein one or more nucleotides on the first strand and / or the second strand are modified to form modified nucleotides.

[0847] 8. A nucleic acid as described in any of the preceding statements, wherein the nucleic acid comprises phosphorothioate bonds between one, two or three terminal 3' nucleotides and / or 5' nucleotides at one or both ends of the first strand and / or the second strand.

[0848] 9. The nucleic acid of any of the preceding statements, wherein the nucleic acid is conjugated to a ligand.

[0849] 10. The nucleic acid of claim 9, wherein the ligand comprises (i) one or more N-acetylgalactosamine (GalNAc) moieties or derivatives thereof, and (ii) a linker, wherein the linker conjugates the at least one GalNAc moiety or derivative thereof to the nucleic acid.

[0850] 11. The nucleic acid of any one of statements 9 to 10, wherein the nucleic acid is conjugated to a ligand comprising a compound of formula (I):

[0851] [SX 1 -PX 2 ]3-AX 3 -(I)

[0852] in:

[0853] S represents a sugar, preferably, wherein the sugar is N-acetylgalactosamine;

[0854] X 1 represents C3-C6 alkylene or (-CH2-CH2-O) m (-CH2)2-, where m is 1, 2 or 3;

[0855] P is phosphate or a modified phosphate, preferably phosphorothioate;

[0856] X 2 is an alkylene group or a group of formula (-CH2) n -O-CH2-alkylene ether, wherein n=1-6;

[0857] A is the branching unit;

[0858] X 3 represents the bridge unit;

[0859] wherein the nucleic acid as defined in any one of statements 1 to 8 is conjugated to X via a phosphate or a modified phosphate (preferably via a phosphorothioate) 3 .

[0860] 12. The nucleic acid of any one of statements 9 to 10, wherein the first RNA strand is a compound of formula (X):

[0861]

[0862] where b is 0 or 1; and

[0863] The second RNA strand is a compound of formula (XI):

[0864]

[0865] in:

[0866] c and d are independently 0 or 1;

[0867] Z1 and Z2 are RNA portions of the first RNA strand and the second RNA strand, respectively;

[0868] Y is O or S;

[0869] n is 0, 1, 2 or 3; and

[0870] L1 is a linker attached to the ligand; and

[0871] Where b+c+d is 2 or 3.

[0872] 13. A nucleic acid for inhibiting the expression of LPA in a cell, the nucleic acid comprising at least one duplex region, the duplex region comprising at least a portion of a first strand and at least a portion of a second strand at least partially complementary to the first strand, wherein the first strand is at least partially complementary to at least a portion of RNA transcribed from an LPA gene, wherein the first strand comprises the nucleotide sequence of SEQ ID NO: 9, and preferably consists of the nucleotide sequence of SEQ ID NO: 9, and optionally, wherein the second strand comprises the nucleotide sequence of SEQ ID NO. 10, and preferably consists of the nucleotide sequence of SEQ ID NO. 10.

[0873] 14. The nucleic acid of claim 13, wherein the nucleic acid is conjugated to a ligand and has the structure

[0874]

[0875] wherein Z is a nucleic acid according to statement 13 and is preferably conjugated to the 5' end of the second strand.

[0876] 15. A nucleic acid as described in statement 14, wherein the nucleic acid comprises two phosphorothioate bonds between each of the terminal three 3' nucleotides on the first chain and between each of the terminal three 5' nucleotides on the first chain, and two phosphorothioate bonds between the terminal three nucleotides at the 3' end of the second chain, and wherein the ligand is conjugated to the 5' end of the second chain.

[0877] 16. The nucleic acid of statement 13, wherein the nucleic acid is conjugated to a ligand, wherein the first RNA strand is a compound of formula (XV):

[0878]

[0879] where b is 0 or 1; and

[0880] The second RNA strand is a compound of formula (XVI):

[0881]

[0882] Where c and d are independently 0 or 1;

[0883] in:

[0884] Z1 and Z2 are RNA portions of the first RNA strand and the second RNA strand, respectively;

[0885] Y is O or S;

[0886] R1 is H or methyl;

[0887] n is 0, 1, 2 or 3; and

[0888] In formula (XV) and (XVI), L is the same or different and is selected from the group consisting of:

[0889] -(CH2) q , where q = 2-12;

[0890] -(CH2) r -C(O)-, wherein r = 2-12;

[0891] -(CH2-CH2-O) s -CH2-C(O)-, where s = 1-5;

[0892] -(CH2) t -CO-NH-(CH2) t -NH-C(O)-, wherein t is independently 1-5;

[0893] -(CH2) u -CO-NH-(CH2) u -C(O)-, wherein u is independently 1-5; and

[0894] -(CH2) v -NH-C(O)-, wherein v is 2-12; and

[0895] wherein if a terminal C(O) is present, it is attached to an NH group;

[0896] And where b+c+d is 2 or 3.

[0897] 17. The nucleic acid of any one of statements 13 to 16, comprising the sequence and modifications shown below:

[0898]

[0899] Where specific modifications are depicted by numbers

[0900] 1 = 2'F-dU;

[0901] 2 = 2'F-dA;

[0902] 3 = 2'F-dC;

[0903] 4 = 2'F-dG;

[0904] 5=2'-OMe-rU;

[0905] 6 = 2'-OMe-rA;

[0906] 7 = 2'-OMe-rC;

[0907] 8 = 2'-OMe-rG.

[0908] 18. A nucleic acid as described in any one of statements 13 to 16, wherein the nucleotides at positions 2 and 14 of the first strand from the 5' end are modified with a 2' fluoro modification, and the nucleotides on the second strand corresponding to positions 11, or 13, or 11 and 13, or 11-13 of the first strand are modified with a 2' fluoro modification.

[0909] 19. A composition comprising a nucleic acid as described in any one of statements 1 to 18, and optionally comprising a delivery vehicle and / or a physiologically acceptable excipient and / or a carrier and / or a diluent and / or a buffer and / or a preservative, for use as a medicament, preferably for preventing or treating a disease or pathology, or reducing the risk of a disease or pathology, wherein the disease or pathology is preferably a cardiovascular disease, wherein the cardiovascular disease is preferably stroke, atherosclerosis, thrombosis, coronary heart disease or aortic valve stenosis and / or any other disease or pathology associated with increased levels of Lp(a)-containing particles.

[0910] 20. A pharmaceutical composition comprising a nucleic acid as described in any one of statements 1 to 18, and further comprising a delivery vehicle, preferably a liposome, and / or a physiologically acceptable excipient and / or carrier and / or diluent.

[0911] 21. Use of a nucleic acid as described in any one of statements 1 to 18 or a pharmaceutical composition as described in statement 20 for preventing or treating a disease or pathology or reducing the risk of a disease or pathology, wherein the disease or pathology is preferably a cardiovascular disease, wherein the cardiovascular disease is preferably stroke, atherosclerosis, thrombosis, coronary heart disease or aortic valve stenosis and any other disease or pathology associated with increased levels of Lp(a) particles.

[0912] 22. A method for preventing or treating a disease, disorder or syndrome, the method comprising administering to an individual in need of treatment a composition comprising a nucleic acid as described in any one of statements 1 to 18 or a composition according to statements 19 to 20, preferably, wherein the nucleic acid or the composition is administered to the subject subcutaneously, intravenously or using any other administration route such as oral, rectal or intraperitoneal administration.

[0913] Overview Sequence Table

[0914]

[0915]

[0916]

[0917]

[0918]

[0919]

[0920]

[0921]

[0922]

[0923]

[0924]

[0925] A single sequence may have more than one name. In those cases, one of those names is given in the summary sequence table.

[0926] When specific linkers and / or modified linkages within a RNA sequence are taught such as (ps) and [ST23(ps)]3ST41(ps) etc., these are optional parts of the sequence but are preferred embodiments of that sequence.

[0927] The following abbreviations may be used, particularly in listed sequences:

[0928]

[0929]

[0930]

Claims

1. A nucleic acid for inhibiting the expression of LPA in a cell, the nucleic acid comprising at least one duplex region, the duplex region comprising at least a portion of a first strand and at least a portion of a second strand at least partially complementary to the first strand, wherein the first strand is at least partially complementary to at least a portion of RNA transcribed from an LPA gene, wherein the first strand comprises the following nucleotide sequence: 5'mA(ps)fU(ps)mA fA mC fU mC fU mG fU mC fC mA fU mU fA mC(ps)fC(ps)mG 3' (SEQ ID NO: 165), And wherein the second strand comprises the following nucleotide sequence: 5'mC mG mG mU mA mA fU fG fG mA mC mA mG mA mG mU mU(ps)mA(ps)mU 3′ (SEQ ID NO: 163); wherein fA, fC, fG and fU represent 2'-deoxy-2'-fluororibonucleotides; mA, mC, mG, and mU represent 2′-O-methyl ribonucleotides; and (ps) represents a phosphorothioate bond, wherein the first strand and the second strand are each 19 nucleotides in length, wherein the nucleic acid comprises phosphorothioate bonds between each of the terminal three 3' nucleotides on the first strand and between each of the terminal three 5' nucleotides on the first strand, and between the terminal three nucleotides at the 3' terminus of the second strand; and wherein the nucleic acid is conjugated to a ligand comprising (i) one or more N-acetylgalactosamine (GalNAc) moieties or derivatives thereof, and (ii) a linker, wherein the linker conjugates at least one GalNAc moiety or derivative thereof to the nucleic acid.

2. The nucleic acid of claim 1, wherein the first strand consists of the following nucleotide sequence: 5'mA(ps)fU(ps)mA fA mC fU mC fU mG fU mC fC mA fU mU fA mC(ps)fC(ps)mG 3′ (SEQ ID NO: 165); wherein fA, fC, fG and fU represent 2'-deoxy-2'-fluororibonucleotides; mA, mC, mG, and mU represent 2′-O-methyl ribonucleotides; and (ps) indicates a phosphorothioate bond.

3. The nucleic acid of claim 1 or claim 2, wherein the second strand consists of the following nucleotide sequence: 5'mC mG mG mU mA mA fU fG fG mA mC mA mG mA mG mU mU(ps)mA(ps)mU 3′ (SEQ ID NO: 163); wherein fA, fC, fG and fU represent 2'-deoxy-2'-fluororibonucleotides; mA, mC, mG, and mU represent 2'-O-methyl ribonucleotides; and (ps) indicates a phosphorothioate bond.

4. A nucleic acid as claimed in any preceding claim, wherein the ligand comprises a compound of formula (I): [S-X 1 -P-X 2 ]3-A-X 3 -(I) in: S represents a sugar, preferably, wherein the sugar is N-acetylgalactosamine; X 1 represents C3-C6 alkylene or (-CH2-CH2-O) m (-CH2)2-, where m is 1, 2 or 3; P is phosphate or a modified phosphate, preferably phosphorothioate; X 2 is an alkylene group or a group of formula (-CH2) n -O-CH2-alkylene ether, wherein n=1-6; A is the branching unit; X 3 represents the bridge unit; wherein the nucleic acid as defined in any one of claims 1 to 3 is conjugated to X via a phosphate or a modified phosphate, preferably via a phosphorothioate 3 , and the nucleic acid is preferably conjugated to X via the 5' end of the second strand 3 .

5. A nucleic acid as described in any preceding claim, wherein the nucleic acid is conjugated to a ligand and has the structure wherein Z is a nucleic acid according to any preceding claim, and the ligand is preferably conjugated to the 5' end of the second strand.

6. A nucleic acid as claimed in any one of the preceding claims, wherein the second strand comprises the nucleotide sequence 5'[ST23(ps)]3C6XLT(ps)mC mG mG mU mA mA fU fG fG mA mC mA mG mA mG mU mU(ps)mA(ps)mU 3′ (SEQ ID NO: 164); wherein fA, fC, fG and fU represent 2'-deoxy-2'-fluororibonucleotides; mA, mC, mG, and mU indicate 2′-O-methyl ribonucleotides; (ps) represents a phosphorothioate bond; and [ST23(ps)]3C6XLT(ps) 7. A nucleic acid as claimed in any one of the preceding claims, wherein the second strand consists of the following nucleotide sequence: 5′[ST23(ps)]3C6XLT(ps)mC mG mG mU mA mA fU fG fG mA mC mA mG mA mG mU mU(ps)mA(ps)mU 3' (SEQ ID NO: 164); wherein fA, fC, fG and fU represent 2'-deoxy-2'-fluororibonucleotides; mA, mC, mG, and mU indicate 2′-O-methyl ribonucleotides; (ps) indicates a phosphorothioate bond; and [ST23(ps)]3C6XLT(ps) 8. A composition comprising a nucleic acid as described in any one of claims 1 to 7, and optionally comprising a delivery vehicle and / or a physiologically acceptable excipient and / or a carrier and / or a diluent and / or a buffer and / or a preservative, for use as a medicament, preferably for preventing or treating a disease or pathology, or reducing the risk of a disease or pathology, wherein the disease or pathology is preferably a cardiovascular disease, wherein the cardiovascular disease is preferably stroke, atherosclerosis, thrombosis, coronary heart disease or aortic valve stenosis and / or any other disease or pathology associated with increased levels of Lp(a) particles.

9. A pharmaceutical composition comprising the nucleic acid according to any one of claims 1 to 7, and further comprising a delivery vehicle and / or a physiologically acceptable excipient and / or a carrier and / or a diluent.

10. Use of a nucleic acid as described in any one of claims 1 to 7 or a composition as described in claim 8 or claim 9 for the manufacture of a medicament for preventing or treating a disease or pathology or reducing the risk of a disease or pathology, wherein the disease or pathology is preferably a cardiovascular disease, wherein the cardiovascular disease is preferably stroke, atherosclerosis, thrombosis, coronary heart disease or aortic valve stenosis and any other disease or pathology associated with increased levels of Lp(a) particles.

11. Use of a nucleic acid as described in any one of claims 1 to 7 or a composition as described in claim 8 or claim 9 in the manufacture of a medicament for preventing or treating a disease, disorder or syndrome, wherein the composition is administered to an individual in need of treatment, preferably, wherein the nucleic acid or the composition is administered to the subject subcutaneously, intravenously or using any other administration route such as oral, rectal or intraperitoneal administration route.

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