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 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 increase in Lp (a)-containing 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 elevated Lp (a)-containing particles.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201880073189.3, application date November 13, 2018, and invention name “Nucleic acid for inhibiting the expression of LPA in cells”.

[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, anti-second-strand 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) is a heterogeneous low-density lipoprotein particle expressed primarily in the liver (Witztum and Ginsberg, J Lipid Res. 2016 March; 57(3): 336-9). It consists of apolipoprotein(a) (Apo(a) is encoded by the LPA gene) connected to LDL via the ApoB polypeptide. Genetically defined high Lp(a) serum levels 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 Lp(a) serum level 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). In addition to indirect standard general LDL lowering measures, there is no currently approved specific Lp(a) lowering therapy. Therefore, there is a 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:

[0006] A first 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 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, 43, 63, 65, 67, 69, 71 or 73.

[0007] In one embodiment, the nucleic acid comprises in the first strand 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 of any one of the reference 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, 43, 63, 65, 67, 69, 71 or 73.

[0008] In one embodiment, the number of single nucleotide mismatches in the first strand sequence relative to the portion with the reference sequence comprised in the first strand sequence is at most three, preferably at most two, more preferably at most one, and most preferably zero.

[0009] 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, 44, 64, 66, 68, 70, 72 or 74.

[0010] The first strand may comprise the nucleotide sequences of SEQ ID NO:5 and SEQ ID NO:9, and / or the second strand may comprise the nucleotide sequences of SEQ ID NO:6 and SEQ ID NO:10.

[0011] The length of the first strand and / or the second strand may each be 17-35 nucleotides, and the length of at least one duplex region may be 10-25 nucleotides. The duplex may comprise two separate strands, or it may comprise a single strand comprising the first strand and the second strand.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] At least one modification may be 2'-O-methyl, and / or at least one modification may be 2'-F.

[0021] As a second aspect, the present invention also provides 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, 43, 63, 65, 67, 69, 71 or 73, wherein the nucleotides of the first strand are modified by a first modification on odd-numbered nucleotides and by a second modification on even-numbered nucleotides, and the nucleotides of the second strand are modified by a third modification on even-numbered nucleotides and by a fourth modification on odd-numbered nucleotides, wherein at least the first modification is different from the second modification, and the third modification is different from the fourth modification. 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, 36, 38, 40, 42, 44, 64, 66, 68, 70, 72, or 74. The third modification and the first modification may be the same, and / or the second modification and the fourth modification may be the same.

[0022] The first modification may be 2'OMe, and the second modification may be 2'F.

[0023] In the nucleic acid of the second aspect, the first strand may comprise the nucleotide sequences of SEQ ID NO:5 and SEQ ID NO:9, and the second strand may comprise the nucleotide sequences of SEQ ID NO:6 and SEQ ID NO:10.

[0024] The sequences and modifications may be as shown in the following table; the table shows preferred sequences based on an excerpt from Table 1 as provided herein:

[0025] SEQ ID NO:5 5'auaacucuguccauuacca 3' 6162717181736152736 SEQ ID NO:6 5'ugguaauggacagaguuau 3' 1845261846364645161 SEQ ID NO:9 5'auaacucuguccauuaccg 3' 6162717181736152738 SEQ ID NO:10 5'cgguaauggacagaguuau 3' 3845261846364645161

[0026] The specific modifications are depicted by the following numbers:

[0027] 1 = 2'F-dU;

[0028] 2 = 2'F-dA;

[0029] 3 = 2'F-dC;

[0030] 4 = 2'F-dG;

[0031] 5=2'-OMe-rU;

[0032] 6 = 2'-OMe-rA;

[0033] 7 = 2'-OMe-rC;

[0034] 8 = 2'-OMe-rG.

[0035] The nucleic acid of the present invention may comprise a phosphorothioate bond between one, two or three terminal 3' nucleotides and / or one, two or three terminal 5' nucleotides of the first and / or second strands. It may comprise two phosphorothioate bonds between each of the three terminal 3' nucleotides on the first strand and between each of the three terminal 5' nucleotides on the first strand, and two phosphorothioate bonds between the three terminal nucleotides at the 3' end of the second strand.

[0036] This nucleic acid can be conjugated to a ligand.

[0037] As a third aspect, the present invention also provides 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: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 63, 65, 67, 69, 71 or 73, and wherein the nucleic acid is conjugated to a ligand.

[0038] The ligand 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.

[0039] The ligand may comprise Formula I:

[0040] [SX 1 -PX 2 ]3-AX 3 -(I)

[0041] in:

[0042] S represents a sugar, wherein the sugar is N-acetylgalactosamine;

[0043] X 1 represents C3-C6 alkylene or (-CH2-CH2-O) m (-CH2)2-, where m is 1, 2 or 3;

[0044] P is phosphate or a modified phosphate (preferably phosphorothioate);

[0045] X 2 is an alkylene group or a group of formula (-CH2) n -O-CH2-alkylene ether, wherein n=1-6;

[0046] A is the branching unit;

[0047] X 3 represents the bridge unit;

[0048] wherein the nucleic acid of the invention is conjugated to X via phosphate or modified phosphate (preferably via phosphorothioate) 3 .

[0049] Therefore, the present invention further provides a conjugated nucleic acid having one of the following structures

[0050]

[0051]

[0052]

[0053]

[0054]

[0055] wherein Z represents a nucleic acid as defined herein before.

[0056] Alternatively, the nucleic acid of the invention may be conjugated to a ligand having the structure

[0057]

[0058] 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 according to any aspect of the present invention and a 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 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:

[0059] (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

[0060] (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, or

[0061] The present invention relates to a conjugate for inhibiting the expression of an LPA gene in a cell, the conjugate comprising a nucleic acid portion and a 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 RNA transcribed from the LPA gene, the ligand portion comprising a 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:

[0062] (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

[0063] (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.

[0064] The linker moiety can be, for example, a serinol-derivatized linker moiety, or one of the other linker types described herein.

[0065] 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 a physiologically acceptable excipient.

[0066] 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.

[0067] 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.

[0068] Following subcutaneous administration, the subject invention can be delivered to the liver (hepatocytes) in a tissue-specific manner and specifically targets LPA to reduce undesirable side effects and achieve lower therapeutic doses necessary to achieve the desired effect.

[0069] The invention or pharmaceutical compositions 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 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 for the reduction of 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.

[0070] Also included is a method of making a nucleic acid or conjugated nucleic acid of the invention. DETAILED DESCRIPTION

[0071] 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.

[0072] A first 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 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, 43, 63, 65, 67, 69, 71 or 73.

[0073] The nucleic acid may comprise: a first strand comprising a nucleotide sequence of SEQ ID NO: 9, and optionally, wherein the second strand comprises a nucleotide sequence of SEQ ID NO: 10; or a first strand comprising a nucleotide sequence of SEQ ID NO: 5, and optionally, wherein the second strand comprises a nucleotide sequence of SEQ ID NO: 6; or a first strand comprising a nucleotide sequence of SEQ ID NO: 1, and optionally, wherein the second strand comprises a nucleotide sequence of SEQ ID NO: 2; or a first strand comprising a nucleotide sequence of SEQ ID NO: 4, and optionally, wherein the second strand comprises a nucleotide sequence of SEQ ID NO: 4; or a first strand comprising a nucleotide sequence of SEQ ID NO: 7, and optionally, wherein the second strand comprises a nucleotide sequence of SEQ ID NO: 8; or a first strand comprising a nucleotide sequence of SEQ ID NO: 11, and optionally, wherein the second strand comprises a nucleotide sequence of SEQ ID NO: 12; or a first strand comprising a nucleotide sequence of SEQ ID NO: 13, and optionally, wherein the second strand comprises a nucleotide sequence of SEQ ID NO: 14; or a first strand comprising a nucleotide sequence of SEQ ID NO: 15, and optionally, wherein the second strand comprises a nucleotide sequence of SEQ ID NO: NO:16; or a first strand containing a nucleotide sequence of SEQ ID NO:17, and optionally, wherein the second strand contains a nucleotide sequence of SEQ ID NO:18; or a first strand containing a nucleotide sequence of SEQ ID NO:19, and optionally, wherein the second strand contains a nucleotide sequence of SEQ ID NO:20; or a first strand containing a nucleotide sequence of SEQ ID NO:21, and optionally, wherein the second strand contains a nucleotide sequence of SEQ ID NO:22; or a first strand containing a nucleotide sequence of SEQ ID NO:23, and optionally, wherein the second strand contains a nucleotide sequence of SEQ ID NO:24; or a first strand containing a nucleotide sequence of SEQ ID NO:25, and optionally, wherein the second strand contains a nucleotide sequence of SEQ ID NO:26; or a first strand containing a nucleotide sequence of SEQ ID NO:27, and optionally, wherein the second strand contains a nucleotide sequence of SEQ ID NO:28; or a first strand containing a nucleotide sequence of SEQ ID NO:29, and optionally, wherein the second strand contains a nucleotide sequence of SEQ ID NO:30; or a first strand containing a nucleotide sequence of SEQ ID NO:31. NO:31, and optionally, wherein the second strand comprises the nucleotide sequence of SEQ ID NO:32; or a first strand comprising the nucleotide sequence of SEQ ID NO:33, and optionally, wherein the second strand comprises the nucleotide sequence of SEQ ID NO:34;or a first strand containing a nucleotide sequence of SEQ ID NO:35, and optionally, wherein the second strand contains a nucleotide sequence of SEQ ID NO:36; or a first strand containing a nucleotide sequence of SEQ ID NO:37, and optionally, wherein the second strand contains a nucleotide sequence of SEQ ID NO:38; or a first strand containing a nucleotide sequence of SEQ ID NO:39, and optionally, wherein the second strand contains a nucleotide sequence of SEQ ID NO:40; or a first strand containing a nucleotide sequence of SEQ ID NO:41, and optionally, wherein the second strand contains a nucleotide sequence of SEQ ID NO:42; or a first strand containing a nucleotide sequence of SEQ ID NO:43, and optionally, wherein the second strand contains a nucleotide sequence of SEQ ID NO:44; or a first strand containing a nucleotide sequence of SEQ ID NO:63, and optionally, wherein the second strand contains a nucleotide sequence of SEQ ID NO:64; or a first strand containing a nucleotide sequence of SEQ ID NO:65, and optionally, wherein the second strand contains a nucleotide sequence of SEQ ID NO:66; or a first strand containing a nucleotide sequence of SEQ ID NO:67. NO:67, and optionally, wherein the second strand comprises the nucleotide sequence of SEQ ID NO:68; or a first strand comprising a nucleotide sequence of SEQ ID NO:69, and optionally, wherein the second strand comprises the nucleotide sequence of SEQ ID NO:70; or a first strand comprising a nucleotide sequence of SEQ ID NO:71, and optionally, wherein the second strand comprises the nucleotide sequence of SEQ ID NO:72; or a first strand comprising a nucleotide sequence of SEQ ID NO:73, and optionally, wherein the second strand comprises the nucleotide sequence of SEQ ID NO:74. ;

[0074] 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.

[0075] A related aspect of the invention 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, 43, 63, 65, 67, 69, 71 or 73, 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.

[0076] 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 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] The portion of the first strand and the portion of the second strand that form at least one duplex region may be fully complementary and at least partially complementary to each other. Depending on the length of the nucleic acid, a complete match in terms of base complementarity between the first strand and the second strand is not necessarily required. However, the first strand and the second strand must be able to hybridize under physiological conditions.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] The nucleic acid may comprise a second sequence comprising the 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, 44, 64, 66, 68, 70, 72 or 74.

[0087] 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.

[0088] Complementarity between the first strand and the target sequence may be perfect (no nucleotide mismatches or extra / missing nucleotides in either nucleic acid).

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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 which may be consecutive.

[0095] The nucleic acid may comprise a first strand sequence of SEQ ID NO: 5 or SEQ ID NO: 9. The nucleic acid may comprise a second strand sequence of SEQ ID NO: 6 or SEQ ID NO: 10.

[0096] Preferably, the nucleic acid mediates RNA interference.

[0097] 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 with any one 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, 43, 63, 65, 67, 69, 71 or 73. At least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95%, and most preferably 100% sequence identity.

[0098] 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.

[0099] The nucleic acid can be blunt ended at both ends; have an overhang at one end and a blunt end at the other end; or have an overhang at both ends.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] One or more nucleotides on the second strand and / or the first strand of the nucleic acid of the invention may be modified.

[0106] 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.

[0107] 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.

[0108] 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. 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.

[0109] 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 connected to cholesterol derivatives or dodecanoic acid bisdecylamide groups.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] As used herein, modified nucleic acids may include one or more of the following:

[0116] (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);

[0117] (ii) a change, such as a substitution, to the composition of the ribose, such as the 2′ hydroxyl group on the ribose;

[0118] (iii) replacement of the phosphate moiety with a "dephospho" linker;

[0119] (iv) modification or substitution of naturally occurring bases;

[0120] (v) substitutions or modifications to the ribose-phosphate backbone;

[0121] (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.

[0122] The terms substitution, modification, alteration indicate a deviation from the naturally occurring molecule.

[0123] Specific modifications are discussed in more detail below.

[0124] 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).

[0125] 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.

[0126] 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.

[0127] 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).

[0128] "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.

[0129] 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.

[0130] 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.

[0131] The 2' modification may be used in combination with one or more phosphate linker modifications (eg, phosphorothioate).

[0132] The phosphate groups can individually be replaced by non-phosphorus containing linkers.

[0133] 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.

[0134] The phosphate linker and ribose sugar can be replaced by nuclease-resistant nucleotides.

[0135] Examples include morpholino, cyclobutyl, pyrrolidine, and peptide nucleic acid (PNA) nucleoside surrogates. In certain embodiments, PNA surrogates may be used.

[0136] 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.

[0137] 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).

[0138] Terminal modifications may be added for a number of reasons, including to modulate activity or to modulate resistance to degradation. Terminal modifications useful for modulating activity include modification of the 5′ 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′-monothioate (phosphorothioate; (HO)2(S)P—O-5′); 5′-monodisulfide Phosphorus acid esters (dithiophosphates; (HO)(HS)(S)P—O-5′), 5′-thiophosphates ((HO)2(O)P—S-5′); any additional combinations 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., such as RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′ vinylphosphonates, 5′-alkyl ether phosphonates (R=alkyl ether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., such as RP(OH)(O)—O-5′-).

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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).

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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'.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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 the second modification.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] At least one modification may be 2'-O-methyl, and / or at least one modification may be 2'-F. Other modifications as described herein may be present on the first strand and / or the second strand.

[0168] 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.

[0169] Throughout the present 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.

[0170] 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.

[0171] 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 that is a 2'-O-methyl (2'OMe) that may be a first modification, and a second modification that is a 2'-F. The nucleic acids of the invention may also include phosphorothioate modifications and / or deoxy modifications that may be present in or between the terminal 1, 2 or 3 nucleotides at each or any end of each or both strands.

[0172] 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, 43, 63, 65, 67, 69, 71 or 73, 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.

[0173] 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, 44, 64, 66, 68, 70, 72 or 74. 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.

[0174] 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.

[0175] 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.

[0176] The asialoglycoprotein receptor complex (ASGP-R) that specifically recognizes the terminal β-galactosyl subunit of glycosylated proteins or other oligosaccharides (Weigel, 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.).

[0177] 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).

[0178] 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.

[0179] The sugar may be selected from N-acetylgalactosamine, mannose, galactose, glucose, glucosamine and trehalose. The sugar may be N-acetylgalactosamine (GalNAc).

[0180] 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.

[0181] "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.

[0182] Thus, the ligand may comprise GalNAc.

[0183] The ligand may include a compound of formula I:

[0184] [SX 1 -PX 2 ]3-AX 3 -(I)

[0185] in:

[0186] S represents a sugar, wherein the sugar is N-acetylgalactosamine;

[0187] X 1 represents C3-C6 alkylene or (-CH2-CH2-O) m (-CH2)2-, where m is 1, 2 or 3;

[0188] P is phosphate or a modified phosphate (preferably phosphorothioate);

[0189] X 2 is an alkylene group or a group of formula (-CH2) n -O-CH2-alkylene ether, wherein n=1-6;

[0190] A is the branching unit;

[0191] X 3 represents the bridge unit;

[0192] wherein the nucleic acid of the invention is conjugated to X via a phosphate or modified phosphate (preferably via a phosphorothioate) 3 .

[0193] 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.

[0194] The branching unit A may have a structure selected from the following:

[0195]

[0196] wherein each A1 independently represents O, S, C=O or NH; and

[0197] Each n independently represents an integer from 1 to 20.

[0198] The branching unit may have a structure selected from the following:

[0199]

[0200] wherein each A1 independently represents O, S, C=O or NH; and

[0201] Each n independently represents an integer from 1 to 20.

[0202] The branching unit may have a structure selected from the following:

[0203]

[0204] wherein A1 is O, S, C=O or NH; and

[0205] Each n independently represents an integer from 1 to 20.

[0206] A branch unit can have the structure:

[0207]

[0208] A branch unit can have the structure:

[0209]

[0210] A branch unit can have the structure:

[0211]

[0212] Optionally, the branching units consist only of carbon atoms.

[0213] "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.

[0214] 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-.

[0215] 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.

[0216] The ligand may include a compound of formula (II):

[0217] [SX 1 -PX 2 ]3-AX 3 -(II)

[0218] in:

[0219] S stands for sugar;

[0220] X 1 represents C3-C6 alkylene or (-CH2-CH2-O) m (-CH2)2-, where m is 1, 2 or 3;

[0221] P is phosphate or a modified phosphate (preferably phosphorothioate);

[0222] X 2 is C1-C8 alkylene;

[0223] A is a branching unit selected from the following:

[0224]

[0225] X 3 It is a bridge unit;

[0226] wherein the nucleic acid of the invention is conjugated to X via a phosphate or modified phosphate (preferably via a phosphorothioate) 3

[0227] The branch unit A may have the structure:

[0228]

[0229] The branch unit A may have the structure:

[0230] Where X 3 Attached to the nitrogen atom.

[0231] 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 -.

[0232] The ligand may include a compound of formula (III):

[0233] [SX 1 -PX 2 ]3-AX 3 -(III)

[0234] in:

[0235] S stands for sugar;

[0236] X 1 represents C3-C6 alkylene or (-CH2-CH2-O) m (-CH2)2-, where m is 1, 2 or 3;

[0237] P is phosphate or a modified phosphate (preferably phosphorothioate);

[0238] X 2 It is of the formula -C3H6-O-CH2-alkylene ether;

[0239] A is the branching unit;

[0240] 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,

[0241] And where X 3 Conjugation to the nucleic acids of the invention is via phosphate or modified phosphate, preferably via phosphorothioate.

[0242] The branching units may comprise carbon. Preferably, the branching units are carbon.

[0243] 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-C6H 12 - and -CH2-O-C8H 16 .

[0244] For any of the above aspects, when P represents a modified phosphate group, P may be represented by:

[0245]

[0246] where Y 1 and Y2 Each independently represents =O, =S, -O - , -OH, -SH, -BH3, -OCH2CO2, -OCH2CO2R x 、-OCH2C(S)OR x AND –OR x , where R x represents a C1-C6 alkyl group, and wherein Indicates the connection to the rest of the compound.

[0247] 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).

[0248] 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.

[0249] For example, Y 1 Can represent -OH, and Y 2 Can represent =O or =S; or

[0250] Y 1 Can represent -O - , and Y 2 Can represent =O or =S;

[0251] Y 1 Can represent =O, and Y 2 Can represent –CH3, -SH, -OR x or –BH3

[0252] Y 1 Can represent =S, and Y 2 Can represent –CH3, OR x or –SH.

[0253] The skilled person will appreciate that in some cases, 1 With Y 2 There will be delocalization between them.

[0254] Preferably, the modified phosphate group is a thiophosphate group. The thiophosphate group includes dithiophosphates (i.e., wherein Y1 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.

[0255] P can also be ethyl phosphate (i.e., where Y 1 represents =O, and Y 2 represents OCH2CH3).

[0256] 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).

[0257] 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.

[0258] "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.

[0259]

[0260] 2-(Acetylamino)-2-deoxy-D-galactopyranose

[0261]

[0262] 2-(Acetylamino)-2-deoxy-β-D-galactopyranose

[0263]

[0264] 2-(Acetylamino)-2-deoxy-α-D-galactopyranose

[0265] 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.

[0266] For the compound of formula (III), X 2 It represents the formula -C3H6-O-CH2-alkylene ether, i.e. C3 alkoxymethylene or -CH2CH2CH2OCH2-.

[0267] The present invention provides a conjugated nucleic acid having one of the following structures:

[0268]

[0269]

[0270]

[0271]

[0272]

[0273] wherein Z is a nucleic acid as defined herein before.

[0274] 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 because a single ligand is sufficient to achieve efficient targeting of the nucleic acid to the target cell.

[0275] 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.

[0276] 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.

[0277] In one embodiment, the nucleic acid is conjugated to a ligand comprising a lipid, and more preferably to a ligand comprising cholesterol.

[0278] The conjugates of the invention may comprise any nucleic acid as disclosed herein conjugated to any one or more ligands as disclosed herein.

[0279] 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:

[0280] (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

[0281] (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.

[0282] In one embodiment of the present invention, the second RNA strand (i.e., the sense strand) is conjugated to a targeting ligand at the 5' end, the first RNA strand (i.e., the antisense strand) is conjugated to a targeting ligand at the 3' end, and the 3' end of the second RNA strand (i.e., the sense strand) is not conjugated, so as to form a target having the following structure: Fig.40A The schematic structure of the conjugate is shown in FIG.

[0283] In one embodiment of the present invention, the second RNA strand (i.e., the sense strand) is conjugated to a targeting ligand at the 5' end, the second RNA strand (i.e., the sense strand) is also conjugated to a targeting ligand at the 3' end, and the 3' end of the first RNA strand (i.e., the antisense strand) is not conjugated, so as to form a RNA having the following structure: Fig.40B The schematic structure of the conjugate is shown in FIG.

[0284] In one embodiment of the present invention, both the second RNA strand (i.e., the sense strand) and the first RNA strand (i.e., the antisense strand) are conjugated to a targeting ligand at the 3' end, and the 5' end of the second RNA strand (i.e., the sense strand) is not conjugated, so as to form a first RNA strand having the following structure: Fig.40C The schematic structure of the conjugate is shown in FIG.

[0285] In one embodiment of the present invention, the second RNA strand (i.e., the sense strand) is conjugated to a targeting ligand at the 5' end, and both the second RNA strand (i.e., the sense strand) and the first RNA strand (i.e., the antisense strand) are also conjugated to a targeting ligand at the 3' end, so as to form a RNA having the following structure: Fig.40D The schematic structure of the conjugate is shown in FIG.

[0286] In any of the above embodiments, A linker is indicated that conjugates the ligand to the terminus of the nucleic acid portion; the ligand may be a GalNAc moiety such as GalNAc; and the schematic structure as shown in 40E represents the nucleic acid portion.

[0287] These schematic diagrams are not intended to limit the number of nucleotides in the first or second strand, nor do the figures represent any kind of limitation regarding complementarity of the bases or any other limitation.

[0288] The ligand may be monomeric or polymeric (eg, dimeric, trimeric, etc.).

[0289] Suitably, the ligand is monomeric and therefore contains a single targeting ligand moiety, for example a single GalNAc moiety.

[0290] 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.

[0291] Alternatively, 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.

[0292] Two or three serinol-derivatized linker moieties can be linked in series, for example as follows:

[0293]

[0294] wherein n is 1 or 2, and Y is S or O.

[0295] Preferably, the ligand is a monomer.

[0296] 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).

[0297] Suitably, the linker moiety is a serinol-derivatized linker moiety.

[0298] 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.

[0299] More suitably, the further linker comprises a PEG chain.

[0300] More preferably, the other linker comprises a saturated, unbranched C 1-15 Alkyl chain.

[0301] More preferably, the other linker comprises a saturated, unbranched C 1-6 Alkyl chain.

[0302] More suitably, the further linker comprises a saturated, unbranched C4 or C6 alkyl chain, such as a C4 alkyl chain.

[0303] In one embodiment, Is a connecting part of formula (V):

[0304]

[0305] wherein n, Y and L1 are defined below, and the O of the phosphate group is linked to the terminal oligonucleotide of the RNA chain.

[0306] Thus, in one embodiment, the targeting ligand moiety is a linking moiety of formula (VI):

[0307]

[0308] wherein n, Y and L1 are defined below, and the O of the phosphate group is linked to the terminal oligonucleotide of the RNA chain.

[0309] Suitably, is a linking moiety of formula (XIV):

[0310]

[0311] wherein n, Y, R1 and L are defined below, L is linked to a targeting ligand such as GalNAc, and the O of the phosphate group is linked to the terminal oligonucleotide of the RNA chain.

[0312] Suitably, the targeting ligand moiety is a linking moiety of formula (IV):

[0313]

[0314] wherein n, Y, R1 and L are defined below, and the O of the phosphate group is linked to the terminal oligonucleotide of the RNA chain.

[0315] Suitably, is a connecting moiety of formula (VII):

[0316]

[0317] wherein n, Y and L2 are defined below, and the O of the phosphate group is linked to the terminal oligonucleotide of the RNA chain.

[0318] Suitably, the targeting ligand moiety is a linking moiety of formula (VIII):

[0319]

[0320] wherein n, Y and L2 are defined below, and the O of the phosphate group is linked to the terminal oligonucleotide of the RNA chain.

[0321] Suitably, is a linking moiety of formula (IX):

[0322]

[0323] wherein F, Y and L are defined below, and the O of the phosphate group is linked to the terminal oligonucleotide of the RNA chain.

[0324] Suitably, the targeting ligand moiety is a linking moiety of formula (IXa):

[0325]

[0326] wherein F, Y and L are defined below, and the O of the phosphate group is linked to the terminal oligonucleotide of the RNA chain.

[0327] Suitably, L is:

[0328]

[0329] In any of the above structures, suitably, the ligand is selected from GalNAc and a galactose moiety, in particular a GalNAc moiety. Alternatively, GalNAc may be replaced by another targeting ligand, such as a sugar.

[0330] In one embodiment of the invention, the first RNA strand is a compound of formula (X):

[0331]

[0332] where b is 0 or 1; and

[0333] The second RNA strand is a compound of formula (XI):

[0334]

[0335] in:

[0336] c and d are independently 0 or 1;

[0337] Z1 and Z2 are RNA moieties of the first RNA strand and the second RNA strand, respectively; Y is O or S;

[0338] n is 0, 1, 2 or 3; and

[0339] L1 is a linker attached to the ligand;

[0340] And where b+c+d is 2 or 3.

[0341] Suitably, the first RNA strand is a compound of formula (XV)

[0342] where b is 0 or 1; and

[0343] The second RNA strand is a compound of formula (XVI):

[0344]

[0345] Where c and d are independently 0 or 1;

[0346] in:

[0347] Z1 and Z2 are RNA portions of the first RNA strand and the second RNA strand, respectively;

[0348] Y is O or S;

[0349] R1 is H or methyl;

[0350] n is 0, 1, 2 or 3; and

[0351] In formula (XV) and (XVId), L is the same or different and is selected from the group consisting of:

[0352] -(CH2) r -C(O)-, wherein r = 2-12;

[0353] -(CH2-CH2-O) s -CH2-C(O)-, where s = 1-5;

[0354] -(CH2) t -CO-NH-(CH2) t -NH-C(O)-, wherein t is independently 1-5;

[0355] -(CH2) u -CO-NH-(CH2) u -C(O)-, wherein u is independently 1-5; and

[0356] -(CH2) v -NH-C(O)-, wherein v is 2-12; and

[0357] wherein if a terminal C(O) is present, it is attached to an NH group;

[0358] And where b+c+d is 2 or 3.

[0359] Suitably, the first RNA strand is a compound of formula (XII):

[0360]

[0361] where b is 0 or 1; and

[0362] The second RNA strand is a compound of formula (XIII):

[0363]

[0364] in:

[0365] c and d are independently 0 or 1;

[0366] Z1 and Z2 are RNA portions of the first RNA strand and the second RNA strand, respectively;

[0367] Y is O or S;

[0368] n is 0, 1, 2 or 3; and

[0369] 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:

[0370] or

[0371] n is 0, and L2 is:

[0372]

[0373] And the terminal OH group is absent, so that the following parts are formed:

[0374]

[0375] in

[0376] 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;

[0377] In formula (I) and (II), L is the same or different and is selected from the group consisting of:

[0378] -(CH2) r -C(O)-, wherein r = 2-12;

[0379] -(CH2-CH2-O) s -CH2-C(O)-, where s = 1-5;

[0380] -(CH2) t -CO-NH-(CH2) t -NH-C(O)-, wherein t is independently 1-5;

[0381] -(CH2) u -CO-NH-(CH2) u -C(O)-, wherein u is independently 1-5; and

[0382] -(CH2) v -NH-C(O)-, wherein v is 2-12; and

[0383] wherein if a terminal C(O) is present, it is attached to an NH group;

[0384] And where b+c+d is 2 or 3.

[0385] 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.

[0386] 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.

[0387] 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.

[0388] Most preferably, b is 0, c is 1, and d is 1.

[0389] In one embodiment, Y is O. In another embodiment, Y is S.

[0390] In one embodiment, R1 is H or methyl. In one embodiment, R1 is H. In another embodiment, R1 is methyl.

[0391] In one embodiment, n is 0, 1, 2 or 3. Suitably, n is 0.

[0392] In one embodiment, L is selected from the group consisting of:

[0393] -(CH2) r -C(O)-, wherein r = 2-12;

[0394] -(CH2-CH2-O) s-CH2-C(O)-, where s = 1-5;

[0395] -(CH2) t -CO-NH-(CH2) t -NH-C(O)-, wherein t is independently 1-5;

[0396] -(CH2) u -CO-NH-(CH2) u -C(O)-, wherein u is independently 1-5; and

[0397] -(CH2) v -NH-C(O)-, wherein v is 2-12;

[0398] The terminal C(O) is connected to the NH group.

[0399] Suitably, L is -(CH2) r -C(O)-, wherein r = 2-12. Suitably, r = 2-6. More suitably, r = 4 or 6, for example 4.

[0400] Suitably, L is:

[0401]

[0402] 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.

[0403] Suitably, L2 is:

[0404]

[0405] Suitably, L2 is:

[0406]

[0407] Suitably, L2 is:

[0408]

[0409] Suitably, L2 is:

[0410]

[0411] Suitably, n is 0, and L2 is:

[0412]

[0413] And the terminal OH group is absent, so that the following parts are formed:

[0414]

[0415] wherein Y is as defined elsewhere herein.

[0416] 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.

[0417] 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:

[0418]

[0419] 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.

[0420] In one embodiment, the targeted cell is a hepatocyte.

[0421] General synthesis process: 1

[0422] Example compounds can be synthesized according to the following description and methods known to those skilled in the art. Although the process illustrates the synthesis of specific conjugates, it should be understood that other claimed conjugates can be prepared by similar methods. The assembly of oligonucleotide chains and joint building blocks can be carried out, for example, by solid phase synthesis using the phosphoramidite method. Solid phase synthesis can start from bases or lcaa CPG loaded with modified building blocks. The phosphoramidite synthesis coupling cycle consists of the following: 1) remove DMT, 2) use the required DMT to mask phosphoramidites and activators for chain extension, and the activator can be benzylthio tetrazolium (BTT), 3) cap the non-extended oligonucleotide chain, then iodine (if phosphodiester bond is required) or EDITH (if thiophosphate bond is required) to oxidize P (III) to P (V), and cap again (capping / oxidation / capping or capping / thio / capping). GalNAc conjugation can be achieved by forming peptide bonds with the oligonucleotides of the previously assembled and purified GalNAc-carboxylic acid building blocks and the amino-modified joint building blocks with the necessary number of connections. The necessary building blocks are commercially available or synthesized as described below. All final single-stranded products were analyzed by AEX-HPLC to verify their purity. Purity is 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 was verified by LC-MS analysis.

[0423] Synthesis of synthons

[0424] Scheme 1: Synthesis of DMT-serinol (TFA) linker synthon

[0425]

[0426] i) Ethyl trifluoroacetate, NEt3, MeOH, 0°C, 16 hours, 2:86% 5:90%, ii) DMTCl, pyridine, 0°C, 16 hours, 74%, iii) LiBH4, EtOH / THF (1 / 1, v / v), 0°C, 1 hour, 76%, iv) 2-Cyanoethyl-N,N-diisopropylchlorophosphoramidite, EtNiPr2, CH2Cl2, 56%, v) Succinic anhydride, DMAP, pyridine, room temperature, 16 hours, 38%, vi) HBTU, DIEA, amino-lcaa CPG (500A), room temperature, 18 hours, 29% (26umol / g loading).

[0427] (S)-DMT-serinol (TFA)-phosphoramidite 7 can be synthesized from (L)-serine methyl ester derivative 1 according to the method disclosed in the literature (Hoevelmann et al. Chem. Sci., 2016, 7, 128-135).

[0428] (S)-DMT-serinol (TFA)-succinate 6 can be prepared by converting intermediate 5 with succinic anhydride in the presence of a catalyst such as DMAP.

[0429] 6 is loaded onto a solid support such as a controlled pore glass (CPG) support and can be realized by using a coupling agent such as HBTU to form a peptide bond with a solid support such as a natural CPG support (500A) modified by amino groups. (S)-DMT-serinol (TFA)-succinate 6 and a coupling agent such as HBTU are dissolved in a solvent such as CH3CN. A base such as diisopropylethylamine is added to the solution, and the reaction mixture is stirred for 2 minutes. A solid support such as a natural amino-lcaa-CPG support (500A, 3g, amine content: 136umol / g) is added to the reaction mixture, and a suspension is formed. The suspension is gently shaken on a wrist-moving oscillator for 16 hours at room temperature, then filtered, and washed with solvents such as DCM and EtOH. The support is dried under vacuum for 2 hours. The unreacted amine on the support can be capped by stirring at room temperature with acetic anhydride / lutidine / N-methylimidazole. The washing of the support can be repeated as above. The solid support is dried under vacuum to produce a solid support 10.

[0430] Scheme 2: Synthesis of GalNAc synthon 9

[0431]

[0432] (vii) TMSOTf, DCM, hexenol, viii) RuCl3, NaIO4, DCM, CH3CN, H2O, 46% (over two steps).

[0433] The synthesis of GalNAc synthon 9 can be prepared starting from commercially available peracetylated galactosamine 8 as described in Nair et al. (2014).

[0434] Synthesis of single-chain serinol-derived GalNAc conjugates

[0435] Scheme 3: General oligonucleotide synthesis procedure for serinol-derivatized linkers

[0436]

[0437] Oligonucleotide synthesis of 3' mono-GalNAc conjugated oligonucleotides (such as compound A0264) is generally described in Fig.13and are summarized in Scheme 3. As in the case of example compound A0264, the synthesis is started using (S)-DMT-serinol (TFA)-succinate-lcaa-CPG 10. Where additional serinol building blocks are required, (S)-DMT-serinol (TFA) subamide (7) is used in an appropriate solid phase synthesis cycle. For example, to prepare compound A0329, after the base sequence is fully assembled, the chain assembly is completed by coupling with additional serinol subamide. In addition, the oligonucleotide synthesis of 5' single GalNAc conjugated oligonucleotides can be started from a solid support loaded with appropriate nucleosides of its relevant sequence. In example compound A0220, this can be 2'fA. The oligonucleotide chain is assembled according to its sequence, and where appropriate, the building block (S)-DMT-serinol (TFA)-subamide (7) is used. After completion of chain elongation, the protective DMT group of the last coupled amidite building block is removed, as in step 1) of the phosphoramidite synthesis cycle.

[0438] After completing the last synthesizer step, the single strand can be removed from the solid support by cleavage with an amine treatment such as a 40% methylamine aqueous solution. Any remaining protecting groups are also removed in this step, and the methylamine treatment also releases the serinol amino functional group. The crude product is then purified by AEX-HPLC and SEC to produce a precursor oligonucleotide for further GalNAc conjugation.

[0439] Scheme 4: GalNAc conjugation synthesis of serinol-derived precursor oligonucleotides

[0440]

[0441] Post-solid phase synthesis GalNAc conjugation is achieved by pre-activating the GalN(Ac4)-C4 acid (9) with a peptide coupling reagent such as HBTU. The pre-activated acid 9 is then reacted with the amino group in 11 (e.g. A0264) to form an intermediate GalN(Ac4) conjugate. The acetyl group protecting the hydroxyl group in the GalNAc moiety is cleaved off by treatment with methylamine to produce the desired example compound 12 (e.g. A0268), which is further purified by AEX-HPLC and SEC. Synthesis of single-chain non-serinol-derivatized GalNAc conjugates

[0442] Amino-modified building blocks other than serinol are commercially available from various suppliers and can be used in place of serinol to provide reactive amino groups that allow for GalNAc conjugation. For example, the commercially available building blocks shown in Table 6 below can be used to provide non-serinol-derivatized amino-modified precursor oligonucleotides 14 (Scheme 5A) by using amino-modifier loaded CPGs such as 10-1 to 10-3, followed by sequence assembly as described above, and finally coupling of amino-modifier phosphoramidites such as 13-1, 13-2, or 13-4.

[0443] For example, to prepare 14 (A0653), GlyC3Am-CPG (10-2) was used in combination with GlyC3Am-imidite 13-2. Structurally different modifiers can be used to prepare 14, for example, for A0651, C7Am-CPG was used in combination with C6Am-imidite as the second amino modification. In a similar manner, commercially available amino modifier-loaded CPG 10-5 and amino-modified phosphoramidite 13-5 can be used to synthesize amino-modified precursor molecule 14 (A0655).

[0444] Table 6: Commercially available building blocks

[0445]

[0446]

[0447] Scheme 5: General oligonucleotide synthesis procedure

[0448]

[0449] The resulting precursor oligonucleotide 14 can then be conjugated with GalN(Ac4)-C4 acid (9) to produce the desired example compound 15 (Scheme 6).

[0450] Scheme 6: GalNAc conjugation synthesis of precursor oligonucleotides

[0451]

[0452] Synthesis of single-chain triantennary GalNAc conjugates in reference conjugates 3-4

[0453] Oligonucleotide synthesis of triantennary GalNAc cluster-conjugated siRNA is outlined in Fig.14In. As in the case of example compound A0006, oligonucleotide chain assembly uses a substrate-loaded carrier such as 5'DMT-2'FdA (bz)-succinate-lcaa-CPG to start. Repeat the following phosphoramidite synthesis coupling cycle until the full length of the product is reached: 1) remove DMT, 2) use the required DMT masking phosphoramidite to carry out chain extension, 3) non-extended oligonucleotide chain is capped, then P (III) is oxidized to P (V) by iodine or EDITH (if phosphorothioate bond is required), and capped again (capping / oxidation / capping or capping / thio / capping). For the post conjugation of trivalent triantennary GalNAc clusters, the same synthesis cycle is applied in the case of using necessary trivalent branched amidite C4XLT-phos, and another round of synthesis cycle is carried out using GalNAc amidite ST23-phos. After completing this last synthesizer step, the oligonucleotide is cracked from the solid support, and the additional protecting group can be removed by methylamine treatment. The crude product was then purified by AEX-HPLC and SEC, respectively.

[0454] General duplex formation procedure

[0455] To obtain a double-stranded conjugate, a single strand is dissolved in H2O at a concentration of 60 OD / mL. Two single oligonucleotide solutions can be added together to a reaction vessel. For reaction monitoring, titration can be performed. The first strand is added in an excess of 25% compared to the second strand, as determined by UV absorption at 260 nm. The reaction mixture is heated, for example, to 80°C for 5 minutes and then slowly cooled to room temperature. Double-stranded formation can be monitored by ion-pair reversed-phase HPLC. Based on the UV area of ​​the residual single strand, the required amount of the second strand can be calculated and added to the reaction mixture. The reaction is heated, for example, to 80°C again and slowly cooled to room temperature. This procedure can be repeated until less than 10% of the residual single strand is detected.

[0456] The above method (including processes 1-6 and Fig.13 and Fig.14 ) can be easily adapted to replace GalNac with another targeting ligand such as a sugar.

[0457] In any of the above aspects, instead of solid phase synthesis post-conjugation, it is possible to prepare preformed serinol (GN)-phosphoramidite and use this for on-column conjugation.

[0458] General synthesis process: 2

[0459] Example compounds can be synthesized according to the following description and methods known to those skilled in the art. Assembly of oligonucleotide chains and linker building blocks can be performed, for example, by solid phase synthesis using the phosphoramidite method. GalNAc conjugation can be achieved by forming a peptide bond between a GalNAc-carboxylic acid building block and a previously assembled and purified oligonucleotide connected to the necessary number of amino-modified linker building blocks.

[0460] Scheme 1: Synthesis of DMT-serinol (TFA) linker synthon

[0461]

[0462] i) Ethyl trifluoroacetate, NEt3, MeOH, 0°C, 16 hours, 2:86% 5:90%, ii) DMTCl, pyridine, 0°C, 16 hours, 74%, iii) LiBH4, EtOH / THF (1 / 1, v / v), 0°C, 1 hour, 76%, iv) 2-Cyanoethyl-N,N-diisopropylchlorophosphoramidite, EtNiPr2, CH2Cl2, 56%, v) Succinic anhydride, DMAP, pyridine, room temperature, 16 hours, 38%, vi) HBTU, DIEA, amino-lcaa CPG (500A), room temperature, 18 hours, 29%.

[0463] DMT-serinol (TFA)-phosphoramidite 7 can be synthesized from serinol derivative 1 according to the method disclosed in the literature (Hoevelmann et al. Chem. Sci., 2016, 7, 128-135).

[0464] DMT-serinol (TFA)-succinate 6 can be prepared by converting intermediate 5 with succinic anhydride in the presence of a catalyst such as DMAP.

[0465] 6 is loaded onto a solid support such as a CPG support and can be realized by using a coupling agent such as HBTU to form a peptide bond with a solid support such as a natural CPG support (500A) modified by amino groups. DMT-serinol (TFA)-succinate 6 and a coupling agent such as HBTU are dissolved in a solvent such as CH3CN. A base such as diisopropylethylamine is added to the solution, and the reaction mixture is stirred for 2 minutes. A solid support such as a natural amino-lcaa-CPG support (500A, 3g, amine content: 136 μmol / g) is added to the reaction mixture, and a suspension is formed. The suspension is gently shaken on a wrist-moving oscillator for 16 hours at room temperature, then filtered, and washed with solvents such as DCM and EtOH. The support is dried under vacuum for 2 hours. The unreacted amine on the support can be capped by stirring at room temperature with acetic anhydride / lutidine / N-methylimidazole. The washing of the support can be repeated as above. The solid support is dried under vacuum to produce a solid support 10.

[0466] Scheme 2: Synthesis of GalNAc synthon 9

[0467]

[0468] (vii) TMSOTf, DCM, hexenol, viii) RuCl3, NaIO4, DCM, CH3CN, H2O, 46% (over two steps).

[0469] The synthesis of GalNAc synthon 9 can be prepared starting from commercially available peracetylated galactosamine 8 according to the method described in Nair et al., J. Am. Chem. Soc., 2014, 136(49), pp. 16958-16961.

[0470] Scheme 3: General oligonucleotide synthesis procedure

[0471]

[0472] All oligonucleotides were synthesized on an AKTA oligopilot 10 synthesizer using standard phosphoramidite chemistry as described in detail below.

[0473] Oligonucleotide synthesis of 3' and 5' GalNAc conjugated oligonucleotide precursors (such as compound X0385B-prec) is generally described in Fig.17 In, and is summarized in flow 3. Synthesis is started using DMT-serinol (TFA)-succinate-lcaa-CPG 10. Phosphoramidite synthesis cycles are applied until the full length of the product is reached. After completing chain extension, the protective DMT group of the last coupled imide building block can be removed in the same manner as in each individual synthesis cycle. To complete the synthesis of example compound X0385B-prec (which has serinol derived linker moieties at the 3' and 5' ends of the second chain), after the base sequence is fully assembled, chain assembly is completed with additional serinol imide coupling.

[0474] After completing the last synthesizer step, the single-stranded can be removed from the solid support cracking by treating with amines such as 40% methylamine aqueous solution. Any remaining protecting groups are also removed in this step, and methylamine treatment also releases the serinol amino functional group. The gained crude oligonucleotide can be purified on AKTA Pure HPLC system using gradients such as sodium chloride gradients by ion exchange chromatography (Resource Q, 6mL, GE Healthcare). Excess salts from IEX purification can be removed by SEC to produce amino-modified precursor oligonucleotides 11. The fractions containing the product are merged, desalted on size exclusion column (Zetadex, EMP Biotech), and lyophilized.

[0475] Scheme 4: General GalNAc conjugation procedure

[0476]

[0477] (i) 9, HBTU, DIPEA, DMSO; 11, H2O, DMSO, DIPEA; thus, activated 9 and 11 were obtained; then 40% MeNH2, H2O

[0478] Conjugation of the GalNAc synthon 9 as described above can be achieved by coupling 9 to the serinol-amino functional group of the corresponding oligonucleotide chain 11 using standard peptide coupling conditions known to those skilled in the art. For example, the corresponding amino-modified precursor molecule 11 is dissolved in H2O, and a polar solvent such as DMSO (e.g., DMSO / H2O, 2 / 1, v / v) is added, followed by a base such as DIPEA (e.g., 2.5% of the total volume). In a separate reaction vessel, pre-activation of the GalNAc synthon 9 can be carried out by reacting 2 equivalents (according to the amino functional group in the amino-modified precursor oligonucleotide) of the carboxylic acid component with 2 equivalents of a coupling reagent such as HBTU in the presence of 8 equivalents of a base such as DIPEA in a polar solvent such as DMSO. After 2 minutes, the activated compound 9 is added to the solution of the corresponding amino-modified precursor molecule 11. The progress of the reaction can be monitored by LCMS or AEX-HPLC. After completion of the conjugation reaction (e.g., 30 minutes), the crude product can be precipitated by adding 10x iPrOH 0.1x2M NaCl and collected by centrifugal decantation. The acetyl hydroxy protecting group is removed under alkaline conditions such as 40% MeNH2 (1 mL / 500 OD). After 15 minutes at room temperature, H2O (1:10 v / v) is added and compound 12 (such as Fig.17 X0385B) shown in , was purified again by anion exchange and size exclusion chromatography, followed by lyophilization.

[0479] General duplex formation procedure

[0480] The single strand is dissolved in H2O at a concentration of 60 OD / mL. Two single oligonucleotide solutions can be added together to the reaction vessel. For reaction monitoring, titration can be performed. The first strand is added in an excess of 25% compared to the second strand, as measured by UV absorption at 260 nm. The reaction mixture is heated to, for example, 80°C for 5 minutes and then slowly cooled to room temperature. Double-stranded formation can be monitored by ion-pair reversed-phase HPLC. Based on the UV area of ​​the residual single strand, the required amount of the second strand can be calculated and added to the reaction mixture. The reaction is heated to, for example, 80°C again and slowly cooled to room temperature. This procedure can be repeated until less than 10% of the residual single strand is detected.

[0481] The above method (including processes 1-4 and Fig.17 ) can be easily adapted to replace GalNac with another targeting ligand such as a sugar.

[0482] The present invention relates to a conjugate for inhibiting the expression of an LPA gene in a cell, the conjugate comprising a nucleic acid portion and a 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 RNA transcribed from the LPA gene, the ligand portion comprising a 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:

[0483] (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

[0484] (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.

[0485] The linker moiety can be, for example, a serinol-derivatized linker moiety, or one of the other linker types described herein.

[0486] As another aspect, the present invention provides 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: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 63, 65, 67, 69, 71 or 73, wherein the nucleic acid is conjugated to a ligand. 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, 36, 38, 40, 42, 44, 64, 66, 68, 70, 72, or 74. The nucleotides of the first strand and / or the second strand may be modified as described herein.

[0487] 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 a 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.

[0488] More preferably, the nucleic acid comprises SEQ ID NO: 5 or SEQ ID NO: 9 and SEQ ID NO: 6 or SEQ ID NO: 10, wherein the nucleic acid is conjugated to a ligand of Formula I (as described above), and further, wherein the nucleic acid has a modification pattern as shown below which is an excerpt from Table 1 provided herein.

[0489]

[0490] where specific modifications are depicted by numbers

[0491] 1 = 2'F-dU;

[0492] 2 = 2'-F-dA;

[0493] 3 = 2'F-dC;

[0494] 4 = 2'F-dG;

[0495] 5=2'-OMe-rU;

[0496] 6 = 2'-OMe-rA;

[0497] 7 = 2'-OMe-rC;

[0498] 8 = 2'-OMe-rG.

[0499] The ligand may comprise GalNAc, and Figure 4A or Figure 4B Examples of the present invention are also described.

[0500] 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.

[0501] The nucleic acids or conjugated nucleic acids of the invention may also be administered in combination with other therapeutic compounds administered separately or simultaneously, for example, in a combined unit dosage form. The invention also includes a pharmaceutical composition comprising one or more nucleic acids or conjugated nucleic acids of the invention in a physiologically / pharmaceutically acceptable excipient such as a stabilizer, preservative, diluent, buffer, etc.

[0502] 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. The dosage level may also be calculated by other parameters such as (for example) body surface area.

[0503] The pharmaceutical composition may be a sterile injectable aqueous suspension or solution, or in lyophilized form.

[0504] 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.

[0505] 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. Treatment may be for the prevention and reduction of the risk of stroke, atherosclerosis, thrombosis or cardiovascular disease such as coronary heart disease or aortic valve stenosis, and any other disease or pathology associated with elevated levels of Lp(a)-containing particles. The invention includes a pharmaceutical composition comprising one or more nucleic acids or conjugated nucleic acids of the invention in a physiologically / pharmaceutically acceptable excipient such as a stabilizer, preservative, diluent, buffer, etc.

[0506] 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.

[0507] 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 acids or conjugated nucleic acids of the present invention or compositions comprising the nucleic acids or conjugated nucleic acids can be used in regimens 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 regimens employing combinations of different dosing frequencies such as the intervals mentioned above. The nucleic acids can be used subcutaneously, intravenously, or using any other route of administration such as oral, rectal, or intraperitoneal administration.

[0508] 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.

[0509] 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.

[0510] Also included in the present invention is a method for treating or preventing a disease, condition or syndrome 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). The nucleic acid composition may include a regimen of 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 for treatment, or a regimen of a combination of different dosing frequencies such as the intervals mentioned above. Nucleic acids or conjugated nucleic acids may be used subcutaneously or intravenously or by other routes of application such as oral, rectal or intraperitoneal administration.

[0511] The nucleic acid or conjugated nucleic acid of the present invention can also be administered to be used in combination with other therapeutic compounds administered separately or simultaneously, for example in the form of a combined unit dosage. 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.

[0512] 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.

[0513] The present invention consists of chemical molecular entities that mediate LPA mRNA degradation by binding to LPA gene transcripts through a cellular RNA interference mechanism. The molecular compounds of the invention can be used in the form of conjugates with, but not limited to, N-acetylgalactosamine (GalNAc) sugar moieties, which ensure hepatocyte-specific cellular uptake by specifically binding to the asialoglycoprotein receptor complex (ASGPR). The present invention can be linked to other different chemical structures, thereby conferring different properties as mentioned below. The use of chemically modified patterns of nucleic acids confers nuclease stability in serum and makes, for example, subcutaneous administration routes feasible.

[0514] The subject invention is characterized by high specificity at the molecular and tissue-directed delivery level, potentially conferring a better safety profile than currently available treatments.

[0515] 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 linked to the second nucleotide in the first strand via a phosphodiester bond.

[0516] In one embodiment, the first strand may include more than one phosphodiester bond.

[0517] In one embodiment, the first strand may comprise phosphodiester bonds between at least three terminal 5' nucleotides and.

[0518] In one embodiment, the first strand may comprise phosphodiester bonds between at least four terminal 5' nucleotides.

[0519] In one embodiment, the first chain may comprise formula (XVII):

[0520] (vp)-N(po)[N(po)] n -

[0521] (XVII)

[0522] 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).

[0523] In one embodiment, the first strand may include at least one phosphorothioate (ps) bond.

[0524] In one embodiment, the first strand may further comprise a phosphorothioate bond between the two terminal 3' nucleotides, or a phosphorothioate bond between the three terminal 3' nucleotides.

[0525] In one embodiment, the linkages between the other nucleotides in the first strand are phosphodiester bonds.

[0526] In one embodiment, the first strand may include more than one phosphorothioate bond.

[0527] In another embodiment, the second strand may comprise phosphorothioate bonds between the two terminal 3' nucleotides, or phosphorothioate bonds between the three terminal 3' nucleotides.

[0528] In another embodiment, the second strand may comprise phosphorothioate bonds between the two terminal 5' nucleotides, or phosphorothioate bonds between the three terminal 5' nucleotides.

[0529] In one embodiment, the terminal 5' (E)-vinylphosphonate nucleotide is an RNA nucleotide.

[0530] 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=):

[0531] Nucleotides with natural phosphates at the 5' end

[0532] Nucleotides with E-vinylphosphonate at the 5' end

[0533] 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.

[0534] One aspect of the present invention is a nucleic acid for inhibiting the expression of a target gene in a cell as disclosed herein, 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 target gene, wherein the first strand comprises modified nucleotides or unmodified nucleotides at multiple positions to facilitate processing of the nucleic acid by RISC.

[0535] In one aspect, "promoting processing by RISC" means that the nucleic acid can be processed by RISC, eg, any modification present will allow processing of the nucleic acid by RISC, suitably such that siRNA activity can occur.

[0536] 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.

[0537] 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.

[0538] 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.

[0539] 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.

[0540] 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.

[0541] 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.

[0542] 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.

[0543] 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.

[0544] 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.

[0545] It is also preferred that:

[0546] 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.

[0547] 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.

[0548] 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 together with any other limitations described herein.

[0549] 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.

[0550] 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.

[0551] 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.

[0552] 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 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.

[0553] 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.

[0554] 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.

[0555] 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%, of nucleotides having 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.

[0556] 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.

[0557] A nucleic acid as disclosed herein, wherein all nucleotides are modified with a 2'O-methyl modification, except for 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 11-13 of the first strand. Preferably, the nucleotides not modified with a 2'O-methyl are modified with a fluorine at the 2' position.

[0558] 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.

[0559] 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.

[0560] In one aspect, there is at most one DNA per nucleic acid of the invention.

[0561] 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.

[0562] In one aspect, the nucleic acid is modified with alternating 2-O-methyl and 2'-fluoro modifications on the first strand, 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 nucleotides corresponding to positions 11, or 13, or 11 and 13, or 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.

[0563] 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.

[0564] 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.

[0565] All features of the nucleic acids can be combined with all other aspects of the invention disclosed herein.

[0566] In particular, preferred are nucleic acids as siRNA molecules, 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:

[0567] (i) an inverted nucleotide at the 3' terminus of the second strand, preferably using a 3'-3' linkage;

[0568] (ii) one or more phosphorodithioate bonds;

[0569] (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;

[0570] (iv) the nucleic acid comprises at least 80% of all nucleotides having a 2'-O-methyl modification;

[0571] (v) The nucleic acid comprises at most 20% of nucleotides having a 2' fluoro modification.

[0572] 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.

[0573] Specific preferred examples of blunt double-stranded 19-base nucleic acids having no overhangs are:

[0574] 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.

[0575] 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.

[0576] 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.

[0577] 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.

[0578] 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.

[0579] 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.

[0580] 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.

[0581] 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.

[0582] 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.

[0583] 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%, of nucleotides having 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.

[0584] 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.

[0585] 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.

[0586] 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.

[0587] 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.

[0588] 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.

[0589] The present invention also relates to the unmodified sequences of all modified sequences disclosed herein.

[0590] The invention will now be described with reference to the following non-limiting figures and examples.

[0591] Attached photos

[0592] Figure 1 Shown are the results of a screening of unconjugated siRNA molecules that inhibit LPA mRNA expression in human RT-4 cells.

[0593] 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.

[0594] 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.

[0595] Figure 4A and Figure 4B Examples of structures of GalNAc ligands with different L1 and L6 linkers to which exemplary oligonucleotides are conjugated are shown, respectively.

[0596] Figure 5 Shown are representative examples of LPA-mRNA knockdown in primary human hepatocytes by the indicated L6-conjugated GalNAc siRNAs delivered via receptor-mediated uptake.

[0597] Figure 6 Conjugate 1 is depicted.

[0598] Figure 7 Conjugate 2 is depicted.

[0599] Figure 8 Conjugate 3 is depicted.

[0600] Fig. 9 Reference conjugate 1 is depicted.

[0601] Fig.10 Reference conjugate 2 is depicted.

[0602] Fig.11 Reference conjugate 3 is depicted.

[0603] Fig.12 Reference conjugate 4 is depicted.

[0604] exist Figure 6-Figure 12 And Figure 19- Fig.30 In each of the , the top strand is the antisense strand, and the bottom strand is the sense strand. In addition, to more clearly show the connection between the nucleic acid and the ligand moiety, the nucleotides at the ends of the corresponding conjugated strands are fully drawn.

[0605] Fig.13 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.

[0606] Fig.14 Shown is the synthesis of A0006, a 5' triantennary GalNAc conjugated single stranded oligonucleotide used to synthesize reference conjugate 4. (ps) indicates phosphorothioate linkage.

[0607] FIG15 illustrates an in vitro assay for TTR knockdown. In particular, Fig.15A In vitro assays showing TTR knockdown achieved by reference conjugates (RC) 1 and 3, as well as untreated control "UT"; Fig. 15B In vitro assays showing TTR knockdown achieved by reference conjugates (RC) 2 and 3, as well as an untreated control "UT"; and Fig. 15C In vitro assays for TTR knockdown by conjugates 1, 2, and 3 and by RC3, as well as an untreated control "UT" are shown. 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.

[0608] Fig.16 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.

[0609] Fig.17 Oligonucleotide synthesis of 3' and 5' GalNAc conjugated oligonucleotide precursors such as compound X0385B-prec is shown.

[0610] Fig.18 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.

[0611] Fig.19A Depicted is conjugate 4. The last three nucleotides at the 5' and 3' ends of the antisense and sense strands are linked by phosphorothioate linkers between each nucleotide. A serinol-GalNAc linker is conjugated to the 3' and 5' ends of the sense strand via phosphodiester bonds.

[0612] Fig.19B Depicted is conjugate 5. The last three nucleotides at the 5' and 3' ends of the antisense strand are linked by phosphorothioate linkers between each nucleotide. A serinol-GalNAc linker is conjugated to the 3' and 5' ends of the sense strand via phosphorothioate bonds.

[0613] Fig. 20 Depicted is conjugate 6. The last three nucleotides at the 5' and 3' ends of the antisense strand are linked by phosphorothioate linkers between each nucleotide. A serinol-GalNAc linker is conjugated to the 3' and 5' ends of the sense strand via phosphodiester bonds.

[0614] Fig.21 Depict conjugate 7. The last three nucleotides at the 5' and 3' ends of the antisense and sense strands are connected by phosphorothioate linkers between each nucleotide. The serinol-GalNAc linker is conjugated to the 3' and 5' ends of the sense strand via a phosphorothioate bond. The serinol-GalNAc linker is connected to each other via a phosphorothioate bond.

[0615] Fig. 22 Depicts conjugate 8. The last three nucleotides at the 5' and 3' ends of the antisense and sense strands are linked by phosphorothioate linkers between each nucleotide. A GalNAc-C6-amino modifier linker is conjugated at the 5' end of the sense strand, and a GalNAc-C7-amino modifier linker is conjugated at the 3' end of the sense strand.

[0616] Fig.23 Depicted is conjugate 9. The last three nucleotides at the 5' and 3' ends of the antisense and sense strands are linked by phosphorothioate linkers between each nucleotide. GalNAc-GlyC3-amino modifier linkers are conjugated at the 5' and 3' ends of the sense strand.

[0617] Fig.24 Depict conjugate 10. The last three nucleotides at the 5' and 3' ends of the antisense and sense strands are linked by phosphorothioate linkers between each nucleotide. And the sense strand is linked by phosphorothioate linkers between each nucleotide. GalNAc-piperidinyl-amino modifier linkers are conjugated at the 5' and 3' ends of the sense strand.

[0618] Fig.25 Depicted is conjugate 11. The last three nucleotides at the 5' and 3' ends of the antisense and sense strands are linked by phosphorothioate linkers between each nucleotide. GalNAc-C3-amino modifier linkers are conjugated at the 5' and 3' ends of the sense strand.

[0619] Fig.26 Depicts conjugate 12. The last three nucleotides at the 5' and 3' ends of the antisense and sense strands are linked by phosphorothioate linkers between each nucleotide. A GalNAc-C6-amino modifier linker is conjugated at the 5' end of the sense strand, and a GalNAc-GlyC3-amino modifier linker is conjugated at the 3' end of the sense strand.

[0620] Fig. 27 Depict conjugates 15, 16, 18, and 19 that differ only by their RNA sequences. In each conjugate, the last three nucleotides at the 5' and 3' ends of the antisense and sense strands are linked by phosphorothioate linkers between each nucleotide. The serinol-GalNAc linker is conjugated to the 3' and 5' ends of the sense strand via phosphorothioate bonds.

[0621] Fig.28 Reference conjugate 5 and reference conjugate 9 are depicted, which differ only by their RNA sequences. In both conjugates, the last three nucleotides at the 5' and 3' ends of the antisense strand and the 3' end of the sense strand are linked by a phosphorothioate linker between each nucleotide. In both conjugates, a trimeric GalNAc linker is conjugated to the 5' end of the sense strand via a phosphorothioate bond.

[0622] Fig.29 Reference conjugate 6 and reference conjugate 7, which differ only by their RNA sequences, are depicted. In both conjugates, the last three nucleotides at the 5' and 3' ends of the antisense strand and the 3' end of the sense strand are linked by a phosphorothioate linker between each nucleotide. In both conjugates, a trimeric GalNAc linker is conjugated to the 5' end of the sense strand via a phosphorothioate bond.

[0623] Fig.30 Reference conjugate 8 is depicted. The last three nucleotides at the 5' and 3' ends of the antisense strand and the 3' end of the sense strand are linked by a phosphorothioate linker between each nucleotide. A trimeric GalNAc linker is conjugated to the 5' end of the sense strand via a phosphorothioate bond.

[0624] Figure 31 illustrates an in vitro assay for TTR knockdown. In particular, Fig.31A 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.31B 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 are normalized to PtenII.

[0625] FIG32 illustrates an in vitro assay for TTR knockdown. In particular, Fig.32A 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.32B 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 are normalized to PtenII.

[0626] Fig.33 Illustrate an in vitro assay for LPA mRNA knockdown achieved by conjugate 19 compared to a control. Ctr represents non-targeting GalNAc siRNA, and "untreated" ("UT") represents untreated cells. Both Ctr and UT are negative controls. mRNA levels are normalized to ACTB.

[0627] Fig.34 Shown is the time course of Aldh2 liver mRNA levels in a cohort 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.

[0628] Fig.35 Shown is the time course of Aldh2 liver mRNA levels in a cohort 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.

[0629] Fig.36 Shown is the time course of Tmprss6 liver mRNA levels in a cohort 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.

[0630] Fig.37 The serum stability of conjugates 4, 5, 6, 7 and 2 and untreated control (UT) over 3 days at 37°C is shown.

[0631] Fig.38 The serum stability of conjugates 8, 9, 10, 11, 12 and 2 and untreated control (UT) over 3 days at 37°C is shown.

[0632] Fig.39 Shown is the inhibition of LPA mRNA expression in human primary hepatocytes by different doses of GalNAc-L6-conjugated siRNA delivered via receptor-mediated uptake.

[0633] Figure 40 shows a schematic representation of various embodiments of nucleic acids conjugated to ligands via linkers.

[0634] Example

[0635] The numbers mentioned in each example are specific to that example.

[0636] Example 1

[0637] Modified and conjugated siRNA molecules are used in the functional examples herein.

[0638] LPA-1038 derivatives:

[0639] GalNAc-LPA-1038-L1

[0640] First strand (SEQ ID NO: 119, based on SEQ ID NO 5)

[0641] OMeA-(ps)-FU-(ps)-OMeA-FA-OMeC-FU-OMeC-FU-OMeG-FU-OMeC-FC-OMeA-FU-OMeU-FA-OMeC-(ps)-FC-(ps)-OMeA 3'

[0642] Second strand (SEQ ID NO: 120, based on SEQ ID NO SEQ ID NO 6)

[0643] 5'[ST23(ps)]3 long triple son(ps)FU-OMeG-FG-OMeU-FA-OMeA-FU-OMeG-FG-OMeA-FC-OMeA-FG-OMeA-FG-OMeU-FU-(ps)-OMeA-(ps)-FU 3'

[0644] GalNAc-LPA-1038-L6

[0645] First strand (SEQ ID NO: 121, based on SEQ ID NO 5)

[0646] OMeA-(ps)-FU-(ps)-OMeA-FA-OMeC-FU-OMeC-FU-OMeG-FU-OMeC-FC-OMeA-FU-OMeU-FA-OMeC-(ps)-FC-(ps)-OMeA 3'

[0647] Second strand (SEQ ID NO: 122, based on SEQ ID NO 6)

[0648] 5'[ST23(ps)]3ST43(ps)FU-OMeG-FG-OMeU-FA-OMeA-FU-O MeG-FG-OMeA-FC-OMeA-FG-OMeA-FG-OMeU-FU-(ps)-OMeA-(ps)-FU 3'

[0649] FN (N = A, C, G, U) represents 2' fluoro 2' deoxynucleoside

[0650] OMeN (N=A,C,G,U) represents 2'O methyl nucleoside

[0651] (ps) indicates phosphorothioate bond

[0652] ST23 and ST43 are as follows.

[0653] Other examples are LPA 1041 derivatives:

[0654] GalNAc-LPA-1041-L1

[0655] First strand (SEQ ID NO: 123, based on SEQ ID NO 9)

[0656] 5'OMeA-(ps)-FU-(ps)-OMeA-FA-OMeC-FU-OMeC-FU-OMeG-F U-OMeC-FC-OMeA-FU-OMeU-FA-OMeC-(ps)-FC-(ps)-OMeG 3'

[0657] Second strand (SEQ ID NO: 124, based on SEQ ID NO 10)

[0658] 5'[ST23(ps)]3 long triple son(ps)FC-OMeG-FG-OMeU-FA-OMeA-F U-OMeG-FG-OMeA-FC-OMeA-FG-OMeA-FG-OMeU-FU-(ps)-OMeA-(ps)-FU 3'

[0659] GalNAc-LPA-1041-L6

[0660] First strand (SEQ ID NO: 125, based on SEQ ID NO 9)

[0661] 5'OMeA-(ps)-FU-(ps)-OMeA-FA-OMeC-FU-OMeC-FU-OMeG-F U-OMeC-FC-OMeA-FU-OMeU-FA-OMeC-(ps)-FC-(ps)-OMeG 3'

[0662] Second strand (SEQ ID NO: 126, based on SEQ ID NO 10)

[0663] 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'

[0664] FN (N = A, C, G, U) represents 2' fluoro 2' deoxynucleoside

[0665] OMeN (N=A,C,G,U) represents 2'O methyl nucleoside

[0666] (ps) indicates phosphorothioate bond

[0667] ST23 and ST43 are as follows.

[0668] ST23 is GalNac C4 phosphoramidite (the structural components are as follows)

[0669]

[0670]

[0671] ST41 is as follows:

[0672]

[0673] ST43 is as follows, and as described in WO2017 / 174657:

[0674]

[0675] 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 and 2'O-methyl RNA phosphoramidites, 2' fluoro DNA phosphoramidites (all standardly protected) and commercially available long triplet phosphoramidites (Glen research) were used. Anhydrous acetonitrile solutions of 0.1M phosphoramidites were used for synthesis, and benzylthio tetrazolium (BTT) was used as an activator (0.3M acetonitrile solution). All other reagents were commercially available standard reagents.

[0676] 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).

[0677] 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.

[0678] 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.

[0679] The sequences of the resulting nucleic acids (siRNAs) are set forth in Table 1 below.

[0680] Table 1 : Non-conjugated nucleic acid sequences tested for inhibition of LPA mRNA expression. The sequences and applied modification patterns are indicated.

[0681]

[0682]

[0683] Table 1

[0684] 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.

[0685] Table 2: Shown below are the sequences of the LPA, APOB, beta-Actin, and PTEN qPCR amplicons used to measure mRNA levels.

[0686]

[0687]

[0688] Table 2

[0689] Example 2

[0690] Screening of unconjugated siRNA molecules (Table 1) for inhibition of LPA mRNA expression in human RT-4 cells.

[0691] 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 bars 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). 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 ​​are normalized to the amount of LPA mRNA detected in untreated cells (in-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.

[0692] Example 3

[0693] Dose response of unconjugated LPA-targeting siRNA compounds on LPA mRNA expression in human RT-4 cells.

[0694] RT4 human bladder transitional cell papilloma cells were reverse transfected as described above (Example 2) and treated with different non-conjugated siRNA compounds (Table 1) as labeled 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.

[0695] Example 4

[0696] 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.

[0697] 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 ​​are normalized to LPA expression in untreated cells. The mean and SD of the normalized triplicate values ​​of the remaining LPA mRNA levels are shown as black bars. Results are shown in Figure 3 middle.

[0698] Example 5

[0699] Knockdown of LPA-mRNA in human primary hepatocytes by different indicated L6-GalNAc-conjugated siRNAs after receptor-mediated delivery.

[0700] 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 represented in pairs as bars (100 nM black bars, 20 nM gray bars). The means and SDs of normalized triplicate values ​​are shown in Figure 5 middle.

[0701] Example 6 - Synthesis of conjugates

[0702] 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.

[0703] Oligonucleotide synthesis, deprotection and purification followed standard procedures known in the art.

[0704] All oligonucleotides were synthesized on an AKTA oligopilot synthesizer using standard phosphoramidite chemistry. Commercially available solid supports were used along with 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). Peracetylated galactosamine 8 is commercially available.

[0705] 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.

[0706] The connection of the serinol derived linker part is achieved by using (S)-DMT-serinol (TFA)-succinate-lcaa-CPG 10 or (S)-DMT-serinol (TFA) phosphoramidite 7 (synthesized as described in Hoevelmann et al. (Chem. Sci., 2016, 7, 128-135)) loaded by substrate. The three antennae GalNAc clusters (ST23 / C4XLT) are introduced by successively coupling the corresponding triple subamid derivatives (C4XLT-phos) and GalNAc subamids (ST23-phos).

[0707] 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.

[0708] 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.

[0709] Synthesis of Compound 2-10

[0710] 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).

[0711] (S)-4-(3-(bis(4-methoxyphenyl)phenyl)methoxy)-2-(2,2,2-trifluoroacetamido)propoxy)-4-oxobutanoic acid (6).

[0712] To a solution of 5 in pyridine 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 of 6 (yield = 38%). m / z (ESI-): 588.2 (100%), (C30H29F3NO8 - Calculated value: [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).

[0713] (S)-DMT-serinol (TFA)-succinate-lcaa-CPG (10)

[0714] (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).

[0715] GalNAc synthon(9)

[0716] 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.

[0717] The representational data were matched with the publicly available data.

[0718] Oligonucleotide synthesis

[0719] All single-stranded oligonucleotides were prepared according to the Fig.13 and Fig.14 The reaction conditions described in the synthesis.

[0720] 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.

[0721] Table 3: Single-stranded unconjugated oligonucleotides

[0722]

[0723] 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.

[0724] Synthesis of Conjugate 1-3 and Reference Conjugate 1-2

[0725] Conjugate 1-2 and conjugated single chain of reference conjugate 1-2

[0726] 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 give the final product 12.

[0727] Table 4: Single-stranded GalNAc-conjugated oligonucleotides

[0728]

[0729] Double strand formation

[0730] Double-strand formation was performed according to the above method.

[0731] 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.

[0732] Table 5: Nucleic acid conjugates

[0733]

[0734] sequence

[0735] Modified index for the following sequence:

[0736] f represents 2'fluoro 2'deoxyribonucleotide or 2'-fluororibonucleotide (the terms are interchangeable)

[0737] m represents 2'O methyl ribonucleotide

[0738] (ps) indicates phosphorothioate bond

[0739] Ser(GN) is a GalNAc-C4 building block attached to a serinol-derived linker moiety:

[0740]

[0741] wherein O--- is a bond between an oxygen atom and, for example, H, a phosphodiester bond, or a phosphorothioate bond.

[0742] The C4XLT is:

[0743]

[0744] ST23 is:

[0745]

[0746] The synthesis of phosphoramidite derivatives of C4XLT (C4XLT-phos) and phosphoramidite derivatives of ST23 (ST23-phos) can be carried out as described in WO2017 / 174657.

[0747] C4XLT-phos:

[0748]

[0749] ST23-phos:

[0750]

[0751] Conjugate 1

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

[0753] 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’

[0754] Sense strand - STS16001BL20 (SEQ ID NO:128)

[0755] 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’

[0756] Conjugate 2

[0757] Antisense strand - STS16001A (SEQ ID NO:129)

[0758] 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

[0759] Sense strand - STS16001BV1L42 (SEQ ID NO:130)

[0760] 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)

[0761] Conjugate 3

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

[0763] 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’

[0764] Sense strand - STS16001BV1L42 (SEQ ID NO:130)

[0765] 5'Ser(GN)(ps)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(ps)Ser(GN)3'

[0766] Reference conjugate 1

[0767] Antisense strand - STS16001A (SEQ ID NO:129)

[0768] 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

[0769] Sense strand – STS16001BL20 (SEQ ID NO:128)

[0770] 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

[0771] Reference conjugate 2

[0772] Antisense strand - STS16001AL33 (SEQ ID NO:127)

[0773] 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)

[0774] Sense strand - STS16001V1B (SEQ ID NO:131)

[0775] 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

[0776] Reference conjugate 3

[0777] Antisense strand - STS18001A (A0130; SEQ ID NO:132)

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

[0779] Sense strand-STS18001BL4 (A0131, SEQ ID NO: 133)

[0780] [(ST23)(ps)]3C4XLT(ps)fC mG fU mA fC mG fC mG fG mA fA mU fA mC fU mUfC(ps)mG(ps)fA

[0781] Reference conjugate 4

[0782] Antisense strand-STS16001AL33 (SEQ ID NO: 127)

[0783] 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

[0784] Sense strand-STS16001BL4 (SEQ ID NO: 134)

[0785] 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

[0786] Example 7 - In vitro determination of TTR knockdown achieved with various TTR siRNA GalNAc conjugates

[0787] 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.

[0788] Compared to the negative control (see the "UT" column and reference conjugate 3), the target gene expression in primary mouse hepatocytes 24 hours after treatment with the conjugates of the present invention, i.e., conjugates 1-3, showed that the target gene expression decreased with increasing doses of the conjugates, as shown in FIG15. This indicates that the first strand binds to the target gene, thereby reducing gene expression. FIG15 also shows the target gene expression levels in the case of reference conjugates 1 and 2, which serve as comparative conjugates. As can be seen from Fig.15A and Fig. 15C ,as well as Fig. 15B and Fig. 15C As seen from the comparison between the data presented in , the conjugates of the invention (Conjugates 1-3) reduced target gene expression compared to reference conjugates 1 and 2. The most effective conjugate at 0.01 nM appeared to be Conjugate 2. The most effective conjugate at 0.1 nM, 0.5 nM, 1 nM and 10 nM appeared to be Conjugate 3.

[0789] Example 8 - In vivo time course of serum TTR in mice

[0790] 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).

[0791] 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 Fig.16 In. Fig.16 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.

[0792] Example 9 - Synthesis of Conjugate 2

[0793] 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.

[0794] Oligonucleotide synthesis, deprotection and purification followed standard procedures known in the art.

[0795] All oligonucleotides were synthesized on an AKTA oligopilot synthesizer using standard phosphoramidite chemistry. Commercially available solid supports were used along with 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). Peracetylated galactosamine 8 is commercially available.

[0796] 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.

[0797] 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).

[0798] 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.

[0799] 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.

[0800] Synthesis of Compound 2-10

[0801] 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).

[0802] (S)-4-(3-(bis(4-methoxyphenyl)phenyl)methoxy)-2-(2,2,2-trifluoroacetamido)propoxy)-4-oxobutanoic acid (6).

[0803] To a solution of 5 in pyridine 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 of 6 (yield = 38%). m / z (ESI-): 588.2 (100%), (C30H29F3NO8 - Calculated value: [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).

[0804] (S)-DMT-serinol (TFA)-succinate-lcaa-CPG (10)

[0805] (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).

[0806] GalNAc synthon(9)

[0807] 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.

[0808] The representational data were matched with the publicly available data.

[0809] Oligonucleotide synthesis

[0810] All single-stranded oligonucleotides were prepared according to the Fig.13 and Fig.14 The reaction conditions described in the synthesis.

[0811] All final single-stranded products are analyzed by AEX-HPLC to prove their purity. Purity is given as FLP% (full-length product%), which is the percentage of UV area under the specified product signal in the case of the ultraviolet trace of the AEX-HPLC analysis of the final product. The identity of the corresponding single-stranded product (non-modified, amino-modified precursor, C4XLT / ST23 or C6XLT / ST23GalNAc conjugated oligonucleotide) is proved by LC-MS analysis.

[0812] Table 7: Single-stranded unconjugated and on-column conjugated oligonucleotides

[0813]

[0814] Synthesis of Conjugate 1-3 and Reference Conjugate 1-2

[0815] Conjugate 1-2 and conjugated single chain of reference conjugate 1-2

[0816] 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 give the final product 12.

[0817] Table 8: Single-stranded GalNAc-conjugated oligonucleotides

[0818]

[0819] Double strand formation

[0820] Double-strand formation was performed according to the above method.

[0821] 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.

[0822] Table 9: Nucleic acid conjugates

[0823]

[0824]

[0825] sequence

[0826] Modified index for the following sequence:

[0827] f represents 2'fluoro 2'deoxyribonucleotide or 2'-fluororibonucleotide (the terms are interchangeable)

[0828] m represents 2'O methyl ribonucleotide

[0829] (ps) indicates phosphorothioate bond

[0830] Ser(GN) is a GalNAc-C4 building block attached to a serinol-derived linker moiety:

[0831]

[0832] wherein O--- is a bond between an oxygen atom and, for example, H, a phosphodiester bond, or a phosphorothioate bond.

[0833] The C4XLT is:

[0834]

[0835] The C6XLT is:

[0836]

[0837] ST23 is:

[0838]

[0839] 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.

[0840] C4XLT-phos:

[0841]

[0842] C6XLT-phos:

[0843]

[0844] ST23-phos:

[0845]

[0846] Example 10

[0847] 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.

[0848] The 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. In conjugate 19, a serinol-GalNAc unit was each linked to the 5' and 3' of the second strand via a PS bond. In conjugate 20, there was a phosphodiester between the two terminal 5' nucleotides of the second strand, and a triantennary GalNAc linker was linked to this end via a PS bond.

[0849] 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 to untreated hepatocytes (named ut (ΔΔct)).

[0850] Data shown in Fig.18 middle.

[0851] Materials and methods:

[0852] siRNA

[0853]

[0854] Chart symbols

[0855]

[0856]

[0857] Primers:

[0858]

[0859] General Approach

[0860] In vitro experiments

[0861] 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 of 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.

[0862] TaqMan Assays

[0863] 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.

[0864] 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.

[0865] In vivo experiments

[0866] 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.

[0867] Decontaminated lysosome stability assay

[0868] 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, 4, 24 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 the 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.

[0869] Example 11 - Synthesis of Conjugate 3

[0870] 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.

[0871] Oligonucleotide synthesis, deprotection and purification followed standard procedures known in the art.

[0872] 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.

[0873] 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.

[0874] Attachment of the serinol-derived linker moiety was achieved by using substrate-supported (S)-DMT-serinol (TFA)-succinate-lcaa-CPG 10 or (S)-DMT-serinol (TFA) phosphoramidite 7 (synthesis was performed as described in Hoevelmann et al. (2016)). The triantennary GalNAc cluster (ST23 / C4XLT) was introduced by sequential coupling of the corresponding triplyl amidite derivative (C4XLT-phos) and the GalNAc amidite (ST23-phos).

[0875] Attachment of amino-modified moieties (non-serinol-derivatized linkers) was achieved by using the corresponding commercially available amino-modified building blocks CPG or amidite.

[0876] 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.

[0877] 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.

[0878] Synthesis of Compound 2-10

[0879] 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).

[0880] (S)-4-(3-(bis(4-methoxyphenyl)phenyl)methoxy)-2-(2,2,2-trifluoroacetamido)propoxy)-4-oxobutanoic acid (6).

[0881] To a solution of 5 in pyridine 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 of 6 (yield = 38%). m / z (ESI-): 588.2 (100%), (C30H29F3NO8 - Calculated value: [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).

[0882] (S)-DMT-serinol (TFA)-succinate-lcaa-CPG (10)

[0883] (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).

[0884] GalNAc synthon(9)

[0885] 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.

[0886] The representational data were matched with the publicly available data.

[0887] Oligonucleotide synthesis

[0888] All single-stranded oligonucleotides were prepared according to the Fig.13 and Fig.14 The reaction conditions described in were used for the synthesis and are summarized in Tables 10 and 11.

[0889] 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.

[0890] Table 10: Single-stranded unconjugated oligonucleotides

[0891]

[0892] 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.

[0893] Table 11: Single-stranded oligonucleotides with 5' and 3' modifications

[0894]

[0895] 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.

[0896] Synthesis of Certain Conjugates of the Invention and Reference Conjugates 1-2

[0897] 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 12).

[0898] Table 12: Single-stranded GalNAc-conjugated oligonucleotides

[0899]

[0900] Synthesis of Certain Conjugates of the Invention

[0901] 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 13).

[0902] Table 13: Single-stranded GalNAc-conjugated oligonucleotides

[0903]

[0904] Double strand formation

[0905] Double strand formation was performed according to the method described above.

[0906] 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 14).

[0907] Table 14: Nucleic acid conjugates

[0908]

[0909] sequence

[0910] Modified index for the following sequence:

[0911] f represents 2'fluoro 2'deoxyribonucleotide or 2'-fluororibonucleotide (the terms are interchangeable)

[0912] m represents 2'O methyl ribonucleotide

[0913] (ps) indicates phosphorothioate bond

[0914] FAM = 6-carboxyfluorescein

[0915] BHQ = Black Hole Quencher 1

[0916] YY=Yakima Yellow

[0917] definition

[0918] Ser(GN) is a GalNAc-C4 building block attached to a serinol-derived linker moiety:

[0919]

[0920] wherein O--- is a bond between an oxygen atom and, for example, H, a phosphodiester bond, or a phosphorothioate bond.

[0921] GN is:

[0922]

[0923] The C4XLT (also known as the ST41) is:

[0924]

[0925] ST23 is:

[0926]

[0927] The synthesis of phosphoramidite derivatives of C4XLT (C4XLT-phos) and phosphoramidite derivatives of ST23 (ST23-phos) can be carried out as described in WO2017 / 174657.

[0928] C4XLT-phos:

[0929]

[0930] ST23-phos:

[0931]

[0932]

[0933] Wherein G=H (before conjugation) or G=GN (after conjugation).

[0934] Conjugate 1

[0935] Antisense strand - STS16001AL33 (SEQ ID NO:127)

[0936]

[0937] 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’

[0938] Sense strand - STS16001BL20 (SEQ ID NO:128)

[0939] 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’

[0940] Conjugate 2

[0941] Antisense strand - STS16001A (SEQ ID NO:129)

[0942] 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

[0943] Sense strand - STS16001BV1L42 (SEQ ID NO:130)

[0944] 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)

[0945] Conjugate 3

[0946] Antisense strand - STS16001AL33 (SEQ ID NO:127)

[0947] 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’

[0948] Sense strand-STS16001BV1L42 (SEQ ID NO: 130)

[0949] 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'

[0950] Conjugate 4

[0951] Antisense strand-STS16001A (SEQ ID NO: 129)

[0952] 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

[0953] Sense strand-STS16001BV1L75 (SEQ ID NO: 142)

[0954] 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'

[0955] Conjugate 5

[0956] Antisense strand-STS16001A (SEQ ID NO: 129)

[0957] 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

[0958] Sense strand-STS16001BV16L42 (SEQ ID NO: 143)

[0959] 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'

[0960] Conjugate 6

[0961] Antisense strand-STS16001A (SEQ ID NO: 129)

[0962] 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

[0963] Sense strand - STS16001BV20L75 (SEQ ID NO:144)

[0964] 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’

[0965] Conjugate 7

[0966] Antisense strand - (SEQ ID NO:129)

[0967] 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

[0968] Sense strand - STS16001BV1L94 (SEQ ID NO:145)

[0969] 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’

[0970] Conjugate 8

[0971] Antisense strand - STS16001A (SEQ ID NO:129)

[0972] 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’

[0973] Sense strand - STS16001V1BL96 (SEQ ID NO:146)

[0974] 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’

[0975] Conjugate 9

[0976] Antisense strand - STS16001A (SEQ ID NO:129)

[0977] 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’

[0978] Sense strand - STS16001V1BL97 (SEQ ID NO:147)

[0979] 5’GlyC3Am(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’

[0980] Conjugate 10

[0981] Antisense strand - STS16001A (SEQ ID NO:129)

[0982] 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’

[0983] Sense strand (SEQ ID NO:148)

[0984] 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’

[0985] Conjugate 11

[0986] Antisense strand - STS16001A (SEQ ID NO:129)

[0987] 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’

[0988] Sense strand - STS16001V1BL88 (SEQ ID NO:149)

[0989] 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’

[0990] Conjugate 12

[0991] Antisense strand - STS16001A (SEQ ID NO:129)

[0992] 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’

[0993] Sense strand - STS16001V1BL87 (SEQ ID NO:150)

[0994] 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’

[0995] Conjugate 15

[0996] Antisense strand (SEQ ID NO:151)

[0997] mU(ps)fC(ps)mU fU mC fU mU fA mA fA mC fU mG fA mG fU mU(ps)fU(ps)mC

[0998] Sense strand (SEQ ID NO:152)

[0999] 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)

[1000] Conjugate 16

[1001] Antisense strand (SEQ ID NO: 153)

[1002] mA(ps)fU(ps)mG fU mA fG mC fC mG fA mG fG mA fU mC fU mU(ps)fC(ps)mU

[1003] Sense strand (SEQ ID NO: 154)

[1004] 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)

[1005] Conjugate 18

[1006] Antisense strand (SEQ ID NO: 155)

[1007] mA(ps)fA(ps)mC fC mA fG mA fA mG fA mA fG mC fA mG fG mU(ps)fG(ps)mA

[1008] Sense strand (SEQ ID NO: 156)

[1009] 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)

[1010] Conjugate 19

[1011] Antisense strand (SEQ ID NO: 135)

[1012] mA(ps)fU(ps)mA fA mC fU mC fU mG fU mC fC mA fU mU fA mC(ps)fC(ps)mG

[1013] Sense strand (SEQ ID NO: 136)

[1014] 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)

[1015] Reference conjugate 1

[1016] Antisense strand-STS16001A (SEQ ID NO: 129)

[1017] 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

[1018] Sense strand - STS16001BL20 (SEQ ID NO: 128)

[1019] 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

[1020] Reference conjugate 2

[1021] Antisense strand-STS16001AL33 (SEQ ID NO: 127)

[1022] 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)

[1023] Sense strand-STS16001BV1 (SEQ ID NO: 157)

[1024] 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

[1025] Reference conjugate 3 – "Luc"

[1026] Antisense strand-STS18001A (A0130, SEQ ID NO: 132)

[1027] mU(ps)fC(ps)mG fA mA fG mU fA mU fU mC fC mG fC mG fU mA(ps)fC(ps)mG

[1028] Sense strand-STS18001BL4 (A0131, SEQ ID NO: 133)

[1029] [(ST23)(ps)]3C4XLT(ps)fC mG fU mA fC mG fC mG fG mA fA mU fA mC fU mUfC(ps)mG(ps)fA

[1030] Reference conjugate 4

[1031] Antisense strand - STS16001AL33 (SEQ ID NO:127)

[1032] 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

[1033] Sense strand - STS16001BL4 (SEQ ID NO:134)

[1034] 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

[1035] Reference conjugate 5 – "Ctr"

[1036] Antisense strand (SEQ ID NO:138)

[1037] mC(ps)fU(ps)mU fA mC fU mC fU mC fG mC fC mC fA mA fG mC(ps)fG(ps)mA

[1038] Sense strand (SEQ ID NO:139)

[1039] [(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

[1040] Reference conjugate 6

[1041] Antisense strand (SEQ ID NO:151)

[1042] mU(ps)fC(ps)mU fU mC fU mU fA mA fA mC fU mG fA mG fU mU(ps)fU(ps)mC

[1043] Sense strand (SEQ ID NO:158)

[1044] [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

[1045] Reference conjugate 7

[1046] Antisense strand (SEQ ID NO:153)

[1047] mA(ps)fU(ps)mG fU mA fG mC fC mG fA mG fG mA fU mC fU mU(ps)fC(ps)mU

[1048] Sense strand (SEQ ID NO:159)

[1049] [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

[1050] Reference conjugate 8

[1051] Antisense strand (SEQ ID NO:160)

[1052] mU(ps)fA(ps)mC fC mA fG mA fA mG fA mA fG mC fA mG fG mU(ps)fG(ps)mA

[1053] Sense strand (SEQ ID NO:161)

[1054] [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

[1055] Reference conjugate 9

[1056] Antisense strand (SEQ ID NO:135)

[1057] mA(ps)fU(ps)mA fA mC fU mC fU mG fU mC fC mA fU mU fA mC(ps)fC(ps)mG

[1058] Sense strand (SEQ ID NO: 162)

[1059] [ST23(ps)]3C6XLT(ps)fC mG fG mU fA mA fU mG fG mA fC mA fG mA fG mUfU(ps)mA(ps)fU

[1060] Example 12 - In vitro determination of TTR knockdown achieved with various TTR siRNA GalNAc conjugates

[1061] Conjugates 1 to 3

[1062] 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.

[1063] Compared to the negative control (see the "UT" column and reference conjugate 3), the target gene expression in primary mouse hepatocytes 24 hours after treatment with the conjugates of the present invention, i.e., conjugates 1-3 at 0.01 nM, 0.1 nM, 0.5 nM, 1 nM, and 10 nM, showed that the target gene expression decreased with increasing doses of the conjugates, as shown in FIG15. This indicates that the first strand binds to the target gene, thereby reducing gene expression. FIG15 also shows the target gene expression levels in the case of reference conjugates 1 and 2, which serve as comparative conjugates. As can be seen from Fig.15A and Fig. 15C ,as well as Fig. 15B and Fig. 15C As seen from the comparison between the data presented in , the conjugates of the invention (Conjugates 1-3) reduced target gene expression compared to reference conjugates 1 and 2. The most effective conjugate at 0.01 nM appeared to be Conjugate 2. The most effective conjugate at 0.1 nM, 0.5 nM, 1 nM and 10 nM appeared to be Conjugate 3.

[1064] Conjugates 4 to 7

[1065] The methods described above under "In vitro experiments" in the General Methods section were followed.

[1066] 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 31. This indicates that the first strand binds to the target gene, thereby reducing gene expression.

[1067] 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 three terminal nucleotides in the sense strand could be varied while maintaining efficacy in reducing target gene expression.

[1068] Conjugates 8 to 12 and 19

[1069] The methods described above under "In vitro experiments" in the General Methods section were followed.

[1070] 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 32. 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.

[1071] 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.33 This indicates that the first strand binds to the target gene, thereby reducing gene expression.

[1072] 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.

[1073] Example 13 - In vivo time course of serum Ttr, Aldh2 and Tmprss6 in mice

[1074] Conjugates 1 to 3

[1075] 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).

[1076] 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 Fig.16 In. Fig.16 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 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 2 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.

[1077] 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.

[1078] Conjugates 15 to 18

[1079] The methods described above under "In vivo experiments" in the General Methods section were followed.

[1080] 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.34 and Fig.35 In. Fig.34 and Fig.35 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.

[1081] 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.36 In. Fig.36 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 the reference conjugate 8.

[1082] 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.

[1083] Example 14 - Serum stability study

[1084] The method described above under "Decontaminated lysosomal stability assay" in the General Methods section was followed.

[1085] Fig.37 Results from serum stability studies for conjugates 2, 4, 5, 6, and 7 are shown. Fig.38 The serum stability of conjugates 2, 8, 9, 10, 11 and 12 is shown.

[1086] All conjugates of the invention tested were more stable in serum compared to the control.

[1087] 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.

[1088] 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, is used to achieve conjugation with both ends of the second chain. In conjugate 2, PS is used 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.

[1089] 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 three terminal nucleotides in the sense strand can be varied while maintaining stability in serum.

[1090] Embodiment 15

[1091] Knockdown of LPA-mRNA in human primary hepatocytes by different indicated L6-GalNAc-conjugated siRNAs after receptor-mediated delivery.

[1092] Table 15 : Other conjugated nucleic acid sequences tested for inhibition of LPA mRNA expression. The sequences and applied modification patterns are indicated.

[1093]

[1094]

[1095] Table 15

[1096] Table 15: 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.

[1097] 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 were added immediately after cell plating at the indicated concentrations. Total RNA was isolated 24 hours after treatment with siRNA 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 ​​were normalized to LPA mRNA expression in untreated cells. Means and SDs of normalized triplicate values ​​are shown in Fig.39 IC estimation using four-parameter nonlinear regression curve fitting 50 value and maximum suppression.

[1098] Claim of Invention

[1099] 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 an RNA transcribed from an LPA gene, wherein the first strand comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 9, 1, 3, 5, 7, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 63, 65, 67, 69, 71 or 73 or any sequence disclosed herein.

[1100] 2. The nucleic acid of claim 1, wherein the second strand comprises the nucleotide sequence of SEQ ID NO: 10, 2, 4, 6, 8, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 64, 66, 68, 70, 72 or 74, or any sequence disclosed herein.

[1101] 3. The nucleic acid of statement 1 or statement 2, wherein the first strand comprises the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 5.

[1102] 4. The nucleic acid of any one of statements 1 to 3, wherein the second strand comprises the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 6.

[1103] 5. The nucleic acid according to any one of statements 1 to 4, wherein the first strand and / or the second strand are each 17-35 nucleotides in length.

[1104] 6. The nucleic acid of any one of statements 1 to 5, wherein the at least one duplex region consists of 19-25 consecutive nucleotide base pairs.

[1105] 7. A nucleic acid as described in any of the previous statements, wherein the nucleic acid

[1106] a) are blunt-ended at both ends; or

[1107] b) has a protruding portion at one end and a blunt end at the other end; or

[1108] c) having overhangs at both ends.

[1109] 8. The nucleic acid according to any preceding statement, wherein one or more nucleotides on the first strand and / or the second strand are modified to form modified nucleotides.

[1110] 9. The nucleic acid of statement 8, wherein one or more of the odd-numbered nucleotides of the first strand are modified.

[1111] 10. The nucleic acid of statement 9, wherein one or more of the even-numbered nucleotides of the first strand is modified by at least a second modification, wherein the at least second modification is different from the modification described in statement 9.

[1112] 11. The nucleic acid of statement 10, wherein at least one of the one or more modified even-numbered nucleotides is adjacent to at least one of the one or more modified odd-numbered nucleotides.

[1113] 12. The nucleic acid of any one of statements 9 to 11, wherein a plurality of odd-numbered nucleotides are modified.

[1114] 13. The nucleic acid of statements 10 to 12, wherein a plurality of even-numbered nucleotides are modified by a second modification.

[1115] 14. The nucleic acid of any one of statements 8 to 13, wherein the first strand comprises adjacent nucleotides modified by a common modification.

[1116] 15. The nucleic acid of any one of statements 9 to 14, wherein the first strand comprises adjacent nucleotides modified by a second modification different from the modification of statement 9.

[1117] 16. The nucleic acid of any one of statements 9 to 15, wherein one or more of the odd-numbered nucleotides of the second strand is modified by a modification different from the modification described in statement 9.

[1118] 17. The nucleic acid of any one of statements 9 to 15, wherein one or more of the even-numbered nucleotides of the second strand is modified by a modification as described in statement 9.

[1119] 18. The nucleic acid of statement 16 or 17, wherein at least one of the one or more modified even-numbered nucleotides of the second strand is adjacent to one or more modified odd-numbered nucleotides of the second strand.

[1120] 19. The nucleic acid of any one of statements 16 to 18, wherein a plurality of odd-numbered nucleotides of the second strand are modified by a common modification.

[1121] 20. The nucleic acid of any one of statements 16 to 19, wherein a plurality of even-numbered nucleotides are modified by a modification according to statement 9.

[1122] 21. The nucleic acid of any one of statements 16 to 20, wherein a plurality of odd-numbered nucleotides on the second strand are modified by a second modification, wherein the second modification is different from the modification described in statement 9.

[1123] 22. The nucleic acid of any one of statements 16 to 21, wherein the second strand comprises adjacent nucleotides modified by a common modification.

[1124] 23. The nucleic acid of any one of statements 16 to 22, wherein the second strand comprises adjacent nucleotides modified by a second modification different from the modification of statement 9.

[1125] 24. The nucleic acid of any one of statements 8 to 23, wherein each of the odd-numbered nucleotides in the first strand and each of the even-numbered nucleotides in the second strand are modified with a common modification.

[1126] 25. The nucleic acid of statement 24, wherein each of the even-numbered nucleotides in the first strand is modified with a second modification, and each of the odd-numbered nucleotides in the second strand is modified with a second modification.

[1127] 26. The nucleic acid of any one of statements 8 to 25, wherein the modified nucleotides of the first strand are shifted by at least one nucleotide relative to the unmodified or differently modified nucleotides of the second strand.

[1128] 27. A nucleic acid according to any one of statements 8 to 26, wherein the one or more modifications are each and individually selected from the group consisting of: 3'-terminal deoxythymine, 2'-O-methyl, 2'-deoxy modification, 2'-amino modification, 2'-alkyl modification, morpholino modification, amino phosphoroate modification, 5'-thiophosphate group modification, 5' phosphate or 5' phosphate mimetic modification and cholesterol derivative or dodecanoic acid bisdecylamide group modification.

[1129] 28. The nucleic acid of any one of statements 8 to 27, wherein the modification is any of a locked nucleotide, an abasic nucleotide, or a nucleotide comprising a non-natural base.

[1130] 29. The nucleic acid of any one of statements 8 to 28, wherein at least one modification is a 2'-O-methyl.

[1131] 30. The nucleic acid of any one of statements 8 to 29, wherein at least one modification is 2'-F.

[1132] 31. 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 group consisting of SEQ ID NO: 9, 1, 3, 5, 7, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 63, 65, 67, 69, 71 or 73, wherein the nucleotides of the first strand are modified by a first modification on odd-numbered nucleotides and by a second modification on even-numbered nucleotides, and the nucleotides of the second strand are modified by a third modification on even-numbered nucleotides and by a fourth modification on odd-numbered nucleotides, wherein at least the first modification is different from the second modification and the third modification is different from the fourth modification.

[1133] 32. The nucleic acid of claim 31, wherein the second sequence comprises the nucleotide sequence of SEQ ID NO: 10, 2, 4, 6, 8, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 64, 66, 68, 70, 72 or 74.

[1134] 33. The nucleic acid of statement 31 or 32, wherein the fourth modification and the second modification are the same.

[1135] 34. The nucleic acid of any one of statements 31 to 33, wherein the first modification and the third modification are the same.

[1136] 35. The nucleic acid of any one of statements 31 to 34, wherein the first modification is 2'O-Me and the second modification is 2'F.

[1137] 36. The nucleic acid of any one of statements 31 to 35, wherein the first strand comprises the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 5, and the second strand comprises the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 6.

[1138] 37. A nucleic acid as described in any one of statements 31 to 36, comprising the sequence and modifications as shown below:

[1139]

[1140] Where specific modifications are depicted by numbers

[1141] 1 = 2'F-dU;

[1142] 2 = 2'F-dA;

[1143] 3 = 2'F-dC;

[1144] 4 = 2'F-dG;

[1145] 5=2'-OMe-rU;

[1146] 6 = 2'-OMe-rA;

[1147] 7 = 2'-OMe-rC;

[1148] 8 = 2'-OMe-rG.

[1149] 38. The nucleic acid of any one of statements 1 to 37, conjugated to a ligand.

[1150] 39. The nucleic acid of any one of statements 1 to 38, comprising phosphorothioate bonds between one, two or three terminal 3' nucleotides and / or 5' nucleotides of the first strand and / or the second strand.

[1151] 40. A nucleic acid according to any one of statements 1 to 39, comprising two phosphorothioate bonds between each of the three terminal 3' nucleotides on the first strand and between each of the three terminal 5' nucleotides on the first strand, and two phosphorothioate bonds between the three terminal nucleotides at the 3' end of the second strand.

[1152] 41. 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 group consisting of SEQ ID NO: 9, 1, 3, 5, 7, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 63, 65, 67, 69, 71 or 73, and wherein the nucleic acid is conjugated to a ligand.

[1153] 42. A nucleic acid according to statement 41, wherein the ligand comprises (i) one or more N-acetylgalactosamine (GalNac) moieties and derivatives thereof, and (ii) a linker, wherein the linker conjugates the GalNac moiety to the nucleic acid as defined in statement 41.

[1154] 43. The nucleic acid according to any one of statements 41 or 42, wherein the linker is a divalent, trivalent or tetravalent branched structure.

[1155] 44. The nucleic acid of any one of statements 41 to 43, wherein the nucleotides are modified as defined in any of the preceding statements.

[1156] 45. A nucleic acid as described in any of the previous statements, conjugated to a ligand comprising formula I:

[1157] [SX 1 -PX 2 ]3-AX 3 -(I)

[1158] in:

[1159] S represents a sugar, wherein the sugar is N-acetylgalactosamine;

[1160] X 1 represents C3-C6 alkylene or (-CH2-CH2-O) m (-CH2)2-, where m is 1, 2 or 3;

[1161] P is phosphate or a modified phosphate (preferably phosphorothioate);

[1162] X 2 is an alkylene group or a group of formula (-CH2) n -O-CH2-alkylene ether, wherein n=1-6;

[1163] A is the branching unit;

[1164] X 3 represents the bridge unit;

[1165] wherein the nucleic acid as defined in any one of statements 1 to 40 is conjugated to X via a phosphate or a modified phosphate, preferably via a phosphorothioate 3 .

[1166] 46. ​​A conjugated nucleic acid having one of the following structures

[1167]

[1168]

[1170]

[1172]

[1173]

[1174] wherein Z is a nucleic acid according to any one of statements 1 to 40.

[1175] 47. A nucleic acid according to any one of statements 1 to 40, wherein the nucleic acid is conjugated to a ligand having the structure

[1176]

[1177] 48. A nucleic acid or conjugated nucleic acid as described in any preceding statement, wherein the duplex comprises individual strands.

[1178] 49. A nucleic acid or conjugated nucleic acid as described in any previous statement, wherein the duplex comprises a single strand comprising a first strand and a second strand.

[1179] 50. A composition comprising a nucleic acid or a conjugated nucleic acid as described in any of the previous statements and a physiologically acceptable excipient.

[1180] 51. A nucleic acid or conjugated nucleic acid according to any preceding statement for use in preventing or treating a disease or pathology, or reducing the risk of a disease or pathology.

[1181] 52. Use of a nucleic acid or conjugated nucleic acid according to any preceding statement for the manufacture of a medicament for the prevention or treatment of a disease, disorder or syndrome.

[1182] 53. A method of 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 or conjugated nucleic acid according to any of the preceding statements.

[1183] 54. The method of statement 53, wherein the nucleic acid or conjugated nucleic acid is administered to the subject subcutaneously, intravenously, or using any other administration route such as oral, rectal, or intraperitoneal administration.

[1184] 55. The use or method of statements 52 to 54, wherein the disease or pathology is cardiovascular disease, stroke, atherosclerosis, thrombosis or cardiovascular diseases such as coronary heart disease or aortic valve stenosis and any other disease or pathology associated with elevated levels of Lp(a) containing particles.

[1185] 56. The use or method of statement 55, wherein the cardiovascular disease is stroke, atherosclerosis, thrombosis, coronary heart disease or aortic valve stenosis and any other disease or pathology associated with elevated levels of Lp(a)-containing particles.

[1186] 57. A method for preparing a nucleic acid or a conjugated nucleic acid as described in any one of statements 1 to 49.

[1187] 58. A nucleic acid for inhibiting the expression of LPA, 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 expression of LPA is reduced to a level that is at least 15% lower than the expression level observed under the same test conditions but in the absence of the nucleic acid or conjugated nucleic acid or in the presence of a non-silencing control.

[1188] 59. A nucleic acid for inhibiting the expression of LPA, 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 group consisting of SEQ ID NO: 9, 1, 3, 5, 7, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 63, 65, 67, 69, 71 or 73, wherein the expression of LPA is reduced to a level that is at least 15% lower than the expression level observed under the same test conditions but in the absence of the nucleic acid or conjugated nucleic acid or in the presence of a non-silencing control.

[1189] 60. 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 NOs: 63, 65, 67 and 73, wherein the first RNA strand has a terminal 5' (E)-vinylphosphonate nucleotide.

[1190] Other provisions of the present invention include:

[1191] 1. A conjugate for inhibiting the expression of an LPA gene in a cell, the conjugate comprising a nucleic acid portion and a 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 ligand portion comprising 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:

[1192] (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

[1193] (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, or

[1194] A conjugate for inhibiting the expression of an LPA gene in a cell, the conjugate comprising a nucleic acid portion and a 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 ligand portion comprising a 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:

[1195] (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

[1196] (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, optionally

[1197] wherein the linker moiety is a serinol-derivatized linker moiety, or one of the other linker types described herein.

[1198] 2. The conjugate according to clause 1, wherein the second RNA strand is conjugated to the targeting ligand at the 5' end, the second RNA strand is also conjugated to the targeting ligand at the 3' end, and the 3' end of the first RNA strand is not conjugated.

[1199] 3. The conjugate according to clause 1, wherein the second RNA strand is conjugated to the targeting ligand at the 5' end, the first RNA strand is conjugated to the targeting ligand at the 3' end, and the 3' end of the second RNA strand is not conjugated.

[1200] 4. The conjugate according to clause 1, wherein the second RNA strand is conjugated to the targeting ligand at the 5' end, and both the second RNA strand and the first RNA strand are also conjugated to the targeting ligand at the 3' end.

[1201] 5. The conjugate according to clause 1, wherein 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.

[1202] 6. The conjugate according to any one of clauses 1 to 5, wherein the ligand is a monomeric ligand.

[1203] 7. The conjugate according to any one of clauses 1 to 6, wherein the ligand is selected from GalNAc and a galactose moiety, in particular a GalNAc moiety.

[1204] 8. A conjugate according to any one of clauses 1 to 7, wherein the conjugated RNA strand is conjugated to the targeting ligand via a serinol-derived 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).

[1205] 9. The conjugate according to clause 8, wherein the further 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.

[1206] 10. The conjugate according to clause 9, wherein the further linker comprises a PEG chain.

[1207] 11. The conjugate according to clause 8, wherein the further linker comprises a saturated, unbranched C 1-15 Alkyl chain.

[1208] 12. The conjugate according to clause 11, wherein the further linker comprises a saturated, unbranched C 1-6 Alkyl chain.

[1209] 13. The conjugate according to clause 12, wherein the further linker comprises a saturated, unbranched C4 or C6 alkyl chain, such as a C4 alkyl chain.

[1210] 14. The conjugate according to clause 1 or statement 1, wherein the first RNA strand is a compound of formula (XV):

[1211]

[1212] where b is 0 or 1; and

[1213] The second RNA strand is a compound of formula (XVI):

[1214]

[1215] (XVI);

[1216] Where c and d are independently 0 or 1;

[1217] in:

[1218] Z1 and Z2 are RNA parts of the first RNA strand and the second RNA strand, respectively;

[1219] Y is O or S;

[1220] R1 is H or methyl;

[1221] n is 0, 1, 2 or 3; and

[1222] In formulae (XV) and (XVI), L is the same or different and is selected from the group consisting of:

[1223] -(CH2) q -, where q = 2-12;

[1224] -(CH2) r -C(O)-, wherein r = 2-12;

[1225] -(CH2-CH2-O) s -CH2-C(O)-, where s = 1-5;

[1226] -(CH2) t -CO-NH-(CH2) t -NH-C(O)-, wherein t is independently 1-5;

[1227] -(CH2) u -CO-NH-(CH2) u -C(O)-, wherein u is independently 1-5; and

[1228] -(CH2) v -NH-C(O)-, wherein v is 2-12; and

[1229] wherein if a terminal C(O) is present, it is attached to an NH group;

[1230] And where b+c+d is 2 or 3.

[1231] 15. The conjugate according to clause 14, wherein b is 0, c is 1, and d is 1.

[1232] 16. The conjugate according to clause 14, wherein b is 1, c is 0, and d is 1.

[1233] 17. The conjugate according to clause 14, wherein b is 1, c is 1, and d is 0.

[1234] 18. The conjugate according to clause 14, wherein b is 1, c is 1, and d is 1.

[1235] 19. The conjugate according to any one of clauses 14-18, wherein Y is O.

[1236] 20. The conjugate according to any one of clauses 14-18, wherein Y is S.

[1237] 21. The conjugate according to any one of clauses 14-20, wherein R1 is H.

[1238] 22. The conjugate according to any one of clauses 14-20, wherein R1 is methyl.

[1239] 23. The conjugate according to any one of clauses 14-22, wherein n is 0.

[1240] 24. The conjugate according to any one of clauses 14-23, wherein L is -(CH2) r -C(O)-, wherein r=2-12.

[1241] 25. The conjugate according to clause 24, wherein r=2-6.

[1242] 26. The conjugate according to clause 25, wherein r=4 or 6, such as 4.

[1243] 27. A conjugate according to any preceding clause, having any feature or combination of features disclosed herein.

[1244]

[1245]

[1246]

[1247]

[1248]

[1249]

[1250]

[1251]

[1252]

[1253]

[1254]

[1255] index 1=2'F-dU2=2'F-dA3=2'F-dC4=2'F-dG5=2'OMe-rU6=2'OMe-rA7=2'OMe-rC8=2'OMe-rG

[1256] mA, mU, mC, mG, OMeA, OMeU, OMeC, OMeG–2'-OMe RNA

[1257] fA, fU, fC, fG, FA, FU, FG, FC–2’deoxy-2’-F RNA

[1258] (ps) – phosphorothioate

[1259] (vp)-vinyl-(E)-phosphonate

[1260] ivA, ivC, ivU, ivG - inverted RNA (3'-3')

[1261] A single sequence may have more than one name. In those cases, one of those names is given in the summary sequence table.

[1262] 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.

[1263] The following abbreviations may be used:

[1264]

[1265]

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 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.

2. The nucleic acid of claim 1, wherein the first strand comprises the nucleotide sequence of SEQ ID NO: 9, and optionally, wherein the second strand comprises the nucleotide sequence of SEQ ID NO: 10; or wherein the first strand comprises the nucleotide sequence of SEQ ID NO: 5, and optionally, wherein the second strand comprises the nucleotide sequence of SEQ ID NO:

6.

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: 63, 65, 67, 69, 71 or 73.

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

5. The nucleic acid according to any one of the preceding claims, wherein the at least one duplex region consists of 17-25, preferably 19-25, consecutive nucleotide base pairs.

6. A nucleic acid as claimed in any one of the preceding claims, wherein the nucleic acid: a) are blunt-ended at both ends; or b) has a protruding portion at one end and a blunt end at the other end; or c) having overhangs at both ends.

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

8. The nucleic acid of any one of the preceding claims, 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.

9. The nucleic acid of any one of the preceding claims, wherein the nucleic acid is conjugated to a ligand.

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.

11. The nucleic acid of any one of claims 9 to 10, wherein the nucleic acid is conjugated to a ligand comprising a compound of formula (I): [SX 1 -PX 2 ]3-AX 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 8 is conjugated to X via a phosphate or a modified phosphate, preferably via a phosphorothioate. 3 .

12. The nucleic acid of any one of claims 9 to 10, wherein the first RNA strand is a compound of formula (X): where b is 0 or 1; and The second RNA strand is a compound of formula (XI): in: c and d are independently 0 or 1; Z1 and Z2 are RNA parts of the first RNA strand and the second RNA strand, respectively; Y is O or S; n is 0, 1, 2 or 3; and L1 is a linker attached to the ligand; and Where b+c+d is 2 or 3.

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.

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

15. The nucleic acid of claim 14, wherein the nucleic acid comprises two phosphorothioate bonds between each of the three terminal 3' nucleotides on the first strand and between each of the three terminal 5' nucleotides on the first strand, and two phosphorothioate bonds between the three terminal nucleotides at the 3' end of the second strand, and wherein the ligand is conjugated to the 5' end of the second strand.

16. The nucleic acid of claim 13, wherein the nucleic acid is conjugated to a ligand, wherein the first RNA strand is a compound of formula (XV): where b is 0 or 1; and The second RNA strand is a compound of formula (XVI): Where c and d are independently 0 or 1; in: Z1 and Z2 are RNA parts of the first RNA strand and the second RNA strand, respectively; Y is O or S; R1 is H or methyl; n is 0, 1, 2 or 3; and In formulae (XV) and (XVI), L is the same or different and is selected from the group consisting of: -(CH2) q , where q = 2-12; -(CH2) r -C(O)-, wherein r = 2-12; -(CH2-CH2-O) s -CH2-C(O)-, where s = 1-5; -(CH2) t -CO-NH-(CH2) t -NH-C(O)-, wherein t is independently 1-5; -(CH2) u -CO-NH-(CH2) u -C(O)-, wherein u is independently 1-5; and -(CH2) v -NH-C(O)-, wherein v is 2-12; and wherein if a terminal C(O) is present, it is attached to an NH group; And where b+c+d is 2 or 3.

17. A nucleic acid according to any one of claims 13 to 16, comprising the sequence and modifications shown below: in, Specific modifications are depicted by numbers 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.

18. The nucleic acid of any one of claims 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 at positions 11, or 13, or 11 and 13, or 11-13 of the second strand corresponding to the first strand are modified with a 2' fluoro modification.

19. A composition comprising a nucleic acid as described in any one of claims 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) particles.

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

21. Use of a nucleic acid as described in any one of claims 1 to 18 or a pharmaceutical composition as described in claim 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.

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 claims 1 to 18 or a composition as described in claims 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.

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