RU2822093C1 - Nucleic acids for inhibiting expression of lpa in cell - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described is a group of inventions comprising a nucleic acid for inhibiting expression of LPA in a cell, a conjugate for inhibiting expression of LPA in a cell (embodiments), composition for preventing, or treating, or reducing the risk of developing cardiovascular disease and use of nucleic acid, or a conjugate, or a composition for preventing, or treating, or reducing the risk of developing a cardiovascular disease. In one embodiment, the nucleic acid comprises at least one duplex region, which contains at least part of first chain and at least part of second chain, which is at least partially complementary to said first chain, wherein said first strand is at least partially complementary to at least a portion of the RNA transcribed from the LPA gene.

EFFECT: invention extends the range of agents for inhibiting expression of LPA in a cell.

26 cl, 40 dwg, 15 tbl, 15 ex

Description

In accordance with the present invention, products and compositions are provided, as well as applications thereof. In particular, the present invention relates to nucleic acid derivatives that interfere with or inhibit the expression of the LPA gene. These Lp(a)-lowering therapies serve to prevent and reduce the risk of stroke, atherosclerosis, thrombosis and cardiovascular diseases such as coronary heart disease and aortic stenosis, or any other disorder, pathology or syndrome associated with increased levels of Lp(a)-containing particles.

State of the art

Double-stranded RNA (dsRNA) capable of complementarily binding to expressed mRNA has been shown to block gene expression (Fire et al., 1998, Nature. 1998 Feb 19; 391(6669):806-11 and Elbashir et al., 2001, Nature . 2001 May 24; 411(6836):494-8) through a mechanism called RNA interference (RNAi). Short dsRNAs direct gene-specific post-transcriptional silencing in many organisms, including vertebrates, and such molecules have become useful tools for studying gene function. RNAi is mediated by the RNA-induced silencing complex (RISC), a sequence-specific multicomponent nuclease that degrades messenger RNAs homologous to the initiation silencing factor loaded into the RISC complex. Interfering RNAs (referred to herein as mRNAs), such as siRNA directed against the second strand of RNA and microRNAs, are oligonucleotides that prevent the formation of proteins by suppressing genes, i.e. inhibit the translation of a protein gene by destroying mRNA molecules. Gene silencing agents are becoming increasingly important for therapeutic applications in medicine.

According to Watts and Corey in the Journal of Pathology (2012; Vol 226, p 365-379), there are algorithms that can be used to design nucleic acid silencing triggers, but they all have serious limitations. Identifying effective mRNAs may require different experimental techniques because the algorithms do not take into account factors such as the tertiary structure of the target mRNA or the involvement of RNA-binding proteins. Thus, discovering an effective initiating factor for nucleic acid silencing with minimal off-target effects is a complex process. Pharmaceutical development of these highly potentially dangerous molecules requires that they be synthesized economically, be distributed to target tissues, enter cells, and function within acceptable toxicity limits.

Lp(a) is a heterogeneous low-density lipoprotein particle expressed predominantly in the liver (Witztum and Ginsberg, J Lipid Res. 2016 Mar; 57(3):336-9). It consists of apolipoprotein (a) (Apo(a), encoded by the LPA gene), linked to LDL by the ApoB polypeptide. High serum Lp(a) levels, genetically determined, are independent of diet and exercise and are associated with an increased risk of cardiovascular disease due to the associated potential for the development of atherosclerosis (Alonso et al., Journal of the American College of Cardiology Vol. 63, No. 19, 2014). From a diagnostic and preventive medicine perspective, a patient's serum Lp(a) level is a common, independent genetic risk factor for coronary artery disease and aortic stenosis (Saeedi and Frohlich Clinical Diabetes and Endocrinology (2016) 2:7). There are currently no approved specific therapies to reduce Lp(a) levels beyond indirect, standard general LDL-lowering measures. Accordingly, there is now a need for methods to effectively treat, prevent and reduce the risk of developing disorders such as stroke, atherosclerosis, thrombosis and or associated cardiovascular diseases such as coronary heart disease, aortic stenosis and other as yet unidentified associated disorders. pathologies or syndromes. The present invention addresses this unmet medical need.

A first aspect of the invention relates to a nucleic acid for inhibiting the expression of LPA in a cell comprising at least one duplex region that contains at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, said first strand the strand is at least partially complementary to at least a portion of the RNA transcribed from the LPA gene, wherein said first strand contains 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.

According to one embodiment of the present invention, the first nucleic acid strand comprises a sequence of at least 15, preferably at least 16, more preferably at least 17, even more preferably at least 18, and most preferably at least 19 nucleotides of any one of 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.

According to one embodiment of the present invention, the number of individual nucleotide mismatches in the first strand sequence relative to the portion of the reference sequence that is contained in the first strand sequence is no more than three, preferably no more than two, more preferably no more than one, and most preferably zero.

The second strand may contain the nucleotide sequence 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.

The first strand may comprise the nucleotide sequence of SEQ ID NO: 5 and SEQ ID NO: 9, and/or the second strand may contain the nucleotide sequence of SEQ ID NO: 6 and SEQ ID NO: 10.

Each of said first strand and/or second strand may be 17-35 nucleotides in length, and at least one duplex region may be 10-25 nucleotides in length. A duplex may contain two separate strands, or it may contain a single strand that contains a first strand and a second strand.

A nucleic acid may: a) have blunt ends at both ends; b) contain a sticky end at one end and a blunt end at the other; or c) contain sticky ends at both ends.

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

A plurality of nucleotides at odd positions in the first strand sequence may be modified in the nucleic acid of the present invention. Multiple nucleotides at even-numbered positions in the first strand sequence may be modified using a second modification. The first strand may contain contiguous nucleotides that are modified using a common modification. The first strand may also contain adjacent nucleotides that are modified with a second different modification.

One or more of the nucleotides at the odd positions of the second strand sequence may be modified using a modification that is different from the modification of the nucleotides at the odd positions of the first strand, and/or one or more of the nucleotides at the even positions of the second strand may be modified using a modification that is similar to that for nucleotides in odd positions of the first chain. At least one of the one or more modified nucleotides at even positions of the second strand may be adjacent to one or more modified nucleotides at odd positions. A plurality of nucleotides at odd positions of the second strand may be modified using a common modification, and/or a plurality of nucleotides at even positions may be modified using a modification similar to that present on the odd nucleotides of the first strand. A plurality of nucleotides at odd positions of the second strand may be modified using a second modification, wherein said second modification is different from the modification of nucleotides at odd positions of the first strand.

The second strand may contain contiguous nucleotides that are modified using a common modification, which may be a second modification that is different from the modification of nucleotides at odd positions of the first strand.

In the nucleic acid of the present invention, each of the nucleotides at the odd positions of the first strand and each of the nucleotides at the even positions of the second strand can be modified using a general modification, and each of the nucleotides at the even positions of the first strand can be modified using a second modification, and each of nucleotides at odd positions in the second strand may be modified using a second different modification.

In the nucleic acid of the present invention, the modified first strand nucleotides may be shifted by at least one nucleotide relative to the unmodified or differently modified second strand nucleotides.

The modification and/or modifications may be selected individually and independently from the group consisting of 3'-terminal deoxythymine, 2'-O-methyl, 2'-deoxy modification, 2'-amino modification, 2'-alkyl modification , morpholino modification, phosphoamidate modification, modification with a 5'-phosphothioate group, modification with a 5'-phosphate or a 5'-phosphate mimetic, as well as modification with a cholesterol derivative or a dodecanoic acid bisdecylamide group, and/or the modified nucleotide may be any of the closed nucleotide , lacking the nitrogenous base of a nucleotide or containing an unnatural nucleotide base. At least one modification may be 2'-O-methyl and/or at least one modification may be 2'-F.

The present invention also provides, as a second aspect, a nucleic acid for inhibiting the expression of LPA in a cell, comprising at least one duplex region that contains at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary a first strand, wherein said first strand is at least partially complementary to at least a portion of the RNA transcribed from the LPA gene, wherein said 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, with the nucleotides of the first strands are modified using a first modification for nucleotides in odd positions and modified using a second modification for nucleotides in even positions, and nucleotides of the second strand are modified using a third modification for nucleotides in even positions and modified using a fourth modification for nucleotides in odd positions, with herein, 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 contain the nucleotide sequence 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.

The first modification may be 2'-OMe and the second modification may be 2'-F.

In the nucleic acid according to the second aspect, the first strand may comprise the nucleotide sequence of SEQ ID NO: 5 and SEQ ID NO: 9, and the second strand may contain the nucleotide sequence of SEQ ID NO: 6 and SEQ ID NO: 10.

The sequence and modifications may be those shown in the table below; where the preferred sequences are presented based on an extract from Table 1 presented in this application:

SEQ ID NO: 5 5' auaacucuguccaauuacca 3' 6162717181736152736 SEQ ID NO: 6 5' uggguaauggacagaguuau 3' 1845261846364645161 SEQ ID NO: 9 5' auaacucuguccaauuaccg 3' 6162717181736152738 SEQ ID NO:10 5' cgguaauggacagaguuau 3' 3845261846364645161

where specific modifications are indicated by the following 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.

The nucleic acid of the present invention may contain a phosphorothioate linkage between one, two or three terminal 3' nucleotides and/or one, two or three 5' nucleotides of the first strand and/or the second strand. It may contain two phosphorothioate bonds between each of the three terminal 3' nucleotides and between each of the three terminal 5' nucleotides on the first strand and two phosphorothioate bonds between the three terminal nucleotides of the 3' end of the second strand.

Such a nucleic acid may be conjugated to a ligand.

The present invention also provides, as a third aspect, a nucleic acid for inhibiting the expression of LPA in a cell, comprising at least one duplex region that contains at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary a first strand, wherein said first strand is at least partially complementary to at least a portion of the RNA transcribed from the LPA gene, wherein said 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 the specified the nucleic acid is conjugated to a ligand.

The ligand may contain (i) one or more N-acetylgalactosamine (GalNAc) groups and derivatives thereof and (ii) a linker, said linker conjugating the GalNAc groups to the sequence defined in any of the preceding aspects. The linker may have a divalent, trivalent or tetravalent branched structure. Nucleotides may be modified as defined herein.

The ligand may contain formula I:

wherein:

S is a saccharide, said saccharide being N-acetylgalactosamine;

X ¹ represents C ₃ -C ₆ alkylene or (-CH ₂ -CH ₂ -O) _m (-CH ₂ ) ₂ -, where m is 1, 2 or 3;

P is phosphate or modified phosphate (preferably thiophosphate);

X ² represents an alkylene or alkylene ether of the formula (-CH ₂ ) _n -O-CH ₂ -, where n=1-6;

A represents a branching block;

X ³ is a bridge block;

wherein the nucleic acid in accordance with the present invention is conjugated to X ³ through a phosphate or modified phosphate (preferably thiophosphate).

Thus, the present invention further provides a conjugated nucleic acid having one of the following structures

where Z represents a nucleic acid as previously defined herein.

In another embodiment, the nucleic acid of the present invention may be conjugated to a ligand having the following structure

The present invention also provides a conjugate for inhibiting LPA gene expression in a cell, wherein the conjugate comprises a nucleic acid portion comprising a nucleic acid according to any aspect of the present invention, and ligand portions, wherein said nucleic acid portion comprises at least one duplex region that contains at least a portion of a first RNA strand and at least a portion of a second RNA strand that is at least partially complementary to the first strand, wherein said first strand is at least partially complementary to at least a portion of the RNA transcribed from said LPA gene, wherein said portions ligands contain a linker group, preferably a serinol-derived linker group, as well as a targeting ligand for in vivo targeting to cells and conjugated exclusively to the 3' and/or 5' ends of one or both RNA strands, the 5' end of the first RNA chains are not conjugated, and:

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

(ii) both the second strand RNA and the first RNA strand are conjugated at the 3' ends to the targeting ligand and the 5' end of the second RNA strand is not conjugated, or

The present invention relates to a conjugate for inhibiting the expression of the LPA gene in a cell, wherein said conjugate comprises a nucleic acid portion and a ligand portion, wherein said nucleic acid portion comprises at least one duplex region that contains at least a portion of a first strand of RNA and at least a portion of a second strand of RNA that is at least partially complementary to a first strand, wherein said first strand is at least partially complementary to at least a portion of the RNA transcribed from said LPA gene, wherein said ligand portions comprise a linker group and a targeting ligand for targeting to conditions in vivo on cells and conjugated exclusively to the 3' and/or 5' ends of one or both RNA strands, wherein the 5' end of the first RNA strand is not conjugated, and:

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

(ii) both the second strand RNA and the first strand RNA are conjugated at the 3' ends to the targeting ligand and the 5' end of the second strand RNA is not conjugated.

The linker group may be, for example, a serinol-derived linker group or one of the other types of linkers described herein.

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

Also provided is a nucleic acid or conjugated nucleic acid in accordance with any aspect of the present invention for use in the treatment of a disease, disorder or syndrome and/or in the manufacture of a medicament for the treatment of a disease, disorder or syndrome.

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

After subcutaneous administration, the subject matter of the invention can be delivered in a tissue-specific manner to the liver (hepatocytes) and specifically target LPA to reduce unwanted side effects and achieve a lower therapeutic dose required to achieve the target effect.

The present invention or a pharmaceutical composition containing a nucleic acid or conjugated nucleic acid according to the present invention can be used in the treatment of a disease, disorder or syndrome. Treatment may involve preventing and reducing the risk of stroke, atherosclerosis, thrombosis or cardiovascular disease such as coronary artery disease or aortic stenosis, and any other disease or pathology associated with elevated levels of Lp(a) containing particles.

Also provided is a method for producing a nucleic acid or conjugated nucleic acid in accordance with the present invention.

Detailed Description of the Invention

The present invention relates to a nucleic acid that is double-stranded and directed to an expressed LPA RNA transcript, and compositions thereof. These nucleic acids or conjugated nucleic acids can be used in the treatment and prevention of various diseases, disorders and syndromes in which reduced expression of the LPA gene product is desired.

A first aspect of the present invention relates to a nucleic acid for inhibiting the expression of LPA in a cell, comprising at least one duplex region that contains at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, said a first strand is at least partially complementary to at least a portion of the RNA transcribed from the LPA gene, wherein said first strand contains 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.

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

The LPA gene contains sequences with a very large number of repeats. Therefore, first-strand nucleic acids with very similar sequences may have perfect sequence complementarity with very different mRNA target regions.

A related aspect of the present invention is a nucleic acid for inhibiting the expression of LPA in a cell, the nucleic acid comprising at least one duplex region which comprises: a first strand; and a second chain, wherein said second chain is at least partially complementary to the first chain, wherein said first chain comprises a sequence of at least 15, preferably at least 16, more preferably at least 17, even more preferably at least 18 and most preferably at least 19 nucleotides of any 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 the number of individual nucleotide mismatches and/or deletions and/or insertions in the first-strand sequence relative to the portion of the reference sequence that is contained in the first-strand sequence chains, is no more than three, preferably no more than two, more preferably no more than one, and most preferably zero.

In one aspect, the first nucleic acid strand comprises a sequence of at least 18 nucleotides of any of the reference sequences, preferably any of the reference sequences of SEQ ID NOs: 9 and 5, and wherein the number of individual nucleotide mismatches and/or deletions and/or insertions in the sequence of the first strand relative to the portion of the reference sequence that is contained in the sequence of the first strand is no more than one, and preferably zero.

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

A certain number of mismatches, deletions or insertions between the first (antisense) strand and the target sequence or between the first strand and the second (sense) strand can be tolerated with siRNA and in some cases can even increase activity.

By nucleic acid we mean a nucleic acid containing two chains containing nucleotides, which is capable of interfering with the expression of genes. Inhibition can be complete or partial and results in decreased gene expression in a targeted manner. Nucleic acid contains two separate polynucleotide chains; a first chain, which may also be a guide chain; and a second circuit, which may also be a passenger circuit. The first strand and the second strand may be part of the same polynucleotide molecule, which is self-complementary and folds back to form a double-stranded molecule. The nucleic acid may be a siRNA molecule.

A nucleic acid may contain ribonucleotides, modified ribonucleotides, deoxynucleotides, deoxyribonucleotides, or non-nucleotide nucleotide analogues that are capable of mimicking nucleotides such that they can “pair” with a corresponding base on a target sequence or complementary strand. The nucleic acid may further comprise a double-stranded nucleic acid portion or a duplex region formed by all or part of a first strand (also known in the art as a guide strand) and all or part of a second strand (also known in the art as a passenger strand). A duplex region is defined as the region 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, inclusive.

By duplex region is meant a region in two complementary or substantially complementary oligonucleotides that are base paired with each other, either by Watson-Crick base pairing or any other method that produces a duplex between oligonucleotide chains that are complementary or substantially complementary. complementary. For example, an oligonucleotide strand containing 21 nucleotide units may pair with another oligonucleotide of 21 nucleotide units, but only 19 nucleotides on each strand are complementary or substantially complementary such that the “duplex region” consists of 19 base pairs. The remaining base pairs may exist as 5' and 3' overhangs or as single-stranded regions. Additionally, 100% complementarity is not required in the duplex region; In the duplex region, significant complementarity is allowed. Essential complementarity refers to such complementarity between chains that they are capable of renaturation under biological conditions. Techniques for empirically determining whether two chains are capable of renaturation under biological conditions are well known in the art. In another embodiment, the two chains can be synthesized and added together under biological conditions to determine whether they renature with each other.

The portions of the first strand and the second strand that form the 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 perfect match between the first strand and the second strand in terms of base complementarity is not necessarily required. However, the first and second strands must be able to hybridize under physiological conditions.

The complementarity between the first strand and the second strand in at least one duplex region may be ideal in that there are no nucleotide mismatches or additional/deleted nucleotides on either strand. In another embodiment, the complementarity may not be ideal. Complementarity can range from about 70% to about 100%. More specifically, the complementarity may be at least 70%, 75%, 80%, 85%, 90%, or 95%, and values in between.

For purposes of the present invention, a “part,” for example, in “one duplex region that contains at least a portion of the first strand,” is to be understood as meaning that the duplex region contains at least 10, preferably at least 12, more preferably at least at least 14, even more preferably at least 16, even more preferably at least 18, and most preferably all of the nucleotides of a given chain reference sequence. A portion of the reference sequence in the duplex region is at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, and most preferably 100% identical to the corresponding portion of the reference sequence . In another embodiment, the number of individual nucleotide mismatches relative to a portion of the reference sequence is no more than three, preferably no more than two, more preferably no more than one, and most preferably zero.

The first strand and the second strand may each contain a region of complementarity that contains at least 15 contiguous nucleotides, differing by no more than 3 nucleotides from any of the sequences listed in Table 1.

The nucleic acid may comprise a second sequence comprising the nucleotide sequence 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.

Use of a nucleic acid in accordance with the present invention involves the formation of a duplex region between all or part of the first strand and a portion of the target nucleic acid. The portion of a target nucleic acid that forms a first-strand duplex region, defined as the region that begins with the first base pair formed between the first strand and the target sequence and ends with the last base pair formed between the first strand and the target sequence, inclusive , is the target nucleic acid sequence or simply the target sequence. The duplex region formed between the first strand and the second strand need not be similar to the duplex region formed between the first strand and the target sequence. This means that the second strand may have a different sequence from the target sequence; however, the first strand must be capable of forming a duplex structure with both the second strand and the target sequence, at least under physiological conditions.

The complementarity between the first strand and the target sequence can be perfect (there are no nucleotide mismatches or additional/deleted nucleotides in each nucleic acid).

The complementarity between the first strand and the target sequence may not be ideal. Complementarity can range from about 70% to about 100%. More specifically, the complementarity may be at least 70%, 80%, 85%, 90%, or 95%, and values in between.

The identity between the first strand and the complementary sequence of the target sequence can range from about 75% to about 100%. More specifically, the complementarity may be at least 75%, 80%, 85%, 90%, or 95%, and values in between, as long as the nucleic acid is capable of reducing or inhibiting LPA expression.

A nucleic acid having less than 100% complementarity between the first strand and the target sequence may be able to reduce LPA expression to the same level as a nucleic acid having perfect complementarity between the first strand and the target sequence. In another embodiment, it may be able to reduce LPA expression to a level that is 15-100% of the reduction achieved with a perfectly complementary nucleic acid.

The nucleic acid may comprise a first strand and a second strand, each of which is 19-25 nucleotides in length. The first chain and the second chain may have different lengths.

A nucleic acid may be 15-25 base pairs in length. A nucleic acid can be 17-23 base pairs in length. A nucleic acid can be 17-25 base pairs in length. A nucleic acid may be 23-24 base pairs in length. A nucleic acid can be 19-21 base pairs in length. A nucleic acid can be 21-23 base pairs in length.

The nucleic acid may contain a duplex region that consists 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 contiguous.

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

Preferably, the nucleic acid mediates RNA interference.

According to one embodiment of the present invention, a nucleic acid for inhibiting the expression of LPA in a cell contains at least one duplex region that contains a first strand and a second strand that is at least partially complementary to the first strand, wherein said first strand contains a sequence of at least 15 , preferably at least 16, more preferably at least 17, even more preferably at least 18 and most preferably at least 19 nucleotides, wherein the sequence is at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, and most preferably 100% identical to any 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.

In a further aspect, the disclosed nucleic acid or conjugated nucleic acid can reduce LPA expression by at least 15% compared to the expression observed in the absence of the nucleic acid or conjugated nucleic acid. All preferred features of any of the previous aspects also apply to this aspect. In particular, LPA expression may be reduced to at least the following predetermined percentages (%) or to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, or less than and intermediate values relative to the expression that was observed in the absence of nucleic acid or conjugated nucleic acid or in the presence of a non-inhibitory control.

The nucleic acid may contain blunt ends at both ends; may contain a sticky end at one end and a blunt end at the other end; or contain a sticky end on both ends.

As used herein, “overhang” has its usual and accepted meaning in the art, i.e. a single-stranded portion of a nucleic acid that extends beyond the terminal nucleotide of the complementary strand in a double-stranded nucleic acid. The term "blunt end" includes a double-stranded nucleic acid in which both strands end at the same position, regardless of whether the terminal nucleotide(s) are paired. The terminal nucleotide of the first strand and the second strand at the blunt end may be paired. The terminal nucleotide of the first strand and the second strand at the blunt end may be unpaired. The two terminal nucleotides of the first strand and the second strand at the blunt end may be paired. The two terminal nucleotides of the first strand and the second strand at the blunt end may be unpaired.

The nucleic acid may contain a sticky end at one end and a blunt end at the other. The nucleic acid may contain a sticky end at both ends. The nucleic acid may contain blunt ends at both ends. The nucleic acid may be blunt ended at the 5' end of the first strand and the 3' end of the second strand, or at the 3' end of the first strand and the 5' end of the second strand.

The nucleic acid may contain a sticky end at the 3' or 5' end. The nucleic acid may contain a 3' overhang on the first strand. The nucleic acid may contain a 3' overhang on the second strand. The nucleic acid may contain a 5' overhang on the first strand. The nucleic acid may contain a 5' overhang on the second strand. The nucleic acid may contain a sticky end at either the 5' end or the 3' end of the first strand. The nucleic acid may contain a sticky end at either the 5' end or the 3' end of the second strand. The nucleic acid may contain a 5' overhang on the first strand and a 3' overhang on the second strand. The nucleic acid may contain a 3' overhang on the first strand and a 5' overhang on the second strand. The nucleic acid may contain a 3' overhang on the first strand and a 3' overhang on the second strand. The nucleic acid may contain a 5' overhang on the first strand and a 5' overhang on the second strand.

The overhang at the 3' end or 5' end of the second strand or the first strand may be selected from 1, 2, 3, 4 and 5 nucleotides in length. Optionally, the overhang can consist of 1 or 2 nucleotides, which can be modified, but not necessarily.

Unmodified polynucleotides, particularly ribonucleotides, may be susceptible to degradation by cellular nucleases, and as such, modifications/modified nucleotides may be incorporated into the nucleic acid of the present invention. Such modifications can help stabilize the nucleic acid, making it more resistant to nucleases. Improved stability ensures that the nucleic acids are active as RNA interference mediators for longer periods of time and is particularly desirable if the nucleic acids are to be used for treatment.

One or more nucleotides on the second strand and/or the first strand of the nucleic acid according to the present invention may be modified.

The nucleic acid modifications of the present invention generally provide an effective tool to overcome potential limitations, including, but not limited to, stability and in vitro and in vivo bioavailability of naturally occurring RNA molecules. The nucleic acid in accordance with the present invention can be modified using chemical modifications. The modified nucleic acid may also minimize the possibility of inducing interferon activity in humans. The modification may further enhance the functional delivery of the nucleic acid to the target cell. The modified nucleic acid of the present invention may contain one or more chemically modified ribonucleotides of either or both of the first strand or the second strand. A ribonucleotide may contain chemical modification of a base, sugar, or phosphate groups. Ribonucleic acid can be modified by substitution or insertion of nucleic acid analogues or bases.

One or more nucleic acid nucleotides of the present invention may be modified. The nucleic acid may contain 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 located in a duplex region. The modified nucleotide may be located outside the duplex region, i.e. in the single-stranded region. The modified nucleotide may be on the first strand and may be located outside the duplex region. The modified nucleotide may be on the second strand and may be located 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.

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

The modified nucleotide may be a purine or a pyrimidine. At least half of the purines can be modified. At least half of the pyrimidines can be modified. All of the purines can be modified. All of the pyrimidines can be modified. The modified nucleotides may be selected from the group consisting of a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl-modified nucleotide, a 2'-modified nucleotide, a 2'-deoxy-modified nucleotide, a closed nucleotide lacking a nitrogenous base nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholino-nucleotide, phosphoamidate containing unnatural base nucleotide, nucleotide containing 5'-phosphothioate group, nucleotide containing 5'-phosphate or 5'-mimetic phosphate, and a terminal nucleotide connected to a cholesterol derivative or dodecanoic acid bisdecylamide group.

The nucleic acid may contain a nucleotide containing a modified nucleotide, the base being 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-bromuridine), 6-azapyrimidine, 6-alkylpyrimidine (e.g. , 6-methyluridine), propine, queuosine, 2-thiouridine, 4-thiouridine, vaibutozin, vaibutoxozin, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5'-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D- galactosylqueuosine, 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-isopentenyl adenosine, beta-D-mannosylqueuosine, uridine-5-hydroxyacetic acid and 2-thiocytidine.

The nucleic acids discussed herein include unmodified RNA, as well as RNA that has been modified, for example, to improve potency, and nucleoside surrogate polymers. Unmodified RNA refers to a molecule in which the nucleic acid components, namely sugars, bases and phosphate groups, are the same or substantially the same as those that occur naturally, such as those that occur naturally in the human body. As used herein, a modified nucleotide refers to a nucleotide in which one or more of the nucleotide components, namely sugars, bases and phosphate groups, are different from those found in nature. Although modified nucleotides are so called because of their modification, the term also includes molecules that are not nucleotides, such as a polynucleotide molecule in which the ribophosphate backbone is replaced by a non-ribophosphate construct that allows hybridization between the strands, i.e. modified nucleotides mimic the ribophosphate backbone.

Many of the modifications described below that occur in a nucleic acid will be repeated in a polynucleotide molecule, such as modification of a base or a phosphate group, or a non-union O from a phosphate group. In some cases the modification will occur at all possible positions/nucleotides in a polynucleotide, but in many cases it will not occur. The modification may occur only at the 3' or 5' terminal position, may occur only in terminal regions, for example, at a position on the terminal nucleotide or in the last 2, 3, 4, 5 or 10 nucleotides of the chain. The modification may occur in a double-stranded region, a single-stranded region, or both. The modification may occur only in the double-stranded region of the nucleic acid according to the present invention, or may occur only in the single-stranded region of the nucleic acid according to the present invention. The phosphothioate modification at the non-connecting O position may occur at only one or both ends, may occur only in a terminal region, such as at a position on a terminal nucleotide or the last 2, 3, 4 or 5 nucleotides, or may occur in a duplex and/or single-stranded areas, particularly at the ends. The 5'-end or 3'-end can be phosphorylated.

The stability of the nucleic acid of the present invention can be increased by including specific bases at the overhangs or by including modified nucleotides at the single-stranded overhangs, for example, at the 5' or 3' overhangs or both. Purine nucleotides may be included in the sticky ends. All or some of the bases at the 3' or 5' overhang may be modified. Modifications may include the use of modifications on the 2'-OH group of the ribose sugar, the use of deoxyribonucleotides instead of ribonucleotides, and modifications on the phosphate group, such as phosphorothioate modifications. The sticky ends do not need to be homologous to the target sequence.

Nucleases can hydrolyze phosphodiester bonds of nucleic acids. However, chemical modifications of nucleic acids can impart improved properties and can make oligoribonucleotides more resistant to nucleases.

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

(i) a change, for example, substitution of one or both of the non-joining phosphate oxygen atoms and/or one or more of the joining phosphate oxygen atoms (referred to as a joining event even if occurring at the 5' and 3' end of the nucleic acid according to the present invention );

(ii) alteration, eg substitution of a ribose sugar component, eg 2'-hydroxyl on the ribose sugar;

(iii) replacement of the phosphate group "dephospho" by linkers;

(iv) modification or replacement of natural substrate;

(v) replacement or modification of the ribose phosphate backbone;

(vi) modification of the 3' end or 5' end of the RNA, for example, removal, modification or replacement of a terminal phosphate group or conjugation of a group, for example a fluorescently labeled group, to the 3' or 5' end of the RNA.

The terms substitution, modification, change indicate a difference from a natural molecule.

Specific modifications are discussed in more detail below.

Examples of modified phosphate groups include phosphorothioate, phosphoselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoamidates, alkyl or arylphosphonates, and phosphotriesters. In phosphodithioates, both non-bonding oxygen atoms are replaced by sulfur. One, each, or both of the non-bonding oxygen atoms in the phosphate group may be independently substituted by any of S, Se, B, C, H, N, or OR (R is alkyl or aryl).

The phosphate linker can also be modified by replacing the connecting oxygen atom with nitrogen (bridged phosphoamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylene phosphonates). Substitution can occur at the terminal oxygen. It is possible to replace non-bonding oxygen atoms with nitrogen.

The modified nucleotide may contain modification of sugar groups. The 2'-hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents.

Examples of "oxy"-2'-hydroxyl group modifications include alkoxy or aryloxy (OR, eg R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethylene glycols (PEG), O(CH2CH2O)nCH2CH2OR; “closed” nucleic acids (LNA), in which the 2'-hydroxyl is connected, for example, by a methylene bridge, to the 4'-carbon of the same ribose sugar; O-AMINE (AMIN=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine, polyamino) and aminoalkoxy, O(CH2)nAMIN, (for example, AMIN=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine, polyamino).

"Deoxy" modifications include hydrogen, halogen, amino (eg, NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH2CH2NH)nCH2CH2-AMINE (AMIN=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino), -NHC(O)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano ; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted, for example, with an amino function. Other substituents from certain embodiments include 2'-methoxyethyl, 2'-OCH3, 2'-O-allyl, 2'-C-allyl, and 2'-fluoro. A sugar group may also contain one or more carbon atoms that have a stereochemical configuration opposite that of the corresponding carbon in ribose. Thus, the modified nucleotides may contain a sugar such as arabinose.

Modified nucleotides may also contain "baseless" sugars, which lack the nucleobase at C-1'. These baseless sugars may further contain modifications on one or more of the sugar atoms.

2' modifications can be used in combination with one or more phosphate linker modifications (eg, phosphorothioate).

Phosphate groups can be individually replaced by connectors that do not contain phosphorus.

Examples of groups that can replace the phosphate group include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formoacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, and methyleneoxymethylimino. In certain embodiments of the present invention, substitutions may include methylenecarbonylamino and methylenemethylimino groups.

The phosphate linker and ribose sugar can be replaced with nuclease-resistant nucleotides.

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

The 3' and 5' ends of the oligonucleotide can be modified. Such modifications may be at the 3' end or the 5' end or at both ends of the molecule. These may involve modification or substitution of the entire terminal phosphate or one or more phosphate group atoms. For example, the 3' and 5' ends of the oligonucleotide may be conjugated to other functional molecular groups, such as labeling groups such as fluorophores (eg, pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes), or protecting groups ( e.g. based on sulfur, silicon, boron or ester). Functional molecular groups can be attached to the sugar via a phosphate group and/or a linker. The terminal linker atom may connect to or replace the connecting atom of a phosphate group or a C-3' or C-5' O, N, S or C sugar group. In another embodiment, the linker can connect to or replace the terminal atom of a surrogate nucleotide (eg, PNA). These spacers or linkers may include, for example, -(CH ₂ ) _n -, -(CH ₂ ) _n N-, -(CH ₂ ) _n O-, -(CH ₂ ) _n S-, -(CH ₂ CH ₂ O) _n CH ₂ CH ₂ O- (for example, n=3 or 6), baseless sugars, amide, carboxy, amine, oxyamine, hydroxyimine, thioether, disulfide, thiourea, sulfonamide or morpholino, or biotin and fluorescein reagents. The 3' end may be an -OH group.

Other examples of terminal modifications include dyes, intercalating agents (eg, acridines), cross-linkers (eg, psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphirin), polycyclic aromatic hydrocarbons (eg, 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 group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic 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, amino, mercapto, PEG (e.g. PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption stimulants (e.g. aspirin, vitamin E, folic acid) , synthetic ribonucleases (eg, imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3 ⁺ and tetraazamacrocycle complexes).

Terminal modifications may be added for a number of reasons, including modulation of activity or modulation of degradation resistance. Terminal modifications that can be used to modulate activity include modification of the 5' end with phosphate or phosphate analogues. The nucleic acids of the present invention may be 5'-phosphorylated on the first or second strands or may contain a phosphoryl analogue at the 5' end. 5'-phosphate modifications include modifications that are compatible with RISC-mediated gene silencing. Suitable modifications include: 5'-monophosphate ((HO) ₂ (O)PO-5');5'-diphosphate ((HO) ₂ (O)POP(HO)(O)-O-5');5'-triphosphate ((HO) ₂ (O)PO-(HO)(O)POP(HO)(O)-O-5');5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5');5'-adenosine cap (Appp) and any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5') ; 5'-monothiophosphate (phosphothioate; (HO) ₂ (S)PO-5');5'-monodithiophosphate(phosphodithioate;(HO)(HS)(S)PO-5'),5'-phosphothiolate((HO)2(O)PS-5'); any additional combination of monophosphate, diphosphate and triphosphates in which oxygen is replaced by sulfur (for example, 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoamidates ((HO)2(O)P -NH-5', (HO)(NH2)(O)PO-5'), 5'-alkylphosphonates (R = alkyl = methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)( O)-O-5′-, (OH) ₂ (O)P-5′-CH ₂ -), 5'-vinylphosphonate, 5'-alkyl ether phosphonates (R = alkyl ether = methoxymethyl (MeOCH ₂ -), ethoxymethyl, etc. .d., for example, RP(OH)(O)-O-5'-).

The nucleic acid of the present invention may contain one or more phosphorothioate modifications at one or more terminal portions of the first and/or second chains. Optionally, each or any end of the first strand may contain one, two or three phosphorothioate-modified nucleotides. Optionally, each or any end of the second strand may contain one, two or three phosphorothioate-modified nucleotides.

Terminal modifications can also be used to monitor distribution, and in such cases the groups that will be added may include fluorophores such as fluorescein or Alexa dye. Terminal modifications can also be used to enhance absorption, suitable modifications include cholesterol. Terminal modifications can also be used to cross-link an RNA agent to another group.

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 using these bases or synthetic and natural nucleic acid bases (eg, inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanisine or tubercidin) and any one of the above modifications. In another embodiment, substituted or modified analogues of any of the above bases and "universal bases" may 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-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5- uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-aminoallyluracyl, 8-halogen, amino, thiol, thioalkyl, 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-propynyluracil 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-aminoallyluracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-hydroxyacetic 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-methylguanines or O-alkylated bases.

As used herein, the term "non-pairing nucleotide analog" means a nucleotide analog that contains a non-base-pairing group, including, but not limited to: 6-deaminoadenosine (nebularine), 4-Me-indole, 3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-Me-ribo-U, N3-Me-ribo-T, N3-Me dC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, N3-Me dC. In certain embodiments of the present invention, the non-base-pairing nucleotide analog is a ribonucleotide. In other embodiments of the present invention, it is a deoxyribonucleotide.

As used herein, the term “terminal functional group” includes, but is not limited to, halogen, alcohol, amine, carbonic, ester, amide, aldehyde, ketone, and ether groups.

Certain groups may be connected to the 5' end of the first strand or the second strand. These include a baseless ribose group, a baseless deoxyribose group, baseless ribose modifications and a baseless deoxyribose group, including 2'O-alkyl modifications; inverted baseless ribose groups and baseless deoxyribose groups and modifications thereof, C6-imino-Pi; mirror nucleotide, including L-DNA and L-RNA; 5'-OMe-nucleotide; and nucleotide analogues, including 4',5'-methylene nucleotide; 1-β-D-erythrofuranosylnucleotide; 4'-thionucleotide, carbocyclic nucleotide; 5'-aminoalkylphosphate; 1,3-diamino-2-propylphosphate, 3-aminopropylphosphate; 6-aminohexylphosphate; 12-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; alpha nucleotide; threopentofuranosyl nucleotide; acyclic 3',4'-seconucleotide; 3,4-dihydroxybutylnucleotide; 3,5-dihydroxypentylnucleotide, 5'-5'-inverted baseless group; 1,4-butanediol phosphate; 5'-amino; and bridged or non-bridged methylphosphonate and 5'-mercapto groups.

The nucleic acids of the present invention may contain one or more inverted nucleotides, for example, inverted thymidine or inverted adenine (eg, see Takei, et al., 2002. JBC 277 (26):23800-06).

As used herein, the term “inhibit,” “suppress,” or “reduce,” with respect to gene expression, means that the expression of a gene or the level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits (e.g., mRNA), or the activity of one or more proteins or protein subunits are reduced below the level observed in the absence of the nucleic acid or conjugated nucleic acid of the present invention, or relative to an siRNA molecule with no known homology to human transcripts (referred to herein as a non-inhibitory control). Such a control can be conjugated and modified in a similar manner to the molecule of the present invention and delivered to the target cell in a similar manner; for example, expression may be reduced to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15% or intermediate values, or to a value less than that observed in the absence of nucleic acid acid or conjugated nucleic acid or in the presence of a non-inhibitory control.

The nucleic acid according to the present invention may contain a nucleotide devoid of a nitrogenous base. As used herein, the term "baseless" refers to groups that do not contain a base or have other chemical groups in place of the base at the 1' position, for example, a 3',3'-linked or 5',5'-linked baseless deoxyribose derivative .

The nucleic acid may contain one or more nucleotides on the second and/or first strands that are modified. Alternating nucleotides can be modified to form modified nucleotides.

Alternation, as described herein, means that it occurs one after another on a regular basis. In other words, alternation means repeating in turns. For example, if one nucleotide is modified, the next adjacent nucleotide is not modified, the next adjacent nucleotide is modified, and so on. One nucleotide can be modified with a first modification, the next adjacent nucleotide can be modified with a second modification, and the next adjacent nucleotide can be modified with a first modification, and so on, with the first and second modifications being different.

One or more of the nucleotides at odd positions in the first strand of the nucleic acid of the present invention may be modified, with the first strand numbered 5' to 3', the 5'most nucleotide being nucleotide number 1 of the first strand. The term "odd" as used herein means a number that is indivisible by two. Examples of odd numbers are 1, 3, 5, 7, 9, 11 and so on. One or more of the nucleotides at even-numbered positions of the first nucleic acid strand of the present invention may be modified, the first strand being numbered 5' to 3'. The term "even" as used herein means a number that is equally divisible by two. Examples of even numbers are 2, 4, 6, 8, 10, 12, 14 and so on. One or more of the nucleotides at odd positions in the second nucleic acid strand of the present invention may be modified, with the second strand numbered 3' to 5', with the 3'-most terminal nucleotide being nucleotide number 1 of the second strand. One or more of the nucleotides at the even-numbered positions of the second nucleic acid strand of the present invention may be modified, the second strand being numbered 3' to 5'.

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

A plurality of nucleotides at odd positions of the first strand may be modified in the nucleic acid of the present invention. Multiple nucleotides at even-numbered positions on the first strand may be modified using a second modification. The first strand may contain contiguous nucleotides that are modified using a common modification. The first strand may also contain adjacent nucleotides that are modified with a second different modification.

One or more of the nucleotides at the odd positions of the second strand may be modified using a modification that is different from the modification of the nucleotides at the odd positions of the first strand, and/or one or more of the nucleotides at the even positions of the second strand may be modified using a modification similar to that for nucleotides in odd positions of the first strand. At least one of the one or more modified nucleotides at even positions of the second strand may be adjacent to one or more modified odd nucleotides. A plurality of nucleotides at odd positions of the second strand may be modified using a common modification, and/or a plurality of nucleotides at even positions may be modified using a modification that is similar to that present on the odd nucleotides of the first strand. A plurality of nucleotides at odd positions of the second strand may be modified using a second modification, wherein the second modification is different from the modification of nucleotides at odd positions of the first strand.

The second strand may contain contiguous nucleotides that are modified using a common modification, which may be a second modification that is different from the modification of nucleotides at odd positions of the first strand.

In the nucleic acid of the present invention, each of the nucleotides at odd positions in the first strand and each of the nucleotides at even positions in the second strand can be modified using a general modification, and each of the nucleotides at even positions in the first strand can be modified using a second modification, and each of the nucleotides at odd positions in the second strand may be modified using a second modification.

The modified first strand nucleic acid nucleotides of the present invention may be shifted by at least one nucleotide relative to the unmodified or otherwise modified second strand nucleotides.

One or more or each of the nucleotides at the odd positions may be modified in the first strand, and one or more or each of the nucleotides at the even positions at the even positions may be modified in the second strand. One or more or each of the alternating nucleotides on either or both strands may be modified using a second modification. One or more or each of the nucleotides at the even-numbered positions may be modified in the first strand, and one or more or each of the nucleotides at the even-numbered positions may be modified in the second strand. One or more or each of the alternating nucleotides on either or both strands may be modified using a second modification. One or more or each of the nucleotides at odd positions in the first strand may be modified, and one or more of the nucleotides at odd positions in the second strand may be modified using a general modification. One or more or each of the alternating nucleotides on either or both strands may be modified using a second modification. One or more or each of the nucleotides at even positions in the first strand may be modified, and one or more or each of the nucleotides at odd positions in the second strand may be modified using a common modification. One or more or each of the alternating nucleotides on either or both strands may be modified using a second modification.

The nucleic acid of the present invention may contain single-stranded or double-stranded constructs that contain at least two regions of alternating modifications in one or both chains. These alternating regions may contain up to about 12 nucleotides, but preferably contain from about 3 to about 10 nucleotides. Regions of alternating nucleotides may be located at the ends of one or both nucleic acid strands of the present invention. A nucleic acid may contain from 4 to about 10 alternating nucleotides at each end (3' and 5'), and these regions can be separated by about 5 to about 12 contiguous unmodified or differently modified or modified using a common nucleotide modification .

The odd nucleotides of the first strand can be modified and the even nucleotides can be modified using a second modification. The second strand may contain adjacent nucleotides that are modified using a general modification that may be similar to the modification of nucleotides at odd positions in the first strand. One or more nucleotides of the second strand may also be modified using a second modification. One or more nucleotides with a second modification may be adjacent to each other and to nucleotides having a modification that is similar to the modification of nucleotides at odd positions of the first strand. The first strand may also contain phosphorothioate bonds between two nucleotides at the 3' end and at the 5' end. The second strand may contain a phosphorothioate bond between two nucleotides at the 5' end. The second strand may also be conjugated to a ligand at the 5' end.

The nucleic acid of the present invention may comprise a first strand containing contiguous nucleotides that are modified using a common modification. One or more such nucleotides may be adjacent to one or more nucleotides that may be modified using a second modification. One or more nucleotides with a second modification may be contiguous. The second strand may contain contiguous nucleotides that are modified using a common modification, which may be similar to one of the modifications of one or more nucleotides of the first strand. One or more nucleotides of the second strand may also be modified using a second modification. One or more nucleotides with a second modification may be contiguous. The first strand may also contain phosphorothioate bonds between two nucleotides at the 5' end and at the 3' end. The second strand may contain a phosphorothioate bond between two nucleotides at the 3' end. The second strand may also be conjugated to a ligand at the 5' end.

Nucleotides numbered 5' to 3' on the first strand and 3' to 5' on the second strand 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 and 25 can be modified using modification on the first chain. Nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 can be modified by applying a second modification on the first strand. Nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 can be modified using modification on the second strand. Nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 can be modified using a second modification on the second strand. In relation to the nucleic acid 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.

Nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 can be modified using modification on the first strand. Nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 can be modified by applying a second modification on the first strand. Nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 can be modified using modification on the second strand. Nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 can be modified using a second modification on the second strand.

It is understood that if the first and/or second strands are shorter than 25 nucleotides in length, for example 19 nucleotides in length, then there are no nucleotides numbered 20, 21, 22, 23, 24 and 25 to be modified. One skilled in the art will understand the above description to apply to shorter circuits accordingly.

One or more modified nucleotides on the first strand may be paired with modified nucleotides on the second strand having a common modification. One or more modified nucleotides on the first strand may be paired with modified nucleotides on the second strand having a different modification. One or more modified nucleotides on the first strand may be paired with unmodified nucleotides on the second strand. One or more modified nucleotides on the second strand may be paired with unmodified nucleotides on the first strand. In other words, alternating nucleotides may be aligned on two strands such that, for example, all modifications in alternating regions of the second strand are paired with identical modifications in the first strand or, alternatively, the modifications may be offset by a single nucleotide sharing modifications in the alternating regions one strand paired with different modifications (i.e., a second or additional modification) on another strand. Another option is to have different modifications in each of the circuits.

Modifications on the first strand can be shifted by one nucleotide relative to modified nucleotides on the second strand so that nucleotides with a common modification do not pair with each other.

The modification and/or modifications may be individually and independently selected from the group consisting of 3'-terminal deoxythymine, 2'-O-methyl, 2'-deoxy modification, 2'-amino modification, 2'-alkyl modification , morpholino modification, phosphoamidate modification, modification with a 5'-phosphothioate group, modification with a 5'-phosphate or a 5'-phosphate mimetic, as well as modification with a cholesterol derivative or a dodecanoic acid bisdecylamide group, and/or the modified nucleotide may be any of the closed nucleotide , lacking the nitrogenous base of a nucleotide or containing an unnatural nucleotide base.

At least one modification may be 2'-O-methyl and/or at least one modification may be 2'-F. Additional modifications described herein may be present on the first and/or second circuits.

The nucleic acid of the present invention may contain an inverted RNA nucleotide at one or more ends of the chain. Such inverted nucleotides ensure the stability of the nucleic acid. Preferably, the nucleic acid contains at least an inverted nucleotide at one or more of the 3' end of at least one of the strands and/or the 5' end of the second strand. More preferably, the nucleic acid contains an inverted nucleotide at the 3' end of the second strand. Most preferably, the nucleic acid contains an inverted RNA nucleotide at the 3' end of the second strand, and this nucleotide is preferably an inverted A. The inverted nucleotide is preferably present at the end of the strand, not as a sticky end, but opposite the corresponding nucleotide on the other strand. Nucleic acid with this modification is stable and easy to synthesize.

As used herein, “similar or common modification” means the same modification for any nucleotide, whether A, G, C or U, modified with a group such as a methyl group or a fluoro group. This does not imply a similar addition at the same nucleotide. For example, 2'-F-dU, 2'-F-dA, 2'-F-dC, 2'-F-dG are all considered analogous or common modifications, as are 2'-OMe-rU, 2'- OMe-rA; 2'-OMe-rC; 2'-OMe-rG. The 2'-F modification is a modification different from the 2'-OMe modification.

Some typical modified nucleic acid sequences according to the present invention are shown in the examples. These examples are intended to be representative and not limiting.

Preferably, the nucleic acid may contain a modification and a second or additional modification that are individually and independently selected from the group consisting of a 2'-O-methyl modification and a 2'-F modification. The nucleic acid may contain a modification that is 2'-O-methyl (2'-OMe), which may be a first modification, and a second modification, which is 2'-F. The nucleic acid of the present invention may also contain a phosphorothioate modification and/or a deoxy modification, which may be present on or between 1, 2 or 3 terminal nucleotides of each or either end of each or both strands.

The present invention provides, as an additional aspect, a nucleic acid for inhibiting the expression of LPA in a cell, comprising the nucleotide sequence 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, in which the nucleotides of the first strand are modified using a first modification on odd nucleotides and modified using a second modifications on even nucleotides, and the second strand nucleotides are modified using a third modification on even nucleotides and modified using a fourth modification on odd nucleotides, wherein at least the first modification is different from the second modification and the third modification is different from the fourth modification. The third and first modifications may be the same or different, the second and fourth modifications may be the same or different. The first and second modifications may be different from each other, and the third and fourth modifications may be different from each other.

The second strand may contain the nucleotide sequence 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. First strand nucleotides can be modified using a first modification on odd nucleotides and modified using a second modification on even nucleotides, and a second strand can be modified on odd nucleotides using the second modification and modified using the first modification on even nucleotides. The first modification may be 2'-OMe and the second modification may be 2'-F. The first strand may contain the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 9, and/or the second strand may contain the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 10. Modifications may be those presented in Table 1 .

The nucleic acid of the present invention may be conjugated to a ligand. Efficient delivery of oligonucleotides, in particular double-stranded nucleic acids of the present invention, into cells in vivo is important and requires specific targeting and significant protection from the extracellular environment, in particular serum proteins. One way to achieve specific targeting is by conjugating a ligand to a nucleic acid. The ligand helps target the nucleic acid to the desired target site. There is a need to conjugate appropriate ligands for target receptor molecules in order for target cells to take up the conjugated molecules through mechanisms such as various receptor-mediated endocytosis pathways or functionally similar processes.

One example is the asialoglycoprotein receptor (ASGP-R) complex, consisting of varying ratios of multimers of the membrane receptors ASGR1 and ASGR2, which is very abundant on hepatocytes and has high affinity for the GalNAc group described herein. US Pat. No. 5,885,968 provides one of the first disclosures of the use of three-antennary cluster glycosides as conjugated ligands. Conjugates having 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 exhibits 50-fold higher affinity for N-acetyl-D-galactosylamine (GalNAc) compared to D-Gal.

The asialoglycoprotein receptor (ASGP-R) complex, which specifically recognizes the terminal β-galactosyl subunits of glycosylated proteins or other oligosaccharides (Weigel, P.H. et. al., Biochim. Biophys. Acta. 2002 Sep 19; 1572(2-3):341- 63), can be used to deliver a drug to liver hepatocytes expressing the receptor complex by covalently linking galactose or galactosamine to the drug (Ishibashi, S.; et. al., J Biol. Chem. 1994 Nov 11;269(45) :27803-6). In addition, binding affinity can be significantly increased by a multivalent effect, which can be achieved by repetition of the targeting group (Biessen EA, et al., J Med Chem. 1995 Apr 28; 38(9):1538-46.).

The ASGP-R complex acts as an intermediary for the active uptake of glycoproteins containing terminal β-galactosyl into cellular endosomes. Thus, ASGPR is very suitable for targeted delivery of drug candidates conjugated to such ligands, such as nucleic acids, into receptor-expressing cells (Akinc et al., Mol Ther. 2010 Jul; 18(7):1357-64).

More generally, the ligand may contain a saccharide that is selected to have affinity for at least one type of receptor on the target cell. In particular, the receptor is located on the surface of the mammalian liver cell, for example, the hepatic asialoglycoprotein receptor complex described previously (ASGP-R).

The saccharide may be selected from N-acetylgalactosamine, mannose, galactose, glucose, glucosamine and fucose. The saccharide may be N-acetylgalactosamine (GalNAc).

Thus, a ligand for use in the present invention may contain (i) one or more N-acetylgalactosamine (GalNAc) groups and derivatives thereof, and (ii) a linker, wherein said linker conjugates GalNAc groups to a sequence defined in any of the preceding aspects. The linker may have a divalent, trivalent or tetravalent branched structure. Nucleotides may be modified as defined herein.

"GalNAc" refers to 2-acetylamino-2-deoxy-D-galactopyranose, commonly referred to in the literature as N-acetylgalactosamine. 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. Both the β-form, 2-acetylamino-2-deoxy-β-D-galactopyranose, and the α-form, 2-acetylamino-2-deoxy-α-D-galactopyranose, can be used interchangeably. Preferably, the compounds of the present invention contain the β-form, 2-acetylamino-2-deoxy-β-D-galactopyranose.

Thus, the ligand may contain GalNAc.

The ligand may contain a compound of formula I:

wherein:

S is a saccharide, said saccharide being N-acetylgalactosamine;

X ¹ represents C ₃ -C ₆ alkylene or (-CH ₂ -CH ₂ -O) _m (-CH ₂ ) ₂ -, where m is 1, 2 or 3;

P is phosphate or modified phosphate (preferably thiophosphate);

X ² represents an alkylene or alkylene ether of the formula (-CH ₂ ) _n -O-CH ₂ -, where n=1-6;

A represents a branching block;

X ³ is a bridge block;

wherein the nucleic acid in accordance with the present invention is conjugated to X ³ through a phosphate or modified phosphate (preferably thiophosphate).

In Formula I, branch block "A" branches into three to accommodate three saccharide ligands. The branching unit is covalently attached to the remaining anchored ligand and nucleic acid portions. The branching unit may contain a branched aliphatic group containing groups selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether and hydroxyamine groups. The branching unit may contain groups selected from alkyl and ether groups.

Branch block A may have a structure selected from:

in which each A ₁ independently represents O, S, C=O or NH; And

each n is independently an integer between 1 and 20.

The branch block may have a structure selected from:

in which each A ₁ independently represents O, S, C=O or NH; And

each n is independently an integer between 1 and 20.

The branch block may have a structure selected from:

in which A ₁ represents O, S, C=O or NH; And

each n is independently an integer between 1 and 20.

A branching block can have the structure:

A branching block can have the structure:

A branching block can have the structure:

Optionally, the branching block consists of only a carbon atom.

The “X ³ ” part is a bridge block. The bridging block is linear and is covalently linked to the branching block and the nucleic acid.

X ³ can be selected from -C ₁ -C ₂₀ alkylene-, -C ₂ -C ₂₀ alkenylene-, alkylene ether of the formula -(C ₁ -C ₂₀ alkylene)-O-(C ₁ -C ₂₀ alkylene)-, -C(O)-C ₁ -C ₂₀ alkylene-, -C ₀ -C ₄ alkylene(Cy)C ₀ -C ₄ alkylene-, where Cy is a substituted or unsubstituted 5 or 6 membered cycloalkylene, arylene, heterocyclylene or heteroarylene ring, -C ₁ -C ₄ alkylene-NHC(O)-C ₁ -C ₄ -alkylene-, -C ₁ -C ₄ alkylene-C(O)NH-C ₁ -C ₄ -alkylene-, -C ₁ -C ₄ alkylene-SC(O)-C ₁ -C ₄ -alkylene-, -C ₁ -C ₄ alkylene-C(O)SC ₁ -C ₄ alkylene-, -C ₁ -C ₄ alkylene-OC( O)-C ₁ -C ₄ alkylene-, -C ₁ -C ₄ alkylene-C(O)OC ₁ -C ₄ alkylene- and -C ₁ -C ₆ alkylene-SSC ₁ -C ₆ alkylene-.

X ³ may be an alkylene ether of the formula -(C ₁ -C ₂₀ alkylene)-O-(C ₁ -C ₂₀ alkylene)-. X ³ may be an alkylene ether of the formula -(C ₁ -C ₂₀ alkylene)-O-(C ₄ -C ₂₀ alkylene)-, wherein said (C ₄ -C ₂₀ alkylene) is linked to Z. X ³ may be selected from the group consisting of -CH ₂ -OC ₃ H ₆ -, -CH ₂ -OC ₄ H ₈ -, -CH ₂ -OC ₆ H ₁₂ - and -CH ₂ -OC ₈ H ₁₆ -, in particular -CH ₂ -OC ₄ H ₈ -, -CH ₂ -OC ₆ H ₁₂ - and -CH ₂ -OC ₈ H ₁₆ -, where in each case the -CH ₂ - group is connected to A.

The ligand may comprise a compound of formula (II):

wherein:

S represents a saccharide;

X ¹ represents C ₃ -C ₆ alkylene or (-CH ₂ -CH ₂ -O) _m (-CH ₂ ) ₂ -, where m is 1, 2 or 3;

P is phosphate or modified phosphate (preferably thiophosphate);

X ² represents C ₁ -C ₈ alkylene;

A represents a branching block selected from:

X ³ is a bridge block;

wherein the nucleic acid in accordance with the present invention is conjugated to X ³ due to phosphate or modified phosphate (preferably thiophosphate)

Branch block A can have the structure:

Branch block A can have the structure:

where X ³ is attached to the nitrogen atom.

X³ may represent C₁-C₂₀ alkylene. Preferred X³ selected from the group consisting of -C₃H₆-, -C₄H₈-_,-C₆H₁₂- and -C₈H₁₆-, specifically -C₄H₈-_,-C₆H₁₂- and -C₈H₁₆-.

The ligand may comprise a compound of formula (III):

wherein:

S represents a saccharide;

X ¹ represents C ₃ -C ₆ alkylene or (-CH ₂ -CH ₂ -O) _m (-CH ₂ ) ₂ -, where m is 1, 2 or 3;

P is phosphate or modified phosphate (preferably thiophosphate);

X ² is an alkylene ether of the formula -C ₃ H ₆ -O-CH ₂ -;

A represents a branching block;

X ³ is an alkylene ether of formula selected from the group consisting of -CH ₂ -O-CH ₂ -, -CH ₂ -OC ₂ H ₄ -, -CH ₂ -OC ₃ H ₆ -, -CH ₂ -OC ₄ H ₈ -, -CH ₂ -OC ₅ H ₁₀ -, -CH ₂ -OC ₆ H ₁₂ -, -CH ₂ -OC ₇ H ₁₄ - and -CH ₂ -OC ₈ H ₁₆ -, in each case group -CH ₂ - connected to A,

and wherein X ³ is conjugated to a nucleic acid according to the present invention using a phosphate or modified phosphate (preferably thiophosphate).

The branch block may contain carbon. Preferably the branching block is carbon.

X ³ may be selected from the group consisting of -CH ₂ -OC ₄ H ₈ -, -CH ₂ -OC ₅ H ₁₀ -, -CH ₂ -OC ₆ H ₁₂ -, -CH ₂ -OC ₇ H ₁₄ - and -CH ₂ -OC ₈ H ₁₆ -. Preferably X ³ is selected from the group consisting of -CH ₂ -OC ₄ H ₈ -, -CH ₂ -OC ₆ H ₁₂ - and -CH ₂ -OC ₈ H ₁₆ .

For any of the above aspects, if P is a modified phosphate group, P may be represented as:

where Y ¹ and Y ² each independently represent =O, =S, -O ^- , -OH, -SH, -BH ₃ , -OCH ₂ CO ₂ , -OCH ₂ CO ₂ R ^x , -OCH ₂ C(S )OR ^x and –OR ^x , where R ^x represents C ₁ -C ₆ alkyl and where indicates joining to the rest of the connection.

By modified phosphate is meant a phosphate group in which one or more of the non-bonding oxygen atoms have been replaced. Examples of modified phosphate groups include phosphorothioate, phosphoselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoamidates, alkyl or arylphosphonates, and phosphotriesters. In phosphodithioates, both non-bonding oxygen atoms are replaced by sulfur. One, each, or both of the non-bonding oxygen atoms in the phosphate group may independently be any of S, Se, B, C, H, N, or OR (R is alkyl or aryl).

Phosphate can also be modified by replacing the connecting oxygen atom with nitrogen (bridged phosphoamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylene phosphonates). The substitution may occur at the terminal oxygen atom. It is possible to replace non-bonding oxygen atoms with nitrogen.

For example, Y ¹ may be -OH and Y ² may be =O or =S; or

Y ¹ may represent -O ^- and Y ² may represent =O or =S;

Y ¹ may be =O and Y ² may be -CH ₃ , -SH, -OR ^x or -BH _{3 ;}

Y ¹ may represent =S and Y ² may represent -CH ₃ , OR ^x or -SH.

One skilled in the art will understand that in certain cases there will be delocalization between Y ¹ and Y ² .

Preferably, the modified phosphate group is a thiophosphate group. Thiophosphate groups include bithiophosphate (i.e., where Y ¹ represents =S and Y ² represents -S ^- ) and monothiophosphate (i.e., where Y ¹ represents -O ^- and Y ² represents =S, or where Y ¹ represents = O and Y ² represents -S ^- ). Preferably P is monothiophosphate. The present inventors have discovered that conjugates having thiophosphate groups instead of phosphate groups have improved potency and duration of action in vivo.

P may also be ethyl phosphate (ie where Y ¹ is =O and Y ² is OCH ₂ CH ₃ ).

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

For any of the above aspects, the saccharide may be selected from N-acetyl with one or more of galactosamine, mannose, galactose, glucose, glucosamine and fructose. Typically, a ligand for use in the present invention may comprise N-acetylgalactosamine (GalNAc). Preferably, the compounds of the present invention may contain 3 ligands, each of which will preferably contain N-acetylgalactosamine.

"GalNAc" refers to 2-acetylamino-2-deoxy-D-galactopyranose, commonly referred to in the literature as N-acetylgalactosamine. 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 of the present invention, the β form, 2-acetylamino-2-deoxy-β-D-galactopyranose, and the α form, 2-acetylamino-2-deoxy-α-D-galactopyranose, can be used interchangeably. Preferably, the compounds of the present invention contain the β-form, 2-acetylamino-2-deoxy-β-D-galactopyranose.

2-acetylamino-2-deoxy-D-galactopyranose

2-acetylamino-2-deoxy-β-D-galactopyranose

2-acetylamino-2-deoxy-α-D-galactopyranose.

For any of the above compounds of formula (III), X ¹ may be (-CH ₂ -CH ₂ -O)(-CH ₂ ) ₂ -. X ¹ may be (-CH ₂ -CH ₂ -O) ₂ (-CH ₂ ) ₂ -. X ¹ may be (-CH ₂ -CH ₂ -O) ₃ (-CH ₂ ) ₂ -. Preferably X ¹ is (-CH ₂ -CH ₂ -O) ₂ (-CH ₂ ) ₂ -. In another embodiment, X ¹ is C ₃ -C ₆ alkylene. X ¹ may be propylene. X ¹ may be butylene. X ¹ may be pentylene. X ¹ may be hexylene. Preferably, alkyl is linear alkylene. In particular, X ¹ may be butylene.

For compounds of formula (III), X ² is an alkylene ether of the formula -C ₃ H ₆ -O-CH ₂ -, i.e. C ₃ alkoxymethylene, or –CH ₂ CH ₂ CH ₂ OCH ₂ -.

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

where Z represents a nucleic acid as previously defined herein.

A ligand of formula (I), (II) or (III) may be attached at the 3' end of the first (antisense) strand and/or at either of the 3' and/or 5' ends of the second (sense) strand. The nucleic acid may contain more than one ligand of formula (I), (II) or (III). However, one ligand of formulas (I), (II) or (III) is preferred because one such ligand is sufficient to effectively target the nucleic acid to target cells.

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

A nucleic acid with one ligand of formula (I), (II) or (III) at the 5' end of the chain has a simpler structure and is therefore less expensive to synthesize than a similar nucleic acid with a similar ligand at the 3' end. Thus, preferably, one ligand of any of formulas (I), (II) or (III) is covalently attached (conjugated thereto) to the 5' end of the second nucleic acid strand.

According to one embodiment of the present invention, the nucleic acid is conjugated to a ligand that contains a lipid, and more preferably to a ligand that contains cholesterol.

The conjugate of the present invention may comprise any nucleic acid described herein conjugated to any ligand or ligands described herein.

The present invention also provides a conjugate for inhibiting LPA gene expression in a cell, wherein the conjugate comprises a nucleic acid moiety comprising a nucleic acid according to any aspect of the present invention, and at least one ligand moiety, wherein said nucleic acid moiety contains at least one duplex a region that contains at least a portion of a first RNA strand and at least a portion of a second RNA strand that is at least partially complementary to the first strand, wherein said first strand is at least partially complementary to at least a portion of the RNA transcribed from said LPA gene, said at least one part of the ligand contains a linker group, preferably a linker group derived from serinol, as well as a targeting ligand for targeting in vivo to cells and conjugated exclusively to the 3' and/or 5' ends of one or both RNA strands, wherein the 5' end of the first strand of RNA is not conjugated, and:

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

(ii) both the second strand RNA and the first strand RNA are conjugated at the 3' ends to the targeting ligand and the 5' end of the second strand RNA is not conjugated.

According to an embodiment of the present invention, the second RNA strand (i.e., the sense strand) is conjugated at the 5' end to a targeting ligand, the first RNA strand (i.e., the antisense strand) is conjugated at the 3' end to the targeting ligand and 3' the end of the second strand of RNA (i.e., the sense strand) is not conjugated, such that a conjugate is formed with the schematic structure shown in Figure 40A.

In an embodiment of the present invention, the second RNA strand (i.e., the sense strand) is conjugated at the 5' end to a targeting ligand, the second RNA strand (i.e., the sense strand) is also conjugated at the 3' end to the targeting ligand, and 3' The -end of the first strand of RNA (ie, the antisense strand) is not conjugated, so that a conjugate is formed with the schematic structure shown in Figure 40B.

In an 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 at their 3' ends to a targeting ligand, and the 5' end of the second RNA strand (i.e., e. sense strand) is not conjugated, such that a conjugate is formed with the schematic structure shown in Figure 40C.

In an embodiment of the present invention, a second RNA strand (i.e., the sense strand) is conjugated at the 5' end to a targeting ligand, and both the second RNA strand (i.e., the sense strand) and the first RNA strand (i.e., the antisense strand ) are also conjugated at the 3' ends to the targeting ligand so that a conjugate is formed with the schematic structure shown in Figure 40D.

In any of the above implementations indicates a linker by which the ligand is conjugated to the ends of the nucleic acid portion; the ligand may be a GalNAc group such as GalNAc; and the schematic structure shown in Figure 40E represents a portion of the nucleic acid.

These schematic diagrams are not intended to limit the number of nucleotides in the first or second strands, nor do the diagrams represent any base complementarity or other limitation.

Ligands may be monomeric or multimeric (eg, dimeric, trimeric, etc.).

It is acceptable when the ligands are monomeric and accordingly contain one targeting ligand group, for example one GalNAc group.

In another embodiment, the ligands may be dimeric ligands in which portions of the ligands contain two linker groups, such as linker groups derived from serinol or linker groups other than serinol, each of which is connected to one group of the targeting ligand.

The ligands may be trimeric ligands, in which portions of the ligands contain three linker groups, such as linker groups derived from serinol or linker groups other than serinol, each of which is connected to one group of the targeting ligand.

Two or three serinol-derived linker groups can be connected in series, for example as shown below:

where n is 1 or 2 and Y is S or O.

Preferably the ligands are monomeric.

It is suitable for the conjugated RNA strands to be conjugated to the targeting ligand through a linker group, including an additional linker, wherein said additional linker is or contains a saturated, straight or branched C _1-15 alkyl chain, optionally one or more carbon atoms (e.g. , 1, 2 or 3 carbon atoms, is suitable when 1 or 2, in particular 1), is replaced by a heteroatom selected from O, N, S(O) _p , where p is 0, 1 or 2 (for example, the group CH ₂ is substituted with O or NH, or S, or SO ₂ , or a -CH ₃ group at the end of the chain or on a branch is substituted with OH or NH ₂ ), and said chain is optionally substituted with one or more oxo groups (for example, 1-3, for example, 1 group).

It is acceptable when the linker group is a linker group derived from serinol.

It is more suitable when the additional linker contains a saturated straight-chain C _1-15 alkyl chain in which one or more carbon atoms (for example, 1, 2 or 3 carbon atoms, is suitable when 1 or 2, in particular 1) is substituted ( s) an oxygen atom.

It is more suitable when the additional linker contains a PEG chain.

It is more suitable when the additional linker contains a saturated straight-chain C _1-15 alkyl chain.

It is more suitable when the additional linker contains a saturated straight-chain C _1-6 alkyl chain.

It is more suitable when the additional linker contains a saturated straight chain C ₄ or C ₆ alkyl chain, for example a C ₄ alkyl chain.

According to an embodiment of the present invention represents a connecting group of formula (V):

in which n, Y and L ₁ are defined below, and the O phosphogroup is attached to the terminal oligonucleoside of the RNA chains.

Thus, in an embodiment, the targeting ligand portion is a connecting group of formula (VI):

in which n, Y and L ₁ are defined below, and the O phosphogroup is attached to the terminal oligonucleoside of the RNA chains.

Is acceptable when represents a connecting group of formula (XIV):

in which n, Y, R ₁ and L are defined below, L is connected to a targeting ligand, for example, GalNAc, and the O phosphogroup is attached to the terminal oligonucleoside of the RNA chains.

It is suitable when part of the targeting ligand is a connecting group of formula (IV):

in which n, Y, R ₁ and L are defined below, and the O phosphogroup is attached to the terminal oligonucleoside of the RNA chains.

Is acceptable when represents a connecting group of formula (VII):

in which n, Y and L ₂ are defined below, and the O phosphogroup is attached to the terminal oligonucleoside of the RNA chains.

It is suitable when part of the targeting ligand is a connecting group of formula (VIII):

in which n, Y and L ₂ are defined below, and the O phosphogroup is attached to the terminal oligonucleoside of the RNA chains.

Is acceptable when represents a connecting group of formula (IX):

in which F, Y and L are defined below, and the O phosphogroup is attached to the terminal oligonucleoside of the RNA strands.

It is suitable when part of the targeting ligand is a connecting group of formula (IXa):

in which F, Y and L are defined below, and the O phosphogroup is attached to the terminal oligonucleoside of the RNA strands.

Is acceptable when L is:

In any of the above structures, it is suitable for the ligands to be selected from GalNAc and galactose groups, in particular GalNAc groups. In another embodiment, GalNac may be replaced by another targeting ligand, such as a saccharide.

According to an embodiment of the present invention, the first strand of RNA is a compound of formula (X):

in which b is 0 or 1; And

the second strand of RNA is a compound of formula (XI):

wherein:

c and d are independently equal to 0 or 1;

Z ₁ and Z ₂ represent the RNA parts of the first and second strands of RNA, respectively;

Y represents O or S;

n is 0, 1, 2 or 3; And

L ₁ represents a linker to which a ligand is attached;

and at the same time b + c + d is equal to 2 or 3.

It is acceptable when the first RNA strand is a compound of formula (XV)

in which b is 0 or 1; And

the second strand of RNA is a compound of formula (XVI):

in which c and d are independently equal to 0 or 1;

wherein:

Z ₁ and Z ₂ represent the RNA parts of the first and second strands of RNA, respectively;

Y represents O or S;

R ₁ represents H or methyl;

n is 0, 1, 2 or 3; And

L is the same or different in formulas (XV) and (XVId and is selected from the group consisting of:

-(CH ₂ ) _r -C(O)-, where r=2-12;

-(CH ₂ -CH ₂ -O) _s -CH ₂ -C(O)-, where s=1-5;

-(CH ₂ ) _t -CO-NH-(CH ₂ ) _t -NH-C(O)-, where t is independently 1-5;

-(CH ₂ ) _u -CO-NH-(CH ₂ ) _u -C(O)-, where u is independently 1-5; And

-(CH ₂ ) _v -NH-C(O)-, where v is 2-12; And

wherein the terminal C(O) (if present) is attached to the NH group;

and at the same time b + c + d is equal to 2 or 3.

It is acceptable when the first RNA strand is a compound of formula (XII):

in which b is 0 or 1; And

the second strand of RNA is a compound of formula (XIII):

wherein:

c and d are independently equal to 0 or 1;

Z ₁ and Z ₂ represent the RNA parts of the first and second strands of RNA, respectively;

Y represents O or S;

n is 0, 1, 2 or 3; And

L ₂ is the same or different in formulas (XII) and (XIII) and is the same or different in the groups enclosed in brackets with the subscript b, c and d, and is selected from the group consisting of:

n is 0 and L ₂ is:

and the terminal OH group is missing, so that the following group is formed:

Where

F is a saturated branched or unbranched (e.g. straight) C _1-8 alkyl (e.g. C _1-6 alkyl) chain in which one of the carbon atoms is optionally replaced by an oxygen atom, provided that said oxygen atom is separated from another heteroatom (eg O or N atom) with at least 2 carbon atoms;

L is the same or different in formulas (I) and (II) and is selected from the group consisting of:

-(CH ₂ ) _r -C(O)-, where r=2-12;

-(CH ₂ -CH ₂ -O) _s -CH ₂ -C(O)-, where s=1-5;

-(CH ₂ ) _t -CO-NH-(CH ₂ ) _t -NH-C(O)-, where t is independently 1-5;

-(CH ₂ ) _u -CO-NH-(CH ₂ ) _u -C(O)-, where u is independently 1-5; And

-(CH ₂ ) _v -NH-C(O)-, where v is 2-12; And

wherein the terminal C(O) (if present) is attached to the NH group;

and at the same time b + c + d is equal to 2 or 3.

In any of the above formulas in which GalNAc is present, GalNAc may be replaced by any other targeting ligand, such as those mentioned herein.

It is acceptable when 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.

It is more acceptable when 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.

It is most acceptable when b is 0, c is 1 and d is 1.

In one embodiment of the present invention, Y is O. In another embodiment, Y is S.

According to one embodiment of the present invention, R ₁ represents H or methyl. In one embodiment of the present invention, R ₁ is H. In another embodiment of the present invention, R ₁ is methyl.

In one embodiment of the present invention, n is 0, 1, 2, or 3. It is acceptable when n is 0.

According to one embodiment of the present invention, L is selected from the group consisting of:

-(CH ₂ ) _r -C(O)-, where r=2-12;

-(CH ₂ -CH ₂ -O) _s -CH ₂ -C(O)-, where s=1-5;

-(CH ₂ ) _t -CO-NH-(CH ₂ ) _t -NH-C(O)-, where t is independently 1-5;

-(CH ₂ ) _u -CO-NH-(CH ₂ ) _u -C(O)-, where u is independently 1-5; And

-(CH ₂ ) _v -NH-C(O)-, where v is 2-12;

in this case, the terminal C(O) is attached to the NH group.

It is acceptable when L is -(CH ₂ ) _r -C(O)-, where r=2-12. Is acceptable when r=2-6. It is more acceptable when r=4 or 6, for example 4.

Is acceptable when L is:

Exemplary F groups include (CH ₂ ) _1-6 , eg (CH ₂ ) _1-4 , eg CH ₂ , (CH ₂ ) ₄ , (CH ₂ ) ₅ or (CH ₂ ) ₆ , or CH ₂ O( CH ₂ ) _2-3 , for example, CH ₂ O(CH ₂ )CH ₃ .

Is acceptable when L ₂ is:

Is acceptable when L ₂ is:

Is acceptable when L ₂ is:

Is acceptable when L ₂ is:

Is acceptable when n is 0 and L ₂ is:

and the terminal OH group is missing, so that the following group is formed:

;

where Y is defined elsewhere in this application.

Typically, L ₂ within the group enclosed in parentheses with the subscript b, c and d is the same. L ₂ may be the same or different in groups enclosed in brackets with subscripts b, c and d. In an embodiment, L ₂ in the group enclosed in brackets with subscript c is the same as L ₂ in the group enclosed in parentheses with subscript d. According to one embodiment of the present invention, the L ₂ in the group enclosed in parentheses with subscript c is different from the L ₂ in the group enclosed in parentheses with subscript d. According to one embodiment of the present invention, L ₂ in the group enclosed in brackets with subscripts b, c and d is the same, for example, when the linker group is a linker group derived from serinol.

Serinol-derived linker groups can be based on serinol with any stereochemistry, i.e. are derived from the L-serine isomer, D-serine isomer, racemic serine, or other combination of isomers. In a preferred aspect of the present invention, the serinol-GalNAc (SerGN) group has the following stereochemistry:

those. is based on a (S)-serinolamidite or (S)-serinolesuccinate solid supported building block derived from the L-serine isomer.

According to one embodiment of the present invention, the target cells are hepatocytes.

General synthesis schemes: 1

Exemplary compounds can be synthesized in accordance with the methods described below and known to one skilled in the art. Although the schemes illustrate the synthesis of specific conjugates, it should be understood that other claimed conjugates can be prepared using similar methods. Assembly of the oligonucleotide chain and linker building blocks can be accomplished, for example, by solid-phase synthesis using phosphoamidite methodology. Solid phase synthesis can be started from a base or modified building block loaded onto an lcaa CPG. The coupling cycle in phosphoamidite synthesis consists of 1) removal of DMT, 2) chain extension using the required DMT-masked phosphoamidite and an activator, which may be benzylthiotetrazole (BTT), 3) capping of non-extended oligonucleotide chains followed by oxidation of P(III) to P (V) either using iodine (if a phosphodiester bond is required) or EDITH (if a phosphorothioate bond is required) and then recapping (Cap/Ox/Cap or Cap/Thio/Cap). GalNAc conjugation can be accomplished by forming a peptide bond between the GalNAc-carboxylic acid building block and a preassembled and purified oligonucleotide having the required number of amino-modified linker building blocks attached. The required building blocks are either commercially available or their synthesis is described below. All finished single chain products were analyzed by LC-HPLC to confirm their purity. Purity is reported as % FLP (% Full Length Product), which is the percentage of UV area under the signal of a given product in the UV LC-HPLC analysis curve of the final product. The identity of the corresponding single-chain products was confirmed by LC/MS analysis.

Synthon synthesis

Scheme 1: Synthesis of synthons with DMT-serinol (TFA) linker

i) ethyl trifluoroacetate, NEt ₃ , MeOH, 0°C, 16 h, 2: 86% 5: 90%, ii) DMTCl, pyridine, 0°C, 16 h, 74%, iii) LiBH4, EtOH/THF (1 /1, v/v), 0°C, 1 h, 76%, iv) 2-cyanoethyl-N,N-diisopropylchlorophosphoamidite, EtNiPr ₂ , CH ₂ Cl ₂ , 56%, v) succinic anhydride, DMAP, pyridine, RT, 16 h, 38%, vi) HBTU, DIEA, amino-Icaa CPG (500 A), bt, 18 h, 29% (load 26 µmol/g).

(S)-DMT-serinol(TFA)-phosphoamidite 7 can be synthesized from the (L)-serine methyl ester derivative 1 according to methods published in the literature (Hoevelmann et al. Chem. Sci., 2016,7, 128-135 ).

(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.

Loading 6 onto a solid support, such as a controlled pore glass (CPG) support, can be accomplished by forming a peptide bond with the solid support, such as an amino-modified native CPG support (500 A), using a coupling reagent such as HBTU . (S)-DMT-serinol (TFA)-succinate 6 and a coupling reagent such as HBTU are dissolved in a solvent such as CH ₃ CN. A base such as diisopropylethylamine is added to the solution and the reaction mixture is stirred for 2 minutes. A solid support such as native amino-Icaa-CPG support (500 A, 3 g, amine content: 136 μmol/g) is added to the reaction mixture to form a suspension. The suspension is shaken gently at room temperature on a wrist shaker for 16 hours, then filtered and washed with a solvent such as DCM and EtOH. The substrate is dried in vacuum for 2 hours. Unreacted amines on the substrate can be capped by stirring with acetic anhydride/lutidine/N-methylimidazole at room temperature. Washing the substrate can be repeated as described above. The solid support is dried in a vacuum to form a solid support 10.

Scheme 2: Synthesis of GalNAc synthon 9

(vii) TMSOTf, DCM, hexenol, viii) RuCl ₃ , NaIO ₄ , DCM, CH ₃ CN, H ₂ O, 46% in two steps.

Synthesis of GalNAc synthon 9 can be performed according to the methods described in Nair et al. (2014), starting with commercially available per-acetylated galactosamine 8.

Synthesis of single-chain GalNAc conjugates derived from serinol

Scheme 3: General procedure for the synthesis of oligonucleotides for serinol-derived linkers

Oligonucleotide synthesis for 3'-mono-GalNAc-conjugated oligonucleotides (such as compound A0264) is shown in Figure 13 and summarized in Scheme 3. The synthesis begins using (S)-DMT-serinol(TFA)-succinate-Icaa-CPG 10. as in example connection A0264. If additional serinol building blocks are required, (S)-DMT-serinol(TFA)amidite (7) is used in the appropriate solid-phase synthesis cycle. For example, to obtain compound A0329, chain assembly is completed using the binding of an additional serinolamidite after the base sequence is completely assembled. In addition, oligonucleotide synthesis for 5'-mono-GalNAc-conjugated oligonucleotides can be started from a solid support loaded with the corresponding nucleoside from its corresponding sequence. In the exemplary compound A0220 this could be 2'-fA. The oligonucleotide chain is assembled according to its sequence and, if necessary, the building block (S)-DMT-serinol(TFA)-amidite (7) is used. Once chain extension is complete, the DMT protecting group of the last bound amidite building block is removed as in step 1) of the phosphoamidite synthesis cycle.

After completion of the last step in the synthesizer, the individual chains can be cleaved from the solid support by treatment with an amine, such as treatment with 40% aq. methylamine. This step also removes any remaining protecting groups, and treatment with methylamine also releases the amino functional group of the serinol. Each of the crude products was then purified by LC-HPLC and size exclusion chromatography (SEC) to obtain an oligonucleotide precursor for further conjugation to GalNAc.

Scheme 4: GalNAc-conjugation synthesis of serinol-derived oligonucleotide precursors

After solid-phase synthesis, conjugation to GalNAc was accomplished by preactivating the GalN(Ac4)-C4 acid (9) with a peptide bond-forming coupling reagent such as HBTU. The preactivated acid 9 was then reacted with the amino groups in 11 (e.g., A0264) to form intermediate GalN(Ac4) conjugates. The acetyl groups protecting the hydroxyl groups on the GalNAc groups were cleaved by treatment with methylamine to give the target example compounds 12 (eg, A0268), which were further purified by AC-HPLC and SEC.

Synthesis of single-chain GalNAc conjugates not derived from serinol

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 enable GalNAc conjugation. For example, the commercially available building blocks shown in Table 6 below can be used to provide amino-modified non-serinol-derived oligonucleotide precursors 14 (Scheme 5A) using an amino-modifier-loaded CPG, e.g., 10-1 to 10-3, followed by sequence assembly as described above, and finally coupling phosphoamidites of amino modifiers such as 13-1, 13-2 or 13-4.

For example, to prepare 14 (A0653), GlyC3Am-CPG (10-2) was used in combination with GlyC3Am-amidite 13-2. Structurally different modifiers can be used to prepare 14, for example, to prepare A0651 C7Am-CPG was used in combination with C6Am-amidite as the second amino modification. Similarly, commercially available amino-modified CPG 10-5 and amino-modified phosphoamidite 13-5 can be used to synthesize amino-modified precursor molecules 14 (A0655).

Scheme 5: General procedure for oligonucleotide synthesis

The resulting oligonucleotide precursors 14 can then be conjugated to GalN(Ac4)-C4 acid (9) to yield the target exemplary compounds 15 (Scheme 6).

Scheme 6: GalNAc-conjugation synthesis of oligonucleotide precursors

Synthesis of single-stranded triple-antennary GalNAc conjugates in reference conjugates 3-4

Synthesis of oligonucleotides for the tri-antennary GalNAc cluster-conjugated siRNA is shown in Figure 14. Assembly of the oligonucleotide chain was initiated using a base-loaded support, for example, 5'DMT-2'-FdA(bz)-succinate-lcaa-CPG, as in exemplary compound A0006 . The coupling cycle of phosphoamidite synthesis, consisting of 1) removal of DMT, 2) chain extension using the required DMT-masked phosphoamidite, 3) capping of unextended oligonucleotide chains followed by oxidation of P(III) to P(V) with either iodine or EDITH ( if a phosphorothioate linkage is required) and finally capping (Cap/Ox/Cap or Cap/Thio/Cap), repeat until the full length of the product is reached. For on-column conjugation of the trivalent triantennary GalNAc cluster, a similar round of synthesis was used using the required trivalent branching amidite C4XLT-phos, followed by another round of synthesis round using the GalNAc amidite ST23-phos. Once this final step in the synthesizer is complete, the oligonucleotide is cleaved from the solid support and additional protecting groups can be removed by treatment with methylamine. Each of the crude products was then purified by HPLC-AC and SEC.

General procedure for double strand formation

To prepare double-stranded conjugates, the individual single strands are dissolved at a concentration of 60 OD/ml in H ₂ O. Both solutions of individual oligonucleotides can be added together to the reaction vessel. Titration can be performed to monitor the reaction. The first strand is added at a 25% excess over the second strand, as determined by UV absorbance at 260 nm. The reaction mixture is heated, for example, to 80°C for 5 minutes and then slowly cooled to room temperature. Double chain formation can be monitored by the formation of ion pairs using reverse phase HPLC. Based on the UV region of the residual single chain, the required amount of second chain can be calculated and added to the reaction mixture. The reaction mixture is heated again, for example to 80°C, and slowly cooled to room temperature. This procedure can be repeated until less than 10% of the residual single chain is detected.

The above method (including Schemes 1-6 and Figures 13 and 14) can be easily adapted to replace GalNAc with another targeting ligand, such as a saccharide.

In any of the above aspects, instead of conjugation after solid-phase synthesis, preformed serinol(GN)-phosphoamidite can be prepared and used for on-column conjugation.

General synthesis schemes: 2

Exemplary compounds can be synthesized in accordance with the methods described below and known to one skilled in the art. Assembly of the oligonucleotide chain and linker building blocks can be accomplished, for example, by solid-phase synthesis using phosphoamidite methodology. GalNAc conjugation can be accomplished by forming a peptide bond between the GalNAc-carboxylic acid building block and a preassembled and purified oligonucleotide containing the required amount of attached amino-modified linker building blocks.

Scheme 1: Synthesis of synthons with DMT-serinol (TFA) linker

i) ethyl trifluoroacetate, NEt ₃ , MeOH, 0°C, 16 h, 2: 86% 5: 90%, ii) DMTCl, pyridine, 0°C, 16 h, 74%, iii) LiBH4, EtOH/THF (1 /1, v/v), 0°C, 1 h, 76%, iv) 2-cyanoethyl-N,N-diisopropylchlorophosphoamidite, EtNiPr ₂ , CH ₂ Cl ₂ , 56%, v) succinic anhydride, DMAP, pyridine, b.t., 16 h, 38%, vi) HBTU, DIEA, amino-lcaa CPG (500A), b.t., 18 h, 29%.

DMT-serinol(TFA)-phosphoamidite 7 can be synthesized from serinol derivative 1 according to methods published in the literature (Hoevelmann et al. Chem. Sci., 2016,7, 128-135).

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

Loading 6 onto a solid support, such as a CPG support, can be accomplished by forming a peptide bond with a solid support, such as an amino-modified native CPG support (500A), using a coupling reagent such as HBTU. DMT-serinol (TFA)-succinate 6 and a coupling reagent such as HBTU are dissolved in a solvent such as CH ₃ CN. A base such as diisopropylethylamine is added to the solution and the reaction mixture is stirred for 2 minutes. A solid support such as native amino-Icaa-CPG support (500 A, 3 g, amine content: 136 μmol/g) is added to the reaction mixture to form a suspension. The suspension is shaken gently at room temperature on a wrist shaker for 16 hours, then filtered and washed with a solvent such as DCM and EtOH. The substrate is dried in vacuum for 2 hours. Unreacted amines on the substrate can be capped by stirring with acetic anhydride/lutidine/N-methylimidazole at room temperature. Washing the substrate can be repeated as described above. The solid support is dried in a vacuum to form a solid support 10.

Scheme 2: Synthesis of GalNAc synthon 9

(vii) TMSOTf, DCM, hexenol, viii) RuCl ₃ , NaIO ₄ , DCM, CH ₃ CN, H ₂ O, 46% in two steps.

Synthesis of GalNAc synthon 9 can be carried out in accordance with the methods described in Nair et al. J. Am. Chem. Soc., 2014, 136(49), pp 16958-16961, starting with commercially available per-acetylated galactosamine 8.

Scheme 3: General procedure for oligonucleotide synthesis

All oligonucleotides can be synthesized on the AKTA oligopilot 10 synthesizer using standard phosphoamidite reactions, which are described in detail below.

Synthesis of oligonucleotides from 3' and 5' GalNAc-conjugated oligonucleotide precursors (such as compound X0385B-prec) is presented in Figure 17 and summarized in Scheme 3. Synthesis is started using DMT-serinol(TFA)-succinate-lcaa-CPG 10 The phosphoamidite synthesis cycle is continued until the full length of the product is reached. Once chain extension is complete, the DMT protecting group of the last bound amidite building block can be removed in the same manner as in each individual round of synthesis. To complete the synthesis of the exemplary compound X0385B-prec (which contains a serinol-derived linker group at the 3' and 5' ends of the second strand), chain assembly was completed using additional serinolamidite coupling after the base sequence was completely assembled.

After completion of the last step in the synthesizer, the single chains can be cleaved from the solid support by treatment with an amine such as 40% aq. methylamine This step also removes any remaining protecting groups, and treatment with methylamine also releases the amino functional group of the serinol. The resulting crude oligonucleotide can be purified by ion exchange chromatography (Resource Q, 6 ml, GE Healthcare) on an AKTA Pure HPLC system using a gradient, such as a sodium chloride gradient. Excess salt from ion exchange chromatography (IC) purification can be removed by SEC to yield the amino-modified oligonucleotide precursor 11. Product-containing fractions are pooled, desalted on a gel permeation chromatography column (Zetadex, EMP Biotech) and lyophilized.

Scheme 4: General procedure for GalNAc conjugation

(i) 9, HBTU, DIPEA, DMSO; 11, H ₂ O, DMSO, DIPEA; then activated 9, 11; then 40% MeNH ₂ , H ₂ O.

Conjugation of GalNAc synthon 9, as described above, can be accomplished by coupling 9 to the amino functional group of the serinol of the corresponding oligonucleotide chain 11 using standard peptide coupling conditions known to one skilled in the art. For example, the appropriate amino-modified precursor molecule 11 is dissolved in H ₂ O and a polar solvent such as DMSO (eg, DMSO/H ₂ O, 2/1, v/v) is added, followed by a base such as DIPEA (for example, 2.5% of the total). In a separate reaction vessel, pre-activation of GalNAc synthon 9 can be performed using a 2 eq reaction. (per amino functional group in the amino-modified oligonucleotide precursor) carboxylic acid component with 2 eq. coupling reagent such as HBTU in the presence of 8 eq. a base such as DIPEA in a polar solvent such as DMSO. After 2 min, activated compound 9 is added to a solution of the corresponding amino-modified precursor molecule 11. The progress of the reaction can be monitored using LC/MS or LC-HPLC. After completion of the conjugation reaction (eg 30 min), the crude product can be precipitated by adding 10× iPrOH 0.1×2 M NaCl and collected by centrifugation and decantation. Acetylhydroxy protecting groups are removed under basic conditions such as 40% MeNH ₂ (1 ml at 500 OD). After 15 minutes at room temperature, H ₂ O (1:10 v/v) is added and compound 12 (such as X0385B shown in Figure 17) is isolated, repurified by anion exchange and gel permeation chromatography and then lyophilized.

General procedure for double chain formation

The individual single strands are dissolved at a concentration of 60 OD/ml in H ₂ O. Both single oligonucleotide solutions can be added together to the reaction vessel. Titration can be performed to monitor the reaction. The first strand is added at a 25% excess over the second strand, as determined by UV absorbance at 260 nm. The reaction mixture is heated, for example, to 80°C for 5 minutes and then slowly cooled to room temperature. Double chain formation can be monitored by the formation of ion pairs using reverse phase HPLC. Based on the UV area of the residual single chain, the required amount of second chain can be calculated and added to the reaction mixture. The reaction mixture is heated again, for example to 80°C, and slowly cooled to room temperature. This procedure can be repeated until less than 10% of the residual single chain is detected.

The above method (including Schemes 1-4 and Figure 17) can be easily adapted to replace GalNac with another targeting ligand, such as a saccharide.

The present invention relates to a conjugate for inhibiting expression of the LPA gene in a cell, wherein said conjugate comprises a nucleic acid portion and a ligand portion, wherein said nucleic acid portion comprises at least one duplex region that contains at least a portion of a first strand of RNA and at least a portion of a second strand of RNA that is at least partially complementary to a first strand, wherein said first strand is at least partially complementary to at least a portion of the RNA transcribed from said LPA gene, wherein said ligand portions comprise a linker group and a targeting ligand for targeting under conditions in vivo onto cells and conjugated exclusively to the 3' and/or 5' ends of one or both RNA strands, with the 5' end of the first RNA strand not being conjugated, wherein:

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

(ii) both the second strand RNA and the first strand RNA are conjugated at the 3' ends to the targeting ligand, and the 5' end of the second strand RNA is not conjugated.

The linker group may be, for example, a serinol-derived linker group or one of the other types of linkers described herein.

The present invention provides, in another aspect, a nucleic acid for inhibiting the expression of LPA in a cell, comprising at least one duplex region that contains at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary a first strand, wherein said first strand is at least partially complementary to at least a portion of the RNA transcribed from the LPA gene, wherein said 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 conjugated to a ligand. The second strand may contain the nucleotide sequence 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 first and/or second chain nucleotides may be modified as described herein.

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 (set forth above), wherein said ligand is conjugated to the described nucleic acid, and wherein the first strand is modified using 2'-OMe modification on odd nucleotides and modified using 2'-F on even nucleotides, and the second strand is modified using 2'-OMe on even nucleotides and modified using 2'-F on odd nucleotides.

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 said nucleic acid is conjugated to a ligand of Formula I (set forth above), and, in addition, the nucleic acid has a modification profile presented below, which is an excerpt from Table 1 presented in this application.

SEQ ID NO: 5 5' auaacucuguccaauuacca 3' 6162717181736152736 SEQ ID NO: 6 5' uggguaauggacagaguuau 3' 1845261846364645161 SEQ ID NO: 9 5' auaacucuguccaauuaccg 3' 6162717181736152738 SEQ ID NO: 10 5' cgguaauggacagaguuau 3' 3845261846364645161

where specific modifications are indicated 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.

The ligand may contain GalNAc, and Figure 4A or Figure 4B further illustrates examples of the present invention.

The present invention also provides pharmaceutical compositions containing a nucleic acid or conjugated nucleic acid according to the present invention. Pharmaceutical compositions can be used as drugs or as diagnostic agents, alone or in combination with other agents. For example, one or more nucleic acid conjugates of the present invention can be combined with a delivery vehicle (eg, liposomes) and/or excipients such as carriers, diluents. Other agents such as preservatives and stabilizers may also be added. Methods for delivering nucleic acids are known in the art and known to one skilled in the art.

The nucleic acid or conjugated nucleic acid of the present invention can also be administered in combination with other therapeutic compounds, either administered separately or simultaneously, for example, as a combination unit dose. The present invention also includes a pharmaceutical composition containing one or more nucleic acids or conjugated nucleic acids in accordance with the present invention in a physiologically/pharmaceutically acceptable excipient such as a stabilizer, preservative, diluent, buffer and the like.

Dosage levels for the drug and pharmaceutical compositions of the present invention can be determined by those skilled in the art through standard experimentation. According to one embodiment of the present invention, a unit dose may contain from about 0.01 mg/kg to about 100 mg/kg body weight of nucleic acid or conjugated nucleic acid. In another embodiment, the dose may be from 10 mg/kg to 25 mg/kg body weight, or from 1 mg/kg to 10 mg/kg body weight, or from 0.05 mg/kg to 5 mg/kg body weight, or from 0.1 mg/kg to 5 mg/kg body weight, or from 0.1 mg/kg to 1 mg/kg body weight, or from 0.1 mg/kg to 0.5 mg/kg body weight, or from 0.5 mg/kg to 1 mg/kg body weight. Dosage levels can also be calculated using other parameters such as body surface area.

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

The pharmaceutical compositions and drugs of the present invention can be administered to a mammalian subject at a pharmaceutically effective dose. The mammal may be selected from human, non-human primate, monkey or arboreal primate, dog, cat, horse, bovine, pig, goat, sheep, mouse, rat, hamster, hedgehog and guinea pig or other suitable species. Based on this, in this application, the wording "LPA" or "LPA" refers to a nucleic acid or protein in any of the above species, if expressed naturally or artificially therein, but preferably this wording refers to human nucleic acids or proteins.

A further aspect of the present invention relates to a nucleic acid or conjugated nucleic acid according to the present invention or a pharmaceutical composition containing a nucleic acid or conjugated nucleic acid according to the present invention, for use in treating a disease, disorder or syndrome. Treatment may involve preventing and reducing the risk of stroke, atherosclerosis, thrombosis or cardiovascular disease such as coronary artery disease or aortic stenosis, and any other disease or pathology associated with elevated levels of Lp(a) containing particles. The present invention includes a pharmaceutical composition containing one or more nucleic acids or conjugated nucleic acids in accordance with the present invention in a physiologically/pharmaceutically acceptable excipient such as a stabilizer, preservative, diluent, buffer and the like.

The pharmaceutical composition may be a sterile injectable aqueous suspension or solution, or may be in lyophilized form, or may be attached, absorbed or incorporated into or into any other suitable galenic vehicle such as dragees, tablets, capsules, nanoparticles, gels, tablets, granules or similar structures.

The nucleic acid described herein may be capable of inhibiting the expression of LPA. The nucleic acid described herein may be capable of partially inhibiting the expression of LPA. Inhibition can be complete, i.e. 0% compared to the expression level of LPA in the absence of the nucleic acid according to the present invention. Inhibition of LPA expression can be partial, i.e. it may be 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or intermediate values of LPA expression in the absence of the nucleic acid according to the present invention. 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 when administered to a subject such as a patient -Human. The nucleic acid or conjugated nucleic acid of the present invention, or compositions containing it, can be administered in a regimen comprising once or twice weekly, every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks or every eight weeks, or in different dosing frequency regimens, such as combinations of the above intervals. The nucleic acid can be administered subcutaneously, intravenously, or any other route of administration such as oral, rectal, or intraperitoneal.

In cells and/or subjects treated with or receiving a nucleic acid or conjugated nucleic acid according to the present invention, LPA expression may be inhibited relative to untreated cells and/or subjects by an amount ranging from 15% to 100%, but at least approximately 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% or intermediate values . The level of inhibition may provide treatment for a disease associated with LPA expression or overexpression, or may serve to further study the functions and physiological roles of the LPA gene product.

A further aspect of the present invention relates to a nucleic acid or conjugated nucleic acid according to the present invention in the manufacture of a medicament for the treatment of a disease, disorder or syndromes, such as those listed above, or additional pathologies associated with elevated Lp(a) levels, or when additional therapeutic approaches where inhibition of LPA expression is desired.

The present invention also includes a method of treating or preventing a disease, disorder or syndrome, such as those listed above, comprising administering a pharmaceutical composition comprising a nucleic acid or conjugated nucleic acid described herein to an individual in need of treatment (to improve such pathologies). ). The nucleic acid composition can be administered in a schedule including treatments twice a week, once a week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks or every eight weeks, or regimens with different dosing frequencies, such as combinations of the above intervals. The nucleic acid or conjugated nucleic acid may be intended for subcutaneous or intravenous administration or other routes of administration such as oral, rectal or intraperitoneal.

The nucleic acid or conjugated nucleic acid of the present invention can also be administered for use in combination with other therapeutic compounds, either administered separately or simultaneously, for example, as a combination unit dose. Molecular conjugation with other biologically active molecular species such as peptides, cellular or artificial ligands, or small and large molecules is also possible.

The nucleic acid or conjugated nucleic acid of the present invention can be prepared using conventional methods in the art, including chemical synthesis or expression of the nucleic acid under both in vitro (eg, interrupted transcription) and in vivo conditions. For example, using solid-phase chemical synthesis or using a nucleic acid-based expression vector, including viral derivatives or partially or fully synthetic expression systems. In one embodiment of the present invention, an expression vector can be used to produce a nucleic acid of the present invention in vitro, within an intermediate host organism or cell type, within an intermediate or terminal organism, or within a desired target cell. Methods for obtaining (synthesis or enzymatic transcription) of nucleic acid described in this application are known to specialists in the art.

The subject matter of the invention consists of chemical molecular particles that mediate the destruction of LPA mRNA by binding to LPA gene transcripts through cellular RNA interference mechanisms. The molecular compounds of the present invention can be used as, but not limited to, conjugates with the sugar moiety of N-acetylgalactosamine (GalNAc), which mediates hepatocyte-specific cellular uptake through specific binding to the asialoglycoprotein receptor (ASGPR) complex. The subject matter of the invention may be combined with various other chemical structures imparting different properties, as indicated below. The use of a chemical modification profile of nucleic acids confers resistance to nucleases in serum and makes possible, for example, a subcutaneous route of administration.

The subject matter of the invention is characterized by high specificity of targeted delivery at the molecular and tissue levels, which potentially provides a better safety profile than currently available treatments.

The present invention also provides a nucleic acid in accordance with any aspect of the present invention described herein, wherein the first strand of RNA has a terminal 5'-(E)-vinylphosphonate nucleotide, and the terminal 5'-(E)-vinylphosphonate nucleotide is connected to the second nucleotide in the first strand via a phosphodiester bond.

According to one embodiment of the present invention, the first chain may contain more than 1 phosphodiester bond.

According to one embodiment of the present invention, the first strand may contain phosphodiester bonds between at least three terminal 5' nucleotides.

According to one embodiment of the present invention, the first strand may contain phosphodiester bonds between at least four terminal 5' nucleotides.

According to one embodiment of the present invention, the first chain may contain formula (XVII):

(vp)-N(po)[N(po)] _n -

(XVII)

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

According to one embodiment of the present invention, the first chain may contain at least one phosphorothioate (ps) bond.

According to one embodiment of the present invention, the first strand may further comprise a phosphorothioate linkage between the two terminal 3' nucleotides or phosphorothioate linkages between the three terminal 3' nucleotides.

According to one embodiment of the present invention, the bonds between other nucleotides in the first strand are phosphodiester bonds.

According to one embodiment of the present invention, the first chain may contain more than 1 phosphorothioate bond.

In a further embodiment, the second strand may comprise a phosphorothioate linkage between the two terminal 3' nucleotides or phosphorothioate linkages between the three terminal 3' nucleotides.

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

According to an embodiment of the present invention, the terminal 5'-(E)-vinylphosphonate nucleotide is an RNA nucleotide.

A terminal 5'-(E)-vinylphosphonate nucleotide is a nucleotide in which the natural phosphate group at the 5' end is replaced by an E-vinylphosphonate in which the bridging 5' oxygen atom of the terminal nucleotide of the 5' phosphorylated chain is replaced by a methynyl (-CH= ) group:

Nucleotides with natural phosphate at the 5' end

Nucleotide with E-vinylphosphonate at the 5' end

5'-(E)-vinylphosphonate is a 5'-phosphate mimic. A biological mimic is a molecule that is capable of performing the same function as and is structurally very similar to the parent molecule it mimics. In relation to the present invention, 5'-(E)-vinylphosphonate mimics the function of normal 5'-phosphate, for example, providing efficient loading of RISC. In addition, due to its slightly modified structure, 5'-(E)-vinylphosphonate is able to stabilize the 5'-terminal nucleotide, protecting it from dephosphorylation by enzymes such as phosphatases.

One aspect of the present invention is a nucleic acid disclosed herein for inhibiting expression of a target gene in a cell, comprising at least one duplex region that contains at least a portion of a first strand and at least a portion of a second strand that is at least is partially complementary to a first strand, wherein said first strand is at least partially complementary to at least a portion of the RNA transcribed from said target gene, wherein said first strand contains modified nucleotides or unmodified nucleotides at a variety of positions to facilitate nucleic acid processing by RISC.

In one aspect, "facilitates processing by RISC" means that the nucleic acid can be processed by RISC, e.g., any modification present allows the nucleic acid to be processed by RISC in an acceptable manner such that siRNA activity can be realized.

The nucleic acid disclosed herein, in which 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, which corresponds to position 13 of the first strand, is not modified using 2'-O-methyl modification.

The nucleotide on the second strand that "corresponds" to a position on the first strand is suitably a nucleotide that pairs with said nucleotide on the first strand.

In one aspect, the nucleotide on the second strand that corresponds to position 13 of the first strand is a nucleotide that base pairs with position 13 of the first strand.

In one aspect, the nucleotide on the second strand that corresponds to position 11 of the first strand is a nucleotide that base pairs with position 11 of the first strand.

In one aspect, the nucleotide on the second strand that corresponds to position 12 of the first strand is a nucleotide that base pairs with position 12 of the first strand.

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

The nucleotide that corresponds to position 13 of the first strand is suitably position 13 of the second strand, when counted 3' of the second strand, starting from the first nucleotide of the double-stranded region. Likewise, position 11 of the second strand is suitably the 11th nucleotide 3' of the second strand, starting from the first nucleotide of the double-stranded region. This nomenclature can be applied to other positions of the second circuit.

In one aspect, in the case of partial complementarity between the first and second strands, a nucleotide on the second strand that "matches" a position on the first strand may not necessarily form a base pair if that position is the position at which there is a mismatch, but the principle of nomenclature is still applicable.

Preferably, first and second strands are completely complementary throughout the duplex region (ignoring any overhanging regions) and there are no mismatches in the double-stranded nucleic acid region.

Also preferred are:

The nucleic acid disclosed herein, in which 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, which corresponds to position 11 of the first strand, is not modified using 2'-O-methyl modification.

The nucleic acid disclosed herein, in which 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 on the second strand that correspond to positions 11 and 13 of the first strand, not modified with 2'-O-methyl modification.

In one aspect, the nucleotide on the second strand that corresponds to position 12 of the first strand is not modified with a 2'-O-methyl modification. This restriction on the nucleic acid can be observed with any other restriction described in this application.

Thus, another aspect of the present invention is the nucleic acid disclosed herein, in which 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 nucleotides on the second strand that correspond to positions 11-13 of the first chain, not modified using 2'-O-methyl modification.

The nucleic acid disclosed herein, in which 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 on the second strand that correspond to position 11 or 13 or 11 and 13, or 11-13 of the first chain, are modified using a 2'-fluoro modification.

The nucleic acid disclosed herein, in which the nucleotides at positions 2 and 14 at the 5' end of the first strand are modified with a 2'-fluoro modification, and the nucleotides at the second strand that correspond to positions 11 or 13, or 11 and 13 , or 11-13 of the first chain, is not modified using the 2'-O-methyl modification.

The nucleic acid disclosed herein, in which the nucleotides at positions 2 and 14 at the 5' end of the first strand are modified with a 2'-fluoro modification, and the nucleotides at the second strand that correspond to positions 11 or 13, or 11 and 13 , or 11-13 of the first chain, are modified using a 2'-fluoro modification.

A nucleic acid disclosed herein in which more than 50% of the first and/or second strand nucleotides contain a 2'-O-methyl modification, e.g., more than 55%, 60%, 65%, 70%, 75%, 80%, or 85% or more of the first and/or second strands contain a 2'-O-methyl modification, preferably as measured as a percentage of the total nucleotides in both the first and second strands.

A nucleic acid disclosed herein in which more than 50% of the first and/or second strand nucleotides contain a naturally occurring modification of RNA, for example in which more than 55%, 60%, 65%, 70%, 75%, 80% or 85% or more of the first and/or second strands contain such modification, preferably as measured as a percentage of the total number of nucleotides of both first and second strands. Suitable natural modifications include, in addition to 2-O'-methyl, other 2'-sugar modifications, in particular the 2'-H modification resulting in a DNA nucleotide.

A nucleic acid disclosed herein containing no more than 20%, e.g. no more than 15%, e.g. more than 10%, of nucleotides that have 2' modifications that are not 2'-O-methyl modifications, on the first and /or second strands, preferably as measured as a percentage of the total number of nucleotides of both first and second strands.

A nucleic acid disclosed herein containing no more than 20% (e.g., no more than 15% or no more than 10%) 2'-fluoro modifications on the first and/or second strands, preferably as measured as a percentage of the total nucleotides of both chains.

A nucleic acid disclosed herein in which all nucleotides are modified using a 2'-O-methyl modification, except positions 2 and 14 from the 5' end of the first strand and nucleotides on the second strand that correspond to position 11 or 13, or 11 and 13, or 11-13 of the first chain. Preferably, nucleotides that are not modified with 2'-O-methyl are modified with fluorine at the 2' position.

Preferred is the nucleic acid disclosed herein in which all nucleotides of the nucleic acid are modified at the 2' sugar position. Preferably, these nucleotides are modified using a 2'-fluoro modification, which modification is not a 2'-O-methyl modification.

The nucleic acids of the present invention may contain one or more nucleotides modified at the 2' position using a 2'-H, and therefore containing a DNA nucleotide within the nucleic acid. The nucleic acids of the present invention may contain DNA nucleotides at positions 2 and/or 14 of the first strand, measured from the 5' end of the first strand. Nucleic acids may contain DNA nucleotides on the second strand that correspond to position 11 or 13, or 11 and 13, or 11-13 of the first strand.

In one aspect, there is no more than one DNA per nucleic acid of the present invention.

The nucleic acids of the present invention may contain one or more LNA nucleotides. The nucleic acids of the present invention may contain LNA nucleotides at positions 2 and/or 14 of the first strand, measured from the 5' end of the first strand. Nucleic acids may contain LNAs on the second strand that correspond to position 11 or 13, or 11 and 13, or 11-13 of the first strand.

In one aspect, the nucleic acid is modified on the first strand using alternating 2-O-methyl modifications and 2-fluoro modifications, and positions 2 and 14 (starting at the 5' end) are modified using 2'-fluoro. Preferably, the second strand is modified using 2'-fluoro modifications in nucleotides on the second strand that correspond to position 11 or 13, or 11 and 13, or 11-13 of the first strand. Preferably, the second chain is modified with 2'-fluoro modifications at positions 11-13, counting from the 3' end, starting from the first position of the complementary (double-stranded) region, and the remaining modifications are natural modifications, preferably 2'-O- methyl.

In one aspect, the nucleic acid of the present invention contains one or more inverted ribonucleotides, preferably an inverted adenine, using a 5'-5' linkage or a 3'-3' linkage, preferably a 3'-3' linkage at the 3' end second chain.

In one aspect, the nucleic acid contains one or more phosphodithioate linkages, such as 1, 2, 3 or 4 phosphodithioate linkages. Preferably, up to 4 phosphodithioate bonds are present, one each at the 5' and 3' ends of the first and second chains.

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

Particularly preferred are nucleic acids that are siRNA molecules in which 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 nucleic acid contains one or more or all from:

(i) an inverted nucleotide, preferably a 3'-3' linkage at the 3' end of the second strand;

(ii) one or more phosphodithioate bonds;

(iii) the second strand nucleotide corresponding to position 11 or 13 of the first strand is not modified with a 2'-O-methyl modification, preferably one or both of these positions containing a 2'-fluoro modification;

(iv) the nucleic acid contains at least 80% of all nucleotides containing a 2'-O-methyl modification;

(v) the nucleic acid contains no more than 20% of nucleotides that contain 2'-fluoro modifications.

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

Specific preferred examples for 19-mer double-stranded nucleic acid with blunt ends and no sticky ends are:

The nucleic acid disclosed herein, in which the nucleotides at positions 2 and 14 at the 5' end of the first strand are not modified with a 2'-O-methyl modification, and the nucleotide at position 7 at the 5' end of the second strand is not modified with using the 2'-O-methyl modification.

The nucleic acid disclosed herein, in which the nucleotides at positions 2 and 14 at the 5' end of the first strand are not modified with a 2'-O-methyl modification, and the nucleotide at position 9 at the 5' end of the second strand is not modified with using the 2'-O-methyl modification.

The nucleic acid disclosed herein, in which the nucleotides at positions 2 and 14 at the 5' end of the first strand are not modified with a 2'-O-methyl modification, and the nucleotides at positions 7 and 9 at the 5' end of the second strand are not modified using 2'-O-methyl modification.

The nucleic acid disclosed herein, in which the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified using a 2'-O-methyl modification, and the nucleotides at positions 7-9 from the 5' end of the second strand are not modified using 2'-O-methyl modification.

The nucleic acid disclosed herein, in which 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' ends of the second strand are modified using a 2'-fluoro modification.

The nucleic acid disclosed herein, in which the nucleotides at positions 2 and 14 from the 5' end of the first strand are modified using a 2'-fluoro modification, and the nucleotides at positions 7 and/or 9, or 7-9 from the 5' The -ends of the second strand are not modified with a 2'-O-methyl modification.

The nucleic acid disclosed herein, in which the nucleotides at positions 2 and 14 from the 5' end of the first strand are modified using a 2'-fluoro modification, and the nucleotides at positions 7 and/or 9, or 7-9 from the 5' -ends of the second chain are modified using a 2'-fluoro modification.

A nucleic acid disclosed herein in which more than 50% of the first and/or second strand nucleotides contain a 2'-O-methyl modification, e.g., more than 55%, 60%, 65%, 70%, 75%, 80%, or 85% or more of the first and/or second strands contain a 2'-O-methyl modification, preferably as measured as a percentage of the total nucleotides of both first and second strands.

A nucleic acid disclosed herein, in which more than 50% of the first and/or second strand nucleotides contain a naturally occurring RNA modification, e.g., more than 55%, 60%, 65%, 70%, 75%, 80%, or 85% or more the first and/or second strands contain such modification, preferably as measured as a percentage of the total number of nucleotides of both first and second strands. Suitable natural modifications include, in addition to 2-O'-methyl, other 2'-sugar modifications, in particular the 2'-H modification resulting in a DNA nucleotide.

A nucleic acid disclosed herein containing no more than 20%, e.g. no more than 15%, e.g. more than 10%, of nucleotides that contain 2' modifications that are not 2'-O-methyl modifications, on the first and /or second strands, preferably as measured as a percentage of the total number of nucleotides of both first and second strands.

A nucleic acid disclosed herein containing no more than 20% (e.g., no more than 15% or no more than 10%) 2'-fluoro modifications on the first and/or second strands, preferably as measured as a percentage of the total nucleotides of both chains.

Nucleic acid disclosed herein, in which all nucleotides are modified using a 2'-O-methyl modification, except positions 2 and 14 from the 5' end of the first strand and nucleotides at positions 7 and/or 9 from the 5' end second chain. Preferably, nucleotides that are not modified with 2'-O-methyl are modified with fluorine at the 2' position.

A nucleic acid disclosed herein in which all nucleotides are modified using a 2'-O-methyl modification except positions 2 and 14 at the 5' end of the first strand and nucleotides at positions 7-9 at the 5' end of the second strand . Preferably, nucleotides that are not modified with 2'-O-methyl are modified with fluorine at the 2' position.

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

For a nucleic acid containing a duplex region of 21 base pairs, the second strand preferably does not contain a 2'-O-methyl group at nucleotides 9 or 10 or 11, counted from the 5' end of the duplex, corresponding to positions 13, 12 and 11 of the first chains, respectively.

The present invention also provides unmodified sequences of all modified sequences disclosed herein.

The present invention will be further described with reference to the following non-limiting drawings and examples.

Blueprints

Figure 1 shows the results of a screen for an unconjugated siRNA molecule to inhibit LPA mRNA expression in human RT-4 cells.

Figures 2A and 2B show the dose-dependent response of unconjugated LPA-targeting siRNA molecules on LPA mRNA expression in human RT-4 cells.

Figure 3 shows the inhibition of LPA mRNA expression in primary human and cynomolgus macaque hepatocytes by varying doses of GalNAc-L1 LPA-1038-conjugated siRNA molecules delivered via receptor-mediated uptake.

Figures 4A and 4B show examples of the structure of GalNAc ligands with various linkers L1 and L6, respectively, to which the exemplary oligonucleotides were conjugated.

Figure 5 shows representative examples of LPA mRNA silencing by the indicated L6-conjugated GalNAc siRNAs in primary human hepatocytes delivered by receptor-mediated uptake.

Figure 6 shows conjugate 1.

Figure 7 shows conjugate 2.

Figure 8 shows conjugate 3.

Figure 9 shows reference conjugate 1.

Figure 10 shows reference conjugate 2.

Figure 11 shows reference conjugate 3.

Figure 12 shows reference conjugate 4.

In each of Figures 6-12 and 19-30, the top strand is the antisense strand and the bottom strand is the sense strand. In addition, to more clearly show the relationship between the nucleic acid and the ligand parts, the nucleotide at the end of the corresponding conjugated strands is drawn in full.

Figure 13 shows the synthesis of A0268, which is a single-stranded oligonucleotide conjugated at the 3' end to mono-GalNAc and is the starting material in the synthesis of conjugate 1 and conjugate 3. (ps) denotes a phosphorothioate bond.

Figure 14 shows the synthesis of A0006, which is a single-stranded oligonucleotide conjugated at the 5' end to a three-antennary GalNAc, used for the synthesis of reference conjugate 4. (ps) denotes a phosphorothioate bond.

Figure 15 illustrates the determination of TTR shutdown under in vitro conditions. Specifically, Figure 15A shows the in vitro determination of TTR shutdown using reference conjugates (RC) 1 and 3, as well as the untreated “UT” control; Figure 15B shows in vitro determination of TTR shutdown using reference conjugates (RC) 2 and 3, as well as the untreated “UT” control; and Figure 15C shows in vitro detection of TTR shutdown by conjugates 1, 2 and 3, as well as by RC3 and the untreated “UT” control. Reference conjugates 1 and 2 are comparison conjugates. Reference conjugate 3 represents non-targeting GalNAc siRNA, and “untreated” (“UT”) represents untreated cells. Both RC3 and UT are negative controls. mRNA levels were normalized against PtenII.

Figure 16 shows the time course of serum TTR in cohorts of n=4 c57BL/6 mice 7, 14 and 27 days after treatment with 1 mg/kg SC - conjugates 1-3, reference conjugates (RC) 1, 2 and 4 and sham-treated (FSB) individuals.

Figure 17 shows the synthesis of oligonucleotides from oligonucleotide precursors conjugated to GalNAc at the 3' and 5' ends (such as compound X0385B-prec).

Figure 18 shows an equal dose-dependent shutdown response for siRNA targeting LPA with two separate GalNAc units conjugated to the second strand, compared to a three-antennary GalNAc unit 5' of the second strand, in primary cynomolgus hepatocytes.

Figure 19A shows conjugate 4. The last three nucleotides at the 5' and 3' ends of the antisense and sense strands are connected by a phosphorothioate linker between each nucleotide. Serinol-GalNAc linkers are conjugated via a phosphodiester bond to the 3' end and 5' end of the sense strand.

Figure 19B shows conjugate 5. The last three nucleotides at the 5' and 3' ends of the antisense strand are connected by a phosphorothioate linker between each nucleotide. Serinol-GalNAc linkers are conjugated via a phosphorothioate bond to the 3' end and 5' end of the sense strand.

Figure 20 shows conjugate 6. The last three nucleotides at the 5' and 3' ends of the antisense strand are connected by a phosphorothioate linker between each nucleotide. Serinol-GalNAc linkers are conjugated via a phosphodiester bond to the 3' end and 5' end of the sense strand.

Figure 21 shows conjugate 7. The last three nucleotides at the 5' and 3' ends of the antisense and sense strands are connected by a phosphorothioate linker between each nucleotide. Serinol-GalNAc linkers are conjugated via a phosphorothioate bond to the 3' end and 5' end of the sense strand. Serinol-GalNAc linkers are connected to each other via a phosphorothioate bond.

Figure 22 shows conjugate 8. The last three nucleotides at the 5' and 3' ends of the antisense and sense strands are connected by a phosphorothioate linker between each nucleotide. The GalNAc-C6-amino-modifier linker is conjugated at the 5' end of the sense strand, and the GalNAc-C7-amino-modifier linker is conjugated at the 3' end of the sense strand.

Figure 23 shows conjugate 9. The last three nucleotides at the 5' and 3' ends of the antisense and sense strands are connected by a phosphorothioate linker between each nucleotide. The GalNAc-GlyC3-amino-modifier linker is conjugated at the 5' and 3' ends of the sense strand.

Figure 24 shows conjugate 10. The last three nucleotides at the 5' and 3' ends of the antisense and sense strands are connected by a phosphorothioate linker between each nucleotide, and the sense strands are connected by a phosphorothioate linker between each nucleotide. The GalNAc-piperidyl-amino-modifier linker is conjugated at the 5' and 3' ends of the sense strand.

Figure 25 shows conjugate 11. The last three nucleotides at the 5' and 3' ends of the antisense and sense strands are connected by a phosphorothioate linker between each nucleotide. The GalNAc-C3-amino-modifier linker is conjugated at the 5' and 3' ends of the sense strand.

Figure 26 shows conjugate 12. The last three nucleotides at the 5' and 3' ends of the antisense and sense strands are connected by a phosphorothioate linker between each nucleotide. The GalNAc-C6-amino-modifier linker is conjugated at the 5' end of the sense strand, and the GalNAc-GlyC3-amino-modifier linker is conjugated at the 3' end of the sense strand.

Figure 27 shows conjugates 15, 16, 18 and 19, which differ only in their RNA sequences. The last three nucleotides at the 5' and 3' ends of the antisense and sense strands are connected by a phosphorothioate linker between each nucleotide in each conjugate. Serinol-GalNAc linkers are conjugated via a phosphorothioate bond to the 3' end and 5' end of the sense strand.

Figure 28 shows reference conjugate 5 and reference conjugate 9, which differ only in their RNA sequences. The last three nucleotides at the 5' and 3' ends of the antisense strand and the 3' end of the sense strand are connected by a phosphorothioate linker between each nucleotide in both conjugates. The trimeric GalNAc linker is conjugated via a phosphorothioate bond to the 5' end of the sense strand in both conjugates.

Figure 29 shows reference conjugate 6 and reference conjugate 7, which differ only in their RNA sequences. The last three nucleotides at the 5' and 3' ends of the antisense strand and the 3' end of the sense strand are connected by a phosphorothioate linker between each nucleotide in both conjugates. The trimeric GalNAc linker is conjugated via a phosphorothioate bond to the 5' end of the sense strand in both conjugates.

Figure 30 depicts reference conjugate 8. The last three nucleotides at the 5' and 3' ends of the antisense strand and the 3' end of the sense strand are connected by a phosphorothioate linker between each nucleotide. The trimeric GalNAc linker is conjugated via a phosphorothioate bond to the 5' end of the sense strand.

Figure 31 illustrates the determination of TTR shutdown under in vitro conditions. Specifically, Figure 31A shows the in vitro determination of TTR silencing with conjugates 4, 5, 6 and 2 compared to "Luc" (reference conjugate 3) as well as the untreated control "UT"; Figure 31B shows the in vitro determination of TTR silencing with conjugates 7 and 2 compared to "Luc" (reference conjugate 3), as well as the untreated control "UT". Luc or reference conjugate 3 (RC3) represents the non-targeting GalNAc siRNA, and “untreated” (“UT”) represents untreated cells. Both RC3 and UT are negative controls. The mRNA level was normalized against PtenII.

Figure 32 illustrates the determination of TTR shutdown in vitro. Specifically, Figure 32A shows the in vitro determination of TTR silencing with conjugates 8, 9, 10, 11 and 2 compared to "Luc" (reference conjugate 3) as well as the untreated control "UT"; Figure 32B shows the in vitro determination of TTR silencing with conjugates 12 and 2 compared to "Luc" (reference conjugate 3), as well as the 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. The mRNA level was normalized against PtenII.

Figure 33 illustrates the determination of LPA mRNA silencing in vitro using conjugate 19 compared to controls. Ctr represents non-targeting GalNAc siRNA, and “untreated” (“UT”) represents untreated cells. Both Ctr and UT are negative controls. The mRNA level was normalized against ACTB.

Figure 34 shows the time course of liver Aldh2 mRNA levels in cohorts of n=6 c57BL/6 mice 14, 28 and 42 days after treatment with 1 mg/kg subcutaneous administration - conjugate 15, reference conjugate (RC) 6 and sham individuals. processing (FSB). The mRNA level was normalized against Pten.

Figure 35 shows the time course of liver Aldh2 mRNA levels in cohorts of n=6 c57BL/6 mice 14, 28 and 42 days after treatment with 1 mg/kg subcutaneous administration - conjugate 16, reference conjugate (RC) 7 and sham individuals. processing (FSB). The mRNA level was normalized against Pten.

Figure 36 shows the time course of liver Tmprss6 mRNA levels in cohorts of c57BL/6 mice with n=6 14, 28 and 42 days after treatment with 1 mg/kg subcutaneous administration - conjugate 18, reference conjugate (RC) 8 and sham individuals. processing (FSB). The mRNA level was normalized against Pten.

Figure 37 shows the serum stability of conjugates 4, 5, 6, 7 and 2, as well as untreated control (UT) at 37°C for 3 days.

Figure 38 shows the serum stability of conjugates 8, 9, 10, 11, 12 and 2, as well as untreated control (UT) at 37°C for 3 days.

Figure 39 shows the inhibition of LPA mRNA expression in primary human hepatocytes by different doses of GalNAc-L6-linked siRNA delivered via receptor-mediated uptake.

Figure 40 schematically illustrates various embodiments of nucleic acids conjugated to ligands via linkers.

Examples

The numbering mentioned in each example is specific to the example mentioned.

Example 1

Modified and conjugated siRNA molecules used for functional examples in this application.

LPA-1038 derivatives:

GalNAc-LPA-1038-L1

First Strain (SEQ ID NO: 119, based on SEQ ID NO: 5)

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

Second Circuit (SEQ ID NO: 120, based on SEQ ID NO SEQ ID NO: 6)

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

GalNAc-LPA-1038-L6

First Strain (SEQ ID NO: 121, based on SEQ ID NO: 5)

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

Second Circuit (SEQ ID NO: 122, based on SEQ ID NO: 6)

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

FN (N=A, C, G, U) stands for 2'-fluoro, 2'-deoxynucleosides

OMeN (N=A, C, G, U) stands for 2'-O-methylnucleosides

(ps) denotes phosphorothioate bond

ST23 and ST43 are shown below.

An additional example are derivatives of LPA 1041:

GalNAc-LPA-1041-L1

First Strain (SEQ ID NO: 123, based on SEQ ID NO: 9)

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

Second Circuit (SEQ ID NO: 124, based on SEQ ID NO: 10)

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

GalNAc-LPA-1041-L6

First Strain (SEQ ID NO: 125, based on SEQ ID NO: 9)

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

Second Circuit (SEQ ID NO: 126, based on SEQ ID NO: 10)

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

FN (N=A, C, G, U) stands for 2'-fluoro, 2'-deoxynucleosides

OMeN (N=A, C, G, U) stands for 2'-O-methylnucleosides

(ps) denotes phosphorothioate bond

ST23 and ST43 are shown below.

ST23 is GalNAc-C4 phosphoamidite (structural components shown below)

ST23

Long Triple (STKS)

ST41 is shown below:

ST43 is presented below and described in WO2017/174657:

All oligonucleotides were either obtained from commercial oligonucleotide manufacturers (Biospring, Frankfurt, Germany, or RiboBio, Guangzhou, Guangdong, China) or synthesized on an AKTA oligopilot synthesizer (in-house) using standard phosphoamidite reactions. A commercially available solid support and 2'-O-methyl-RNA phosphoamidites, 2'-fluoro-DNA phosphoamidites (all with standard protection) and a commercially available long triple phosphoamidite (Glen research) were used. The synthesis was performed using 0.1 M solutions of phosphoamidite in dry acetonitrile, and benzylthiotetrazole (BTT) (0.3 M in acetonitrile) was used as an activator. All other reagents were commercially available standard reagents.

Conjugation of the appropriate GalNac synthon (eg, ST23, ST41, or ST43) was accomplished by binding the appropriate phosphoamidite to the 5' end of the oligochain under standard phosphoamidite binding conditions. Phosphothioates were introduced using standard commercially available thiolation reagents (EDITH, Link technologies).

Single strands were cleaved from CPG using methylamine (40% aq) and the resulting crude oligonucleotide was purified by ion exchange chromatography (Resource Q, 6 mL, GE Healthcare) on an AKTA Pure HPLC system using a sodium chloride gradient. Product-containing fractions were pooled, desalted on a gel permeation chromatography column (Zetadex, EMP Biotech) and lyophilized.

For renaturation, equimolar amounts of the corresponding single chains were dissolved in water and heated to 80°C for 5 minutes. After gradual cooling to room temperature, the resulting duplex was lyophilized.

The sequences of the resulting nucleic acids (miRNAs) are shown in Table 1 below.

Table 1: Unconjugated nucleic acid sequences tested for inhibition of LPA mRNA expression. The sequences and the modification profile used are indicated.

SEQ ID NO: Ident. siRNA Chain Subsequence Modifications 1 LPA-1014 first chain 5'ucguauaacaauaaggggc 3' 5381616272616284847 2 second chain 5'gccccuuauuguuauacga 3' 4737351615451616382 3 LPA-1024 first chain 5'gauaacucuguccauuacc 3' 8252635354537251637 4 second chain 5'gguaauggacagaguuauc 3' 4816254827282815253 5 LPA-1038 first chain 5'auaacucuguccaauuacca 3' 6162717181736152736 6 second chain 5'ugguaauggacagaguuau 3' 1845261846364645161 7 LPA-1040 first chain 5'uaacucuguccaauuaccgu 3' 5263535453725163745 8 second chain 5'acgguaauggacagaguua 3' 2748162548272828152 9 LPA-1041 first chain 5'auaacucuguccaauuaccg 3' 6162717181736152738 10 second chain 5'cgguaauggacagaguuau 3' 3845261846364645161 eleven LPA-1055 first chain 5'agaaugugccucgauaacu 3' 6462545473538252635 12 second chain 5'aguuaucgaggcacauucu 3' 2815253828472725171 13 LPA-1057 first chain 5'auaacucuguccaucacca 3' 6162717181736172736 14 second chain 5'uggugaggacagaguuau 3' 1845461846364645161 15 LPA-1058 first chain 5'auaacucuguccaucaccu 3' 6162717181736172735 16 second chain 5'aggugaggacagaguuau 3' 2845461846364645161 17 LPA-1061 first chain 5'uaacucucucccaauuaccau 3' 5263535453725163725 18 second chain 5'augguaauggacagaguua 3' 2548162548272828152 19 LPA-1086 first chain 5'augugccuugauaacucug 3' 6181837154616271718 20 second chain 5'cagaguuaucaaggcacau 3' 3646451617264836361 21 LPA-1099 first chain 5'aguuggugcugcuucagaa 3' 6451845471835172826 22 second chain 5'uucugaagcagcaccaacu 3' 1535462836472736271 23 LPA-1102 first chain 5'aauaaggggcugccacagg 3' 6252648483547363648 24 second chain 5'ccugggcagccccuuauu 3' 3718184728373715251 25 LPA-1116 first chain 5'uaacucuguccaucaccau 3' 5263535453725363725 26 second chain 5'auggugaggacagaguua 3' 2548182548272828152 27 LPA-1127 first chain 5'augagccucgauaacucug 3' 6182837174616271718 28 second chain 5'cagaguuaucgaggcucau 3' 3646451617464835361 29 LPA-1128 first chain 5'aaugagccucgauaacucu 3' 6254647353825263535 thirty second chain 5'agaguuaucgaggcucauu 3' 2828152538284717251 31 LPA-1141 first chain 5'aaugcuuccaggacauuuc 3' 6254715372846361517 32 second chain 5'gaaauguccuggaagcauu 3' 4626181735482647251 33 LPA-1151 first chain 5'acagugguggagaaugugc 3' 6364548184646254547 34 second chain 5'gcacauucucccaccacugu 3' 4727251717363727181 35 LPA-1171 first chain 5'guaugugccucgauaacuc 3' 8161818371746162717 36 second chain 5'gaguuaucgaggcacauaac 3' 4645161746483636163 37 LPA-1177 first chain 5'ucgauaacucuguccauca 3' 5382526353545372536 38 second chain 5'ugaggacagaguuaucga 3' 1825482728281525382 39 LPA-1189 first chain 5'ugucacuggacauuguguc 3' 5453635482725181817 40 second chain 5'gacacaauguccagugaca 3' 4636362545372818272 41 LPA-1244 first chain 5'cugggauccaugguguaac 3' 7184825372548181627 42 second chain 5'guuacaccauggaucccag 3' 4516363725482537364 43 LPA-1248 first chain 5'agaugaccaagcuuggcag 3' 6461827362835184728 44 second chain 5'cugccaagcuuggucaucu 3' 3547362835184536171

Table 1

Table 1: nucleotide modifications are indicated 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.

Table 2: The sequences of the LPA, APOB, beta-actin and Pten qPCR amplicon sets that were used to measure mRNA levels are shown below.

Gene View Sequences SEQ ID NO: LPA: (upper) Human 5' AAGTGTCCTTGGCGACGTCC 3' 45 LPA: (lower) 5' CCTGGACTGTGGGGCTTT 3' 46 LPA: (probe) 5' CTGTTTCTGAACAAGCACCAACGGAGC 3' 47 LPA (upper) Cynomolgus macaque 5' GTGTCCTCGCAACGTCCA 3' 48 LPA (lower) 5' GACCCCGGGGGCTTTG 3' 49 LPA (probe) 5'TGGCTGTTTCTGAACAAGCACAATGG 3' 50 APOB (upper) Human 5' TCATTCCTTCCCCCAAAGAGACC 3' 51 APOB (lower) 5' CACCTCCGTTTTGGTGGTAGAG 3' 52 APOB (probe) 5' CAAGCTGCTCAGTGGAGGCAACACATTA 3' 53 Beta actin (top) Human 5' GCATGGGTCAGAAGGATTCCTAT 3' 54 Beta actin (bottom) 5' TGTAGAAGGTGTGGTGCCAGATT 3' 55 Beta actin (probe) 5' TCGAGCACGGCATCGTCACCAA 3' 56 Beta actin (top) Cynomolgus macaque 5'AAGGCCAACCGCGAGAAG 3' 57 Beta actin (bottom) 5'AGAGCGTACAGGGACAGCA 3' 58 Beta actin (probe) 5' TGAGACCTTCAACACCCCAGCCATGTAC 3' 59 PPIB (upper) Human 5'AGATGTAGGCCGGGTGATCTTT 3' 60 PPIB (lower) 5' GTAGCCAAATCCTTTCTCTCCTGT 3' 61 PPIB (probe) 5' TGTTCCAAAACAGTGGATAATTTTGTGGCC 3' 62

table 2

Example 2

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

Liposomal transfection complexes were prepared in triplicate at a ratio of 1.5 μl RNAiMax (ThermoFisher)/80 pmol of the indicated siRNA molecules. The complex was diluted to the indicated concentrations of 2.5 nM and 25 nM, respectively (values are presented in pairs as lighter and darker gray bars). Human bladder transitional papilloma RT4 cells endogenously expressing LPA were seeded at a density of 125,000 cells per well in a 24-well format on top of pre-plated transfection complexes (reverse transfection) at the indicated concentration. 24 hours after transfection, total RNA was isolated using the Spin Cell Mini Kit 250 (Stratec). LPA mRNA levels were determined by quantitative real-time PCR against PPIB mRNA expression in the corresponding samples as a constitutive gene transcript. Values were normalized to the amount of LPA mRNA detected in untreated cells (intraplate). A non-silencing siRNA compound was transfected as an additional control. Means and standard deviations (SD) of normalized triplicates are presented. The results are shown in Figure 1.

Example 3

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

RT4 human bladder transitional papilloma cells were reverse transfected as described above (Example 2) and treated at the indicated concentration (ranging from 100 nM to 0.2 nM) with various unconjugated siRNA compounds (Table 1) as noted. 24 h after transfection, total RNA was isolated using the Spin Cell Mini Kit 250 (Stratec). LPA mRNA levels were determined by real-time qPCR relative to PPIB mRNA expression in the corresponding samples as a constitutive gene transcript. Values were normalized to the amount of LPA mRNA detected in untreated cells. Bars represent residual LPA mRNA expression for each data point. The results are shown in Figure 2.

Example 4

Inhibition of LPA mRNA expression in primary human and cynomolgus macaque hepatocytes by varying doses of GalNAc-L1 LPA-1038-conjugated siRNA molecule delivered via receptor-mediated uptake.

Primary hepatocytes (ThermoFisher) were seeded onto 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 at the indicated concentrations (nM) immediately after plating. 24 hours after siRNA treatment, total RNA was isolated using the InviTrap RNA cell HTS kit (Stratec) for 96-well plate. LPA mRNA levels were determined by real-time qPCR relative to actin (cynomolgus) or APOB (human) mRNA levels in the corresponding samples as the constitutive gene transcript. Values were normalized to LPA expression in untreated cells. The means and standard deviations of normalized triplicate residual LPA mRNA levels are shown as black bars. The results are shown in Figure 3.

Example 5

Silencing of LPA mRNA in primary human hepatocytes by various indicated L6-GalNAc-conjugated siRNAs in primary human hepatocytes after receptor-mediated delivery.

Primary human hepatocytes (ThermoFisher) were seeded onto collagen-coated 96-well plates at 30,000 cells per well (96-well format). GalNAc-L6-conjugated siRNAs, including a non-silencing control, were added at the two indicated concentrations immediately after cell seeding. 24 hours after siRNA treatment, total RNA was isolated using the InviTrap RNA cell HTS kit (Stratec) for 96-well plate. LPA mRNA expression levels were determined by qRT-PCR against APOB mRNA as the constitutive gene transcript. Values were normalized to LPA mRNA expression in untreated cells, and residual LPA mRNA levels are presented in pairs as bars (100 nM black bars, 20 nM gray bars). The means and standard deviations of the normalized triplicates are shown in Figure 5 .

Example 6 - Synthesis of Conjugates

Exemplary compounds were synthesized in accordance with the methods described below and methods known to one skilled in the art. Assembly of the oligonucleotide chain and linker building blocks was performed using solid-phase synthesis using phosphoamidite methodology. GalNAc conjugation was accomplished by forming a peptide bond between the GalNAc-carboxylic acid building block and a preassembled and purified oligonucleotide containing the required amount of attached amino-modified linker building blocks.

Synthesis, deprotection, and purification of oligonucleotides were performed according to standard procedures known in the art.

All oligonucleotides were synthesized on an AKTA oligopilot synthesizer using standard phosphoamidite reactions. A commercially available solid support and 2'-O-methyl-RNA phosphoamidites, 2'-fluoro, 2'-deoxy-RNA phosphoamidites (all with standard protection, ChemGenes, LinkTech) and a commercially available 3'-amino TFA modifier were used -amino-C-6-lcaa CPG 500 Å (Chemgenes). Per-acetylated galactosamine 8 is available commercially.

Auxiliary reagents were purchased from EMP Biotech. The synthesis was performed using a 0.1 M solution of phosphoamidite in dry acetonitrile, and benzylthiotetrazole (BTT) (0.3 M in acetonitrile) was used as an activator. The binding time was 15 min. The Cap/OX/Cap or Cap/Thio/Cap cycle was used (Cap: Ac ₂ O/NMI/lutidine/acetonitrile, oxidizing agent: 0.1 M I ₂ in pyridine/H ₂ O). Phosphothioates were introduced using a standard commercially available thiolation reagent (EDITH, Link technologies). DMT cleavage was accomplished by treatment with 3% dichloroacetic acid in toluene. After completion of the programmed synthesis cycles, washes with diethylamine (DEA) were performed. All oligonucleotides were synthesized in DMT-off mode.

Attachment of the serinol-derived linker group was accomplished using either base-loaded (S)-DMT-serinol(TFA)-succinate-lcaa-CPG 10 or (S)-DMT-serinol(TFA)phosphoamidite 7 (synthesis was performed as described in Hoevelmann et al. (Chem. Sci., 2016, 7, 128-135)). Trichantine GalNAc clusters (ST23/C4XLT) were introduced by sequential coupling of the corresponding tripling amidite derivatives (C4XLT-phos) followed by the addition of GalNAc amidite (ST23-phos).

Single chains were cleaved from CPG by treatment with 40% aq. methylamine. The resulting crude oligonucleotide was purified by ion exchange chromatography (Resource Q, 6 mL, GE Healthcare) on an AKTA Pure HPLC system using a sodium chloride gradient. Product-containing fractions were pooled, desalted on a gel permeation chromatography column (Zetadex, EMP Biotech) and lyophilized.

The individual single strands were dissolved at a concentration of 60 OD/ml in H ₂ O. Both single oligonucleotide solutions were added together to the reaction vessel. Titration was performed to facilitate reaction monitoring. The first chain was added at a 25% excess over the second chain, as determined by UV absorbance at 260 nm. The reaction mixture was heated to 80°C for 5 minutes and then slowly cooled to room temperature. Double chain formation was monitored by ion pair formation using reverse phase HPLC. Based on the UV area of the residual single chain, the required amount of the second chain was calculated and added to the reaction mixture. The reaction mixture was again heated to 80°C and slowly cooled to room temperature. This procedure was repeated until less than 10% of the residual single chain was detected.

Synthesis of compounds 2-10

Compounds 2-5 and (S)-DMT-serinol(TFA)-phosphoamidite 7 were synthesized according to published methods (Hoevelmann et al. Chem. Sci., 2016,7, 128-135).

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

Succinic anhydride was added to a solution of 5 in pyridine, followed by DMAP. The resulting mixture was stirred at room temperature overnight. All starting material was consumed as assessed by TLC. The reaction was concentrated. The crude material was subjected to silica gel chromatography using a gradient of 0% to 5% methanol in DCM (+ 1% triethylamine) to give 1.33 g of 6 (yield = 38%). m/z (ESI-): 588.2 (100%), (calculated for C30H29F3NO8 ^- [MH] ^- 588.6). 1H-NMR: (400 MHz, CDCl3)δ[ppm]=7.94 (d, 1H, NH), 7.39-7.36 (m, 2H, CHaryl), 7.29-7.25 (m , 7H, CHaril), 6.82-6.79 (m, 4H, CHaril), 4.51-4.47 (m, 1H), 4.31-4.24 (m, 2H), 3.77 (s, 6H, 2xDMTr-OMe), 3.66-3.60 (m, 16H, ^HNEt ₃₊ ), 3.26-3.25 (m, 2H), 2.97-2.81 (m, 20H, NEt ₃ ), 2.50-2.41 (4H, m), 1.48-1.45 (m, 26H, HNEt ₃ ⁺ ), 1.24-1.18 (m, 29H, NEt ₃ ).

(S)-DMT-serinol(TFA)-succinate-Icaa-CPG (10)

(S)-DMT-serinol (TFA)-succinate (159 mg, 270 µmol) and HBTU (113 mg, 299 µmol) were dissolved in CH ₃ CN (10 ml). Diisopropylethylamine (DIPEA, 94 μL, 540 μmol) was added to the solution and the mixture was stirred for 2 min, followed by the addition of native amino-Icaa-CPG (500 A, 3 g, amine content: 136 μmol/g). The suspension was shaken gently at room temperature on a wrist shaker for 16 hours, then filtered and washed with DCM and EtOH. The solid support was dried in vacuum for 2 hours. Unreacted amines on the support were capped by stirring with acetic anhydride/lutidine/N-methylimidazole at room temperature. Washing the substrate was repeated as described above. The solid was dried in vacuo to obtain solid support 10 (3 g, loading 26 μmol/g).

GalNAc synthon (9)

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 stages.

Characteristic data correspond to published data.

Oligonucleotide synthesis

All single-stranded oligonucleotides were synthesized according to the reaction conditions described above and in Figures 13 and 14.

All finished single chain products were analyzed by LC-HPLC to confirm their purity. Purity is reported as % FLP (% Full Length Product), which is the percentage of UV area under the signal of a given product in the UV LC-HPLC analysis curve of the finished product. The identity of the corresponding single-stranded products (unmodified, amino-modified precursors, or GalNAc-conjugated oligonucleotides) was confirmed by LC/MS analysis.

Table 3: Single-stranded unconjugated oligonucleotides

Product (11) Name Calculated mol. weight Installed pier mass (ESI-) %FLP
(AC-HPLC) A0002 STS16001A 6943.3 Yes 6943.0 Yes 86.6% A0006 STS16001BL4 8387.5 Yes 8387.5 Yes 94.1% A0130 STS18001A 6259.9 Yes 6259.8 Yes 76.5% A0131 STS18001BL4 7813.2 Yes 7813.1 Yes 74.3% A0220 STS16001B-5'1xNH2 6982.2 Yes 6982.1 Yes 95.7% A0237 STS16001A 6943.3 Yes 6943.3 Yes 95.6% A0244 STS16001BV1 6845.2 Yes 6844.9 Yes 98.2% A0264 STS16001AV4-3'1xNH2 7112.4 Yes 7112.2 Yes 95.4% A0329 STS16001BV6-3'5'1xNH2 7183.3 Yes 7183.2 Yes 88.8%

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

Synthesis of conjugates 1-3 and reference conjugates 1-2

Conjugated single chains for conjugates 1-2 and reference conjugates 1-2

Conjugation of the GalNAc synthon (9) was accomplished by binding to the amino functional group of the serinol of the corresponding oligonucleotide chain 11 using a coupling reagent to form a peptide bond. Thus, the corresponding amino-modified precursor molecule 11 was dissolved in H ₂ O (500 OD/ml) and DMSO (DMSO/H ₂ O, 2/1, v/v) was added, followed by DIPEA (2.5 % of total volume). In a separate reaction vessel, pre-activation of GalN(Ac4)-C4 acid (9) was performed using 2 eq. (per amino functional group in the amino-modified precursor of oligonucleotide 11) carboxylic acid component with 2 eq. HBTU in the presence of 8 eq. DIPEA in DMSO. After 2 min, preactivated compound 9 was added to the solution of the corresponding amino-modified precursor molecule. After 30 min, the progress of the reaction was monitored using LC/MS or LC-HPLC. After completion of the conjugation reaction, the crude product was precipitated by the addition of 10× iPrOH and 0.1×2 M NaCl and collected by centrifugation and decantation. To release the acetylated hydroxyl groups in the GalNAc groups, the resulting precipitate was dissolved in 40% MeNH2 (1 ml at 500 OD) and after 15 minutes at room temperature diluted in H ₂ O (1:10), finally repurified by anion exchange and gel permeation chromatography and lyophilized to obtain the final product 12.

Table 4: Single-stranded GalNAc-conjugated oligonucleotides

Product (12) Raw material Name Calculated mol. weight Installed pier mass (ESI-) % FLP (AC-HPLC) A0241 A0220 STS16001BL20 7285.5 Yes 7285.3 Yes 91.8% A0268 A0264 STS16001AV4L33 7415.7 Yes 7415.4 Yes 96.9% A0330 A0329 STS16001BV6L42 7789.8 Yes 7789.8 Yes 95.5%

Double chain formation

Double strand formation was performed according to the methods described above.

Double chain purity is expressed as % double chain, which is the percentage of UV area under the signal of a given product in the UV ion pairing assay curve by reverse phase HPLC (RP-RP-HPLC).

Table 5: Nucleic acid conjugates

Product Source materials Name % double chain First chain Second chain Reference conjugate 1 A0237 A0241 STS16001L20 97.7% Reference conjugate 2 A0268 A0244 STS16001L33 97.8% Reference conjugate 3 A0130 A0131 STS18001L4 96.8% Reference conjugate 4 A0002 A0006 STS16001L4 90.1% Conjugate 1 A0268 A0241 STS16001L24 96.0% Conjugate 2 A0237 A0330 STS16001V1L42 98.5% Conjugate 3 A0268 A0330 STS16001V1L43 98.2%

Sequences

Modification designations for the following sequences:

f stands for 2'-fluoro-2'-deoxyribonucleotide or 2'-fluororibonucleotide (the terms are interchangeable)

m stands for 2'-O-methylribonucleotide

(ps) denotes phosphorothioate bond

Ser(GN) is a building block of GalNAc-C4 attached to a serinol-derived linker group:

where O--- represents a bond between an oxygen atom and, for example, H, a phosphodiester bond or a phosphorothioate bond.

C4XLT is:

ST23 is:

The synthesis of phosphoamidite derivatives C4XLT (C4XLT-phos) as well as ST23 (ST23-phos) can be performed as described in WO2017/174657.

C4XLT-phos:

ST23-phos:

Conjugate 1

Antisense Strand-STS16001AL33 (SEQ ID NO: 127)

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'

Sense Circuit - STS16001BL20 (SEQ ID NO: 128)

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'

Conjugate 2

Antisense Strand-STS16001A (SEQ ID NO: 129)

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

Sense Circuit - STS16001BV1L42 (SEQ ID NO: 130)

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)

Conjugate 3

Antisense Strand-STS16001AL33 (SEQ ID NO: 127)

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'

Sense Circuit - STS16001BV1L42 (SEQ ID NO: 130)

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 '

Reference conjugate 1

Antisense Strand—STS16001A (SEQ ID NO: 129)

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

Sense Circuit - STS16001BL20 (SEQ ID NO: 128)

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

Reference conjugate 2

Antisense Strand-STS16001AL33 (SEQ ID NO: 127)

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)

Sense Circuit - STS16001V1B (SEQ ID NO: 131)

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

Reference conjugate 3

Antisense Strand-STS18001A (A0130; SEQ ID NO: 132)

mU (ps) fC (ps) mG fA mA fG mU fA mU fU mC fC mG fC mG fU mA (ps) fC (ps) mG

Sense Circuit - STS18001BL4 (A0131, SEQ ID NO: 133)

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

Reference conjugate 4

Antisense Strand-STS16001AL33 (SEQ ID NO: 127)

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

Sense Circuit - STS16001BL4 (SEQ ID NO: 134)

5'[(ST23) (ps)] ₃ C4XLT(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

Example 7 - Determination of TTR silencing in vitro for various GalNAc TTR siRNA conjugates

Primary mouse hepatocytes were seeded in collagen pre-coated 96-well plates (Thermo Fisher Scientific, catalog number A1142803) at a cell density of 30,000 cells per well and treated with siRNA conjugates at concentrations that ranged from 10 nM to 0.0001 nM. 24 hours after treatment, cells were lysed and RNA was extracted using InviTrap® RNA Cell HTS 96/C24×96 preparative kits (Stratec, catalog number 7061300400) according to the manufacturer's protocol. TTR transcript and constitutive gene (PtenII) mRNA levels were quantified using TaqMan assay.

Target gene expression in primary mouse hepatocytes 24 hours after treatment with the conjugates of the present invention, conjugates 1-3, showed that target gene expression decreased with increasing conjugate dose compared to negative controls (see bar "UT" and reference conjugate 3), as shown in Figure 15. This indicates that the first strand binds to the target gene, which reduces gene expression. Figure 15 also shows the target gene expression levels for reference conjugates 1 and 2, which act as comparison conjugates. As can be seen from a comparison of the data presented in Figure 15A and 15C, and 15B and 15C, the conjugates of the present invention (conjugates 1-3) reduce target gene expression compared to reference conjugates 1 and 2. The most effective conjugate at 0.01 nM appears to be conjugate 2. The most effective conjugate at 0.1 nM, 0.5 nM, 1 nM and 10 nM appears to be conjugate 3.

Example 8 - Temporal dynamics of serum TTR in mice in vivo

c57BL/6 mice were treated with 1 mg/kg siRNA conjugates subcutaneously on day 0. Serum samples were collected on days 7, 14, and 27 by collecting blood from the orbital sinus and stored at −20°C until analysis. Serum TTR quantification was performed using a mouse prealbumin ELISA (ALPCO, 41-PALMS/lot 22, 2008003B) according to the manufacturer's protocol (sample dilution 1:8000 or 1:800).

Results for time course of serum TTR in cohorts of c57BL/6 n=4 mice 7, 14 and 27 days after treatment with 1 mg/kg subcutaneous administration of conjugates 1-3, reference conjugates 1, 2 and 4 and in individuals with sham treatment (PBS) are shown in Figure 16. As evidenced by the data in Figure 16, 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, in particular, 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. Thus, the dosage level of conjugate 3 would be expected to be approximately three times lower to achieve a similar initial shutdown and would also result. to a longer shutdown duration compared to reference conjugate 4.

Example 9 - Synthesis of Conjugates 2

Exemplary compounds were synthesized in accordance with the methods described below and methods known to one skilled in the art. Assembly of the oligonucleotide chain and linker building blocks was performed using solid-phase synthesis using phosphoamidite methodology. GalNAc conjugation was accomplished by forming a peptide bond between the GalNAc-carboxylic acid building block and a preassembled and purified oligonucleotide containing the required amount of attached amino-modified linker building blocks.

Synthesis, deprotection, and purification of oligonucleotides were performed according to standard procedures known in the art.

All oligonucleotides were synthesized on an AKTA oligopilot synthesizer using standard phosphoamidite reactions. A commercially available solid support and 2'-O-methyl-RNA phosphoamidites, 2'-fluoro, 2'-deoxy-RNA phosphoamidites (all with standard protection, ChemGenes, LinkTech) and a commercially available 3'-amino TFA modifier were used -amino-C-6-lcaa-CPG 500 Å (Chemgenes). Per-acetylated galactosamine 8 is available commercially.

Auxiliary reagents were purchased from EMP Biotech. The synthesis was performed using a 0.1 M solution of phosphoamidite in dry acetonitrile, and benzylthiotetrazole (BTT) (0.3 M in acetonitrile) was used as an activator. The binding time was 15 min. The Cap/OX/Cap or Cap/Thio/Cap cycle was used (Cap: Ac ₂ O/NMI/lutidine/acetonitrile, oxidizing agent: 0.1 M I ₂ in pyridine/H ₂ O). Phosphothioates were introduced using a standard commercially available thiolation reagent (EDITH, Link technologies). DMT cleavage was accomplished by treatment with 3% dichloroacetic acid in toluene. After completion of the programmed synthesis cycles, washes with diethylamine (DEA) were performed. All oligonucleotides were synthesized in DMT-off mode.

Attachment of the serinol-derived linker group was accomplished using either base-loaded (S)-DMT-serinol(TFA)-succinate-lcaa-CPG 10 or (S)-DMT-serinol(TFA)-phosphoamidite 7 (synthesis performed , as described in the literature Hoevelmann et al. Chem., 2016,7, 128-135). Trihantane GalNAc clusters (ST23/C4XLT or ST23/C6XLT) were introduced by sequential coupling of the corresponding tripling amidite derivatives (C4XLT-phos or C6XLT-phos) followed by the addition of GalNAc amidite (ST23-phos).

Single chains were cleaved from CPG by treatment with 40% aq. methylamine. The resulting crude oligonucleotide was purified by ion exchange chromatography (Resource Q, 6 mL, GE Healthcare) on an AKTA Pure HPLC system using a sodium chloride gradient. Product-containing fractions were pooled, desalted on a gel permeation chromatography column (Zetadex, EMP Biotech) and lyophilized.

The individual single strands were dissolved at a concentration of 60 OD/ml in H ₂ O. Both single oligonucleotide solutions were added together to the reaction vessel. Titration was performed to facilitate reaction monitoring. The first chain was added at a 25% excess over the second chain, as determined by UV absorbance at 260 nm. The reaction mixture was heated to 80°C for 5 minutes and then slowly cooled to room temperature. The formation of the double chain was monitored by the formation of ion pairs using reverse phase HPLC. Based on the UV area of the residual single chain, the required amount of the second chain was calculated and added to the reaction mixture. The reaction mixture was again heated to 80°C and slowly cooled to room temperature. This procedure was repeated until less than 10% of the residual single chain was detected.

Synthesis of compounds 2-10

Compounds 2-5 and (S)-DMT-serinol(TFA)-phosphoamidite 7 were synthesized according to published methods (Hoevelmann et al. Chem. Sci., 2016,7, 128-135).

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

Succinic anhydride was added to a solution of 5 in pyridine, followed by DMAP. The resulting mixture was stirred at room temperature overnight. All starting material was consumed as assessed by TLC. The reaction was concentrated. The crude material was subjected to silica gel chromatography using a gradient of 0% to 5% methanol in DCM (+ 1% triethylamine) to give 1.33 g of 6 (yield=38%). m/z (ESI-): 588.2 (100%), (calculated for C30H29F3NO8 ^- [MH] ^- 588.6), 1H-NMR: (400 MHz, CDCl3) δ [ppm]=7.94 (d , 1H, NH), 7.39-7.36 (m, 2H, CHaril), 7.29-7.25 (m, 7H, CHaril), 6.82-6.79 (m, 4H, CHaril) , 4.51-4.47 (m, 1H), 4.31-4.24 (m, 2H), 3.77 (s, 6H, 2×DMTr-OMe), 3.66–3.60 ( m, 16H, HNEt ₃ ⁺ ), 3.26-3.25 (m, 2H), 2.97-2.81 (m, 20H, NEt ₃ ), 2.50-2.41 (4H, m) , 1.48–1.45 (m, 26H, HNEt ₃ ⁺ ), 1.24–1.18 (m, 29H, NEt ₃ ).

(S)-DMT-serinol(TFA)-succinate-Icaa-CPG (10)

(S)-DMT-serinol (TFA)-succinate (159 mg, 270 µmol) and HBTU (113 mg, 299 µmol) were dissolved in CH ₃ CN (10 ml). Diisopropylethylamine (DIPEA, 94 μL, 540 μmol) was added to the solution and the mixture was stirred for 2 min, followed by the addition of native amino-Icaa-CPG (500 A, 3 g, amine content: 136 μmol/g). The suspension was shaken gently at room temperature on a wrist shaker for 16 hours, then filtered and washed with DCM and EtOH. The solid support was dried in vacuum for 2 hours. Unreacted amines on the support were capped by stirring with acetic anhydride/lutidine/N-methylimidazole at room temperature. Washing the substrate was repeated as described above. The solid was dried in vacuo to obtain solid support 10 (3 g, loading 26 μmol/g).

GalNAc synthon (9)

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 stages.

Characteristic data correspond to published data.

Oligonucleotide synthesis

All single-stranded oligonucleotides were synthesized according to the reaction conditions described above and in Figures 13 and 14.

All finished single chain products were analyzed by LC-HPLC to confirm their purity. Purity is expressed as % FLP (% Full Length Product), which is the percentage of UV area under the signal of a given product in the UV LC-HPLC analysis curve of the final product. The identity of the corresponding single-stranded products (unmodified, amino-modified precursors, C4XLT/ST23 or C6XLT/ST23 GalNAc conjugated oligonucleotides) was confirmed by LC/MS analysis.

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

Product (11) Calculated mol. weight Installed pier mass (ESI-) %FLP (AC-HPLC) X0385A 6315.0 Yes 6314.6 Yes 91.0% X0385B-prec 6593.1 Yes 6593.1 Yes 87.5% X038BA 6315.0 Yes 6314.6 Yes 91.0% X0386B-prec 6547.1 Yes 6546.9 Yes 87.5% X0383A 6315.0 Yes 6314.5 Yes 91.9% X0383B-prec 6508.8 Yes 6508.6 Yes 84.6% X0371A 6416.1 Yes 6416.1 Yes 88.4% X0371B-prec 6522.0 Yes 6521.8 Yes 91.9% X0320A 6143.8 Yes 6143.7 Yes 94.6% X0320B-prec 6665.0 Yes 6664.8 Yes 87.0% X0477A 6143.8 Yes 6143.4 Yes 85.6% X0477B-prec 6749.3 Yes 6749.2 Yes 83.1% X0027A 6416.1 Yes 6415.8 Yes 92.8% X0027B 7642.0 Yes 7641.8 Yes 88.2%

Synthesis of conjugates 1-3 and reference conjugates 1-2

Conjugated single chains for conjugates 1-2 and reference conjugates 1-2

Conjugation of the GalNAc synthon (9) was accomplished by binding to the amino functional group of the serinol of the corresponding oligonucleotide chain 11 using a coupling reagent to form a peptide bond. Thus, the corresponding amino-modified precursor molecule 11 was dissolved in H ₂ O (500 OD/ml) and DMSO (DMSO/H ₂ O, 2/1, v/v) was added, followed by DIPEA (2.5 % of total volume). In a separate reaction vessel, pre-activation of GalN(Ac4)-C4 acid (9) was performed using 2 eq. (per amino functional group in the amino-modified precursor of oligonucleotide 11) carboxylic acid component with 2 eq. HBTU in the presence of 8 eq. DIPEA in DMSO. After 2 min, preactivated compound 9 was added to the solution of the corresponding amino-modified precursor molecule. After 30 min, the progress of the reaction was monitored using LC/MS or LC-HPLC. After completion of the conjugation reaction, the crude product was precipitated by the addition of 10× iPrOH and 0.1× 2 M NaCl and collected by centrifugation and decantation. To release the acetylated hydroxyl groups in the GalNAc groups, the resulting precipitate was dissolved in 40% MeNH2 (1 ml at 500 OD) and after 15 minutes at room temperature diluted in _H2O (1:10), and finally repurified by anion exchange and gel permeation chromatography and lyophilized to obtain the final product 12.

Table 8: Single-stranded GalNAc-conjugated oligonucleotides

Product (12) Source material (11) Calculated mol. weight Installed pier mass (ESI-) %FLP
(AC-HPLC) X0385B X0385B-prec 7199.8 Yes 7199.3 Yes 93.2% X0386B X0386B-prec 7153.8 Yes 7153.0 Yes 86.2% X0383B X0383B-prec 7115.5 Yes 7115.4 Yes 93.7% X0320B X0320B-prec 7271.7 Yes 7271.7 Yes 90.0% X0371B X0371B-prec 7128.8 Yes 7128.3 Yes 95.0% X0477B X0477B-prec 7356.0 Yes 7355.7 Yes 91.4%

Double chain formation

The double strand was prepared according to the methods described above.

Double chain purity is expressed as % double chain, which is the percentage of UV area under the signal of a given product in the UV curve of the IP-RP-HPLC analysis.

Table 9: Nucleic acid conjugates

Product Source materials % double chain First chain Second chain X0385 X0385A X0385B 97.5% X0386 X0386A X0386B 96.9% X0383 X0383A X0383B 91.9% X0371 X0371A X0371B 97.7% X0027 X0027A X0027B 93.4% X0320 X0320A X0320B 98.6% X0477 X0477A X0477B 96.0%

Sequences

Modification designations for the following sequences:

f stands for 2'-fluoro-2'-deoxyribonucleotide or 2'-fluororibonucleotide (the terms are interchangeable)

m stands for 2'-O-methylribonucleotide

(ps) denotes phosphorothioate bond

Ser(GN) is a building block of GalNAc-C4 attached to a serinol-derived linker group:

where O--- represents a bond between an oxygen atom and, for example, H, a phosphodiester bond or a phosphorothioate bond.

C4XLT is:

C6XLT is:

ST23 is:

The synthesis of phosphoamidite derivatives C4XLT (C4XLT-phos), C6XLT (C6XLT-phos), as well as ST23 (ST23-phos) can be performed as described in WO2017/174657.

Example 10

Equal dose-dependent knockout for siRNA targeting LPA with two distinct GalNAc units conjugated to the second strand compared with a three-antennary GalNAc unit 5' of the second strand in primary cynomolgus macaque hepatocytes.

The miRNAs are modified using alternating 2′-OMe/2′-F and each contains two phosphorothioate (PS) internucleotide linkages at its two 5′ and 3′ internucleotide linkages. In conjugate 19, each serinol-GalNAc block is attached via a PS bond to the 5' and 3' of the second strand. In conjugate 20, the two terminal 5' internal nucleotides of the second strand are phosphodiesters, and a three-antennary GalNAc linker is attached via a PS bond to this end.

Dose-dependent silencing of LPA in primary cynomolgus macaque hepatocytes was assessed 24 h after treatment with 100, 20, 4, 0.8, 0.16, 0.032, and 0.006 nM siRNA. The reference control is design 2, the non-targeting control is called Cte. The transcript ct value for each treatment group was normalized to the constitutive ACTB gene transcript ct value (Δct) and to untreated hepatocytes called ut (ΔΔct).

Data shown in Figure 18

Material and methods:

siRNA

SEQ ID NO: Name Series Chain Subsequence 135 Conjugate 19 X0373 X0373A mA (ps) fU (ps) mA fA mC fU mC fU mG fU mC fC mA fU mU fA mC (ps) fC (ps) mG 136 X0373B 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) 135 Reference conjugate 9 STS20041L6 STS2041A mA (ps) fU (ps) mA fA mC fU mC fU mG fU mC fC mA fU mU fA mC (ps) fC (ps) mG 137 STS2041B ST23 (ps) ST23 (ps) ST23 (ps) C6XLT (ps) fC mG fG mU fA mA fU mG fG mA fC mA fG mA fG mU fU (ps) mA (ps) fU 138 Conjugate Reference 5 (CTR) X0125 X0125A mC (ps) fU (ps) mU fA mC fU mC fU mC fG mC fC mC fA mA fG mC (ps) fG (ps) mA 139 X0125B [(ST23) (ps)] ₃ (C6XLT) (ps) fU mC fG mC fU mU fG mG fG mC fG mA fG mA fG mU fA (ps) mA (ps) fG

Signature

mA, mU, mC, mG 2'-O-methyl RNA fA, fU, fC, fG 2'-deoxy-2'-fluoro-RNA

(ps) - phosphorothioate

(po) - phosphodiester

Primer:

SEQ ID NO: LPA Straight GTGTCCTCGCAACGTCCA 48 Back GACCCCGGGGCTTTG 49 Probe BHQ1-TGGCTGTTTCTGAACAAGCACAATGG-FAM 140 ACTB Straight GCATGGGTCAGAAGGATTCCTAT 54 Back TGTAGAAGGTGTGGTGCCAGATT 55 Probe BHQ1-TCGAGCACGGCATCGTCACCAA-VIC 141

General methods

In vitro experiments

Primary mouse hepatocytes (Thermo Scientific: GIBCO lot #MC798) were thawed and cryopreservation medium was replaced with Williams E medium supplemented with 5% fetal bovine serum (FBS), 1 μM dexamethasone, 2 mM GlutaMax, 1% PenStrep, 4 mg/ml human recombinant insulin, 15 mM Hepes. The cell density was adjusted to 250,000 cells per 1 ml. 100 μl per well of this cell suspension was seeded into collagen-pre-coated 96-well plates. The test article was pre-diluted in the same medium (5-fold concentrated) for each concentration, and 25 μl of this pre-diluted siRNA or medium alone was added to the cells. Cells were cultured at 37°C and 5% CO ₂ . 24 h after treatment, the supernatant was removed, and the cells were washed in cold PBS and 250 μl of RNA lysis buffer S (Stratec) was added. After 15 min of incubation at room temperature, the plates were stored at −80°C until RNA extraction according to the manufacturer's protocol.

TaqMan Assay

For the mTTR&PTEN MultiPlex TaqMan assay, 10 μl of isolated RNA for each treatment group was mixed with 10 μl of a common PCR reaction mixture (TAKYON low Rox) containing 600 nM mTTR primer, 400 nM ApoB primer, and 200 nM each probe, plus 0.5 Euroscript II RT polymerase units with 0.2 units. RNase inhibitor. The TaqMan assay was performed in a 384-well plate with a 10 min RT step at 48°C, a 3 min initial denaturation at 95°C, and 40 cycles of 95°C for 10 s and 60°C for 1 min. The primers contain two of BHQ1, FAM and YY, one at each end of the sequence.

For the TMPRSS6&ApoB MultiPlex TaqMan assay, 10 μl of isolated RNA for each treatment group was mixed with 10 μl of a common PCR reaction mix (TAKYON low Rox) containing 800 nM TMPRSS6 primer, 100 nM ApoB primer, and 200 nM of each probe, plus 0.5 Euroscript II RT polymerase units with 0.2 units. RNase inhibitor. The TaqMan assay was performed in a 384-well plate with a 10 min RT step at 48°C, a 3 min initial denaturation at 95°C, and 40 cycles of 95°C for 10 s and 60°C for 1 min.

In vivo experiments

To compare the in vivo efficacy of different siRNA conjugates, 1 mg/kg siRNA dissolved in PBS was injected subcutaneously into the scapula region of c57BL/6 mice. Cohorts of n=6 were treated with siRNA targeting Aldh2 or Tmprss6 on day 1 and sacrificed at selected time points post-treatment. Liver samples were snap frozen in liquid nitrogen and stored at −80°C until RNA extraction using the InviTrap Spin Tissue RNA Mini Kit (Stratec) according to the manufacturer's instructions. The transcript levels of Aldh2, Tmprss6 and Pten were then quantified as described above.

Stability analysis in tritosomes

To examine RNase stability in the endosomal/lysosomal compartment of liver cells in vitro, siRNA was incubated for 0 h, 4 h, 24 h, or 72 h in Sprague Dawley rat liver tritosomes (Tebu-Bio, catalog number R0610.LT, lot : 1610405, pH: 7.4, 2.827 units/ml). To simulate an acidified environment, tritosomes were mixed in a ratio of 1:10 with a low pH buffer (1.5 M acetic acid, 1.5 M sodium acetate, pH = 4.75). 30 µl of acidified tritosomes. Then, 10 μl of siRNA (20 μM) was mixed and incubated for the indicated time points at 37°C. After incubation, RNA was isolated using Clarity OTX starter kit cartridges (Phenomenex, catalog number KSO-8494) according to the manufacturer's protocol for biological fluids. Lyophilized RNA was reconstituted in 30 μl H ₂ O, mixed with 4× loading buffer, and 5 μl loaded on a 20% TBE-polyacrylamide electrophoresis gel (PAGE) for separation in a qualitative semiquantitative analysis. PAGE was performed at 120 V for 2 h, and RNA was visualized by ethidium bromide staining followed by digital imaging using a Biorad imaging system.

Example 11 - Synthesis of Conjugates 3

Exemplary compounds were synthesized in accordance with the methods described below and methods known to one skilled in the art. Assembly of the oligonucleotide chain and linker building blocks was performed using solid-phase synthesis using phosphoamidite methodology. GalNAc conjugation was accomplished by forming a peptide bond between the GalNAc-carboxylic acid building block and a preassembled and purified oligonucleotide containing the required amount of attached amino-modified linker building blocks.

Synthesis, deprotection, and purification of oligonucleotides were performed according to standard procedures known in the art.

All oligonucleotides were synthesized on an AKTA oligopilot synthesizer using standard phosphoamidite reactions. A commercially available solid support and 2'-O-methyl-RNA phosphoamidites, 2'-fluoro, 2'-deoxy-RNA phosphoamidites (all with standard protection, ChemGenes, LinkTech) and a commercially available 3'-amino TFA modifier were used -amino-C-6-lcaa-CPG 500 Å (Chemgenes), Fmoc-amino-DMT-C-7-CE-phosphoamidite (GlyC3Am), 3'-amino modifier C-3-lcaa-CPG 500 Å (C3Am ), Fmoc-amino-DMT-C-3-CED-phosphoamidite (C3Am) and TFA-amino-C-6-CED-phosphoamidite (C6Am) (Chemgenes), 3'-amino C7 CPG modifier (C7Am) (Glen Research), non-nucleoside TFA amino phosphoamidite (Pip), non-nucleoside TFA amino solid support (PipAm) (AM Chemicals). Per-acetylated galactosamine 8 is available commercially.

Auxiliary reagents were purchased from EMP Biotech. The synthesis was performed using a 0.1 M solution of phosphoamidite in dry acetonitrile, and benzylthiotetrazole (BTT) (0.3 M in acetonitrile) was used as an activator. The binding time was 15 min. The Cap/OX/Cap or Cap/Thio/Cap cycle was used (Cap: Ac ₂ O/NMI/lutidine/acetonitrile, oxidizing agent: 0.1 M I ₂ in pyridine/H ₂ O). Phosphothioates were introduced using a standard commercially available thiolation reagent (EDITH, Link technologies). DMT cleavage was accomplished by treatment with 3% dichloroacetic acid in toluene. After completion of the programmed synthesis cycles, washes with diethylamine (DEA) were performed. All oligonucleotides were synthesized in DMT-off mode.

Attachment of the serinol-derived linker group was accomplished using either base-loaded (S)-DMT-serinol(TFA)-succinate-lcaa-CPG 10 or (S)-DMT-serinol(TFA)phosphoamidite 7 (synthesis was performed as described in Hoevelmann et al. (2016)). Trichantine GalNAc clusters (ST23/C4XLT) were introduced by sequential coupling of the corresponding tripling amidite derivatives (C4XLT-phos) followed by the addition of GalNAc amidite (ST23-phos).

Attachment of amino-modified groups (non-serinol linkers) was accomplished by using either an appropriate commercially available amino-modified CPG building block or amidite.

Single chains were cleaved from CPG by treatment with 40% aq. methylamine. The resulting crude oligonucleotide was purified by ion exchange chromatography (Resource Q, 6 mL, GE Healthcare) on an AKTA Pure HPLC system using a sodium chloride gradient. Product-containing fractions were pooled, desalted on a gel permeation chromatography column (Zetadex, EMP Biotech) and lyophilized.

The individual single strands were dissolved at a concentration of 60 OD/ml in H ₂ O. Both single oligonucleotide solutions were added together to the reaction vessel. Titration was performed to facilitate reaction monitoring. The first chain was added at a 25% excess over the second chain, as determined by UV absorbance at 260 nm. The reaction mixture was heated to 80°C for 5 minutes and then slowly cooled to room temperature. The formation of the double chain was monitored by the formation of ion pairs using reverse phase HPLC. Based on the UV area of the residual single chain, the required amount of the second chain was calculated and added to the reaction mixture. The reaction mixture was again heated to 80°C and slowly cooled to room temperature. This procedure was repeated until less than 10% of the residual single chain was detected.

Synthesis of compounds 2-10

Compounds 2-5 and (S)-DMT-serinol(TFA)-phosphoamidite 7 were synthesized according to published methods (Hoevelmann et al. Chem. Sci., 2016,7, 128-135).

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

Succinic anhydride was added to a solution of 5 in pyridine, followed by DMAP. The resulting mixture was stirred at room temperature overnight. All starting material was consumed as assessed by TLC. The reaction was concentrated. The crude material was subjected to silica gel chromatography using a gradient of 0% to 5% methanol in DCM (+ 1% triethylamine) to give 1.33 g of 6 (yield = 38%). m/z (ESI-): 588.2 (100%), (calculated for C30H29F3NO8 ^- [MH] ^- 588.6), 1H-NMR: (400 MHz, CDCl3) δ [ppm]=7.94 (d , 1H, NH), 7.39-7.36 (m, 2H, CHaril), 7.29-7.25 (m, 7H, CHaril), 6.82-6.79 (m, 4H, CHaril) , 4.51-4.47 (m, 1H), 4.31-4.24 (m, 2H), 3.77 (s, 6H, 2xDMTr-OMe), 3.66-3.60 (m, 16H, HNEt ₃ ⁺ ), 3.26-3.25 (m, 2H), 2.97-2.81 (m, 20H, NEt ₃ ), 2.50-2.41 (4H, m), 1 .48-1.45 (m, 26H, HNEt ₃ ⁺ ), 1.24-1.18 (m, 29H, NEt ₃ ).

(S)-DMT-serinol(TFA)-succinate-Icaa-CPG (10)

(S)-DMT-serinol (TFA)-succinate (159 mg, 270 µmol) and HBTU (113 mg, 299 µmol) were dissolved in CH ₃ CN (10 ml). Diisopropylethylamine (DIPEA, 94 μl, 540 μmol) was added to the solution and the mixture was stirred for 2 minutes, followed by the addition of native amino-Icaa-CPG (500 A, 3 g, amine content: 136 μmol/g). The suspension was shaken gently at room temperature on a wrist shaker for 16 hours, then filtered and washed with DCM and EtOH. The solid support was dried in vacuum for 2 hours. Unreacted amines on the support were capped by stirring with acetic anhydride/lutidine/N-methylimidazole at room temperature. Washing the substrate was repeated as described above. The solid was dried in vacuo to obtain solid support 10 (3 g, loading 26 μmol/g).

GalNAc synthon (9)

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 stages.

Characteristic data correspond to published data.

Oligonucleotide synthesis

All single-stranded oligonucleotides were synthesized according to the reaction conditions described above and in Figures 13 and 14 and are presented in Tables 10 and 11.

All finished single chain products were analyzed by LC-HPLC to confirm their purity. Purity is expressed as % FLP (% Full Length Product), which is the percentage of UV area under the signal of a given product in the UV LC-HPLC analysis curve of the finished product. The identity of the corresponding single-stranded products (unmodified, amino-modified precursors, or GalNAc-conjugated oligonucleotides) was confirmed by LC/MS analysis.

Table 10: Single-stranded unconjugated oligonucleotides

Product (11) Name Calculated mol. weight Installed pier mass (ESI-) %FLP (AC-HPLC) A0002 STS16001A 6943.3 Yes 6943.0 Yes 86.6% A0006 STS16001BL4 8387.5 Yes 8387.5 Yes 94.1% A0114 STS22006A 6143.8 Yes 6143.7 Yes 94.3% A0115 STS22006BL1 7855.1 Yes 7855.1 Yes 92.8% A0122 STS22009A 6260.9 Yes 6260.6 Yes 92.8% A0123 STS22009BL1 7783.0 Yes 7782.9 Yes 87.1% A0130 STS18001A 6259.9 Yes 6259.8 Yes 76.5% A0131 STS18001BL4 7813.2 Yes 7813.1 Yes 74.3% A0220 STS16001B-5'1xNH2 6982.2 Yes 6982.1 Yes 95.7% A0237 STS16001A 6943.3 Yes 6943.3 Yes 95.6% A0244 STS16001BV1 6845.2 Yes 6844.9 Yes 98.2% A0264 STS16001AV4-3'1xNH2 7112.4 Yes 7112.2 Yes 95.4% A0329 STS16001BV6-3'5'1xNH2 7183.3 Yes 7183.2 Yes 88.8% A0560 STS16001A 6943.3 Yes 6943.3 Yes 96.7% A0541 STS16001BV1-3'5'NH2 7151.3 Yes 7151.0 Yes 85.6% A0547 STS16001BV16-3'5'NH2 7119.3 Yes 7119.1 Yes 89.9% A0617 STS16001BV20-3'5'NH2 7087.3Yes 7086.7 Yes 90.1% A0619 STS16001BV1-3'5'2xNH2 7521.3 Yes 7521.3 Yes 93.4% A0680 STS16001A 6943.3 Yes 6942.9 Yes 91.2% A0514 STS22006A 6143.8 Yes 6143.7 Yes 94.6% A0516 STS22009BV11-3'5'NH2 6665.0 Yes 6664.8 Yes 87.0% A0517 STS22009BV11-3'5'NH2 6593.0 Yes 6593.0 Yes 86.0% A0521 STS12009BV1-3'5'NH2 6437.7 Yes 6437.8 Yes 91.1% A0303 STS12209BL4 7665.0 Yes 7664.9 Yes 90.4% A0304 STS12209A 6393.1 Yes 6392.9 Yes 77.6% A0319 STS22009A 6260.9 Yes 6260.5 Yes 86.9% A0353 STS12009A 6416.1 Yes 6416.1 Yes 94.1% A0216 STS17001A 6178.8 Yes 6178.7 Yes 87.2% A0217 STS17001BL6 7937.2 Yes 7937.2 Yes 78.3%

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

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

Product Name 5'-mod. 3'-mod. Calculated pier. weight Installed pier mass (ESI-) %FLP (AC-HPLC) A0561 STS16001BV1-3'5'1xNH2 C6Am GlyC3Am 7267.5 Yes 7267.5 Yes 66.7% A0563 STS16001BV1-3'5'1xNH2 C3Am C3Am 7183.4 Yes 7183.1 Yes 75.1% A0651 STS16001BV1-3'5'1xNH2 C6Am C7Am 7265.6 Yes 7265.2 Yes 99.6% A0653 STS16001BV1-3'5'1xNH2 GlyC3Am GlyC3Am 7299.5 Yes 7299.3 Yes 88.1% A0655 STS16001BV1-3'5'1xNH2 PipAm PipAm 7517.7 Yes 7517.5 Yes 89.8%

Likewise, 3'5'1×NH2 refers to the position (3' and 5' end) and number (1×NH2 each) of free amino groups that are available for conjugation. For example, 3'5'1×NH2 on A0561 means 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 the A0561 chain.

Synthesis of some conjugates according to the present invention and reference conjugates 1-2

Conjugation of the GalNAc synthon (9) was accomplished by binding to the amino functional group of the serinol of the corresponding oligonucleotide chain 11 using a coupling reagent to form a peptide bond. Thus, the corresponding amino-modified precursor molecule 11 was dissolved in H ₂ O (500 OD/ml) and DMSO (DMSO/H ₂ O, 2/1, v/v) was added, followed by DIPEA (2.5 % of total volume). In a separate reaction vessel, pre-activation of GalN(Ac4)-C4 acid (9) was performed using 2 eq. (per amino functional group in the amino-modified precursor of oligonucleotide 11) carboxylic acid component with 2 eq. HBTU in the presence of 8 eq. DIPEA in DMSO. After 2 min, preactivated compound 9 was added to the solution of the corresponding amino-modified precursor molecule. After 30 min, the progress of the reaction was monitored using LC/MS or LC-HPLC. After completion of the conjugation reaction, the crude product was precipitated by the addition of 10× iPrOH and 0.1× 2 M NaCl and collected by centrifugation and decantation. To release the acetylated hydroxyl groups in the GalNAc groups, the resulting precipitate was dissolved in 40% MeNH2 (1 ml at 500 OD) and after 15 minutes at room temperature diluted in _H2O (1:10), and finally repurified by anion exchange and gel permeation chromatography and lyophilized to obtain the final product 12 (Table 12).

Table 12: Single-stranded GalNAc-conjugated oligonucleotides

Product (12) Raw material Name Calculated mol. weight Installed pier mass (ESI-) %FLP (AC-HPLC) A0241 A0220 STS16001BL20 7285.5 Yes 7285.3 Yes 91.8% A0268 A0264 STS16001AV4L33 7415.7 Yes 7415.4 Yes 96.9% A0330 A0329 STS16001BV6L42 7789.8 Yes 7789.8 Yes 95.5% A0544 A0541 STS16001BV1L75 7757.9 Yes 7757.7 Yes 93.3% A0550 A0547 STS16001BV16L42 7725.9 Yes 7725.7 Yes 88.5% A0620 A0617 STS16001BV20L75 7693.91 Yes 7693.2 Yes 90.9% A0622 A0619 STS16001BV1L94 8734.3 Yes 8734.6 Yes 82.9% A0519 A0516 STS22006BV11L42 7271.7 Yes 7271.7 Yes 90.0% A0520 A0517 STS22009BV11L42 7199.6 Yes 7199.7 Yes 92.9% A0522 A0521 STS12009BV1L42 7044.4 Yes 7044.4 Yes 96.0% A0603 A0602 STS20041BV1L42 7280.7 Yes 7280.4 Yes 93.4%

Synthesis of some conjugates according to the present invention

Conjugation of the GalNAc synthon (9) was accomplished by binding to the amino functional group of the corresponding oligonucleotide chain 14 using a coupling reagent to form a peptide bond. Thus, the corresponding amino-modified precursor molecule 14 was dissolved in H ₂ O (500 OD/ml) and DMSO (DMSO/H ₂ O, 2/1, v/v) was added, followed by DIPEA (2.5 % of total volume). In a separate reaction vessel, pre-activation of GalN(Ac4)-C4 acid (9) was performed using 2 eq. (per amino functional group in the amino-modified precursor of oligonucleotide 14) carboxylic acid component with 2 eq. HBTU in the presence of 8 eq. DIPEA in DMSO. After 2 min, preactivated compound 9 was added to the solution of the corresponding amino-modified precursor molecule. After 30 min, the progress of the reaction was monitored using LC/MS or LC-HPLC. After completion of the conjugation reaction, the crude product was precipitated by the addition of 10× iPrOH and 0.1× 2 M NaCl and collected by centrifugation and decantation. To release the acetylated hydroxyl groups in the GalNAc groups, the resulting precipitate was dissolved in 40% MeNH2 (1 ml at 500 OD) and after 15 minutes at room temperature diluted in _H2O (1:10), and finally repurified by anion exchange and gel permeation chromatography and lyophilized to obtain the final product 15 (Table 13).

Table 13: Single-stranded GalNAc-conjugated oligonucleotides

Product (15) Raw material Name Calculated mol. weight Installed pier mass (ESI-) %FLP (AC-HPLC) A0562 A0561 STS16001BV1L87 7874.2 Yes 7874.0 Yes 82.7% A0564 A0563 STS16001BV1L88 7790.0 Yes 7789.4 Yes 90.4% A0652 A0651 STS16001BV1L96 7872.2 Yes 7871.8 Yes 94.6% A0654 A0653 STS16001BV1L97 7906.2 Yes 7905.6 Yes 89.9% A0656 A0655 STS16001BV1L98 8124.3 Yes 8124.0 Yes 93.6%

Double chain formation

Double chains were prepared according to the methods described above.

Double chain purity is expressed as % double chain, which is the percentage of UV area under the signal of a given product in the UV curve of the IP-RP-HPLC analysis (Table 14).

Table 14: Nucleic acid conjugates

Product Source materials Name % double chain First chain Second chain Reference conjugate 1 A0237 A0241 STS16001L20 97.7% Reference conjugate 2 A0268 A0244 STS16001L33 97.8% Reference conjugate 3 A0130 A0131 STS18001L4 96.8% Reference conjugate 4 A0002 A0006 STS16001L4 90.1% Reference conjugate 5 A0216 A0217 STS17001L6 88.4% Conjugate 1 A0268 A0241 STS16001L24 96.0% Conjugate 2 A0237 A0330 STS16001V1L42 98.5% Conjugate 3 A0268 A0330 STS16001V1L43 98.2% Conjugate 4 A0560 A0544 STS16001V1L75 92.5% Conjugate 5 A0560 A0550 STS16001V16L42 95.3% Conjugate 6 A0237 A0620 STS16001V20L75 97.8% Conjugate 7 A0237 A0622 STS16001V1L94 93.7% Conjugate 8 A0680 A0652 STS16001V1L96 98.4% Conjugate 9 A0680 A0654 STS16001V1L97 95.8% Conjugate 10 A0680 A0656 STS16001V1L98 97.6% Conjugate 11 A0560 A0564 STS16001V1L88 95.0% Conjugate 12 A0237 A0562 STS16001V1L87 96.8% Conjugate 13 A0114 A0115 STS22006L1 85.6% Conjugate 14 A0122 A0123 STS22009L1 96.4% Conjugate 15 A0514 A0519 STS22006V11L42 98.6% Conjugate 16 A0319 A0520 STS22009V11L42 97.0% Conjugate 17 A0304 A0303 STS12209L4 93.0% Conjugate 18 A0353 A0522 STS12009V1L42 98.0% Conjugate 19 A0601 A0603 STS20041BL42 97.6%

Sequences

Modification designations for the following sequences:

f stands for 2'-fluoro-2'-deoxyribonucleotide or 2'-fluororibonucleotide (the terms are interchangeable)

m stands for 2'-O-methylribonucleotide

(ps) denotes phosphorothioate bond

FAM=6-carboxyfluorescein

BHQ = Black Hole Quencher 1

YY = Yakima Yellow

Definitions

Ser(GN) is a building block of GalNAc-C4 attached to a serinol-derived linker group:

where O--- represents a bond between an oxygen atom and, for example, H, a phosphodiester bond or a phosphorothioate bond.

GN represents:

C4XLT (also known as ST41) is:

ST23 is:

The synthesis of phosphoamidite derivatives C4XLT (C4XLT-phos) as well as ST23 (ST23-phos) can be performed as described in WO2017/174657.

where G=H (before conjugation) or G=GN (after conjugation).

Conjugate 1

Antisense Strand-STS16001AL33 (SEQ ID NO: 127)

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'

Sense Circuit - STS16001BL20 (SEQ ID NO: 128)

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'

Conjugate 2

Antisense Strand-STS16001A (SEQ ID NO: 129)

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

Sense Circuit - STS16001BV1L42 (SEQ ID NO: 130)

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)

Conjugate 3

Antisense Strand-STS16001AL33 (SEQ ID NO: 127)

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'

Sense Circuit - STS16001BV1L42 (SEQ ID NO: 130)

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 '

Conjugate 4

Antisense Strand-STS16001A (SEQ ID NO: 129)

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

Sense Circuit - STS16001BV1L75 (SEQ ID NO: 142)

5' Ser(GN) 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 Ser(GN) 3'

Conjugate 5

Antisense Strand-STS16001A (SEQ ID NO: 129)

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

Sense Circuit - STS16001BV16L42 (SEQ ID NO: 143)

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

Conjugate 6

Antisense Strand-STS16001A (SEQ ID NO: 129)

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

Sense Circuit - STS16001BV20L75 (SEQ ID NO: 144)

5' Ser(GN) fA mA fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA fU mA fA Ser(GN) 3'

Conjugate 7

Antisense Strand-(SEQ ID NO: 129)

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

Sense Circuit - STS16001BV1L94 (SEQ ID NO: 145)

5' Ser(GN) (ps) 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) (ps) Ser(GN) 3'

Conjugate 8

Antisense Strand-STS16001A (SEQ ID NO: 129)

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'

Sense Circuit - STS16001V1BL96 (SEQ ID NO: 146)

5' C6Am(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) C7Am(GN) 3 '

Conjugate 9

Antisense Strand-STS16001A (SEQ ID NO: 129)

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'

Sense Circuit - STS16001V1BL97 (SEQ ID NO: 147)

5' GlyC3Am(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) GlyC3Am(GN) 3 '

Conjugate 10

Antisense Strand-STS16001A (SEQ ID NO: 129)

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'

Semantic Chain (SEQ ID NO: 148)

5' PipAm(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) PipAm(GN) 3 '

Conjugate 11

Antisense Strand-STS16001A (SEQ ID NO: 129)

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'

Sense Circuit - STS16001V1BL88 (SEQ ID NO: 149)

5' C3Am(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) C3Am(GN) 3 '

Conjugate 12

Antisense Strand-STS16001A (SEQ ID NO: 129)

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'

Sense Circuit - STS16001V1BL87 (SEQ ID NO: 150)

5' C6Am(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) GlyC3Am(GN) 3 '

Conjugate 15

Antisense Strand (SEQ ID NO: 151)

mU (ps) fC (ps) mU fU mC fU mU fA mA fA mC fU mG fA mG fU mU (ps) fU (ps) mC

Semantic Chain (SEQ ID NO: 152)

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)

Conjugate 16

Antisense Strand (SEQ ID NO: 153)

mA (ps) fU (ps) mG fU mA fG mC fC mG fA mG fG mA fU mC fU mU (ps) fC (ps) mU

Semantic Chain (SEQ ID NO: 154)

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)

Conjugate 18

Antisense Strand (SEQ ID NO: 155)

mA (ps) fA (ps) mC fC mA fG mA fA mG fA mA fG mC fA mG fG mU (ps) fG (ps) mA

Semantic Chain (SEQ ID NO: 156)

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)

Conjugate 19

Antisense Strand (SEQ ID NO: 135)

mA (ps) fU (ps) mA fA mC fU mC fU mG fU mC fC mA fU mU fA mC (ps) fC (ps) mG

Semantic Chain (SEQ ID NO: 136)

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)

Reference conjugate 1

Antisense Strand-STS16001A (SEQ ID NO: 129)

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

Sense Circuit - STS16001BL20 (SEQ ID NO: 128)

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

Reference conjugate 2

Antisense Strand-STS16001AL33 (SEQ ID NO: 127)

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)

Sense Circuit - STS16001BV1 (SEQ ID NO: 157)

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

Reference conjugate 3 - “Luc”

Antisense Strand-STS18001A (A0130, SEQ ID NO: 132)

mU (ps) fC (ps) mG fA mA fG mU fA mU fU mC fC mG fC mG fU mA (ps) fC (ps) mG

Sense Circuit - STS18001BL4 (A0131, SEQ ID NO: 133)

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

Reference conjugate 4

Antisense Strand-STS16001AL33 (SEQ ID NO: 127)

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

Sense Circuit - STS16001BL4 (SEQ ID NO: 134)

5'[(ST23) (ps)] ₃ C4XLT(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

Reference conjugate 5 - “Ctr”

Antisense Strand (SEQ ID NO: 138)

mC (ps) fU (ps) mU fA mC fU mC fU mC fG mC fC mC fA mA fG mC (ps) fG (ps) mA

Semantic Chain (SEQ ID NO: 139)

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

Reference conjugate 6

Antisense Strand (SEQ ID NO: 151)

mU (ps) fC (ps) mU fU mC fU mU fA mA fA mC fU mG fA mG fU mU (ps) fU (ps) mC

Semantic Chain (SEQ ID NO: 158)

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

Reference conjugate 7

Antisense Strand (SEQ ID NO: 153)

mA (ps) fU (ps) mG fU mA fG mC fC mG fA mG fG mA fU mC fU mU (ps) fC (ps) mU

Semantic Chain (SEQ ID NO: 159)

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

Reference conjugate 8

Antisense Strand (SEQ ID NO: 160)

mU (ps) fA (ps) mC fC mA fG mA fA mG fA mA fG mC fA mG fG mU (ps) fG (ps) mA

Semantic Chain (SEQ ID NO: 161)

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

Reference conjugate 9

Antisense Strand (SEQ ID NO: 135)

mA (ps) fU (ps) mA fA mC fU mC fU mG fU mC fC mA fU mU fA mC (ps) fC (ps) mG

Semantic Chain (SEQ ID NO: 162)

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

Example 12 - Determination of TTR silencing in vitro for various GalNAc TTR siRNA conjugates

Conjugates 1-3

Primary mouse hepatocytes were seeded in collagen precoated 96-well plates (Thermo Fisher Scientific, catalog number 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 RNA was extracted using InviTrap® RNA Cell HTS 96/C24×96 preparative kits (Stratec, catalog number 7061300400) according to the manufacturer's protocol. TTR and constitutive gene (PtenII) mRNA transcript levels were quantified by TaqMan assay.

Expression of the target gene in primary mouse hepatocytes 24 hours after treatment with 0.01 nM, 0.1 nM, 0.5 nM, 1 nM and 10 nM of the conjugates of the present invention, conjugates 1-3, showed that the expression of the target gene decreases with increasing conjugate dose compared to negative controls (see bar “UT” and reference conjugate 3), as shown in Figure 15. This indicates that the first strand binds to the target gene, which reduces gene expression. Figure 15 also shows the target gene expression levels for reference conjugates 1 and 2, which act as comparison conjugates. As can be seen from a comparison of the data presented in Figure 15A and 15C, as well as 15B and 15C, the conjugates according to the present invention (conjugates 1-3) reduce the expression of the target gene compared to reference conjugates 1 and 2. The most effective conjugate at 0.01 nM appears to be conjugate 2. The most effective conjugate at 0.1 nM, 0.5 nM, 1 nM and 10 nM appears to be conjugate 3.

Conjugates 4-7

The method described above under the heading “In vitro experiments” in the “General methods” section was used.

Expression of the target gene in primary murine hepatocytes 24 hours after treatment with 0.01 nM, 0.1 nM, 0.5 nM, 1 nM and 10 nM of the conjugates of the present invention, conjugates 4-7, showed that the expression of the target gene decreases with increasing conjugate dose compared to negative controls (see bar "UT" and Luc [reference conjugate 3]), as shown in Figure 31. This indicates that the first strand binds to the target gene, reducing gene expression.

In vitro data indicate that in the case of one or two serinol-derived linker groups provided at the 5' and 3' ends of the sense strand in conjugates 4-7, the number of phosphorothioate (PS) linkages between the terminal nucleotide and linker, and/or between the three terminal nucleotides in the sense strand can be changed while maintaining the effectiveness of reducing target gene expression.

Conjugates 8-12 and 19

The method described above under the heading “In vitro experiments” in the “General methods” section was used.

Expression of the target gene in primary murine hepatocytes 24 hours after treatment with 0.01 nM, 0.1 nM, 0.5 nM, 1 nM and 10 nM of the conjugates of the present invention, conjugates 8-12, showed that the expression of the target gene decreases with increasing conjugate dose compared to negative controls (see bar "UT" and Luc [reference conjugate 3]), as shown in Figure 32. This indicates that the first strand binds to the target gene, which reduces gene expression . In particular, conjugates 8, 9, 10, and 11 appear to be comparable to or superior to conjugate 2, which was previously shown to be the most effective conjugate at 0.01 nM.

Conjugate 19 was also shown to reduce target gene expression compared to negative controls (see bar "UT" and Ctr, which is a non-targeting siRNA and also called reference conjugate 5), as shown in Figure 33. This indicates that that the first strand binds to the target gene, which reduces gene expression.

In vitro data for conjugates 8-12 and 19 indicate that a number of structurally distinct linkers that are conjugated at both ends of the sense strand are effective in reducing target gene expression. Conjugates 8-12 and 19 reduce target gene expression more effectively than "Luc", which is reference conjugate 3 (for conjugates 8-12), "Ctr", which is reference conjugate 5 (for conjugate 19), and raw control.

Example 13 - Temporal dynamics of serum Ttr, Aldh2 and Tmprss6 in mice in vivo

Conjugates 1-3

c57BL/6 mice were treated by subcutaneous administration of 1 mg/kg siRNA conjugates on day 0. Serum samples were collected on days 7, 14, and 27 by collecting blood from the orbital sinus and stored at −20°C until analysis. Serum TTR quantification was performed using a mouse prealbumin ELISA (ALPCO, 41-PALMS/lot 22, 2008003B) according to the manufacturer's protocol (sample dilution 1:8000 or 1:800).

Results for the time course of serum TTR in n=4 c57BL/6 mice on days 7, 14 and 27 post-treatment with 1 mg/kg subcutaneous administration of conjugates 1-3, reference conjugates 1, 2 and 4 and sham-treated individuals ( PBS) are shown in Figure 16. The data in Figure 16 indicate that 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, in particular, 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. Thus, it can be expected that the dosage level of conjugate 2 could be approximately three times lower to achieve a similar initial shutdown. and will also result in longer shutdown times compared to reference conjugate 4.

More specifically, in wild-type mice, conjugate 2 resulted in a 3-fold decrease in serum target protein levels on day seven and a 4-fold decrease in serum target protein levels on day 27 compared to the reference conjugate 4 at an equimolar dose. In addition, conjugate 2 resulted in an 85% reduction in serum target protein levels at day 27 after a single injection, compared to a 36% reduction with an equimolar amount of reference conjugate 4.

Conjugates 15-18

The method described above under the heading “In Vivo Experiments” in the “General Methods” section was used.

Results for the time course of serum Aldh2 in cohorts of n=6 c57BL/6 mice on days 14, 28 and 42 after treatment with 1 mg/kg subcutaneous administration of conjugates 15 and 16, reference conjugates 6 and 7, and sham-treated individuals ( PBS) are shown in Figures 34 and 35. The data in Figures 34 and 35 indicate that 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.

Results for the time course of serum Tmprss6 in n=6 c57BL/6 mouse cohorts on days 14, 28 and 42 post-treatment with 1 mg/kg SC of conjugate 18, reference conjugate 8 and sham-treated (PBS) individuals are shown in Figure 36. The data in Figure 36 indicate that the conjugates of the present invention are particularly effective in reducing target gene expression compared to the negative control (PBS) and reference conjugate 8.

Overall, the data obtained in vivo demonstrate that various exemplary linkers that are conjugated at both ends of the second strand are effective in reducing target gene expression in vivo. The location of the linker improves the performance of the conjugates in vivo compared to the control three-antennary GalNAc linker at the 5' end of the second strand (reference conjugates 6, 7 and 8).

Example 14 - Serum Stability Studies

The method described above under the heading “Tritosome Stability Assay” in the “General Methods” section was used.

Figure 37 shows the results of serum stability studies for conjugates 2, 4, 5, 6 and 7. Figure 38 shows the serum stability of conjugates 2, 8, 9, 10, 11 and 12.

All tested conjugates according to the present invention are more stable in serum compared to the control.

Each of all conjugates tested contains one GalNAc linker block at the 5' end and another at the 3' end of the second strand. The siRNAs are modified using alternating 2′-OMe/2′-F and each contain two phosphorothioate (PS) internucleotide linkages at their 5′ and 3′ terminal two internucleotide linkages unless otherwise noted.

In conjugate 4, the serinol-GalNAc blocks are attached via a phosphodiester bond. In conjugate 5, the serinol-GalNAc blocks are conjugated by PS, while all internucleotide bonds in the second strand are phosphodiester. In conjugate 6, the second strand does not contain PS. In conjugate 7, two serinol-GalNAc units are attached to each end of the second strand and to each other via PS bonds at their respective ends. In conjugate 8, a C6-amino modifier at the 5' and a C7-amino modifier at the 3' end of the second strand were used to attach the ligand. In conjugate 9, Gly-C3-amino modifiers were used as linkers for conjugation to both ends of the second chain, piperidyl-amino modifiers were used in conjugate 10, C3-amino modifiers were used in conjugate 11, and serinol-GalNAc blocks were used in conjugate 2 . In conjugate 2, both terminal internucleotide bonds as well as the nucleotide-serinol bonds are PS. In conjugate 12, a C6 amino modifier at the 5' and a GlyC3 amino modifier at the 3' end of the second strand were used to attach the ligand. “Ut” denotes the untreated sample to which the other samples were normalized. “Luc” denotes the luciferase-targeting siRNA (reference conjugate 3) that was used as a non-targeting control and which does not reduce target mRNA levels.

The data shows that in the case of a serinol-derived linker group 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, maintaining stability in serum.

Example 15

Silencing of LPA mRNA in primary human hepatocytes by various indicated L6-GalNAc-conjugated siRNAs in primary human hepatocytes after receptor-mediated delivery.

Table 15: Additional conjugated nucleic acid sequences tested for inhibition of LPA mRNA expression. The sequences and the modification profile used are indicated.

SEQ ID NO: Ident. siRNA Chain Subsequence Modifications 63 LPA-1301 first chain 5'uuaacucuguccaauuaccg 3' 5162717181736152738 64 second chain 5'cgguaauggacagaguuaa 3' 3845261846364645162 65 LPA-1302 first chain 5'uuaacucuguccaauuaccc 3' 5162717181736152737 66 second chain 5'ggguaauggacagaguuaa 3' 4845261846364645162 67 LPA-1303 first chain 5'uuaacucucucccaauuaccu 3' 5162717181736152735 68 second chain 5'agguaauggacagaguuaa 3' 2845261846364645162 69 LPA-1304 first chain 5'auaacucucucccaauuaccc 3' 6162717181736152737 70 second chain 5'ggguaauggacagaguuau 3' 4845261846364645161 71 LPA-1305 first chain 5'auaacucucucccaauuaccu 3' 6162717181736152735 72 second chain 5'agguaauggacagaguuau 3' 2845261846364645161 73 LPA-1306 first chain 5'uuaacucuguccaauuacca 3' 5162717181736152736 74 second chain 5'ugguaauggacagaguuaa 3' 1845261846364645162

Table 15: Nucleotide modifications are indicated 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.

Primary human hepatocytes (ThermoFisher) were seeded onto collagen-coated 96-well plates at 30,000 cells per well (96-well format). GalNAc-L6-conjugated siRNAs were added at the indicated concentrations immediately after cell seeding. 24 hours after siRNA treatment, total RNA was isolated using the InviTrap RNA cell HTS kit (Stratec) for 96-well plate. LPA mRNA expression levels were determined by qRT-PCR against APOB mRNA as the constitutive gene transcript. Values were normalized to LPA mRNA expression in untreated cells. The means and standard deviations of normalized triplicate values are shown in Figure 39. IC ₅₀ values and maximum inhibition were estimated by curve fitting using four-parameter nonlinear regression.

Summary of the invention

1. A nucleic acid for inhibiting the expression of LPA in a cell, comprising at least one duplex region that contains at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said first strand is at least is at least partially complementary to at least a portion of the RNA transcribed from the LPA gene, wherein said first strand contains a nucleotide sequence selected from the following sequences: 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.

2. The nucleic acid of claim 1, wherein said second strand contains the nucleotide sequence 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.

3. A nucleic acid according to claim 1 or claim 2, wherein said first strand contains the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 5.

4. The nucleic acid according to any one of statements 1-3, wherein said second strand contains the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 6.

5. The nucleic acid as claimed in any one of claims 1-4, wherein each of said first strand and/or said second strand is 17-35 nucleotides in length.

6. The nucleic acid according to any of claims 1-5, wherein said at least one duplex region consists of 19-25 consecutive nucleotide base pairs.

7. Nucleic acid according to any of the preceding statements, which

a) has blunt ends at both ends; or

b) has a sticky end on one end and a blunt end on the other; or

c) has a sticky end on both ends.

8. A nucleic acid as claimed in any of the preceding claims, wherein one or more nucleotides on said first and/or second strand are modified to form modified nucleotides.

9. A nucleic acid as defined in Claim 8, in which one or more of the nucleotides in the odd positions of the first strand are modified.

10. A nucleic acid as claimed in Claim 9, wherein one or more of the nucleotides at the even-numbered positions of the first strand are modified with at least a second modification, wherein said at least second modification is different from the modification in Claim 9.

11. The nucleic acid of claim 10, wherein at least one of the one or more modified nucleotides at even positions is adjacent to at least one of the one or more modified nucleotides at odd positions.

12. The nucleic acid according to any of statements 9-11, in which a plurality of nucleotides at odd positions are modified.

13. Nucleic acid according to statement 10-12, in which a plurality of nucleotides at even-numbered positions are modified using a second modification.

14. The nucleic acid according to any one of statements 8-13, wherein said first strand contains contiguous nucleotides that are modified using a common modification.

15. The nucleic acid of any one of statements 9-14, wherein said first strand contains contiguous nucleotides that are modified using a second modification that is different from the modification of statement 9.

16. The nucleic acid of any one of claims 9 to 15, wherein one or more of the nucleotides at the odd positions of the second strand are modified using a modification that is different from the modification of claim 9.

17. The nucleic acid of any one of claims 9-15, wherein one or more of the nucleotides at the even-numbered positions of the second strand are modified using the modification of claim 9.

18. The nucleic acid of claim 16 or 17, wherein at least one of the one or more modified nucleotides at the even positions of the second strand is adjacent to one or more modified odd nucleotides of the second strand.

19. The nucleic acid of any one of claims 16-18, wherein the plurality of nucleotides at odd positions of the second strand are modified using a general modification.

20. The nucleic acid of any one of claims 16-19, wherein the plurality of nucleotides at even-numbered positions are modified using the modification of claim 9.

21. The nucleic acid of any one of claims 16-20, wherein the plurality of nucleotides at odd positions on the second strand are modified using a second modification, wherein said second modification is different from the modification of claim 9.

22. The nucleic acid according to any one of statements 16-21, wherein said second strand contains contiguous nucleotides that are modified using a common modification.

23. The nucleic acid of any one of statements 16-22, wherein said second strand contains contiguous nucleotides that are modified using a second modification that is different from the modification of statement 9.

24. The nucleic acid as claimed in any one of claims 8-23, wherein each of the nucleotides at odd positions in the first strand and each of the nucleotides at even positions in the second strand are modified using a general modification.

25. The nucleic acid of claim 24, wherein each of the nucleotides at even positions in the first strand is modified using a second modification, and each of the nucleotides at odd positions in the second strand is modified using a second modification.

26. The nucleic acid of any one of claims 8-25, wherein the modified first strand nucleotides are shifted by at least one nucleotide relative to the unmodified or otherwise modified second strand nucleotides.

27. The nucleic acid according to any of claims 8-26, wherein said modification and/or modifications are individually and independently selected from the group consisting of a 3'-terminal deoxythymine, a 2'-O-methyl, a 2'-deoxy modification, 2'-amino modifications, 2'-alkyl modifications, morpholino modifications, phosphoamidate modifications, 5'-phosphothioate modifications, modifications with 5'-phosphate or 5'-phosphate mimic, and modifications with cholesterol derivatives or dodecanoic acid bisdecylamide group .

28. The nucleic acid according to any one of statements 8-27, wherein said modification is any of a closed nucleotide, a nucleotide deprived of a nitrogenous base, or a nucleotide containing a non-natural base.

29. The nucleic acid of any one of claims 8-28, wherein at least one modification is 2'-O-methyl.

30. The nucleic acid of any one of claims 8-29, wherein at least one modification is 2'-F.

31. A nucleic acid for inhibiting LPA expression in a cell, comprising at least one duplex region that contains at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said first strand is at least is at least partially complementary to at least a portion of the RNA transcribed from the LPA gene, wherein said first strand contains a nucleotide sequence selected from the following sequences: 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 first strand nucleotides are modified using the first modification on the odd numbered ones nucleotides and modified using a second modification on even nucleotides, and the nucleotides of the second strand are modified using a third modification on even nucleotides and modified using a fourth modification on odd nucleotides, wherein at least said first modification is different from said second modification, and said third the modification is different from the specified fourth modification.

32. A nucleic acid as claimed in Claim 31, wherein the second sequence comprises the nucleotide sequence 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.

33. The nucleic acid of claim 31 or 32, wherein said fourth modification and said second modification are the same.

34. The nucleic acid according to any one of statements 31-33, in which the specified first modification and the specified third modification are the same.

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

36. The nucleic acid as claimed in any one of claims 31-35, wherein said first strand contains the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 5, and said second strand contains the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 6

37. Nucleic acid according to any of statements 31-36, containing the sequence and modifications presented below:

SEQ ID NO: 5 5' auaacucuguccaauuacca 3' 6162717181736152736 SEQ ID NO: 6 5' uggguaauggacagaguuau 3' 1845261846364645161 SEQ ID NO: 9 5' auaacucuguccaauuaccg 3' 6162717181736152738 SEQ ID NO: 10 5' cgguaauggacagaguuau 3' 3845261846364645161

where specific modifications are indicated 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.

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

39. A nucleic acid according to any one of statements 1-38, containing a phosphorothioate bond between one, two or three terminal 3'-nucleotides and/or 5'-nucleotides of the first and/or second strands.

40. Nucleic acid according to any of statements 1-39, containing two phosphorothioate bonds between each of the three terminal 3' and between each of the three terminal 5' nucleotides on the first strand and two phosphorothioate bonds between the three terminal nucleotides of the 3' end of the second chains.

41. A nucleic acid for inhibiting LPA expression in a cell, comprising at least one duplex region that contains at least a portion of a first chain and at least a portion of a second chain that is at least partially complementary to the first chain, wherein said first chain is at least is at least partially complementary to at least a portion of the RNA transcribed from the LPA gene, wherein said first strand contains a nucleotide sequence selected from the following sequences: 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 said nucleic acid is conjugated to a ligand.

42. Nucleic acid according to statement 41, in which the specified ligand contains (i) one or more groups of N-acetylgalactosamine (GalNac) and their derivatives, and (ii) a linker, and with the help of the specified linker GalNac groups are conjugated with the nucleic acid according to statement 41 .

43. The nucleic acid of any one of claims 41 or 42, wherein the linker may have a divalent or trivalent or tetravalent branched structure.

44. Nucleic acid according to any of statements 41-43, in which the specified nucleotides are modified according to any of the preceding statements.

45. Nucleic acid according to any of the preceding statements, which is conjugated to a ligand containing formula I:

[SX ¹ -PX ² ] ₃ -AX ³ -, (I)

wherein:

S is a saccharide, said saccharide being N-acetylgalactosamine;

X ¹ represents C ₃ -C ₆ alkylene or (-CH ₂ -CH ₂ -O) _m (-CH ₂ ) ₂ -, where m is 1, 2 or 3;

P is phosphate or modified phosphate (preferably thiophosphate);

X ² represents an alkylene or alkylene ether of the formula (-CH ₂ ) _n -O-CH ₂ -, where n=1-6;

A represents a branching block;

X ³ is a bridge block;

wherein the nucleic acid of any one of claims 1-40 is conjugated to X ³ via a phosphate or modified phosphate (preferably a thiophosphate).

46. Conjugated nucleic acid having one of the following structures

where Z represents a nucleic acid according to any of statements 1-40.

47. Nucleic acid according to any of statements 1-40, which is conjugated to a ligand of the following structure

48. A nucleic acid or conjugated nucleic acid according to any of the preceding statements, wherein said duplex contains separate strands.

49. A nucleic acid or conjugated nucleic acid according to any of the preceding statements, wherein said duplex comprises a single strand comprising a first strand and a second strand.

50. A composition comprising a nucleic acid or conjugated nucleic acid according to any of the preceding claims and a physiologically acceptable excipient.

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

52. The use of a nucleic acid or conjugated nucleic acid according to any of the foregoing claims in the manufacture of a medicinal product for the prevention or treatment of a disease, disorder or syndrome.

53. A method of preventing or treating a disease, disorder or syndrome, comprising administering a composition comprising a nucleic acid or conjugated nucleic acid according to any of the foregoing claims to an individual in need of treatment.

54. The method of claim 53, wherein said nucleic acid or conjugated nucleic acid is administered to a subject subcutaneously, intravenously or by any other route of administration such as oral, rectal or intraperitoneal.

55. The use or method of claims 52-54, wherein said disease or pathology is cardiovascular disease, stroke, atherosclerosis, thrombosis or cardiovascular disease such as coronary artery disease or aortic stenosis, and any other disease or pathology , associated with increased levels of Lp(a) containing particles.

56. The use or method of claim 55, wherein said cardiovascular disease is stroke, atherosclerosis, thrombosis, coronary artery disease or aortic stenosis and any other disease or pathology associated with elevated levels of Lp(a) containing particles.

57. A method for producing a nucleic acid or conjugated nucleic acid according to any of statements 1-49.

58. A nucleic acid for inhibiting LPA expression, comprising at least one duplex region that contains at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said first strand is at least partially is complementary to at least a portion of the RNA transcribed from the LPA gene, wherein LPA expression is reduced to levels that are at least 15% lower than expression levels observed under similar test conditions, but in the absence of the specified nucleic acid or conjugated nucleic acid or in the presence non-inhibitory control.

59. A nucleic acid for inhibiting LPA expression, comprising at least one duplex region that contains at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said first strand is at least partially is complementary to at least a portion of the RNA transcribed from the LPA gene, wherein said first strand contains a nucleotide sequence selected from the following sequences: 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, where LPA expression is reduced to levels that are at least 15% below expression levels observed under similar test conditions, but in the absence of the indicated nucleic acid or conjugated nucleic acid or in the presence of a non-inhibitory control.

60. A nucleic acid for inhibiting LPA expression in a cell, comprising at least one duplex region that contains at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said first strand is at least is at least partially complementary to at least a portion of the RNA transcribed from the LPA gene, wherein said first strand contains a nucleotide sequence selected from the following sequences: SEQ ID NO: 63, 65, 67 and 73, wherein said first RNA strand contains a terminal 5' -(E)-vinylphosphonate nucleotide.

Other provisions of the present invention include:

A conjugate for inhibiting expression of the LPA gene in a cell, wherein said conjugate comprises a nucleic acid portion and a ligand portion, wherein said nucleic acid portion comprises at least one duplex region that contains at least a portion of a first strand of RNA and at least a portion of a second strand of RNA , which is at least partially complementary to a first strand, wherein said first strand is at least partially complementary to at least a portion of the RNA transcribed from said LPA gene, wherein said ligand portions comprise a serinol-derived linker group and a targeting ligand for targeting conditions in vivo on cells and conjugated exclusively to the 3' and/or 5' ends of one or both RNA strands, wherein the 5' end of the first RNA strand is not conjugated, and:

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

both the second RNA strand and the first RNA strand are conjugated at the 3' ends to the targeting ligand, and the 5' end of the second RNA strand is not conjugated,

or

a conjugate for inhibiting expression of the LPA gene in a cell, wherein said conjugate comprises a nucleic acid portion and a ligand portion, wherein said nucleic acid portion comprises at least one duplex region that comprises at least a portion of a first strand of RNA and at least a portion of a second strand of RNA , which is at least partially complementary to a first strand, wherein said first strand is at least partially complementary to at least a portion of the RNA transcribed from said LPA gene, wherein said ligand portions comprise a linker group and a targeting ligand for in vivo targeting to cells and conjugated exclusively to the 3' and/or 5' ends of one or both RNA strands, wherein the 5' end of the first RNA strand is not conjugated, and:

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

both the second strand RNA and the first RNA strand are conjugated at the 3' ends to the targeting ligand, and the 5' end of the second RNA strand is not conjugated, optionally

wherein said linker group is a serinol-derived linker group or one of the other types of linkers described herein.

A conjugate at position 1, in which said second RNA strand is conjugated at the 5' end to a targeting ligand, said second RNA strand is also conjugated at the 3' end to a targeting ligand, and the 3' end of said first RNA strand is not conjugated.

A conjugate at position 1, in which said second RNA strand is conjugated at the 5' end to a targeting ligand, said first RNA strand is conjugated at the 3' end to a targeting ligand, and the 3' end of said second RNA strand is not conjugated.

A conjugate at position 1, in which said second strand RNA is conjugated at the 5' end to a targeting ligand, and both said second strand RNA and first strand RNA are also conjugated at the 3' end to the targeting ligand.

A conjugate at position 1, in which both the second RNA strand and the first RNA strand are conjugated at the 3' ends to a targeting ligand, and the 5' end of the second RNA strand is not conjugated.

The conjugate of any one of positions 1-5, wherein said ligands are monomeric ligands.

A conjugate at any of positions 1-6, wherein said ligands are selected from GalNAc and galactose groups, in particular GalNAc groups.

A conjugate at any one of positions 1-7, wherein said conjugated RNA strands are conjugated to a targeting ligand via a linker group derived from serinol, including an additional linker, wherein said additional linker is or contains a saturated, straight or branched C _1-15 alkyl chain, wherein optionally one or more carbon atoms (e.g. 1, 2 or 3 carbon atoms are acceptable when 1 or 2, in particular 1) is replaced by heteroatom(s) selected from O, N, S(O) _p , where p is 0, 1 or 2 (for example, a CH ₂ group is substituted with O or NH, or S, or SO ₂ , or a –CH ₃ group at the end of a chain or on a branch is substituted with OH or NH ₂ ), and the specified chain is optional substituted with one or more oxo groups (eg 1-3, eg 1 group).

A conjugate at position 8, wherein said additional linker contains a saturated straight-chain C _1-15 alkyl chain having one or more carbon atoms (e.g. 1, 2 or 3 carbon atoms, suitable when 1 or 2, in particular 1 ) is replaced by an oxygen atom.

A conjugate at position 9, in which said additional linker contains a PEG chain.

A conjugate at position 8, in which said additional linker contains a saturated straight-chain C _1-15 alkyl chain.

A conjugate at position 11, in which said additional linker contains a saturated straight-chain C _1-6 alkyl chain.

A conjugate at position 12, wherein said additional linker contains a saturated straight-chain C ₄ or C ₆ alkyl chain, for example a C ₄ alkyl chain.

A conjugate of position 1 or statement 1, wherein said first RNA strand is a compound of formula (XV):

in which b is 0 or 1; And

said second RNA strand is a compound of formula (XVI):

in which c and d are independently equal to 0 or 1;

wherein:

Z ₁ and Z ₂ represent RNA portions of said first and second RNA strands, respectively;

Y represents O or S;

R ₁ represents H or methyl;

n is 0, 1, 2 or 3; And

L is the same or different in formulas (XV) and (XVI) and is selected from the group consisting of:

-(CH ₂ ) _q -, where q=2-12;

-(CH ₂ ) _r -C(O)-, where r=2-12;

-(CH ₂ -CH ₂ -O) _s -CH ₂ -C(O)-, where s=1-5;

-(CH ₂ ) _t -CO-NH-(CH ₂ ) _t -NH-C(O)-, where t is independently 1-5;

-(CH ₂ ) _u -CO-NH-(CH ₂ ) _u -C(O)-, where u is independently 1-5; And

-(CH ₂ ) _v -NH-C(O)-, where v is 2-12; And

wherein the terminal C(O) (if present) is attached to the NH group;

and at the same time b + c + d is equal to 2 or 3.

Conjugate at position 14 in which b is 0, c is 1 and d is 1.

A conjugate at position 14 in which b is 1, c is 0 and d is 1.

A conjugate at position 14 in which b is 1, c is 1 and d is 0.

A conjugate at position 14 in which b is 1, c is 1 and d is 1.

The conjugate at any one of positions 14-18, wherein Y is O.

The conjugate at any one of positions 14-18, wherein Y is S.

The conjugate according to any of positions 14-20, in which R ₁ represents H.

The conjugate at any of positions 14-20, in which R ₁ represents methyl.

Conjugate at any of positions 14-22, in which n is 0.

The conjugate at any one of positions 14-23, in which L represents -(CH ₂ ) _r -C(O)-, where r=2-12.

Conjugate at position 24, in which r=2-6.

Conjugate at position 25, in which r=4 or 6, for example, 4.

A conjugate according to any of the preceding provisions with any feature or combination of features disclosed in this application.

SEQ ID NO Name Sequence (5'-3') Unmodified sequence analogue (5'-3') 1 LPA-1014 first chain UCUAUAACAAUAAGGGGC UCUAUAACAAUAAGGGGC 2 LPA-1014 second circuit GCCCCUUAUUGUUAUACGA GCCCCUUAUUGUUAUACGA 3 LPA-1024 first chain GAUAACUCUGUCCAUUACC GAUAACUCUGUCCAUUACC 4 LPA-1024 second circuit GGUAAUGGACAGAGUUAUC GGUAAUGGACAGAGUUAUC 5 LPA-1038 first chain AUAACUCUGUCCAUUACCA AUAACUCUGUCCAUUACCA 6 LPA-1038 second circuit UGGUAAUGGACAGAGUUAU UGGUAAUGGACAGAGUUAU 7 LPA-1040 first chain UAACUCUGUCCAUUACCGU UAACUCUGUCCAUUACCGU 8 LPA-1040 second circuit ACGGUAAUGGACAGAGUUA ACGGUAAUGGACAGAGUUA 9 LPA-1041 first chain AUAACUCUGUCCAUUACCG AUAACUCUGUCCAUUACCG 10 LPA-1041 second circuit CGGUAAUGGACAGAGUUAU CGGUAAUGGACAGAGUUAU eleven LPA-1055 first chain AGAAUGUGCCUCGAUAACU AGAAUGUGCCUCGAUAACU 12 LPA-1055 second circuit AGUUAUCGAGGCACAUUCU AGUUAUCGAGGCACAUUCU 13 LPA-1057 first chain AUAACUCUGUCCAUCACCA AUAACUCUGUCCAUCACCA 14 LPA-1057 second circuit UGGUGAUGGACAGAGUUAU UGGUGAUGGACAGAGUUAU 15 LPA-1058 first chain AUAACUCUGUCCAUCACCU AUAACUCUGUCCAUCACCU 16 LPA-1058 second circuit AGGUGAUGGACAGAGUUAU AGGUGAUGGACAGAGUUAU 17 LPA-1061 first chain UAACUCUGUCCAUUACCAU UAACUCUGUCCAUUACCAU 18 LPA-1061 second circuit AUGGUAAUGGACAGAGUUA AUGGUAAUGGACAGAGUUA 19 LPA-1086 first chain AUGUGCCUUGAUAACUCUG AUGUGCCUUGAUAACUCUG 20 LPA-1086 second circuit CAGAGUUAUCAAGGCACAU CAGAGUUAUCAAGGCACAU 21 LPA-1099 first chain AGUUGGUGCUGCUUCAGAA AGUUGGUGCUGCUUCAGAA 22 LPA-1099 second circuit UUCUGAAGCAGCACCACU UUCUGAAGCAGCACCACU 23 LPA-1102 first chain AAUAAGGGGCUGCCACAGG AAUAAGGGGCUGCCACAGG 24 LPA-1102 second circuit CCUGUGGCAGCCCCUUAUU CCUGUGGCAGCCCCUUAUU 25 LPA-1116 first chain UAACUCUGUCCAUCACCAU UAACUCUGUCCAUCACCAU 26 LPA-1116 second circuit AUGGUGAUGGACAGAGUUA AUGGUGAUGGACAGAGUUA 27 LPA-1127 first chain AUGAGCCUCGAUAACUCUG AUGAGCCUCGAUAACUCUG 28 LPA-1127 second circuit CAGAGUUAUCGAGGCUCAU CAGAGUUAUCGAGGCUCAU 29 LPA-1128 first chain AAUGAGCCUCGAUAACUCU AAUGAGCCUCGAUAACUCU thirty LPA-1128 second circuit AGAGUUAUCGAGGCUCAUU AGAGUUAUCGAGGCUCAUU 31 LPA-1141 first chain AAUGCUUCCAGGACAUUUC AAUGCUUCCAGGACAUUUC 32 LPA-1141 second circuit GAAAUGUCCUGGAAGCAUU GAAAUGUCCUGGAAGCAUU 33 LPA-1151 first chain ACAGUGGUGGAGAAUGUGC ACAGUGGUGGAGAAUGUGC 34 LPA-1151 second circuit GCACAUUCUCCCACCACUGU GCACAUUCUCCCACCACUGU 35 LPA-1171 first chain GUAUGUGCCUCGAUAACUC GUAUGUGCCUCGAUAACUC 36 LPA-1171 second circuit GAGUUAUCGAGGCACAUAC GAGUUAUCGAGGCACAUAC 37 LPA-1177 first chain UCGAUAACUCUGUCCAUCA UCGAUAACUCUGUCCAUCA 38 LPA-1177 second circuit UGAUGGACAGAGUUAUCGA UGAUGGACAGAGUUAUCGA 39 LPA-1189 first chain UGUCACUGGACAUUGUGUC UGUCACUGGACAUUGUGUC 40 LPA-1189 second circuit GACACAAUGUCCAGUGACA GACACAAUGUCCAGUGACA 41 LPA-1244 first circuit CUGGGAUCCAUGGGUGUAAC CUGGGAUCCAUGGGUGUAAC 42 LPA-1244 second circuit GUUACACCAUGGAUCCCAG GUUACACCAUGGAUCCCAG 43 LPA-1248 first chain AGAUGACCAAAGCUUGGCAG AGAUGACCAAAGCUUGGCAG 44 LPA-1248 second circuit CUGCCAAGCUUGGUCAUCU CUGCCAAGCUUGGUCAUCU 45 LPA: (upper) person AAGTGTCCTTGGCGACGTCC AAGTGTCCTTGGCGACGTCC 46 LPA: (lower) person CCTGGACTGTGGGGCTTT CCTGGACTGTGGGGCTTT 47 LPA: (probe) person CTGTTTCTGAACAAGCACCAACGGAGC CTGTTTCTGAACAAGCACCAACGGAGC 48 LPA (upper) cynomolgus macaque GTGTCCTCGCAACGTCCA GTGTCCTCGCAACGTCCA 49 LPA (lower) cynomolgus macaque GACCCCGGGGCTTTG GACCCCGGGGCTTTG 50 LPA (probe) cynomolgus macaque TGGCTGTTTCTGAAACAAGCACCAATGG TGGCTGTTTCTGAAACAAGCACCAATGG 51 APOB (upper) man TCATTCCTTCCCCCAAAGAGACC TCATTCCTTCCCCCAAAGAGACC 52 APOB (bottom) person CACCTCCGTTTTGGTGGTAGAG CACCTCCGTTTTGGTGGTAGAG 53 APOB (probe) man CAAGCTGCTCAGTGGAGGCAACACATTA CAAGCTGCTCAGTGGAGGCAACACATTA 54 Beta actin (upper) man GCATGGGTCAGAAGGATTCCTAT GCATGGGTCAGAAGGATTCCTAT 55 Beta actin (bottom) man TGTAGAAGGTGTGGTGCCAGATT TGTAGAAGGTGTGGTGCCAGATT 56 Beta actin (probe) human TCGAGCACGGCATCGTCACCAA TCGAGCACGGCATCGTCACCAA 57 Beta-actin (upper) cynomolgus monkey AAGGCCAACCGCGAGAAG AAGGCCAACCGCGAGAAG 58 Beta actin (lower) cynomolgus monkey AGAGGCGTACAGGGACAGCA AGAGGCGTACAGGGACAGCA 59 Beta actin (probe) cynomolgus monkey TGAGACCTTCAACACCCCAGCCATGTAC TGAGACCTTCAACACCCCAGCCATGTAC 60 PPIB (upper) person AGATGTAGGCCGGGGTGATCTTT AGATGTAGGCCGGGGTGATCTTT 61 PPIB (lower) person GTAGCCAAATCCTTTCTCTCCTGT GTAGCCAAATCCTTTCTCTCCTGT 62 PPIB (probe) man TGTTCCAAAACAGTGGATAATTTTGTGGCC TGTTCCAAAACAGTGGATAATTTTGTGGCC 63 LPA-1301 first chain UUAACUCUGUCCAUUACCG UUAACUCUGUCCAUUACCG 64 LPA-1301 second circuit CGGUAAUGGACAGAGUUAA CGGUAAUGGACAGAGUUAA 65 LPA-1302 first chain UUAACUCUGUCCAUUACCC UUAACUCUGUCCAUUACCC 66 LPA-1302 second circuit GGGUAAUGGACAGAGUUAA GGGUAAUGGACAGAGUUAA 67 LPA-1303 first chain UUAACUCUGUCCAUUACCU UUAACUCUGUCCAUUACCU 68 LPA-1303 second circuit AGGUAAUGGACAGAGUUAA AGGUAAUGGACAGAGUUAA 69 LPA-1304 first chain AUAACUCUGUCCAUUACCC AUAACUCUGUCCAUUACCC 70 LPA-1304 second circuit GGGUAAUGGACAGAGUUAU GGGUAAUGGACAGAGUUAU 71 LPA-1305 first chain AUAACUCUGUCCAUUACCU AUAACUCUGUCCAUUACCU 72 LPA-1305 second circuit AGGUAAUGGACAGAGUUAU AGGUAAUGGACAGAGUUAU 73 LPA-1306 first chain UUAACUCUGUCCAUUACCA UUAACUCUGUCCAUUACCA 74 LPA-1306 second circuit UGGUAAUGGACAGAGUUAA UGGUAAUGGACAGAGUUAA 75 Modified SEQ ID NO: 1 5381616272616284847 UCUAUAACAAUAAGGGGC 76 Modified SEQ ID NO: 2 4737351615451616382 GCCCCUUAUUGUUAUACGA 77 Modified SEQ ID NO: 3 8252635354537251637 GAUAACUCUGUCCAUUACC 78 Modified SEQ ID NO: 4 4816254827282815253 GGUAAUGGACAGAGUUAUC 79 Modified SEQ ID NO: 5 6162717181736152736 AUAACUCUGUCCAUUACCA 80 Modified SEQ ID NO: 6 1845261846364645161 UGGUAAUGGACAGAGUUAU 81 Modified SEQ ID NO: 7 5263535453725163745 UAACUCUGUCCAUUACCGU 82 Modified SEQ ID NO: 8 2748162548272828152 ACGGUAAUGGACAGAGUUA 83 Modified SEQ ID NO: 9 6162717181736152738 AUAACUCUGUCCAUUACCG 84 Modified SEQ ID NO: 10 3845261846364645161 CGGUAAUGGACAGAGUUAU 85 Modified SEQ ID NO: 11 6462545473538252635 AGAAUGUGCCUCGAUAACU 86 Modified SEQ ID NO: 12 2815253828472725171 AGUUAUCGAGGCACAUUCU 87 Modified SEQ ID NO: 13 6162717181736172736 AUAACUCUGUCCAUCACCA 88 Modified SEQ ID NO: 14 1845461846364645161 UGGUGAUGGACAGAGUUAU 89 Modified SEQ ID NO: 15 6162717181736172735 AUAACUCUGUCCAUCACCU 90 Modified SEQ ID NO: 16 2845461846364645161 AGGUGAUGGACAGAGUUAU 91 Modified SEQ ID NO: 17 5263535453725163725 UAACUCUGUCCAUUACCAU 92 Modified SEQ ID NO: 18 2548162548272828152 AUGGUAAUGGACAGAGUUA 93 Modified SEQ ID NO: 19 6181837154616271718 AUGUGCCUUGAUAACUCUG 94 Modified SEQ ID NO: 20 3646451617264836361 CAGAGUUAUCAAGGCACAU 95 Modified SEQ ID NO: 21 6451845471835172826 AGUUGGUGCUGCUUCAGAA 96 Modified SEQ ID NO: 22 1535462836472736271 UUCUGAAGCAGCACCACU 97 Modified SEQ ID NO: 23 6252648483547363648 AAUAAGGGGCUGCCACAGG 98 Modified SEQ ID NO: 24 3718184728373715251 CCUGUGGCAGCCCCUUAUU 99 Modified SEQ ID NO: 25 5263535453725363725 UAACUCUGUCCAUCACCAU 100 Modified SEQ ID NO: 26 2548182548272828152 AUGGUGAUGGACAGAGUUA 101 Modified SEQ ID NO: 27 6182837174616271718 AUGAGCCUCGAUAACUCUG 102 Modified SEQ ID NO: 28 3646451617464835361 CAGAGUUAUCGAGGCUCAU 103 Modified SEQ ID NO: 29 6254647353825263535 AAUGAGCCUCGAUAACUCU 104 Modified SEQ ID NO: 30 2828152538284717251 AGAGUUAUCGAGGCUCAUU 105 Modified SEQ ID NO: 31 6254715372846361517 AAUGCUUCCAGGACAUUUC 106 Modified SEQ ID NO: 32 4626181735482647251 GAAAUGUCCUGGAAGCAUU 107 Modified SEQ ID NO: 33 6364548184646254547 ACAGUGGUGGAGAAUGUGC 108 Modified SEQ ID NO: 34 4727251717363727181 GCACAUUCUCCCACCACUGU 109 Modified SEQ ID NO: 35 8161818371746162717 GUAUGUGCCCUCGAUAACUC 110 Modified SEQ ID NO: 36 4645161746483636163 GAGUUAUCGAGGCACAUAC 111 Modified SEQ ID NO: 37 5382526353545372536 UCGAUAACUCUGUCCAUCA 112 Modified SEQ ID NO: 38 1825482728281525382 UGAUGGACAGAGUUAUCGA 113 Modified SEQ ID NO: 39 5453635482725181817 UGUCACUGGACAUUGUGUC 114 Modified SEQ ID NO: 40 4636362545372818272 GACACAAUGUCCAGUGACA 115 Modified SEQ ID NO: 41 7184825372548181627 CUGGGAUCCAUGGGUGUAAC 116 Modified SEQ ID NO: 42 4516363725482537364 GUUACACCAUGGAUCCCAG 117 Modified SEQ ID NO: 43 6461827362835184728 AGAUGACCAAAGCUUGGCAG 118 Modified SEQ ID NO: 44 3547362835184536171 CUGCCAAGCUUGGUCAUCU 119 GalNAc-LPA-1038-L1 first chain OMeA-(ps)-FU-(ps)-OMeA-FA-OMeC-FU-OMeC-FU-OMeG-FU-OMeC-FC-OMeA-FU-OMeU-FA-OMeC-(ps)-FC-(ps )-OMeA AUAACUCUGUCCAUUACCA 120 GalNAc-LPA-1038-L1 second chain [ST23 (ps)]3 long triple (ps)FU-OMeG-FG-OMeU-FA-OMeA-FU-OMeG-FG-OMeA-FC-OMeA-FG-OMeA-FG-OMeU-FU-(ps)- OMeA-(ps)-FU UGGUAAUGGACAGAGUUAU 121 GalNAc-LPA-1038-L6 first chain OMeA-(ps)-FU-(ps)-OMeA-FA-OMeC-FU-OMeC-FU-OMeG-FU-OMeC-FC-OMeA-FU-OMeU-FA-OMeC-(ps)-FC-(ps )-OMeA AUAACUCUGUCCAUUACCA 122 GalNAc-LPA-1038-L6 second chain [ST23 (ps)]3 ST43 (ps)FU-OMeG-FG-OMeU-FA-OMeA-FU-OMeG-FG-OMeA-FC-OMeA-FG-OMeA-FG-OMeU-FU-(ps)-OMeA -(ps)-FU UGGUAAUGGACAGAGUUAU 123 GalNAc-LPA-1041-L1 first chain OMeA-(ps)-FU-(ps)-OMeA-FA-OMeC-FU-OMeC-FU-OMeG-FU-OMeC-FC-OMeA-FU-OMeU-FA-OMeC-(ps)-FC-(ps )-OMeG AUAACUCUGUCCAUUACCG 124 GalNAc-LPA-1041-L1 second chain [ST23 (ps)]3 long triple (ps) FC-OMeG-FG-OMeU-FA-OMeA-FU-OMeG-FG-OMeA-FC-OMeA-FG-OMeA-FG-OMeU-FU-(ps)- OMeA-(ps)-FU CGGUAAUGGACAGAGUUAU 125 GalNAc-LPA-1041-L6 first chain OMeA-(ps)-FU-(ps)-OMeA-FA-OMeC-FU-OMeC-FU-OMeG-FU-OMeC-FC-OMeA-FU-OMeU-FA-OMeC-(ps)-FC-(ps )-OMeG AUAACUCUGUCCAUUACCG 126 GalNAc-LPA-1041-L6 second chain [ST23 (ps)]3 ST43 (ps) FC-OMeG-FG-OMeU-FA-OMeA-FU-OMeG-FG-OMeA-FC-OMeA-FG-OMeA-FG-OMeU-FU-(ps)-OMeA -(ps)-FU CGGUAAUGGACAGAGUUAU 127 STS16001AL33 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) UUAUAGAGCAAGAACACUGUU 128 STS16001BL20 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 AACAGUGUUCUUGCUCUAUAA 129 STS16001A 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 UUAUAGAGCAAGAACACUGUU 130 STS16001BV1L42 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) AACAGUGUUCUUGCUCUAUAA 131 STS16001V1B 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 AACAGUGUUCUUGCUCUAUAA 132 STS18001A mU (ps) fC (ps) mG fA mA fG mU fA mU fU mC fC mG fC mG fU mA (ps) fC (ps) mG UCGAAGUAUUCCGCGUACG 133 STS18001BL4 [(ST23) (ps)] ₃ C4XLT (ps) fC mG fU mA fC mG fC mG fG mA fA mU fA mC fU mU fC (ps) mG (ps) fA CGUACGCGGAAUACUUCGA 134 STS16001BL4 [(ST23) (ps)]3 C4XLT(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 AACAGUGUUCUUGCUCUAUAA 135 X0373A mA (ps) fU (ps) mA fA mC fU mC fU mG fU mC fC mA fU mU fA mC (ps) fC (ps) mG AUAACUCUGUCCAUUACCG 136 X0373B 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) CGGUAAUGGACAGAGUUAU 137 STS2041B ST23 (ps) ST23 (ps) ST23 (ps) C6XLT (ps) fC mG fG mU fA mA fU mG fG mA fC mA fG mA fG mU fU (ps) mA (ps) fU CGGUAAUGGACAGAGUUAU 138 X0125A mC (ps) fU (ps) mU fA mC fU mC fU mC fG mC fC mC fA mA fG mC (ps) fG (ps) mA CUUACUCUCGCCCAAGCGA 139 X0125B [(ST23) (ps)] ₃ (C6XLT) (ps) fU mC fG mC fU mU fG mG fG mC fG mA fG mA fG mU fA (ps) mA (ps) fG UCGCUUGGGCGAGAGUAAG 140 Probe based on SEQ ID NO: 50 BHQ1-TGGCTGTTTCTGAACAAGCACAATGG-FAM TGGCTGTTTCTGAAACAAGCACCAATGG 141 Probe based on SEQ ID NO: 56 BHQ1-TCGAGCACGGCATCGTCACCAA-VIC TCGAGCACGGCATCGTCACCAA 142 STS16001BV1L75 Ser(GN) 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 Ser(GN) AACAGUGUUCUUGCUCUAUAA 143 STS16001BV16L42 Ser(GN) (ps) fA mA fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA fU mA fA (ps) Ser(GN) AACAGUGUUCUUGCUCUAUAA 144 STS16001BV20L75 Ser(GN) fA mA fC mA fG mU fG mU fU mC fU mU fG mC fU mC fU mA fU mA fA Ser(GN) AACAGUGUUCUUGCUCUAUAA 145 STS16001BV1L94 Ser(GN) (ps) 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) (ps) Ser(GN) AACAGUGUUCUUGCUCUAUAA 146 STS16001V1BL96 C6Am(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) C7Am(GN) AACAGUGUUCUUGCUCUAUAA 147 STS16001V1BL97 GlyC3Am(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) GlyC3Am(GN) AACAGUGUUCUUGCUCUAUAA 148 Conjugate 10 second chain PipAm(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) PipAm(GN) AACAGUGUUCUUGCUCUAUAA 149 STS16001V1BL88 C3Am(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) C3Am(GN) AACAGUGUUCUUGCUCUAUAA 150 STS16001V1BL87 C6Am(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) GlyC3Am(GN) AACAGUGUUCUUGCUCUAUAA 151 Conjugate 15 antisense strand mU (ps) fC (ps) mU fU mC fU mU fA mA fA mC fU mG fA mG fU mU (ps) fU (ps) mC UCUUCUUAAACUGAGUUUC 152 Conjugate 15 sense strand 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) GAAACUCAGUUUAAGAAGA 153 Conjugate 16 antisense strand mA (ps) fU (ps) mG fU mA fG mC fC mG fA mG fG mA fU mC fU mU (ps) fC (ps) mU AUGUAGCCGAGGAUCUUCU 154 Conjugate 16 antisense strand 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) AGAAGAUCCUCGGCUACAU 155 Conjugate 18 antisense strand mA (ps) fA (ps) mC fC mA fG mA fA mG fA mA fG mC fA mG fG mU (ps) fG (ps) mA AACCAGAAGAAGCAGGUGA 156 Conjugate 18 sense strand 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) UCACCUGCUUCUUCUGGUU 157 STS16001BV1 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 AACAGUGUUCUUGCUCUAUAA 158 Reference conjugate 6 sense strand [ST23 (ps)]3 ltrb (ps) fG mA fA mA fC mU fC mA fG mU fU mU fA mA fG mA fA (ps) mG (ps) fA GAAACUCAGUUUAAGAAGA 159 Reference conjugate 7 sense strand [ST23 (ps)]3 ltrb (ps) fA mG fA mA fG mA fU mC fC mU fC mG fG mC fU mA fC (ps) mA (ps) fU AGAAGAUCCUCGGCUACAU 160 Reference conjugate 8 antisense strand mU (ps) fA (ps) mC fC mA fG mA fA mG fA mA fG mC fA mG fG mU (ps) fG (ps) mA UACCAGAGAAGCAGGUGA 161 Reference conjugate 8 sense chain [ST23 (ps)]3 ST41 (ps)fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mG fG (ps) mU (ps) fA UCACUGCUUCUUCUGGUA 162 Reference conjugate 9 sense strand [ST23 (ps)]3 C6XLT (ps) fC mG fG mU fA mA fU mG fG mA fC mA fG mA fG mU fU (ps) mA (ps) fU CGGUAAUGGACAGAGUUAU

Designation

One sequence can have more than one name. In these cases, one of these names is listed in the sequence summary table.

If specific linkers and/or modified links are specified in the RNA sequence, such as PS and [ST23 (ps)]3 ST41 (ps), etc., these are optional parts of the sequence, but are a preferred embodiment of the sequence.

The following abbreviations may be used:

--->

LIST OF SEQUENCES

<110> Silence Therapeutics GmbH

<120> NUCLEIC ACIDS FOR INHIBITION OF LPA EXPRESSION IN THE CELL

<130>S109PCT

<150>EP17201449.0

<151> 2017-11-13

<150>EP18179175.7

<151> 2018-06-21

<150> GB1815915.2

<151> 2018-09-28

<160> 162

<170> Patent In version 3.5

<210> 1

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 1

ucguauaaca auaaggggc 19

<210> 2

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 2

gcccccuuauu guuauacga 19

<210> 3

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 3

gauaacucug uccauuacc 19

<210> 4

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 4

gguaauggac agaguuauc 19

<210> 5

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 5

auaacucugu ccauuacca 19

<210> 6

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 6

ugguaaugga cagaguuau 19

<210> 7

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 7

uaacucuguc cauuaccgu 19

<210> 8

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 8

acgguaaugg acagaguua 19

<210> 9

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 9

auaacucugu ccauuaccg 19

<210> 10

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 10

cgguaaugga cagaguuau 19

<210> 11

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 11

agaaugugcc ucgauaacu 19

<210> 12

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 12

aguuaucgag gcacauucu 19

<210> 13

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 13

auaacucugu ccaucacca 19

<210> 14

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 14

uggugaugga cagaguuau 19

<210> 15

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 15

auaacucugu ccaucaccu 19

<210> 16

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 16

aggugaugga cagaguuau 19

<210> 17

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 17

uaacucuguc cauuaccau 19

<210> 18

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 18

auggaaugg acagaguua 19

<210> 19

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 19

augugccuug auaacucug 19

<210> 20

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 20

cagaguuauc aaggcacau 19

<210> 21

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 21

aguuggugcu gcuucagaa 19

<210> 22

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 22

uucugaagca gcaccaacu 19

<210> 23

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 23

aauaaggggc ugccacagg 19

<210> 24

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 24

ccuguggcag ccccuuauu 19

<210> 25

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 25

uaacucuguc caucaccau 19

<210> 26

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 26

auggugagg acagaguua 19

<210> 27

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 27

augagccucg auaacucug 19

<210> 28

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 28

cagaguuauc gaggcucau 19

<210> 29

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 29

aaugagccuc gauaacucu 19

<210> 30

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 30

agaguuaucg aggcucauu 19

<210> 31

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 31

aaugcuucca ggacauuuc 19

<210> 32

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 32

gaaauguccu ggaagcauu 19

<210> 33

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 33

acaguggugg agaaugugc 19

<210> 34

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 34

gcacauucuc caccacugu 19

<210> 35

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 35

guaugugccu cgauaacuc 19

<210> 36

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 36

gaguuaucga ggcacauaac 19

<210> 37

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 37

ucgauaacuc uguccauca 19

<210> 38

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 38

ugauggacag aguuaucga 19

<210> 39

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 39

ugucacugga cauuguguc 19

<210> 40

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 40

gacacaaugu ccagugaca 19

<210> 41

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 41

cugggaucca ugguguaac 19

<210> 42

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 42

guuacaccau ggaucccag 19

<210> 43

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 43

agaugaccaa gcuuggcag 19

<210> 44

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 44

cugccaagcu uggucaucu 19

<210> 45

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 45

aagtgtcctt gcgacgtcc 19

<210> 46

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 46

cctggactgt ggggcttt 18

<210> 47

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 47

ctgtttctga acaagcacca acggagc 27

<210> 48

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 48

gtgtcctcgc aacgtcca 18

<210> 49

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 49

gaccccgggg ctttg 15

<210> 50

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 50

tggctgtttc tgaacaagca ccaatgg 27

<210> 51

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 51

tcattccttc cccaaagaga cc 22

<210> 52

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 52

cacctccgtt ttggtggtag ag 22

<210> 53

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 53

caagctgctc agtggaggca acacatta 28

<210> 54

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 54

gcatgggtca gaaggattcc tat 23

<210> 55

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 55

tgtagaaggt gtggtgccag att 23

<210> 56

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 56

tcgagcacgg catcgtcacc aa 22

<210> 57

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 57

aaggccaacc gcgagaag 18

<210> 58

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 58

aggagcgtac agggacagca 20

<210> 59

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 59

tgagaccttc aacaccccag ccatgtac 28

<210> 60

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 60

agatgtaggc cgggtgatct tt 22

<210> 61

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 61

gtagccaaat cctttctctc ctgt 24

<210> 62

<211> 31

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 62

tgttccaaaa acagtggata attttgtggc c 31

<210> 63

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 63

uuaacucugu ccauuaccg 19

<210> 64

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 64

cgguaaugga cagaguuaa 19

<210> 65

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 65

uuaacucugu ccauuaccc 19

<210> 66

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 66

ggguaaugga cagaguuaa 19

<210> 67

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 67

uuaacucugu ccauuaccu 19

<210> 68

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 68

agguaaugga cagaguuaa 19

<210> 69

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 69

auaacucugu ccauuaccc 19

<210> 70

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 70

ggguaaugga cagaguuau 19

<210> 71

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 71

auaacucugu ccauuaccu 19

<210> 72

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 72

agguaaugga cagaguuau 19

<210> 73

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 73

uuaacucugu ccauuacca 19

<210> 74

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 74

ugguaaugga cagaguuaa 19

<210> 75

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA – modified_base, modified according to

summary table of sequences at the end of the description

<400> 75

ucguauaaca auaaggggc 19

<210> 76

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 76

gcccccuuauu guuauacga 19

<210> 77

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 77

gauaacucug uccauuacc 19

<210> 78

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 78

gguaauggac agaguuauc 19

<210> 79

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 79

auaacucugu ccauuacca 19

<210> 80

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 80

ugguaaugga cagaguuau 19

<210> 81

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 81

uaacucuguc cauuaccgu 19

<210> 82

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 82

acgguaaugg acagaguua 19

<210> 83

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

sequence summary table at the end of the description

<400> 83

auaacucugu ccauuaccg 19

<210> 84

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 84

cgguaaugga cagaguuau 19

<210> 85

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 85

agaaugugcc ucgauaacu 19

<210> 86

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 86

aguuaucgag gcacauucu 19

<210> 87

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 87

auaacucugu ccaucacca 19

<210> 88

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 88

uggugaugga cagaguuau 19

<210> 89

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 89

auaacucugu ccaucaccu 19

<210> 90

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 90

aggugaugga cagaguuau 19

<210> 91

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 91

uaacucuguc cauuaccau 19

<210> 92

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 92

auggaaugg acagaguua 19

<210> 93

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 93

augugccuug auaacucug 19

<210> 94

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 94

cagaguuauc aaggcacau 19

<210> 95

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 95

aguuggugcu gcuucagaa 19

<210> 96

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 96

uucugaagca gcaccaacu 19

<210> 97

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 97

aauaaggggc ugccacagg 19

<210> 98

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 98

ccuguggcag ccccuuauu 19

<210> 99

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 99

uaacucuguc caucaccau 19

<210> 100

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 100

auggugagg acagaguua 19

<210> 101

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 101

augagccucg auaacucug 19

<210> 102

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

sequence summary table at the end of the description

<400> 102

cagaguuauc gaggcucau 19

<210> 103

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

sequence summary table at the end of the description

<400> 103

aaugagccuc gauaacucu 19

<210> 104

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 104

agaguuaucg aggcucauu 19

<210> 105

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 105

aaugcuucca ggacauuuc 19

<210> 106

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 106

gaaauguccu ggaagcauu 19

<210> 107

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 107

acaguggugg agaaugugc 19

<210> 108

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 108

gcacauucuc caccacugu 19

<210> 109

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 109

guaugugccu cgauaacuc 19

<210> 110

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 110

gaguuaucga ggcacauaac 19

<210> 111

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 111

ucgauaacuc uguccauca 19

<210> 112

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 112

ugauggacag aguuaucga 19

<210> 113

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 113

ugucacugga cauuguguc 19

<210> 114

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

sequence summary table at the end of the description

<400> 114

gacacaaugu ccagugaca 19

<210> 115

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

sequence summary table at the end of the description

<400> 115

cugggaucca ugguguaac 19

<210> 116

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

sequence summary table at the end of the description

<400> 116

guuacaccau ggaucccag 19

<210> 117

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

sequence summary table at the end of the description

<400> 117

agaugaccaa gcuuggcag 19

<210> 118

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 118

cugccaagcu uggucaucu 19

<210> 119

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 119

auaacucugu ccauuacca 19

<210> 120

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 120

ugguaaugga cagaguuau 19

<210> 121

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 121

auaacucugu ccauuacca 19

<210> 122

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 122

ugguaaugga cagaguuau 19

<210> 123

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

sequence summary table at the end of the description

<400> 123

auaacucugu ccauuaccg 19

<210> 124

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 124

cgguaaugga cagaguuau 19

<210> 125

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 125

auaacucugu ccauuaccg 19

<210> 126

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 126

cgguaaugga cagaguuau 19

<210> 127

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 127

uuauagagca agaacacugu u 21

<210> 128

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 128

aacaguguuc uugcucuaua a 21

<210> 129

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 129

uuauagagca agaacacugu u 21

<210> 130

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

sequence summary table at the end of the description

<400> 130

aacaguguuc uugcucuaua a 21

<210> 131

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 131

aacaguguuc uugcucuaua a 21

<210> 132

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 132

ucgaaguauu ccgcguacg 19

<210> 133

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 133

cguacgcgga auacuucga 19

<210> 134

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

sequence summary table at the end of the description

<400> 134

aacaguguuc uugcucuaua a 21

<210> 135

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 135

auaacucugu ccauuaccg 19

<210> 136

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 136

cgguaaugga cagaguuau 19

<210> 137

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 137

cgguaaugga cagaguuau 19

<210> 138

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 138

cuuacucucg cccaagcga 19

<210> 139

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 139

ucgcuugggc gagaguaag 19

<210> 140

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Probe - modified_base, modified according to

summary table of sequences at the end of the description

<400> 140

tggctgtttc tgaacaagca ccaatgg 27

<210> 141

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Probe - modified_base, modified according to

summary table of sequences at the end of the description

<400> 141

tcgagcacgg catcgtcacc aa 22

<210> 142

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 142

aacaguguuc uugcucuaua a 21

<210> 143

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 143

aacaguguuc uugcucuaua a 21

<210> 144

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

sequence summary table at the end of the description

<400> 144

aacaguguuc uugcucuaua a 21

<210> 145

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 145

aacaguguuc uugcucuaua a 21

<210> 146

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

sequence summary table at the end of the description

<400> 146

aacaguguuc uugcucuaua a 21

<210> 147

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 147

aacaguguuc uugcucuaua a 21

<210> 148

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 148

aacaguguuc uugcucuaua a 21

<210> 149

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 149

aacaguguuc uugcucuaua a 21

<210> 150

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 150

aacaguguuc uugcucuaua a 21

<210> 151

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 151

ucuucuuaaa cugaguuuc 19

<210> 152

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 152

gaaacucagu uuaagaaga 19

<210> 153

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 153

auguagccga ggaucuucu 19

<210> 154

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 154

agaagauccu cggcuacau 19

<210> 155

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 155

aaccagaaga agcagguga 19

<210> 156

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 156

ucaccugcuu cuucugguu 19

<210> 157

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

sequence summary table at the end of the description

<400> 157

aacaguguuc uugcucuaua a 21

<210> 158

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

sequence summary table at the end of the description

<400> 158

gaaacucagu uuaagaaga 19

<210> 159

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

sequence summary table at the end of the description

<400> 159

agaagauccu cggcuacau 19

<210> 160

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

sequence summary table at the end of the description

<400> 160

uaccagaaga agcagguga 19

<210> 161

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

summary table of sequences at the end of the description

<400> 161

ucaccugcuu cuucugggua 19

<210> 162

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> miRNA - modified_base, modified according to

sequence summary table at the end of the description

<400> 162

cgguaaugga cagaguuau 19

<---

Claims (78)

1. A nucleic acid for inhibiting the expression of LPA in a cell, comprising at least one duplex region that contains at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to said first strand, wherein said first strand is is at least partially complementary to at least a portion of the RNA transcribed from the LPA gene, wherein said first strand contains the nucleotide sequence SEQ ID NO: 9, wherein the portion of the first strand in the duplex region contains at least 10 nucleotides of SEQ ID NO: 9 and wherein the portion of the second strand is 70-100% complementary to the portion of the first strand. 2. The nucleic acid according to claim 1, wherein said portion of the first strand in the duplex region contains at least 12 nucleotides of SEQ ID NO: 9, preferably at least 14 nucleotides of SEQ ID NO: 9. 3. The nucleic acid according to claim 1 or 2, wherein said portion of the first strand in the duplex region contains at least 16 nucleotides of SEQ ID NO: 9, preferably at least 18 nucleotides of SEQ ID NO: 9. 4. A nucleic acid as claimed in any one of the preceding claims, wherein said portion of the first strand in the duplex region contains all of the nucleotides of SEQ ID NO: 9. 5. A nucleic acid according to any of the preceding claims, characterized in that said second strand contains the nucleotide sequence of SEQ ID NO: 10. 6. Nucleic acid according to any of the preceding claims, characterized in that said first strand is 19-35 nucleotides in length and/or said second strand is 17-35 nucleotides in length. 7. Nucleic acid according to any of the preceding claims, characterized in that said at least one duplex region consists of 17-25, preferably 19-25, consecutive nucleotide base pairs. 8. Nucleic acid according to any of the preceding claims, characterized in that said nucleic acid: a) has blunt ends at both ends; or b) has a sticky end on one end and a blunt end on the other; or c) has a sticky end on both ends. 9. Nucleic acid according to any of the preceding claims, characterized in that one or more nucleotides on said first and/or second strands are modified to form modified nucleotides. 10. Nucleic acid according to any of the preceding claims, characterized in that said nucleic acid contains a phosphorothioate bond between one, two or three terminal 3'-nucleotides and/or 5'-nucleotides of one or both ends of said first and/or second strands. 11. A conjugate for inhibiting the expression of LPA in a cell, containing the nucleic acid according to any of the preceding paragraphs, conjugated to a ligand. 12. The conjugate according to claim 11, characterized in that said ligand contains (i) one or more N-acetylgalactosamine (GalNAc) groups or derivatives thereof and (ii) a linker, wherein by means of said linker at least one GalNAc group or its the derivative is conjugated to the specified nucleic acid. 13. Conjugate according to any one of paragraphs. 11, 12, characterized in that the ligand contains a compound of formula (I) [SX ¹ -PX ² ] ₃ -AX ³ -, (I) wherein S is a saccharide, preferably said saccharide being N-acetylgalactosamine; X ¹ represents C ₃ -C ₆ alkylene or (-CH ₂ -CH ₂ -O) _m (-CH ₂ ) ₂ -, where m is 1, 2 or 3; P is phosphate or modified phosphate, preferably thiophosphate; X ² represents an alkylene or alkylene ether of the formula (-CH ₂ ) _n -O-CH ₂ -, where n=1-6; A represents a branching block; X ³ is a bridge block; wherein the nucleic acid according to any one of claims. 1-8 is conjugated to X ³ by a phosphate or modified phosphate, preferably a thiophosphate. 14. The conjugate according to claim 11 or 12, characterized in that said first strand is RNA and is a compound of formula (X) in which b is 0 or 1; And said second strand is RNA and is a compound of formula (XI) in which c and d are independently equal to 0 or 1; Z ₁ and Z ₂ are RNA portions of said first and second RNA strands, respectively; Y represents O or S; n is 0, 1, 2 or 3; And L ₁ represents a linker to which a ligand is attached; And in this case b+c+d is equal to 2 or 3. 15. The nucleic acid of claim 1, wherein said first strand consists of the nucleotide sequence of SEQ ID NO: 9, and optionally said second strand contains and preferably consists of the nucleotide sequence of SEQ ID NO: 10. 16. A conjugate for inhibiting the expression of LPA in a cell, containing the nucleic acid according to claim 15, conjugated to a ligand. 17. The conjugate according to claim 11 or 16, characterized in that the conjugate has the following structure: where Z represents a nucleic acid according to claim 1 or 15. 18. The conjugate according to claim 17, characterized in that said nucleic acid contains two phosphorothioate bonds between each of the three 3'-terminal and between each of the three 5'-terminal nucleotides on said first strand and two phosphorothioate bonds between the three terminal nucleotides 3 '-end of said second strand, and wherein said ligand is conjugated to the 5'-end of said second strand. 19. Conjugate according to claim 17, characterized in that the first strand is RNA and is a compound of formula (XV) in which b is 0 or 1; And said second strand is RNA and is a compound of formula (XVI) in which c and d are independently equal to 0 or 1; wherein Z ₁ and Z ₂ represent RNA parts of said first and second RNA strands, respectively; Y represents O or S; R ₁ represents H or methyl; n is 0, 1, 2 or 3; And L is the same or different in formulas (XV) and (XVI) and is selected from the group consisting of: -(CH ₂ ) _q , where q=2-12; -(CH ₂ ) _r -C(O)-, where r=2-12; -(CH ₂ -CH ₂ -O) _s -CH ₂ -C(O)-, where s=1-5; -(CH ₂ ) _t -CO-NH-(CH ₂ ) _t -NH-C(O)-, where t independently represents 1-5; -(CH ₂ ) _u -CO-NH-(CH ₂ ) _u -C(O)-, where u independently represents 1-5; And -(CH ₂ ) _v -NH-C(O)-, where v represents 2-12; And wherein the terminal C(O), if present, is attached to the NH group; and b+c+d is equal to 2 or 3. 20. Nucleic acid according to any one of paragraphs. 1-10 or 15 or a conjugate according to any one of paragraphs. 11-14 or 16-19, containing the sequence and modifications presented below: where specific modifications are indicated 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. 21. Nucleic acid according to any one of paragraphs. 1-10 or 15 or a conjugate according to any one of paragraphs. 11-14 or 16-19, characterized in that nucleotides at positions 2 and 14 from the 5' end of said first strand are modified using a 2'-fluoro modification and nucleotides on said second strand that correspond to position 11 or 13, or 11 and 13, or 11-13 of said first chain are modified using a 2'-fluoro modification. 22. A composition for preventing, or treating, or reducing the risk of developing cardiovascular disease, where said composition contains a pharmaceutically effective dose of a nucleic acid or conjugate according to any one of paragraphs. 1-21. 23. The composition according to claim 22, further containing a delivery vehicle, preferably liposomes, and/or a physiologically acceptable excipient, and/or a carrier, and/or a diluent, and/or a buffer, and/or a preservative. 24. The composition according to claim 22 or 23, wherein said cardiovascular disease is stroke, atherosclerosis, thrombosis, coronary heart disease or aortic stenosis and/or any other disease or pathology associated with elevated levels of particles containing Lp(a) . 25. Use of a nucleic acid or conjugate according to any one of paragraphs. 1-21 or compositions according to any one of paragraphs. 22-24 to prevent, or treat, or reduce the risk of developing cardiovascular disease. 26. Use according to claim 25, wherein said cardiovascular disease is stroke, atherosclerosis, thrombosis, coronary heart disease or aortic stenosis and any other disease or pathology associated with elevated levels of Lp(a) containing particles.