Compositions and methods for inhibiting the expression of the protein LPA (Apo(a))

Abstract

Provided herein are compositions and methods useful for reducing gene expression of LPA (Apo(a)) and treating LPA-related diseases or conditions. Further provided are LPA dsRNA agents, LPA antisense polynucleotide agents, compositions comprising LPA dsRNA agents, and compositions comprising LPA antisense polynucleotide agents useful for reducing expression of LPA in cells and subjects.

Description

[Technical field]

[0001] Some embodiments of the present invention relate to compositions and methods that can be used to inhibit LPA (Apo(a)) protein expression. [Background technology]

[0002] Lp(a) particles are heterogeneous low-density lipoprotein particles that are predominantly expressed in the liver (Witztum and Ginsberg, J Lipid Res. March 2016;57(3):336-9). They consist of apolipoprotein(a) (Apo(a) or Lp(a) is encoded by the LPA gene) linked to LDL-like particles via the ApoB polypeptide. Genetically defined high Lp(a) particle serum levels are unaffected by diet and exercise and are associated with an increased risk of developing cardiovascular disease through the associated atherosclerotic potential (Alonso et al., Journal of the American College of Cardiology Vol.63,No.19,2014). According to Diagnostic and Preventive Medicine, the level of Lp(a) particles in serum in patients is a highly prevalent, independent genetic risk factor for coronary heart disease and aortic valve stenosis (Saeedi and Frohlich Clinical Diabetes and Endocrinology (2016) 2:7). Analysis of Lp(a) levels in multiple studies suggests that high Lp(a) levels are an independent risk factor for cardiovascular disease, stroke, and other related conditions, including atherosclerotic stenosis. In addition, genome-wide association studies have also identified LPA as a genetic risk factor for diseases such as atherosclerotic stenosis. Significant reductions in cardiovascular events are observed when both Lp(a) and LDL levels are reduced using therapeutic lipoprotein phlebotomy in hyperlipidemic patients. Thus, there is a need for therapeutic agents and treatments related to these and other LPA-related diseases.

[0003] However, other than indirect standard general LDL-reducing measures, there is currently no approved specific Lp(a) particle reduction therapy.Therefore, there is currently a need for effective methods of treatment, prevention and reduction of the risk of suffering from the following and conditions associated with: Berge's disease, peripheral artery disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic stenosis, aortic regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperapolipoprotein betalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease and venous thrombosis, stroke, atherosclerosis, thrombosis, coronary heart disease or aortic stenosis and / or any other disease or condition associated with elevated levels of Lp(a)-containing particles and other related conditions, conditions or syndromes yet to be identified.The present invention addresses this unmet medical need. Summary of the Invention

[0004] According to aspects of the invention, a double-stranded ribonucleic acid (dsRNA) agent is provided that inhibits LPA (Apo(a)) expression, the dsRNA agent comprising a sense strand and an antisense strand, and optionally comprising a targeting ligand. The region complementary to the LPA RNA transcript is comprised in nucleotide positions 2-18 in the antisense strand, the complementary region comprising at least 15 contiguous nucleotides that differ from one of the antisense sequences listed in Tables 1-3 by 0, 1, 2 or 3 nucleotides. In some embodiments, the region complementary to the LPA RNA transcript comprises at least 15, 16, 17, 18 or 19 contiguous nucleotides that differ from one of the antisense sequences listed in Tables 1-3 by 3 or less nucleotides. In certain embodiments, the antisense strand of the dsRNA is at least substantially complementary to any target region in the mRNA of the human LPA gene and provided in one of Tables 1-3. In some embodiments, the antisense strand of the dsRNA is fully complementary to any target region in the mRNA of the human LPA gene and is provided in one of Tables 1-3. In some embodiments, the dsRNA agent comprises any of the sense strand sequences listed in Tables 1-3, where the sense strand sequence is at least substantially complementary to the antisense strand sequence in the dsRNA agent. In certain embodiments, the dsRNA agent comprises any of the sense strand sequences listed in Tables 1-3, where the sense strand sequence is fully complementary to the antisense strand sequence in the dsRNA agent. In some embodiments, the dsRNA agent comprises any of the antisense strand sequences listed in Tables 1-3. In some embodiments, the dsRNA agent comprises any of the sequences listed as duplex sequences in Tables 1-3. In some embodiments, a dsRNA agent comprises a sense strand that differs from Formula (A) by 0, 1, 2 or 3 nucleotides: 5'-Z1GUUAUCGAGGCACAUAZ2-3' Formula (A), where Z1 is a nucleotide sequence comprising 0-15 nucleotide motifs and Z2 is selected from one of A, U, C and G or is absent. In certain embodiments, Z2 is A.In some embodiments, the Z1 nucleotide sequence is selected from one of the following motifs: A, AA, UA, GA, CA, AGA, UGA, GGA, CGA, UAGA, CAGA, AAGA, ACAGA, GACAGA, GGACAGA, UGGACAGA, AUGGACAGA, AAUGGACAGA, UAAUGGACAGA, GUAAUGGACAGA, GGUAAUGGACAGA, UGGUAAUGGACAGA, and AUGGUAAUGGACAGA. In some embodiments, Z1 is a nucleotide sequence that includes one, two, three, or four nucleotide motifs selected from the following motifs: A, AA, UA, GA, CA, AGA, UGA, GGA, CGA, UAGA, CAGA, AAGA, and ACAGA. In some embodiments, the dsRNA agent comprises an antisense strand that differs from Formula (B) by 0, 1, 2 or 3 nucleotides: 5'-Z3UAUGUGCCUCGAUAACZ4-3' Formula (B), where Z3 is selected from one of A, U, C and G or is absent, and Z4 is a nucleotide sequence comprising 0-15 nucleotide motifs. In certain embodiments, Z3 is U. In some embodiments, the Z4 nucleotide sequence is selected from the following motifs: U, UU, UA, UC, UG, UCU, UCA, UCC, UCG, UCUC, UCUA, UCUG, UCUU, UCUGU, UCUGUC, UCUCUU, UCUCGA, UCUGUCC, UCUGUCCA, UCUGUCCAU, UCUGUCCAU, UCUGUCCAUU, UCUGUCCAUUA, UCUGUCCAUUAC, UCUGUCCAUUACC, UCUGUCCAUUACCA, and UCUGUCCAUUACCAU, or is absent. In some embodiments, Z4 is a nucleotide sequence that comprises one, two, three, or four nucleotide motifs selected from the following motifs: U, UU, UA, UC, UG, UCU, UCA, UCC, UCG, UCUC, UCUA, UCUG, and UCUU. In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand that comprise nucleotide sequences described herein that differ from Formula (A) and Formula (B) by 0, 1, 2, or 3 nucleotides, respectively, and optionally comprises a targeting ligand.In certain embodiments, the length of each of the sense strand (A) and antisense strand (B) of the dsRNA agent does not exceed 35 nucleotides. In certain embodiments, the Z1 and Z4 nucleotide motifs are fully or partially complementary. In certain embodiments, the Z2 and Z3 nucleotide motifs are fully or partially complementary. In certain embodiments, the sense strand is complementary or substantially complementary to the antisense strand, and the length of the complementary region is 16-23 nucleotides. In some embodiments, the complementary region is 19-21 nucleotides long. In some embodiments, the length of the sense strand does not exceed 35 nucleotides and includes a region complementary to the antisense strand that includes at least 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the dsRNA agent comprises a sense strand that differs from Formula (C) by 0, 1, 2 or 3 nucleotides: 5'-Z5CCAAGCUUGGUCAUCUZ6-3' Formula (C), where Z5 is a nucleotide sequence comprising 0-15 nucleotide motifs and Z6 is selected from one of A, U, C and G or is absent. In particular embodiments, Z6 is A. In some embodiments, the Z5 nucleotide sequence is selected from one of the following motifs: G, AG, UG, GG, CG, AUG, UUG, GUG, CUG, UUUG, CUUG, AUUG, ACUUG, AACUUG, GAACUUG, AGAACUUG, AAGAACUUG, GAAGAACUUG, GGAAGAACUUG, AGGAAGAACUUG, CAGGAAGAACUUG, ACAGGAAGAACUUG and CACAGGAAGAACUUG. In some embodiments, Z5 is a nucleotide sequence that includes 1, 2, 3, or 4 nucleotide motifs selected from the following motifs: G, AG, UG, GG, CG, AUG, UUG, GUG, CUG, UUUG, CUUG, and AUUG. In some embodiments, the dsRNA agent includes an antisense strand that differs from Formula (D) by 0, 1, 2, or 3 nucleotides: 5'-Z7AGAUGACCAAGCUUGGZ8-3' Formula (D), where Z7 is selected from one of A, U, C, and G or is absent, and Z8 is a nucleotide sequence that includes 0-15 nucleotide motifs.In certain embodiments, Z7 is U. In some embodiments, the Z8 nucleotide sequence is selected from the following motifs: C, CU, CA, CC, CG, CAU, CAA, CAC, CAG, CAAC, CAAA, CAAG, CAAU, CAAGU, CAAGUU, CAACUU, CAACGA, CAAGUUC, CAAGUUCU, CAAGUUCUU, CAAGUUCUUC, CAAGUUCUUCC, CAAGUUCUUCCU, CAAGUUCUUCCUG, CAAGUUCUUCCUGU and CAAGUUCUUCCUGUG, or is absent. In some embodiments, Z8 is a nucleotide sequence that comprises one, two, three or four nucleotide motifs selected from the following motifs: C, CU, CA, CC, CG, CAU, CAA, CAC, CAG, CAAC, CAAA, CAAG and CAAU. In some embodiments, a dsRNA agent comprises a sense strand and an antisense strand comprising nucleotide sequences described herein that differ from Formula (C) and Formula (D) by 0, 1, 2, or 3 nucleotides, respectively, and optionally comprises a targeting ligand. In certain embodiments, the length of each of the sense strand (C) and antisense strand (D) of a dsRNA agent does not exceed 35 nucleotides. In certain embodiments, the Z5 and Z8 nucleotide motifs are fully or partially complementary. In certain embodiments, the Z6 and Z7 nucleotide motifs are fully or partially complementary. In certain embodiments, the sense strand is complementary or substantially complementary to the antisense strand, and the length of the region of complementarity is 16-23 nucleotides. In some embodiments, the region of complementarity is 19-21 nucleotides long. In some embodiments, the length of the sense strand does not exceed 35 nucleotides and includes a region of complementarity to the antisense strand that includes at least 15, 16, 17, 18, or 19 nucleotides. In some embodiments, a dsRNA agent includes a sense strand that differs from formula (E) by 0, 1, 2 or 3 nucleotides: 5'-Z9GACAGAGUUAUCGAGGZ. 10 -3' Formula (E) (wherein Z9 is a nucleotide sequence containing 0 to 15 nucleotide motifs, Z 10is selected from one of A, U, C and G, or is absent. In certain embodiments, Z 10 is A. In some embodiments, the Z9 nucleotide sequence is selected from one of the following motifs: G, AG, UG, GG, CG, AUG, UUG, GUG, CUG, CAUG, UAUG, GAUG, AAUG, UGAUG, GUGAUG, GGUGAUG, UGGUGAUG, AUGGUGAUG, CAUGGUGAUG, CCAUGGUGAUG, ACCAUGGUGAUG, UACCAUGGUGAUG, CUACCAUGGUGAUG, and GCUACCAUGGUGAUG. In some embodiments, Z9 is a nucleotide sequence that includes 1, 2, 3, or 4 nucleotide motifs selected from the following motifs: G, AG, UG, GG, CG, AUG, UUG, GUG, CUG, CAUG, UAUG, GAUG, and AAUG. In some embodiments, the dsRNA agent includes an antisense strand that differs from formula (F) by 0, 1, 2, or 3 nucleotides: 5'-Z 11 CCUCGAUAACUCUGUCZ 12 -3' Formula (F) (where Z 11 is selected from one of A, U, C and G or is absent; Z 12 is a nucleotide sequence containing 0 to 15 nucleotide motifs. 11 is U. In some embodiments, Z 12 The nucleotide sequence is selected from the following motifs: C, CU, CA, CC, CG, CAU, CAA, CAC, CAG, CAUA, CAUG, CAUC, CAUU, CAUCA, CAUCAC, CAUGUU, CAUGGA, CAUCACC, CAUCACCA, CAUCACCAU, CAUCACCAUG, CAUCACCAUGG, CAUCACCAUGGU, CAUCACCAUGGUA, CAUCACCAUGGUAG, and CAUCACCAUGGUAGC. 12is a nucleotide sequence that includes one, two, three, or four nucleotide motifs selected from the following motifs: C, CU, CA, CC, CG, CAU, CAA, CAC, CAG, CAUA, CAUG, CAUC, and CAUU. In some embodiments, the dsRNA agent includes a nucleotide sequence described herein that differs from Formula (E) and Formula (F) by zero, one, two, or three nucleotides, respectively. In certain embodiments, the sense strand (F) and the antisense strand (F) of the dsRNA agent each have a length of no more than 35 nucleotides. In certain embodiments, the Z and Z 12 The nucleotide motifs are fully or partially complementary. In certain embodiments, Z 10 and Z 11 The nucleotide motifs are fully or partially complementary. In certain embodiments, the sense strand is complementary or substantially complementary to the antisense strand, and the length of the complementary region is 16-23 nucleotides. In some embodiments, the complementary region is 19-21 nucleotides long. In some embodiments, the length of the sense strand does not exceed 35 nucleotides, and includes a region of complementarity to the antisense strand that includes at least 15, 16, 17, 18, or 19 nucleotides.

[0005] In some embodiments, the dsRNA agent comprises at least one modified nucleotide. In certain embodiments, all or substantially all nucleotides of the antisense strand are modified nucleotides. In some embodiments, the at least one modified nucleotide includes 2'-O-methyl nucleotide, 2'-fluoro nucleotide, 2'-deoxy nucleotide, 2',3'-seco nucleotide mimic, locked nucleotide, non-locked nucleic acid (UNA) nucleotide, glycol nucleic acid nucleotide (GNA), 2'-F-arabinose nucleotide, 2'-methoxyethyl nucleotide, abasic nucleotide, ribitol, inverted nucleotide, inverted abasic nucleotide, inverted 2'-OMe nucleotide, inverted 2'-deoxy nucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholino nucleotide and 3'-OMe nucleotide, nucleotide containing 5'-phosphorothioate group or terminal nucleotide linked to cholesterol derivative or dodecanoic acid bisdecylamide group, 2'-amino modified nucleotide, phosphoramidate or nucleotide containing unnatural base. In some embodiments, the antisense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, and there are less than 6 modified nucleotides of 2'-fluoro nucleotides. In some embodiments, the antisense strand comprises 3 or 5 2'-fluoro nucleotides, and preferably, the antisense strand comprises 5 2'-fluoro nucleotides. In some embodiments, the sense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, and there are less than 4 modified nucleotides of 2'-fluoro nucleotides. In certain embodiments, the sense strand comprises 3 2'-fluoro nucleotides.In some embodiments, the antisense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, at least 16 modified nucleotides are 2'-O-methyl nucleotides, and positions 2, 7, 12, 14 and / or 16 at the 5' end of the antisense strand are 2'-fluoro nucleotide modified nucleotides (counting from the first paired nucleotide at the 5' end of the antisense strand). In some embodiments, the sense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, at least 18 modified nucleotides are 2'-O-methyl nucleotides, and positions 9, 11 and / or 13 at the 3' end of the sense strand are 2'-fluoro nucleotide modified nucleotides (counting from the first paired nucleotide at the 3' end of the sense strand). In some embodiments, the antisense strand comprises 2'-fluoro modified nucleotides at positions 2, 7, 12, 14, and 16 of the antisense strand in a 5' to 3' direction, counting from the first paired nucleotide at the 5' end of the antisense strand, and each other nucleotide position in each antisense strand is independently a non-fluoro modified nucleotide. In some embodiments, the antisense strand comprises 2'-fluoro modified nucleotides at positions 2, 5, 12, 14, and 18 of the antisense strand in a 5' to 3' direction, counting from the first paired nucleotide at the 5' end of the antisense strand, and each other nucleotide position in the antisense strand is independently a non-fluoro modified nucleotide. In some embodiments, the sense strand comprises 2'-fluoro modified nucleotides at positions 9, 11, and 13 of the sense strand in a 3' to 5' direction, counting from the first paired nucleotide at the 3' end of the sense strand, and each other nucleotide position in the sense strand is independently a non-fluoro modified nucleotide. In some embodiments, the dsRNA agent comprises an E-vinyl phosphonate nucleotide at the 5'-end of the guide strand. In certain embodiments, the dsRNA agent comprises at least one phosphorothioate internucleoside linkage.In certain embodiments, the sense strand comprises at least one phosphorothioate internucleoside linkage. In some embodiments, the antisense strand comprises at least one phosphorothioate internucleoside linkage. In some embodiments, the sense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages. In certain embodiments, all or substantially all nucleotides of the sense and antisense strands are modified nucleotides. In some embodiments, the modified sense strand is a modified sense strand sequence listed in Tables 2-3. In some embodiments, the modified antisense strand is a modified antisense strand sequence listed in Tables 2-3. In certain embodiments, the sense strand is complementary or substantially complementary to the antisense strand, and the length of the complementary region is 16-23 nucleotides. In some embodiments, the complementary region is 19-21 nucleotides in length. In some embodiments, the complementary region is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long. In some embodiments, each strand is 40 or less nucleotides long. In some embodiments, each strand is 30 or less nucleotides long. In some embodiments, each strand is 25 or less nucleotides long. In some embodiments, each strand is 23 or less nucleotides long. In certain embodiments, the dsRNA agent comprises at least one modified nucleotide and further comprises one or more targeting groups or linking groups. In some embodiments, the one or more targeting groups or linking groups are conjugated to the sense strand. In some embodiments, the targeting group or linking group comprises N-acetyl-galactosamine (GalNAc). In some embodiments, the targeting portion of the targeting group is the following structural fragment: [ka] (p is 1 or 2).

[0006] In some embodiments, the targeting group has the following structure: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] has.

[0007] In certain embodiments, the dsRNA agent comprises a targeting group conjugated to the 5' end of the sense strand. In some embodiments, the dsRNA agent comprises a targeting group conjugated to the 3' end of the sense strand. In some embodiments, the antisense strand comprises one inverted abasic residue at the 3' end. In certain embodiments, the sense strand comprises one or two inverted abasic residues at the 3' end or / and 5' end. In certain embodiments, the sense strand comprises one or two isomannitol residues at the 3' end or / and 5' end. In certain embodiments, the sense strand comprises, independently, one isomannitol residue at each of the 3' end and 5' end. In some embodiments, the sense strand comprises, independently, one isomannitol residue at each of the 3' end and 5' end, and further comprises a targeting group conjugated to the 5' end, preferably GLS-15 as described above. In some embodiments, the dsRNA agent has two blunt ends. In some embodiments, at least one strand comprises a 3' overhang having at least one nucleotide. In some embodiments, at least one strand includes a 3' overhang having at least two nucleotides.

[0008] In certain embodiments, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting LPA (Apo(a)) expression comprises a sense strand and an antisense strand, wherein nucleotide positions 2-18 in the antisense strand comprise a region complementary to an LPA RNA transcript, and the antisense strand is fully or partially complementary to the sense strand, the agent optionally comprises a targeting ligand, each strand is 14-30 nucleotides in length, and the sense strand sequence is represented by Formula (I): 5'-(N' L ) n’ N' L N' L N' L N' N1 N' N2 N' F N' L N' F N' L N' N3 N' N4 N' L N'L N' L N' L N' L N' L (N' L ) m’ -3'(I) (In the formula, each N' F represents a 2'-fluoro modified nucleotide; N' N1 , N' N2 , N' N3 and N' N4 each independently represents a modified or unmodified nucleotide; L may be represented by the formula: independently, a modified or unmodified nucleotide, but does not represent a 2'-fluoro modified nucleotide, n' is an integer from 0 to 7, and m' is an integer from 0 to 3. In some embodiments, each N' N3 represents a 2'-fluoro modified nucleotide, and N' N1 , N' N2 and N' N4 represents independently a modified or unmodified nucleotide, but does not represent a 2'-fluoro modified nucleotide, and m is 1. In some embodiments, each N' N4 represents a 2'-fluoro modified nucleotide, and N' N1 , N' N2 and N' N3 independently represent a modified or unmodified nucleotide, but does not represent a 2'-fluoro modified nucleotide, and m is 1. In some embodiments, n' is 3 and m' is 1; or n' is 0 and m' is 0; or n' is 3 and m' is 3. In certain embodiments, there are only three 2'-fluoro modified nucleotides in Formula (I).

[0009] In a particular embodiment, the present invention relates to non-locked nucleic acid (UNA) oligomers for therapeutic use. Non-locked nucleic acids (UNA) are acyclic analogs of RNA, in which the bond between the C2' and C3' atoms of the ribose ring is broken. It has been demonstrated that the incorporation of UNA is well tolerated and in some cases even enhances siRNA gene silencing activity (Meghan A. et al. "Locked vs. unlocked nucleic acids (LNA vs. UNA): contrasting structures work towards common therapeutic goals". Chem. Soc. Rev., 2011, 40, 5680-5689).

[0010] UNA is a heat-labile modification, and substitution of ribonucleotides with UNA reduces pairing strength and duplex stability. Strategically placing UNA in the seed region of siRNA antisense strand reduces off-target activity in the mechanism of microRNA (miRNA)-mediated gene silencing. miRNAs primarily identify target genes by base pairing between the antisense seed region (positions 2-8 starting from the 5' end) and the target mRNA for gene suppression. Each miRNA potentially regulates a large number of genes. siRNA antisense strands loaded by the RNA-induced silencing complex (RISC) can also potentially regulate a large number of unintended genes through the miRNA-mediated mechanism. Thus, incorporation of heat-labile nucleotides such as UNA in the seed region of siRNA can reduce off-target activity (Lam JK, Chow MY, Zhang Y, Leung SW. siRNA Versus miRNA as Therapeutics for Gene Silencing. Mol Ther Nucleic Acids. 2015 Sep 15; 4(9): e252. doi: 10.1038 / mtna. 2015.23. PMID: 26372022; PMCID: PMC4877448.). In particular, such RNA oligonucleotides or RNA oligonucleotide complexes contain at least one UNA nucleotide monomer in the seed region (Narendra Vaish et al. "Improved specificity of gene silencing by siRNAs containing unlocked nucleobase analog". Nucleic Acids Research, 2011, Vol. 39, No. 5 1823-1832).

[0011] According to the technical solution of the present invention, the potential advantages of incorporating UNA into RNA oligonucleotides or RNA oligonucleotide complexes include, but are not limited to: 1. Off-target activity is reduced. The addition of UNA in the siRNA seed region reduces the base pairing strength at the seed region, thereby reducing potential off-target activity mediated by the microRNA machinery. 2. UNA was well tolerated in terms of siRNA activity. In some cases, UNA can result in enhanced activity.

[0012] Exemplary UNA monomers that can be used in the technical solution of the present invention include: [ka] These include, but are not limited to:

[0013] According to one aspect of the present invention, a composition is provided that comprises any of the above-mentioned dsRNA agents of the present invention.In certain embodiments, the composition further comprises a pharma- ceutically acceptable carrier.In some embodiments, the composition further comprises one or more additional therapeutic agents, such as HMg Co-A reductase inhibitors (statins), ezetimibe, PCSK-9 inhibitors, CTEP inhibitors, ANGPTL3 targeting therapy, AGT targeting therapy, APOC3 targeting therapy and niacin or any combination thereof.In some embodiments, the composition is packaged in a kit, container, packaging, dispenser, pre-filled syringe or vial.In some embodiments, the composition is formulated for subcutaneous administration or formulated for intravenous (IV) administration.

[0014] According to another aspect of the invention, there is provided a cell comprising any of the above dsRNA agent embodiments of the invention. In some embodiments, the cell is a mammalian cell, and optionally a human cell.

[0015] According to another aspect of the present invention, a method for inhibiting LPA gene expression in a cell is provided, the method comprising (i) preparing a cell containing an effective amount of any of the above-mentioned dsRNA agents or compositions of the present invention. In certain embodiments, the method further comprises (ii) maintaining the prepared cell for a sufficient time to obtain degradation of the mRNA transcript of the LPA gene, thereby inhibiting the expression of the LPA gene in the cell. In some embodiments, the cell is in a subject, and the dsRNA agent is administered to the subject subcutaneously. In some embodiments, the cell is in a subject, and the dsRNA agent is administered to the subject via IV administration. In certain embodiments, the method further comprises evaluating the inhibitory effect on the LPA gene after administration of the dsRNA agent to the subject, the means for evaluation comprising (i) determining one or more physiological characteristics of the LPA-related disease or condition in the subject, and (ii) comparing the identified physiological characteristics with the baseline physiological characteristics of the LPA-related disease or condition before treatment and / or the physiological characteristics of a control of the LPA-related disease or condition, the comparison result indicating the presence or absence of inhibition of LPA gene expression in the subject. In some embodiments, the identified physiological characteristic is Lp(a) level in blood. A reduction in LPA level in blood indicates a reduction in LPA gene expression in the subject.

[0016] According to another aspect of the present invention, a method of inhibiting LPA gene expression in a subject is provided, the method comprising administering to the subject an effective amount of the above dsRNA embodiment or the above composition embodiment. In some embodiments, the dsRNA agent is administered to the subject subcutaneously. In certain embodiments, the dsRNA agent is administered to the subject via IV administration. In some embodiments, the method further comprises evaluating the inhibitory effect on the LPA gene after administration of the dsRNA agent, the means for evaluation comprising (i) determining one or more physiological characteristics of the LPA-related disease or condition in the subject, and (ii) comparing the identified physiological characteristics with the baseline physiological characteristics of the LPA-related disease or condition before treatment and / or the physiological characteristics of a control of the LPA-related disease or condition, the comparison result indicating the presence or absence of inhibition of LPA gene expression in the subject. In some embodiments, the identified physiological characteristic is the Lp(a) level in blood. A reduction in the LPA level in blood indicates a reduction in LPA gene expression in the subject.

[0017] According to another aspect of the present invention, there is provided a method for treating disease or condition related to LPA protein, comprising administering to a subject an effective amount of any of the above-mentioned dsRNA agents of the present invention or any of the above-mentioned compositions of the present invention to inhibit LPA gene expression.In a particular embodiment, the LPA-related disorder is a cardiovascular disease, and the cardiovascular disease comprises Berge's disease, peripheral artery disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic stenosis, aortic regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperapolipoprotein betalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease and venous thrombosis, stroke, atherosclerosis, thrombosis, coronary heart disease or aortic stenosis and / or any other disease or condition associated with elevated levels of Lp(a)-containing particles. In some embodiments, the method further comprises administering an additional therapeutic regimen to the subject. In some embodiments, the additional therapeutic regimen comprises treating LPA-related disease or condition. In some embodiments, the additional therapeutic regimen comprises administering one or more LPA antisense polynucleotides of the present invention to the subject; administering a non-LPA dsRNA therapeutic agent to the subject; and effecting behavioral change in the subject. In some embodiments, the non-LPA dsRNA therapeutic agent is one of the additional therapeutic agents, such as HMg Co-A reductase inhibitors (statins), ezetimibe, PCSK-9 inhibitors, CTEP inhibitors, ANGPTL3 targeting therapy, APOC3 targeting therapy and niacin or any combination thereof.

[0018] In some embodiments, the dsRNA agent is administered to the subject subcutaneously. In certain embodiments, the dsRNA agent is administered to the subject via IV administration. In some embodiments, the method further comprises determining the effectiveness of the administered double-stranded ribonucleic acid (dsRNA) agent in the subject. In some embodiments, the means for determining the effectiveness of treatment in the subject comprises (i) determining one or more physiological characteristics of the LPA-related disease or condition in the subject; (ii) comparing the determined physiological profile to a baseline physiological characteristic of the LPA-related disease or condition before treatment, the comparison result indicating one or more of the presence, absence, and level of effectiveness of the double-stranded ribonucleic acid (dsRNA) agent administered to the subject. In some embodiments, the identified physiological characteristic is the Lp(a) level in blood. A reduction in the LPA level in blood indicates the presence of the effectiveness of the administration of the double-stranded ribonucleic acid (dsRNA) agent to the subject.

[0019] According to another aspect of the present invention, there is provided a method for reducing the LPA protein level in a subject, compared to the baseline level of LPA protein in the subject before treatment, comprising administering to the subject an effective amount of any of the above-mentioned dsRNA agent embodiments of the present invention or any of the above-mentioned composition embodiments of the present invention to reduce the level of LPA gene expression.In some embodiments, the dsRNA agent is administered to the subject subcutaneously or via IV.

[0020] According to another aspect of the present invention, there is provided a method for modifying the physiological characteristics of an LPA-related disease or condition in a subject, compared to the baseline physiological characteristics of the LPA-related disease or condition in the subject before treatment, comprising administering to the subject an effective amount of any of the above-mentioned dsRNA agent embodiments of the present invention or any of the above-mentioned compositions of the present invention to modify the physiological characteristics of the LPA-related disease or condition in the subject.In some embodiments, the dsRNA agent is administered to the subject subcutaneously or via IV.In certain embodiments, the physiological characteristics is the Lp(a) level in blood.

[0021] Array Description Duplexes AV00122-AD00484-1, AD00474-2, AV01867-AV01968 are shown in Table 1, with their sense strand sequences indicated.

[0022] The duplexes AV00122-AD00484-1, AD00474-2, AV01867-AV01968 are shown in Table 1, along with their antisense strand sequences.

[0023] In the sequences shown in Table 2, chemical modifications are indicated as follows: upper case: 2'-fluoro; lower case: 2'-OMe; phosphorothioate: *.

[0024] In the sequences shown in Table 3, the delivery molecules used in the in vivo studies are designated as "GLO-0" at the 3' end of each sense strand. The delivery molecules used in the in vivo studies are designated as "GLS-5" or "GLS-15" at the 5' end of each sense strand, with chemical modifications indicated as uppercase: 2'-fluoro; lowercase: 2'-OMe; phosphorothioate: *, and non-locked nucleic acid: UNA.

[0025] The following is the mRNA sequence of human Lp(a) (SEQ ID NO:1): NM_005577.4 Homo sapiens lipoprotein(a) (LPA), mRNA [ka] [ka] [ka] [ka] [Brief description of the drawings]

[0026] [Figure 1] FIG. 1 shows a schematic diagram of serum LPA protein levels in monkeys. [Diagram 2] 1 shows a schematic diagram of serum LPA protein levels of AD00480-8 at a dose of 2 mpk in monkeys. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Some embodiments of the invention include RNAi agents capable of inhibiting LPA (Apo(a)) gene expression, such as, but not limited to, double-stranded (ds) RNAi agents. Some embodiments of the invention further include compositions comprising LPA RNAi agents and methods of using the compositions. The LPA RNAi agents disclosed herein can be coupled to a delivery compound for delivery to cells, including delivery to hepatocytes. The pharmaceutical compositions of the invention can include at least one dsRNA agent and a delivery compound. In some embodiments of the invention, the delivery compound is a GalNAc-containing delivery compound. The LPA RNAi agent delivered to cells can inhibit LPA gene expression, thereby decreasing the LPA protein product of the gene. The dsRNAi agents of the invention can be used to treat LPA-related diseases and conditions. Such dsRNAi agents include, for example, the duplex AV00122-AD00484-1, AD00474-2, AV01867-AV01968 shown in Table 1. In other embodiments, such dsRNAi agents include duplex variants, such as variants of duplexes AV00122-AD00484-1, AD00474-2, and AV01867-AV01968.

[0028] In some embodiments of the present invention, by reducing LPA expression in cells or subjects, the disease or condition associated with LPA expression in cells or subjects is treated, respectively.Non-limiting examples of the disease and condition that can be treated by reducing LPA expression are cardiovascular disease, including Berge's disease, peripheral artery disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic stenosis, aortic regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperapolipoprotein betalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease, and venous thrombosis, stroke, atherosclerosis, thrombosis, coronary heart disease or aortic stenosis and / or any other disease or condition associated with elevated levels of Lp(a)-containing particles.

[0029] The methods of preparing and using compositions containing LPA single-stranded (ssRNA) and double-stranded (dsRNA) agents that inhibit LPA gene expression, as well as compositions and methods for treating diseases and conditions caused or regulated by LPA gene expression, are described below. The term "RNAi" is also known in the art and can be referred to as "siRNA".

[0030] As used herein, the term "RNAi" refers to agents that contain RNA and mediate targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. As known in the art, an RNAi target region refers to a contiguous portion of nucleotide sequence of an RNA molecule formed during gene transcription, including messenger RNA (mRNA), which is the processed product of the primary transcript RNA. The target portion of the sequence will be at least long enough to be used as a substrate for RNAi-directed cleavage at or near that portion. The target sequence may be 8-30 nucleotides (inclusive), 10-30 nucleotides (inclusive), 12-25 nucleotides (inclusive), 15-23 nucleotides (inclusive), 16-23 nucleotides (inclusive), or 18-23 nucleotides (inclusive), including all shorter lengths within each defined range. In some embodiments of the invention, the target sequence is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length. In certain embodiments, the length of the target sequence is 9-25 nucleotides (inclusive), including all subranges and integers therebetween. For example, and not intended to be limiting, in some embodiments of the invention, the target sequence is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length and is fully or at least substantially complementary to at least a portion of an RNA transcript of an LPA gene. Some aspects of the invention include pharmaceutical compositions comprising one or more LPA dsRNA agents and a pharma- ceutically acceptable carrier. In some embodiments of the present invention, LPA RNAi as described herein inhibits expression of the LPA protein.

[0031] As used herein, "dsRNA agent" refers to a composition that contains RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules that can degrade target mRNA transcripts or inhibit the translation of target mRNA transcripts.Without wishing to be limited to a particular theory, the dsRNA agent of the present invention can function by RNA interference mechanism (i.e., by interacting with the RNA interference pathway mechanism (RNA-induced silencing complex or RISC) in mammalian cells to induce the production of RNA interference) or any alternative mechanism or pathway. Methods for achieving gene silencing in plant, invertebrate and vertebrate cells are well known in the art (see, for example, Sharp et al., Genes Dev. 2001, 15:485; Bernstein, et al., (2001) Nature 409:363; Nykanen, et al., (2001) Cell 107:309; and Elbashir, et al., (2001) Genes Dev. 15:188), the disclosures of each of which are incorporated herein by reference in their entirety. Gene silencing means known in the art can be used in combination with the disclosure provided herein to achieve inhibition of expression of LPA.

[0032] The dsRNA agent disclosed herein is composed of a sense strand and an antisense strand, including, but not limited to, small interfering RNA (siRNA), RNAi agent, microRNA (miRNA), small hairpin RNA (shRNA) and dicer substrate.The antisense strand of the dsRNA agent described herein is at least partially complementary to the mRNA to be targeted, and it is understood in the art that dsRNA duplex structures of various lengths can be used to inhibit target gene expression.For example, dsRNA with duplex structures of 19, 20, 21, 22 and 23 base pairs are known to efficiently induce RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888).It is also known in the art that shorter or longer RNA duplex structures can also efficiently induce RNA interference. In certain embodiments of the invention, the LPA dsRNA may comprise at least one strand at least 21 nt in length, or the duplex may have a length minus 1, 2, 3 nt or less based on the length of any one of the sequences listed in Tables 1-3. Reducing four nucleotides at one or both ends of the dsRNA may also be effective compared to the dsRNAs listed in Tables 1-3, respectively. In some embodiments of the invention, the LPA dsRNA agent may have a subsequence of at least 15, 16, 17, 18, 19, 20 or more contiguous nucleotides from one or more sequences in Tables 1-3, and their ability to inhibit LPA gene expression differs by only 5%, 10%, 15%, 20%, 25% or 30% from the level of inhibition provided by a dsRNA comprising the complete sequence (also referred to herein as the "parent" sequence).

[0033] Certain embodiments of the compositions and methods of the invention include administering single-stranded RNA and / or single-stranded RNA in a composition to a subject. For example, the antisense strands listed in any of Tables 1-3 can be used as or in compositions that, when administered to a subject, reduce expression of an LPA polypeptide and / or an LPA gene in the subject. Tables 1-3 show the core extension base sequences of the antisense and sense strands of several LPA dsRNA agents. Single-stranded antisense molecules that may be included in certain compositions of the invention and / or administered in certain methods of the invention are referred to herein as "single-stranded antisense agents" or "antisense polynucleotide agents." Single-stranded sense molecules that may be included in certain compositions of the invention and / or administered in certain methods of the invention are referred to herein as "single-stranded sense agents" or "sense polynucleotide agents." The term "base sequence" is used herein to refer to a polynucleotide sequence without chemical modifications or delivery compounds. For example, the sense strands shown in Table 1 correspond to the corresponding base sequences in Table 3; however, the respective chemical modifications and delivery compounds are shown in the corresponding sequences in Table 3. The sequences disclosed herein may be assigned identifiers. For example, a single-stranded sense sequence may be identified by "sense strand SS#"; a single-stranded antisense sequence may be identified by "antisense strand AS#"; a duplex comprising a sense strand and an antisense strand may be identified by "duplex AD#".

[0034] Table 1 includes both the sense and antisense strands and provides identification numbers for the duplex formed by the sense and antisense strands in the same row of Table 1. In some embodiments of the invention, the antisense sequence contains either nucleobase u or nucleobase a at position 1 of the antisense sequence. In some embodiments of the invention, the antisense sequence contains nucleobase u at position 1 of the antisense sequence. As used herein, the term "matching position" in sense refers to the position in each strand that "pairs" with each other when the two strands form a duplex. For example, in a 21 nucleobase sense strand and a 21 nucleobase antisense strand, position 1 of the sense strand is in a "matching position" with the nucleobase at position 21 of the antisense strand. In another non-limiting example, for a 23 nucleobase sense strand and a 23 nucleobase antisense strand, the nucleobase at position 2 of the sense strand is in a "matching position" with the nucleobase at position 22 of the antisense strand. In another non-limiting example, in an 18 nucleobase sense strand and an 18 nucleobase antisense strand, the nucleobase at position 1 in the sense strand is in a matching position with the nucleobase at position 18 in the antisense strand; the nucleobase at position 4 in the sense strand is in a matching position with the nucleobase at position 15 in the antisense strand. One of skill in the art will understand how to identify matching positions between the sense and antisense strands of a duplex and paired strands.

[0035] The columns of Table 1 represent the duplex AV#, the duplex AD#, and contain both sense and antisense sequences in the same row of the table. For example, Table 1 discloses a duplex designated as "duplex AV00122" and contains the corresponding sense and antisense strand sequences. Thus, each row in Table 1 identifies a duplex of the invention, each duplex containing the sense and antisense sequences shown in the same row, and the designated identifier for each duplex is shown in the last column of that row.

[0036] In some embodiments of the methods of the invention, an RNAi agent comprising a polynucleotide sequence shown in Table 1 is administered to a subject. In some embodiments of the invention, the RNAi agent administered to a subject comprises a duplex that comprises at least one of the base sequences listed in Table 1 and that comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 sequence modifications. Some embodiments of the methods of the invention also include conjugating an RNAi agent of a polynucleotide sequence shown in Table 1 to a delivery molecule, the non-limiting example of which is a GalNAc-containing delivery compound.

[0037] [Table 1]

[0038] [Table 2]

[0039] [Table 3]

[0040] [Table 4]

[0041] [Table 5]

[0042] [Table 6]

[0043] [Table 7]

[0044] [Table 8]

[0045]

Table 9

[0046]

Table 10

[0047]

Table 11

[0048]

Table 12

[0049]

Table 13

[0050]

Table 14

[0051]

Table 15

[0052]

Table 16

[0053] Table 2 shows the antisense and sense strand sequences of certain chemically modified LPA RNAi agents of the invention. In some embodiments of the methods of the invention, an RNAi agent having a polynucleotide sequence shown in Table 2 is administered to a cell and / or subject. In some embodiments of the methods of the invention, an RNAi agent having a polynucleotide sequence shown in Table 2 is administered to a subject. In some embodiments of the invention, the RNAi agent administered to a subject comprises a duplex labeled in the first column of Table 2 and a sequence modification in the sense and antisense strand sequences shown in the third and sixth columns of the same row of Table 2, respectively. In some embodiments of the methods of the invention, the sequences shown in Table 2 can be conjugated (also referred to herein as "conjugated") to a compound capable of delivering the RNAi agent to cells and / or tissues of a subject. A non-limiting example of a delivery compound that can be used in certain embodiments of the invention is a GalNAc-containing compound. In Table 2, the first column represents the duplex AD# of base sequences corresponding to Table 1. The base sequence identified by duplex AD# not only indicates the base sequence contained in the sense strand and the antisense strand, but also has the designated chemical modification shown in the same row of Table 2. For example, the first row of Table 1 indicates the sense and antisense base single strand sequences, which together form a duplex identified as duplex AV00122; duplex AV00122, listed in Table 2 as a duplex, contains the base sequences AV00122-SS and AV00122-AS, which contain the chemical modifications in the sense and antisense sequences shown in columns 3 and 6, respectively. The "sense strand SS#" in column 2 of Table 2 is the designated identifier of the sense sequence (including modifications) shown in column 3 of the same row. The "antisense strand AS#" in column 5 of Table 2 is the designated identifier of the antisense sequence (including modifications) shown in column 6.

[0054] [Table 17]

[0055] [Table 18]

[0056]

Table 19

[0057]

Table 20

[0058]

Table 21

[0059]

Table 22

[0060]

Table 23

[0061]

Table 24

[0062]

Table 25

[0063]

Table 26

[0064]

Table 27

[0065]

Table 28

[0066]

Table 29

[0067] Table 3 shows the antisense and sense strand sequences of certain chemically modified LPA RNAi agents of the invention. In some embodiments of the methods of the invention, the RNAi agents shown in Table 3 are administered to cells and / or subjects. In some embodiments of the methods of the invention, the RNAi agents having polynucleotide sequences shown in Table 3 are administered to subjects. In some embodiments of the invention, the RNAi agents administered to the subject comprise the duplexes identified in the first column of Table 3, and include the sequence modifications and / or delivery compounds shown in the sense and antisense strand sequences in the third and sixth columns of the same row of Table 3, respectively. This sequence is used in some in vivo testing studies described elsewhere herein. In some embodiments of the methods of the invention, the sequences shown in Table 3 can be linked (also referred to herein as "conjugated") to a compound for delivery (a non-limiting example is a GalNAc-containing compound, i.e., having a compound for delivery labeled "GLX-n" on the sense strand in the third column of Table 3). As used herein and as shown in Table 3, "GLX-n" is used to denote a linked GalNAc-containing compound, which is any of the compounds GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16, the structures of each of which are provided elsewhere herein. The first column of Table 3 provides the duplex AD# assigned to the duplex of the sense and antisense sequences in a row of the table. For example, duplex AD00122 is a duplex composed of sense strand AD00122-SS and antisense strand AD00122-AS. Each row of Table 3 provides the sense and antisense strands and discloses the duplex formed by the indicated sense and antisense strands. The "Sense Strand SS#" in the second column of Table 3 is the designated identifier of the sense sequence (including modifications) shown in the third column of the same row.The "Antisense strand AS#" in column 5 of Table 3 is the designated identifier of the antisense sequence (including modifications) shown in column 6. The particular identifier of the linked GalNAc-containing GLO compound is designated GLO-0, and it should be understood that another GLO-n or GLS-n compound can be substituted for the compound designated GLO-0, and the resulting compound is still included in the embodiments of the methods and / or compositions of the invention.

[0068] Table 3 provides antisense and sense strand sequences of chemically modified LPA RNAi agents for in vivo testing. All sequences are shown 5' to 3'. These sequences are used in several in vivo testing studies described elsewhere herein. Delivery molecules used in the in vivo studies are designated as "GLO-0" at the 3' end of each sense strand. Delivery molecules used in the in vivo studies are designated as "GLS-5" or "GLS-15" at the 5' end of each sense strand. Chemical modifications are: uppercase: 2'-fluoro; lowercase: 2'-OMe; phosphorothioate: *; non-locked nucleic acid: UNA; invab = inverted abasic; imann: if at the end of each strand: [ka] Or when further coupled to a delivery molecule: [ka]

[0069] [Table 30]

[0070] [Table 31]

[0071] [Table 32]

[0072] [Table 33]

[0073] mismatch It is known to those skilled in the art that mismatches are tolerated in terms of the efficacy of dsRNA, especially when the mismatch is in the terminal region of the dsRNA. Some mismatches, such as mismatches with wobble base pairs G:U and A:C, are better tolerated and better tolerated in terms of efficacy (Du et al., A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites. Nucleic Acids Res. 2005 Mar 21; 33(5): 1671-7. Doi: 10.1093 / nar / gki312. Nucleic Acids Res. 2005; 33(11): 3698). In some embodiments of the methods and compounds of the present invention, the LPA dsRNA agent may contain one or more mismatches to the LPA target sequence. In some embodiments, the LPA dsRNA agent of the present invention does not contain a mismatch. In certain embodiments, the LPA dsRNA agent of the present invention contains no more than one mismatch. In some embodiments, the LPA dsRNA agent of the present invention comprises no more than two mismatches. In certain embodiments, the LPA dsRNA agent of the present invention comprises no more than three mismatches. In some embodiments of the present invention, the antisense strand of the LPA dsRNA agent comprises a mismatch to the LPA target sequence that is not in the center of the complementary region. In some embodiments, the antisense strand of the LPA dsRNA agent comprises 1, 2, 3, 4 or more mismatches located within the last 5, 4, 3, 2 or 1 nucleotide of either or both of the 5' end or 3' end of the complementary region. The methods described herein and / or known in the art can be used to determine whether the LPA dsRNA agent that comprises a mismatch to the LPA target sequence is effective in inhibiting LPA gene expression.

[0074] Complementarity As used herein, unless otherwise specified, the term "complementary" when used to describe the relationship of a first nucleotide sequence (e.g., an LPA dsRNA agent sense strand or a target LPA mRNA) and a second nucleotide sequence (e.g., an LPA dsRNA agent antisense strand or a single-stranded antisense polynucleotide) refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize [forming hydrogen bonds between base pairs under mammalian physiological conditions (or similar conditions in vitro)] with an oligonucleotide or polynucleotide comprising a second nucleotide sequence and form a duplex or double helix structure under certain conditions. Other conditions, such as physiologically relevant conditions that may occur in an organism, may also be applied. A skilled artisan will be able to determine the optimal set of conditions for testing the complementarity of two sequences based on the ultimate application of the hybridized nucleotides. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, and include natural or modified nucleotides or nucleotide mimetics, so long as they are at least as extensive as described above for hybridization. The sequence identity or complementarity is independent of the modification.

[0075] For example, a complementary sequence in an LPA dsRNA as described herein includes base pairing between an oligonucleotide or polynucleotide containing a first nucleotide and an oligonucleotide or polynucleotide containing a second nucleotide sequence over the entire length of one or two nucleotide sequences. Such sequences may be referred to herein as "fully complementary" to each other. In embodiments in which two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, it will be recognized that such overhangs are not considered mismatches as determined herein based on complementarity. For example, an LPA dsRNA agent may include an oligonucleotide that is 19 nucleotides long and another oligonucleotide that is 20 nucleotides long, where the longer oligonucleotide contains a 19 nucleotide sequence that is fully complementary to the shorter oligonucleotide, and may be referred to as "fully complementary" for the purposes described herein. Thus, as used herein, "fully complementary" means that all (100%) bases in a contiguous sequence of a first polynucleotide hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. The contiguous sequence can include all or a portion of the first or second nucleotide sequence.

[0076] As used herein, the term "substantially complementary" means that in a hybrid pair of nucleic acid base sequences, at least about 85% (but not all) of the bases in a contiguous sequence of a first polynucleotide hybridize with the same number of bases in a contiguous sequence of a second polynucleotide.When two sequences contain one or more mismatched base pairs, such as at least 1, 2, 3, 4 or 5 mismatched base pairs during hybridization, the term "substantially complementary" can be used to refer to the first sequence forming a duplex of up to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs (bp) with the second sequence, while retaining the ability to hybridize under the conditions most suitable for its final application, such as the inhibition of LPA gene expression by the RISC pathway. The term "partially complementary" may be used herein to mean that in a hybrid pair of nucleobase sequences, at least 75% (but not all) of the bases in a contiguous sequence of a first polynucleotide hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. In some embodiments, "partially complementary" means that at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the bases in a contiguous sequence of a first polynucleotide hybridize with the same number of bases in a contiguous sequence of a second polynucleotide.

[0077] The terms "complementary," "fully complementary," "substantially complementary," and "partially complementary," as used herein, may be used to refer to base matches between the sense and antisense strands of an LPA dsRNA agent, base matches between the antisense strand of an LPA dsRNA agent and the sequence of a target LPA mRNA, or base matches between a single-stranded antisense oligonucleotide and the sequence of a target LPA mRNA. It will be understood that the term "antisense strand of an LPA dsRNA agent" may refer to the same sequence as an "LPA antisense polynucleotide agent."

[0078] As used herein, the term "substantially identical" or "substantial identity" when referring to a nucleic acid sequence means that the nucleic acid sequence comprises a sequence having at least 85% or more sequence identity compared to a reference sequence, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity. The percentage of sequence identity is determined by comparing the optimal alignment of the two sequences over an alignment window. The percentage is calculated by determining the number of positions at which the same nucleic acid base occurs in both sequences to obtain the number of matching positions; dividing the number of matching positions by the total number of positions in the alignment window and multiplying the result by 100 to arrive at the percentage of sequence identity. The invention disclosed herein includes nucleotide sequences that are substantially identical to those disclosed herein (e.g., in Tables 1-5). In some embodiments, the nucleotide sequence is completely identical or has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a sequence disclosed herein (e.g., in Tables 1-3).

[0079] As used herein, the term "sequence-containing strand" refers to an oligonucleotide containing a nucleotide strand described by a sequence shown using standard nucleotide nomenclature. As used herein, the term "double-stranded RNA" or "dsRNA" refers to a sequence comprising an RNA molecule or RNAi molecule complex having a hybrid double-stranded region that includes two antiparallel and substantially or completely complementary nucleic acid strands, each of which is referred to as having a "sense" and an "antisense" orientation relative to the target LPA RNA. The double-stranded region can have any desired length that allows for specific degradation of the target LPA RNA via the RISC pathway, but is typically 9-30 base pairs long, e.g., 15-30 base pairs long. Considering a duplex of 9-30 base pairs, the duplex may be any combination within this range, e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs and 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-2 ...30 base pairs, 18-40 base pairs, 18-42 base pairs, 18-44 base pairs, 18-46 base pairs, 18-48 base pairs, 18-49 base pairs, 18-50 base pairs, 18-51 base pairs, 18-52 base pairs, 18-53 base pairs, 18-54 base pairs, 18-55 base pairs, 18-56 base pairs, 18-57 base pairs, 18-58 base pairs, 18-59 base pairs, 19-60 base pairs, 20-61 base pairs, 20-62 base pairs, 20-63 base pairs, 20-64 base pairs, 20-65 base pairs, 20-66 base pairs, 20-67 base pairs, 20-6 The length of the LPA dsRNA agent may be any length within any subrange thereof, including but not limited to 21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs. The length of LPA dsRNA agents produced in cells by processing by Dicer and similar enzymes is generally in the range of 19-22 base pairs. One strand of the double-stranded region of the LPA dsDNA agent contains a sequence that is substantially complementary to a region of the target LPA RNA. The two strands forming the duplex structure can be derived from a single RNA molecule having at least one self-complementary region, or can be formed from two or more individual RNA molecules.When the double-stranded region is formed by a single molecule, the molecule may have a double-stranded structure (referred to herein as a "hairpin loop") formed by one strand of single-stranded nucleotides at the 3' end and the corresponding other strand at the 5' end. In some embodiments of the present invention, the hairpin conformation comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more unpaired nucleotides. When the essentially complementary two strands of an LPA dsRNA agent are composed of individual RNA molecules, these molecules do not have to be covalently linked, but may be covalently linked. When the two strands are covalently linked by means other than a hairpin loop, the linking structure is referred to as a "linker". The term "siRNA" is also used herein to refer to a dsRNA agent as described herein.

[0080] In some embodiments of the present invention, the LPA dsRNA agent may comprise sense and antisense sequences with unpaired nucleotides or nucleotide analogs at one or both ends of the dsRNA agent. The ends without unpaired nucleotides are called "blunt ends" and do not have nucleotide overhangs. If both ends of the dsRNA agent are blunt ends, the dsRNA is called "blunt-ended". In some embodiments of the present invention, the first end of the dsRNA agent is blunt-ended, in some embodiments, the second end of the dsRNA is blunt-ended, and in certain embodiments of the present invention, both ends of the LPA dsRNA agent are blunt-ended.

[0081] In some embodiments of the dsRNA agent of the present invention, the dsRNA does not have one or two blunt ends. In this case, there is at least one unpaired nucleotide at the end of one strand of the dsRNA agent. For example, a nucleotide overhang exists when the 3' end of one strand of the dsRNA extends beyond the 5' end of the other strand, or vice versa. The dsRNA may include an overhang of at least 1, 2, 3, 4, 5, 6 or more nucleotides. The nucleotide overhang may include or consist of a nucleotide / nucleoside analog, including a deoxynucleotide / nucleoside. In some embodiments, the nucleotide overhang exists at the sense strand of the dsRNA agent, the antisense strand of the dsRNA agent, or both ends of the dsRNA agent, and it should be understood that the nucleotides of the overhang may exist at the 5' end, 3' end, or both ends of the antisense strand or the sense strand of the dsRNA. In some embodiments of the present invention, one or more nucleotides in the overhang are replaced by nucleoside phosphorothioates.

[0082] As used herein, the term "antisense strand" or "guide strand" refers to the strand of an LPA dsRNA agent that contains a region that is substantially complementary to an LPA target sequence.As used herein, the term "sense strand" or "passenger strand" refers to the strand of an LPA dsRNA agent that contains a region that is substantially complementary to a region of the antisense strand of the LPA dsRNA agent.

[0083] qualification In some embodiments of the present invention, the RNA of the LPA RNAi agent is chemically modified to enhance stability and / or obtain one or more beneficial properties. The nucleic acids in certain embodiments of the present invention can be synthesized and / or modified by methods well known in the art. See, for example, "Current protocols in Nucleic Acid Chemistry," Beaucage, SLet al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications that may be present in certain embodiments of the LPA dsRNA agent of the present invention include (a) terminal modifications such as 5'-end modifications (phosphorylation, conjugation, reverse linkage, etc.), 3'-end modifications (conjugation, DNA nucleotide, reverse linkage, etc.); (b) base modifications, such as base substitutions, base deletions (abasic nucleotides) or base conjugation, for example, stabilized bases, destabilized bases or expanding the pool of partners for base pairing; (c) sugar modifications (e.g., at 2' or 4' position) or sugar substitutions; and (d) backbone modifications, including modifications or substitutions of phosphodiester bonds. Specific examples of RNA compounds that can be used in certain embodiments of the LPA dsRNA agent, LPA antisense polynucleotide and LPA sense polynucleotide of the present invention include, but are not limited to, RNAs containing modified backbones or RNAs without natural internucleoside linkages. As a non-limiting example, RNAs with backbone modifications may not have a phosphorus atom in the backbone. RNAs that do not have a phosphorus atom in their internucleoside backbone may be called oligonucleosides. In some embodiments of the invention, a modified RNA has a phosphorus atom in its internucleoside backbone.

[0084] The term "RNA molecule" or "RNA" or "ribonucleic acid molecule" should be understood to include not only RNA molecules expressed or found in nature, but also analogs and derivatives of RNA that contain one or more ribonucleotide / ribonucleoside analogs or derivatives as described herein or known in the art. The terms "ribonucleoside" and "ribonucleotide" are used interchangeably herein. RNA molecules may be modified in the nucleobase structure or the ribose-phosphate backbone structure (e.g., as described below), and molecules containing ribonucleoside analogs or derivatives must retain the ability to form duplexes. As non-limiting examples, the RNA molecule may also include at least one modified ribonucleoside, including, but not limited to, a 2'-O-methyl modified nucleoside, a nucleoside containing a 5' phosphorothioate group, a terminal nucleoside linked to a cholesterol derivative or a dodecanoic acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, a 2'-deoxy-2'-fluoro modified nucleoside, a 2'-amino modified nucleoside, a 2'-alkyl modified nucleoside, a morpholino nucleoside, a phosphoramidate or a non-natural base containing nucleoside, or any combination thereof. In some embodiments of the invention, an RNA molecule comprises the following number of modified ribonucleosides: at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or up to the full length of the LPA dsRNA agent molecule. For each of the multiple modified ribonucleosides in such an RNA molecule, the modifications need not be the same.

[0085] In some embodiments, the dsRNA agents, LPA antisense polynucleotides and / or LPA sense polynucleotides of the invention may include one or more independently selected modified nucleotides and / or one or more independently selected non-phosphodiester linkages. The term "independently selected" as used herein means selected elements, such as modified nucleotides, non-phosphodiester linkages, and is used to mean that two or more selected elements can be identical to each other, but need not be identical to each other. As used herein, a "nucleotide base", "nucleotide" or "nucleobase" is a heterocyclic pyrimidine or purine compound that is a standard building block of all nucleic acids and includes the nucleotide-forming bases: adenine (a), guanine (g), cytosine (c), thymine (t) and uracil (u). Nucleobases may be further modified to include, but are not intended to be limited to, universal bases, hydrophobic bases, ambiguous bases, size-expanded bases and fluorinated bases. The term "ribonucleotide" or "nucleotide" may be used herein to refer to unmodified nucleotides, modified nucleotides or alternative moieties. Those of skill in the art will recognize that guanine, cytosine, adenine and uracil may be replaced by other moieties without significantly altering the base pairing properties of oligonucleotides containing nucleotides having such substituted moieties.

[0086] In one embodiment, the modified RNA expected to be used in the methods and compositions described herein is peptide nucleic acid (PNA) that has the ability to form desired double-stranded structure and enable or mediate the specific degradation of target RNA via RISC pathway.In some embodiments of the present invention, LPA RNA interference agent comprises single-stranded RNA that interacts with target LPA RNA sequence to guide the cleavage of target LPA RNA.

[0087] Modified RNA backbones may include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates (including 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-amino phosphoramidates and aminoalkyl phosphoramidates), thionophosphoramidates, thionoalkyl phosphoramidates, thionoalkyl phosphotriesters and boranophosphates (including standard 3'-5' linkages and their 2'-5' linked analogs as well as those with inverted polarity, where adjacent nucleoside unit pairs are linked in a 3'-5' to 5'-3' or 2'-5' to 5'-2' configuration), etc. Various salts, mixed salts and free acid forms are also included. Methods for preparing phosphorus-containing linkages are conventional in the art, and such methods may be used to prepare the specific modified LPA dsRNA agents, specific modified LPA antisense polynucleotides and / or specific modified LPA sense polynucleotides of the invention.

[0088] Modified RNA backbones that do not contain phosphorus atoms include morpholine bonds (partially formed from the sugar moiety of nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and backbones formed by short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heteroatom or heterocyclic internucleoside linkages, including those with N, O, S and CH2 moieties mixed with other moieties.The method of preparing modified RNA backbones that do not contain phosphorus atoms is conventional in the art, and such methods can be used to prepare the specific modified LPA dsRNA agents, specific modified LPA antisense polynucleotides and / or specific modified LPA sense polynucleotides of the present invention.

[0089] In certain embodiments of the present invention, RNA mimics are included in LPA dsRNA, LPA antisense polynucleotide and / or LPA sense polynucleotide, for example, but not limited to, the sugar and internucleoside bond (i.e. backbone) of nucleotide units are replaced with new groups. In such embodiments, the base units are maintained to hybridize with suitable LPA nucleic acid target compounds. One such oligomeric compound that is an RNA mimic that has been shown to have excellent hybridization properties is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by an amide-containing backbone, particularly an aminoethylglycine backbone. Nucleic acid bases are retained and are directly or indirectly linked to the aza nitrogen atom of the amide portion of the backbone. Methods for preparing RNA mimics are routinely practiced in the art, and such methods can be used to prepare certain modified LPA dsRNA agents of the present invention.

[0090] Some embodiments of the present invention include RNA with phosphorothioate backbones and oligonucleosides with heteroatom backbones, particularly -CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [referred to as methylene (methylimino) or MMI backbones], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2-- and --N(CH3)--CH2---- [wherein the natural phosphodiester backbone is represented as --O--P--O--CH2--]. Methods for preparing RNA with phosphorothioate backbones and oligonucleotides with heteroatom backbones are routinely practiced in the art, and such methods can be used to prepare the specific modified LPA dsRNA agents, specific LPA antisense polynucleotides and / or specific LPA sense polynucleotides of the present invention.

[0091] Modified RNAs may also contain one or more substituted sugar moieties. The LPA dsRNA, LPA antisense polynucleotide and / or LPA sense polynucleotide of the invention may contain one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 (Alkenyl and alkynyl). Exemplary suitable modifications include O[(CH2) n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON [(CH2) n In another embodiment, the dsRNA includes one of the following at the 2' position: C1 to C3 10lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino; substituted silyl, RNA cleaving groups, reporter groups, intercalating agents; groups used to enhance the pharmacokinetic properties of an LPA dsRNA agent; or groups used to enhance an LPA dsRNA agent, groups to enhance the pharmacodynamic properties of an LPA dsRNA agent, an LPA antisense polynucleotide and / or an LPA sense polynucleotide, and other substituents with similar properties. In some embodiments, the modification comprises 2'-methoxyethoxy (2'-O-CH2CHOCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., alkoxy-alkoxy. Another exemplary modification is 2'-dimethylaminoethoxyethoxy, i.e., O(CH2)2ON(CH3)2, also referred to as 2'-DMAOE, as described in the examples below; and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2). The methods of preparing those modified RNAs described are routinely practiced in the art, and such methods can be used to prepare the specific modified LPA dsRNA agents of the present invention.

[0092] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can also be made in the LPA dsRNA agents of the present invention at other positions on the RNA of the LPA antisense polynucleotide, LPA sense polynucleotide and / or LPA sense polynucleotide, particularly at the 3' position of the sugar on the 3' terminal nucleotide or the 5' position of the 5' terminal nucleotide of the 2'-5' linked LPA dsRNA, LPA antisense polynucleotide or LPA sense polynucleotide. The LPA dsRNA agents, LPA antisense polynucleotide and / or LPA sense polynucleotide can have, for example, a sugar mimic in place of the cyclobutyl moiety of the pentofuranosyl. For example, methods for preparing those modified RNAs described are routinely practiced in the art, and such methods can be used to prepare the specific modified LPA dsRNA agents, LPA antisense polynucleotides and / or LPA sense polynucleotides of the present invention.

[0093] In some embodiments, LPA dsRNA agents, LPA antisense polynucleotides and / or LPA sense polynucleotides may include nucleobase (commonly referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), douracil), 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines; 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines; and other synthetic and natural nucleobases such as 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-azaguanine and 7-azaadenine and 3-azaguanine and 3-azaadenine.Additional nucleobases that may be included in certain embodiments of the LPA dsRNA agents of the invention are known in the art, see, for example, Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. Ed. Wiley-VCH, 2008; The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JL, Ed. John Wiley & Sons, 1990; English et al., Angewandte Chemie, International Edition, 1991, 30, 613, Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Methods for preparing dsRNAs, LPA antisense polynucleotides and / or LPA sense polynucleotides (such as those described herein) containing nucleobase modifications and / or substitutions are routinely practiced in the art, and such methods may be used to prepare certain modified LPA dsRNA agents, LPA sense polynucleotides and / or LPA antisense polynucleotides of the invention.

[0094] Certain embodiments of the LPA dsRNA agents, LPA antisense polynucleotides and / or LPA sense polynucleotides of the present invention include RNAs modified to include one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides with such modified ribose moieties that contain an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-internal conformation. The addition of a locked nucleic acid to the LPA dsRNA agent, LPA antisense polynucleotide and / or LPA sense polynucleotide of the present invention can increase stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Methods for preparing dsRNA agents, LPA antisense polynucleotides and / or LPA sense polynucleotides that include locked nucleic acids are routinely practiced in the art, and such methods can be used to prepare certain modified LPA dsRNA agents of the present invention. Particular embodiments of the LPA dsRNA compounds, sense polynucleotides and / or antisense polynucleotides of the present invention comprise at least one modified nucleotide, including 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-seconucleotide mimics, locked nucleotides, 2'-F-arabinose nucleotides, 2'-methoxyethyl nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides and 3'-Ome nucleotides, nucleotides containing a 5'-phosphorothioate group or a terminal nucleotide linked to a cholesterol derivative or a dodecanoic acid bisdecylamide group, 2'-amino modified nucleotides, phosphoramidates or nucleotides containing unnatural bases.In some embodiments, the LPA dsRNA compound comprises an E-vinyl phosphonate nucleotide at the 5' end of the antisense strand (also referred to herein as the guide strand).

[0095] In certain embodiments of the present invention, at least one modified nucleotide is included in the LPA dsRNA compound at the 3'-end and 5'-end of the sense polynucleotide and / or the 3'-end of the antisense polynucleotide, and the at least one modified nucleotide includes abasic nucleotide, ribitol, inverted nucleotide, inverted abasic nucleotide, inverted 2'-OMe nucleotide, inverted 2'-deoxy nucleotide. Those skilled in the art know that the inclusion of abasic or inverted abasic nucleotide at the end of an oligonucleotide can enhance stability (Czauderna et al.Structural variations and stabilizing modifications of synthetic siRNAs in mammalian cells.Nucleic Acids Res.2003;31(11):2705-2716.doi:10.1093 / nar / gkg393).

[0096] In some embodiments of the present invention, the LPA dsRNA comprises one or two isomannitol residues at the 3'-end and 5'-end of the sense strand. In certain embodiments, the sense strand independently comprises one isomannitol residue at the 3'-end and 5'-end, respectively. Examples of isomannitol residues include the following examples: [ka] where each term "Olig" independently represents a polynucleotide moiety. Exemplary isomannitol residues (imann) include the following: [ka] In some embodiments, isomannitol residues, stereoisomers thereof, non-limiting examples include: [ka] In some embodiments, the sense strand independently comprises one isomannitol residue (imann) at the 3' or 5' end, respectively, and a targeting group conjugated to the 5' end having the following exemplary structure, e.g., the targeting group N-acetyl-galactosamine, preferably GLS-15 as described above: [ka] wherein each term "Olig" independently represents a polynucleotide portion.

[0097] In certain embodiments of the invention, the LPA dsRNA compound and / or antisense polynucleotide comprises at least one modified nucleotide, the at least one modified nucleotide comprises a non-locked nucleic acid (UNA) nucleotide and / or a glycol nucleic acid nucleotide (GNA). UNA and GNA are known to those skilled in the art to be thermolabile chemical modifications that can significantly enhance the off-target profile of siRNA compounds (Janas, et al., Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity. Nat Commun. 2018; 9(1): 723. doi: 10.1038 / s41467-018-02989-4; Laursen et al., Utilization of unlocked nucleic acid (UNA) to enhance siRNA performance in vitro and in vivo. Mol BioSyst. 2010; 6: 862-70).

[0098] Another modification may be included in the RNA of the LPA dsRNA agent, LPA antisense polynucleotide and / or LPA sense polynucleotide of certain embodiments of the present invention, and includes one or more ligands, moieties or chemically linked conjugates for the RNA, LPA antisense polynucleotide and / or LPA sense polynucleotide, which enhance one or more characteristics of the LPA dsRNA agent, respectively. Non-limiting examples of characteristics that may be enhanced are the activity of the LPA dsRNA agent, LPA antisense polynucleotide and / or LPA sense polynucleotide, the cellular distribution, the delivery of the LPA dsRNA agent, the pharmacokinetic properties of the LPA dsRNA agent and the cellular uptake of the LPA dsRNA agent. In some embodiments of the present invention, the LPA dsRNA agent includes one or more targeting or linking groups that are conjugated to the sense strand in some embodiments of the LPA dsRNA agent of the present invention. A non-limiting example of a targeting group is a compound that includes N-acetyl-galactosamine (GalNAc). The terms "targeting group", "targeting agent", "linking agent", "targeting compound" and "targeting ligand" are used interchangeably herein. In some embodiments of the invention, the LPA dsRNA agent comprises a targeting compound conjugated to the 5' end of the sense strand. In some embodiments of the invention, the LPA dsRNA agent comprises a targeting compound conjugated to the 3' end of the sense strand. In some embodiments of the invention, the LPA dsRNA agent comprises a targeting group containing GalNAc. In some embodiments of the invention, the LPA dsRNA agent does not comprise a targeting compound conjugated to one or both of the 3' and 5' ends of the sense strand. In some embodiments of the invention, the LPA dsRNA agent does not comprise a GalNAc-containing targeting compound conjugated to one or both of the 5' and 3' ends of the sense strand.

[0099] Additionally, targeting agents and linking agents are well known in the art, and examples of targeting agents and linking agents that may be used in certain embodiments of the present invention include lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Sci., 1997, 1:131-132), and the like. Res., 1992,20:533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991,10:1111-1118; Kabanov et al., FEBS Lett., 1990,259:327-330; Svinarchuk et al., Biochimie, 1993,75:49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycerol-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995,36:3651-3654; Shea et al., Nucl. Acids, 1997,10:1111-1118; Res., 1990,18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995,14:969-973) or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651-3654), palmitoyl moieties (Mishra et al., Biochim. Biophys. Acta, 1995,1264:229-237) or octadecylamine or hexaamino-carbonyloxycholesterol moieties (Crooke et al., J.Pharmacol. Exp. Ther., 1996, 277:923-937), but are not limited to these.

[0100] Certain embodiments of compositions comprising LPA dsRNA agents, LPA antisense polynucleotides and / or LPA sense polynucleotides may include ligands that modify the distribution, targeting, and other properties of the LPA dsRNA agents. In some embodiments of compositions comprising LPA dsRNA agents of the present invention, the ligand increases the affinity for a selected target (molecule, cell or cell type, compartment, such as a cell or organ compartment, tissue, organ or body region, etc.) compared to a species in which such ligand is not present. Ligands useful in the compositions and / or methods of the present invention may be naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low density lipoprotein (LDL) or globulins), carbohydrates (e.g., dextran, amylopectin, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid) or lipids. Ligands may also be recombinant or synthetic molecules such as synthetic polymers, e.g., synthetic polyamino acids or polyamines. Examples of polyamino acids are polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic anhydride copolymer, poly (L-lactide-co-glycolic acid) copolymer, divinyl ether-maleic anhydride copolymer, N- (2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly (2-ethylacrylic acid), N-isopropylacrylamide polymer or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of polyamine or alpha helical peptide.

[0101] The ligands included in the compositions and / or methods of the invention may include targeting groups, non-limiting examples of which are cell or tissue targeting agents such as lectins, glycoproteins, lipids or proteins, such as antibodies that bind to specific cell types such as kidney or liver cells. The targeting group may be thyrotropin, melanocyte stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamates, polyaspartates, lipids, cholesterol, steroids, bile acids, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic.

[0102] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantane acetic acid, 1-pyrenebutanoic acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleic acid), and the like. oil)lithocholic acid, O3-(oleoyl)cholic 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 enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, tetraaza macrocycles of Eu 3+complex), dinitrophenyl, HRP or AP.

[0103] The ligand included in the composition and / or method of the present invention can be a protein such as a glycoprotein or peptide, a protein such as a molecule having a specific affinity for a co-ligand, or an antibody, such as an antibody that binds to a specific cell type, such as a cancer cell, an endothelial cell, a cardiomyocyte, or a bone cell. The ligand useful in the composition and / or method embodiments of the present invention can be a hormone or a hormone receptor. The ligand useful in the composition and / or method embodiments of the present invention can be a lipid, a lectin, a carbohydrate, a vitamin, a coenzyme, a multivalent lactose, a multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, a multivalent mannose, or a multivalent fucose. The ligand useful in the composition and / or method embodiments of the present invention can be, for example, a substance that increases the uptake of an LPA dsRNA agent into a cell by disrupting the cytoskeleton of the cell (e.g., by disrupting the microtubules, microfilaments, and / or intermediate filaments of the cell). Non-limiting examples of such agents are taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine and myoservin.

[0104] In some embodiments, the ligand linked to the LPA dsRNA agent of the present invention is used as a pharmacokinetic (PK) modifier. Examples of PK modifiers that can be used in the compositions and methods of the present invention include, but are not limited to, lipophilic drugs, bile acids, steroids, phospholipid analogs, peptides, protein conjugates, PEG, vitamins, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkyl glycerides, diacyl glycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, aptamers that bind to serum proteins, etc. It is also known that oligonucleotides that contain many phosphorothioate bonds bind to serum proteins, and therefore short oligonucleotides that contain multiple phosphorothioate bonds in the backbone, such as oligonucleotides of about 5 bases, 10 bases, 15 bases, or 20 bases, can also be used as ligands in the compositions and / or methods of the present invention.

[0105] LPA dsRNA agent composition In some embodiments of the present invention, the LPA dsRNA agent is present in a composition. The composition of the present invention may include one or more LPA dsRNA agents and optionally one or more pharma- ceutically acceptable carriers, delivery agents, targeting agents, detectable labels, etc. A non-limiting example of a targeting agent that can be used according to some embodiments of the method of the present invention is an agent that guides the LPA dsRNA agent of the present invention into the cell and / or cells to be treated. The selection of the targeting agent depends on the following factors: the nature of the LPA-related disease or condition and the type of target cell. In one non-limiting example, in some embodiments of the present invention, it may be necessary to target the LPA dsRNA agent to and / or into liver cells. It will be recognized that in some embodiments of the method of the present invention, the therapeutic agent includes an LPA dsRNA agent that has only a delivery agent, such as a delivery agent that includes N-acetylgalactosamine (GalNAc) without any additional linking element. For example, in some aspects of the invention, an LPA dsRNA agent can be linked to a delivery compound that includes GalNAc, included in a composition that includes a pharma- ceutically acceptable carrier, and administered to a cell or subject without any detectable label or targeting agent or the like linked to the LPA dsRNA agent.

[0106] When the LPA dsRNA agent of the present invention is administered in conjunction with and / or linked to one or more delivery agents, targeting agents, labeling agents, etc., those skilled in the art can understand, select and use suitable agents for use in the methods of the present invention. Labeling agents can be used in certain methods of the present invention to determine the location of LPA dsRNA agents in cells and tissues, and can be used to determine the location of therapeutic compositions containing LPA dsRNA agents administered in the methods of the present invention in cells, tissues or organs. Means of binding and using labeling agents, such as enzyme labels, dyes, radioactive labels, etc., are well known in the art. It will be recognized that in some embodiments of the compositions and methods of the present invention, labeling agents are linked to one or both of the sense and antisense polynucleotides contained in the LPA dsRNA agent.

[0107] Delivery of LPA dsRNA agents and LPA antisense polynucleotide agents Some embodiments of the method of the present invention include delivering LPA dsRNA agent to cells. As used herein, the term "delivery" refers to promoting or affecting cellular uptake or absorption. Absorption or uptake of LPA dsRNA agent can occur by independent diffusion or active cellular process or by using a delivery agent, targeting agent, etc. that can be associated with the LPA dsRNA agent of the present invention. Delivery modes suitable for the method of the present invention include, but are not limited to, in vivo delivery, where the LPA dsRNA agent is injected or gradually administered into tissue site. In some embodiments of the present invention, the LPA dsRNA agent is linked to a delivery agent.

[0108] Non-limiting examples of methods that can be used to deliver LPA dsRNA agents to cells, tissues and / or subjects include LPA dsRNA-GalNAc conjugates, SAMiRNA technology, LNP-based delivery methods and naked RNA delivery.These and other delivery methods have been successfully used in the art to deliver therapeutic RNAi agents to treat various diseases and conditions, including but not limited to liver disease, acute intermittent porphyria (AIP), hemophilia, pulmonary fibrosis, etc. Details of multiple delivery modes can be found in articles such as Nikam, RR & KR Gore (2018) Nucleic Acid Ther, 28(4), 209-224 Aug 2018; Springer AD & SFDowdy (2018) Nucleic Acid Ther. Jun 1; 28(3):109-118; Lee, K. et al., (2018) Arch Pharm Res, 41(9), 867-874; and Nair, JK et al., (2014) J. Am. Chem. Soc. 136:16958-16961, the contents of which are incorporated herein by reference.

[0109] Some embodiments of the present invention include the use of lipid nanoparticles (LNPs) to deliver the LPA dsRNA agent of the present invention to cells, tissues and / or subjects. LNPs are commonly used for in vivo delivery of LPA dsRNA agents, including therapeutic LPA dsRNA agents. One advantage of using LNPs or other delivery agents is that the stability of the LPA RNA agent is increased when it is delivered to a subject using LNPs or other delivery agents. In some embodiments of the present invention, the LNPs include cationic LNPs loaded with one or more LPA RNAi molecules of the present invention. The LNPs containing LPA RNAi molecules are administered to a subject, and the LNPs and their bound LPA RNAi molecules are taken up by cells via endocytosis. Their presence induces RNAi, thereby causing the release of molecules that mediate RNAi.

[0110] Another non-limiting example of a delivery agent that can be used in the embodiments of the present invention to deliver the LPA dsRNA agent of the present invention to cells, tissues and / or subjects is an agent that contains GalNAc that is linked to the LPA dsRNA agent of the present invention and delivers the LPA dsRNA agent to cells, tissues and / or subjects. Certain examples of other delivery agents that contain GalNAc that can be used in certain embodiments of the methods and compositions of the present invention are disclosed in PCT Application WO2020191183A1. A non-limiting example of a GalNAc targeting ligand that can be used in the compositions and methods of the present invention to deliver LPA dsRNA agents to cells is a targeting ligand cluster. Examples of targeting ligand clusters provided herein are GalNAc ligands with phosphodiester bonds (GLO) and GalNAc ligands with phosphorothioate bonds (GLS). The term "GLX-n" may be used herein to indicate that the GalNAC-containing compound being linked is any of the following compounds: GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16, each of which has a structure as shown below. In the diagram below, the linkage position of the GalNAc targeting ligand to the RNAi agent of the invention is at the right end of each targeting ligand. It will be appreciated that any of the RNAi and dsRNA molecules of the present invention may be linked to GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15 and GLO-16.Below are the structures of GLO-1 to GLO-16 and GLS-1 to GLS-16. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0111] In some embodiments of the present invention, in vivo delivery can also be by beta-dextran delivery system, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677 and U.S. Patent Publication No. 2005 / 0281781, the entirety of which is incorporated herein by reference. LPA RNAi agents can also be introduced into cells in vitro using methods known in the art, such as electroporation and lipid transfection. In some embodiments of the method of the present invention, LPA dsRNA is delivered without a targeting agent. These RNAs can be delivered as "naked" RNA molecules. As a non-limiting example, the LPA dsRNA of the present invention can be administered to a subject in a pharmaceutical composition comprising an RNAi agent but not a targeting agent (e.g., a GalNAc targeting compound) to treat an LPA-related disease or condition in a subject, such as cardiovascular disease, including Berge's disease, peripheral artery disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic stenosis, aortic regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperapolipoprotein betalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease, and venous thrombosis, stroke, atherosclerosis, thrombosis, coronary heart disease or aortic stenosis, and / or any other disease or condition associated with elevated levels of Lp(a)-containing particles.

[0112] In addition to the specific delivery modes described herein, it will be appreciated that other RNAi delivery methods may be used in combination with the embodiments of the LPA RNAi agents and treatment methods described herein, including, but not limited to, those described herein and those used in the art.

[0113] The LPA dsRNA agent of the present invention can be administered to a subject in an amount and manner that effectively reduces the level of LPA polypeptide in cells and / or subjects.In some embodiments of the method of the present invention, one or more LPA dsRNA agents are administered to cells and / or subjects to treat diseases or conditions associated with LPA expression.In some embodiments, the method of the present invention comprises administering one or more LPA dsRNA agents to a subject in need thereof to alleviate diseases or conditions associated with LPA expression in the subject.The LPA dsRNA agent or LPA antisense polynucleotide agent of the present invention can be administered to reduce LPA expression in one or more cells in vitro, ex vivo and in vivo.

[0114] In some embodiments of the present invention, the level of LPA polypeptide in a cell is reduced by delivery (e.g., introduction) of an LPA dsRNA agent or an LPA antisense polynucleotide agent to the cell. Targeting agents and methods can be used to facilitate delivery of an LPA dsRNA agent or an LPA antisense polynucleotide agent to a specific cell type, cell subtype, organ, spatial region and / or subcellular region within a cell in a subject. The LPA dsRNA agent can be administered alone or in combination with one or more additional LPA dsRNA agents in certain methods of the present invention. In some embodiments, two, three, four or more independently selected LPA dsRNA agents are administered to a subject. In some embodiments of the present invention, the LPA dsRNA agent is administered to a subject in combination with one or more additional therapeutic regimens for treating an LPA-related disease or condition to treat the LPA-related disease or condition. Other non-limiting examples of therapeutic regimens are administration of one or more LPA antisense polynucleotides of the present invention, administration of a non-LPA dsRNA therapeutic agent and behavior modification. The additional therapeutic regimen can be administered at one or more of the following times: before, simultaneously and after administration of the LPA dsRNA agent of the present invention. It will be recognized that "simultaneous" as used herein means within the 5th minute of zero, within the 10th minute of zero, within the 30th minute of zero, within the 45th minute of zero and within the 60th minute of zero ("zero time" is the time when the LPA dsRNA agent of the present invention is administered to the subject). Non-limiting examples of non-LPA dsRNA therapeutic agents are additional therapeutic agents such as HMg Co-A reductase inhibitors (statins), ezetimibe, PCSK-9 inhibitors, CTEP inhibitors, ANGPTL3 targeting therapy, APOC3 targeting therapy and niacin or any combination thereof. Non-limiting examples of behavioral modification are dietary regimens, counseling and exercise regimens. These and other therapeutic agents and behavioral modifications are known in the art and can be used to treat an LPA disease or condition in a subject and can be combined with one or more LPA dsRNA agents of the invention administered to a subject to treat the LPA disease or condition.The LPA dsRNA agents of the present invention administered to a cell or subject to treat an LPA-associated disease or condition may act in a synergistic manner with one or more other therapeutic agents or active ingredients, thereby increasing the effectiveness of the one or more therapeutic agents or active ingredients and / or increasing the effectiveness of the LPA dsRNA agent in treating an LPA-associated disease or condition.

[0115] The treatment methods of the present invention include administration of an LPA dsRNA agent that can be used when an LPA-related disease or condition is present, including before the onset of the LPA-related disease or condition and / or at early, middle, late stages of the disease or condition, as well as all time points before and after any of these stages.The methods of the present invention can also treat subjects who have previously been treated with one or more other therapeutic agents and / or therapeutically effective ingredients for an LPA-related disease or condition, where the one or more other therapeutic agents and / or therapeutically effective ingredients have been unsuccessful, minimally successful, and / or are no longer successful in treating the LPA-related disease or condition in the subject.

[0116] Vector-encoded dsRNA In some embodiments of the present invention, the LPA dsRNA agent can be delivered into cells using a vector. The LPA dsRNA agent transcription unit can be contained in a DNA or RNA vector. The preparation and use of vectors encoding such transgenes for delivery of sequences into cells and / or subjects is well known in the art. For example, vectors that result in transient expression of LPA dsRNA for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours or more, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more can be used in the methods of the present invention. The length of transient expression can be determined using conventional methods based on factors such as, but not limited to, the specific vector construct selected and the target cell and / or tissue. Such transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrative or non-integrative vectors. The transgene can also be constructed to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0117] One or more single strands of the LPA dsRNA agent can be transcribed from a promoter on an expression vector. When two individual strands are expressed, for example, to generate dsRNA, two individual expression vectors can be co-introduced into a cell, for example, using transfection or infection. In certain embodiments, each individual strand of the LPA dsRNA agent of the present invention can be transcribed by a promoter contained on the same expression vector. In some embodiments of the present invention, the LPA dsRNA agent is expressed as an inverted repeat polynucleotide linked by a linker polynucleotide sequence so that the LPA dsRNA agent has a stem-loop structure.

[0118] Non-limiting examples of RNA expression vectors are DNA plasmids or viral vectors. The expression vectors useful in the embodiments of the present invention can be compatible with eukaryotic cells. Eukaryotic cell expression vectors are conventionally used in the art and are available from many commercial sources. The delivery of LPA dsRNA expression vectors can be systemic, such as by intravenous or intramuscular administration to target cells that have been removed from a subject, followed by reintroduction of target cells into the subject, or any other means that allows the introduction of desired target cells.

[0119] The viral vector systems that may be included in the embodiments of the method include, but are not limited to, (a) adenoviral vectors; (b) retroviral vectors, including but not limited to lentiviral vectors, Moloney murine leukemia virus, etc.; (c) adeno-associated viral vectors; (d) herpes simplex viral vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) poxvirus vectors, such as orthopoxvirus vectors, e.g., vaccinia virus vectors or avian poxvirus vectors, e.g., canarypox virus or fowlpox virus vectors; (j) helper-dependent or gutless adenovirus vectors. The constructs of recombinant expression of LPA dsRNA agents may include regulatory elements, such as promoters, enhancers, etc., that may be selected to provide constitutive or regulated / inducible expression. The use of viral vector systems and promoters and enhancers, etc., is conventional in the art and may be used in conjunction with the methods and compositions described herein.

[0120] Some embodiments of the present invention include using viral vector to deliver LPA dsRNA agent into cells.Many adenovirus-based delivery systems are conventionally used in the art for delivery to, for example, lung, liver, central nervous system, endothelial cells and muscle.Non-limiting examples of viral vectors that can be used in the method of the present invention are AAV vectors, poxviruses such as vaccinia virus, modified Ankara virus (MVA), NYVAC, avipox virus or canarypox virus.

[0121] Certain embodiments of the present invention include the method of using vector to deliver LPA dsRNA agent into cells, and such vector can be present in a pharma- ceutically acceptable carrier, which may not necessarily include the sustained release matrix that gene delivery vector is incorporated into.In some embodiments, the vector for delivering LPA dsRNA can be produced from recombinant cell, and the pharmaceutical composition of the present invention can include one or more cells that produce LPA dsRNA delivery system.

[0122] Pharmaceutical compositions containing LPA dsRNA or ssRNA agents Certain embodiments of the present invention include the use of pharmaceutical compositions comprising an LPA dsRNA agent or an LPA antisense polynucleotide agent and a pharma- ceutical acceptable carrier. Pharmaceutical compositions comprising an LPA dsRNA agent or an LPA antisense polynucleotide agent can be used in the methods of the present invention to reduce LPA gene expression in cells and can be used in the treatment of LPA-related diseases or conditions. Such pharmaceutical compositions can be formulated based on the mode of delivery. Non-limiting examples of formulations for the mode of delivery are compositions formulated for subcutaneous delivery, compositions formulated for systemic administration by parenteral delivery, compositions formulated for intravenous (IV) delivery, compositions formulated for intrathecal delivery, compositions formulated for direct delivery to the brain, etc. The pharmaceutical compositions of the present invention can be administered using one or more modes of delivery of the LPA dsRNA agent or LPA antisense polynucleotide agent into cells, such as, for example, surface (e.g., by transdermal patch); pulmonary, such as by inhalation or insufflation of powder or aerosol, including via a nebulizer; intra-airway, intranasal, epithelial and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous, for example, by implantation device; or intracranially, for example, via intraparenchymal; intrathecal or intraventricular administration. LPA dsRNA agents or LPA antisense polynucleotide agents can also be delivered directly to target tissues, for example, directly to the liver, directly to the kidney, etc. Of course, "delivery of LPA dsRNA agents" or "delivery of LPA antisense polynucleotide agents" into cells includes delivery of LPA dsRNA agents or LPA antisense polynucleotide agents, direct expression of LPA dsRNA agents in cells, and expression of LPA dsRNA agents from coding vectors delivered into cells, or any suitable manner of making LPA dsRNA or LPA antisense polynucleotide agents appear in cells, respectively. Preparation and use of formulations and means for delivering inhibitory RNA are well known and routinely used in the art.

[0123] As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of an LPA dsRNA agent or an LPA antisense polynucleotide agent of the present invention and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier used to administer a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, glucose, water, glycerin, ethanol, and combinations thereof. This term explicitly excludes cell culture media. For orally administered drugs, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavorings, colorings, and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while cornstarch and alginates are suitable disintegrants. Binders may include starch and gelatin, while lubricants, when present, are usually magnesium stearate, stearic acid, or talc. If necessary, the tablet may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract. The agents contained in the pharmaceutical formulation are further described below. Terms used herein, such as "pharmacologically effective amount", "therapeutically effective amount" and "effective amount", refer to the amount of the LPA dsRNA agent or LPA antisense polynucleotide agent of the present invention that produces the desired pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when a measurable parameter associated with a disease or disorder is reduced by at least 10%, then the therapeutically effective amount of a drug for treating a disease or condition is the amount required to reduce that parameter by at least 10%. For example, a therapeutically effective amount of an LPA dsRNA agent or LPA antisense polynucleotide agent may reduce LPA polypeptide levels by at least 10%. A pharmaceutical composition may include such a dsRNAi agent, for example, including a duplex shown in Table 1. In some other embodiments, such a dsRNAi agent includes a variant of the duplex in Table 1.

[0124] Effective dose In some aspects, the method of the present invention comprises contacting a cell with an effective amount of an LPA dsRNA agent or an LPA antisense polynucleotide agent to reduce LPA gene expression in the contacted cell. Some embodiments of the method of the present invention comprise administering an LPA dsRNA agent or an LPA antisense polynucleotide agent to a subject in an amount that effectively reduces the LPA gene expression of the subject and effectively treats the LPA-related disease or condition. The "effective amount" used to reduce the expression of LPA and / or for treating an LPA-related disease or condition is an amount necessary or sufficient to achieve a desired biological effect. For example, an effective amount of an LPA dsRNA agent or an LPA antisense polynucleotide agent for treating an LPA-related disease or condition can be (i) the amount required to slow or stop the progression of the disease or condition; or (ii) reverse, reduce or eliminate one or more symptoms of the disease or condition. In some aspects of the invention, an effective amount is an amount of an LPA dsRNA agent or an LPA antisense polynucleotide agent that, when administered to a subject in need of treatment of an LPA-related disease or condition, results in a therapeutic response of prevention and / or treatment of the disease or condition. According to some aspects of the invention, an effective amount is an amount of an LPA dsRNA agent or an LPA antisense polynucleotide agent of the invention that, when combined or co-administered with another therapeutic treatment of an LPA-related disease or condition, results in a therapeutic response of prevention and / or treatment of the disease or condition. In some embodiments of the invention, the biological effect of treating a subject with an LPA dsRNA agent or an LPA antisense polynucleotide agent of the invention can be an improvement and / or complete disappearance of symptoms caused by an LPA-related disease or condition. In some embodiments of the invention, the biological effect is a complete disappearance of an LPA-related disease or condition, for example, as demonstrated by a diagnostic test showing that the subject does not have an LPA-related disease or condition. Non-limiting examples of detectable physiological symptoms include a reduction in lipid accumulation in the liver of a subject following administration of an agent of the invention. Other methods of assessing the status of an LPA-associated disease or condition known in the art can be used to determine the effect of the agents and / or methods of the present invention on an LPA-associated disease or condition.

[0125] The effective amount of an LPA dsRNA agent or an LPA antisense polynucleotide agent that reduces LPA polypeptide to a level for the treatment of an LPA-related disease or condition is generally determined in a clinical trial that establishes effective doses for a test population and a control population in a blinded study. In some embodiments, the effective amount is an amount that produces a desired response, such as an amount that reduces an LPA-related disease or condition in a cell, tissue, and / or subject having the disease or condition. Thus, an effective amount of an LPA dsRNA agent or an LPA antisense polynucleotide agent for the treatment of an LPA-related disease or condition that can be treated by reducing an LPA polypeptide can be an amount that, when administered, reduces the amount of LPA polypeptide in a subject to less than the amount present in a cell, tissue, and / or subject without administration of an LPA dsRNA agent or an LPA antisense polynucleotide agent. In some aspects of the present invention, the level of LPA polypeptide and / or LPA gene expression present in a cell, tissue, and / or subject that has not been contacted with or administered an LPA dsRNA agent or an LPA antisense polynucleotide agent of the present invention is referred to as a "control" amount. In some embodiments of the method of the present invention, the control amount for the subject is the amount for the subject before treatment; in other words, the level for the subject before administration of the LPA agent can be the control level for the subject and can be used for comparison with the LPA polypeptide and / or LPA gene expression level after administration of the siRNA to the subject. In the case of treatment for an LPA-related disease or condition, the desired response can be to reduce or eliminate one or more symptoms of the disease or condition in a cell, tissue, and / or subject. The reduction or elimination can be temporary or persistent. It will be appreciated that the status of an LPA-related disease or condition can be monitored using methods such as determining LPA polypeptide and LPA gene expression, symptom assessment, clinical testing, and the like. In some aspects of the present invention, the desired response to treatment of an LPA-related disease or condition is to delay the onset of the disease or condition or to prevent the onset of the disease or condition.

[0126] The effective amount of a compound that reduces LPA polypeptide can also be determined by evaluating the physiological effect of administration of the LPA dsRNA agent or LPA antisense polynucleotide agent on a cell or subject, such as the reduction of an LPA-related disease or condition after administration. Subject assays and / or symptom monitoring can be used to determine the effectiveness of the LPA dsRNA agent or LPA antisense polynucleotide agent of the present invention that can be administered in the pharmaceutical compound of the present invention and to determine whether there is a response to treatment. One non-limiting example is one or more serum lipid profile tests known in the art. Another non-limiting example is that one or more liver function tests known in the art can be used to determine the status of a subject's LPA-related disease or condition before and after treating the subject with the LPA dsRNA agent of the present invention. In another non-limiting example, one or more cholesterol accumulation tests in the liver known in the art are used to determine the status of an LPA-related disease in a subject. In this embodiment, the disease includes cholesterol accumulation, and the test is used to determine the cholesterol level in the subject before and after treating the subject with the LPA dsRNA agent of the present invention.

[0127] Some embodiments of the present invention include a method for determining the effectiveness of a dsRNA agent or LPA antisense polynucleotide agent of the present invention administered to a subject to treat an LPA-related disease or condition by assessing and / or monitoring one or more "physiological characteristics" of the LPA-related disease or condition in the subject. Non-limiting examples of physiological characteristics of an LPA-related disease or condition are the subject's serum LPA level, the subject's serum lipid level, the subject's low-density lipoprotein level, the subject's HDL level, the subject's LDL:HDL ratio, the subject's triglyceride level, fat present in the subject's liver, physical symptoms, etc. Standard methods for determining such physiological characteristics are known in the art and include, but are not limited to, blood tests, imaging tests, and physical examinations.

[0128] It will be appreciated that the amount of LPA dsRNA agent or LPA antisense polynucleotide agent administered to a subject may be modified at least in part based on the results of a determination of the status and / or physiological characteristics of the subject's disease and / or condition. The amount of therapeutic agent may be modified, for example, by increasing or decreasing the amount of LPA dsRNA agent or LPA antisense polynucleotide agent by modifying the composition in which the LPA dsRNA agent or LPA antisense polynucleotide agent is administered, by modifying the route of administration, by modifying the time of administration, etc. The effective amount of LPA dsRNA agent or LPA antisense polynucleotide agent will vary depending on the particular condition being treated, the age and health of the subject being treated, the severity of the condition, the duration of treatment, the nature of concurrent treatment (if any), the particular route of administration, and other factors within the knowledge and expertise of the medical practitioner. For example, the effective amount may depend on the level of LPA polypeptide and / or the desired level of LPA gene expression that is effective in treating an LPA-related disease or condition. Those skilled in the art can empirically determine the effective amount of a particular LPA dsRNA agent or LPA antisense polynucleotide agent for use in the method of the present invention without undue experimentation.In combination with the teachings provided herein, an effective prophylactic or therapeutic regimen can be designed to effectively treat a particular subject by selecting from a plurality of LPA dsRNA agents or LPA antisense polynucleotide agents of the present invention and comparing factors such as efficacy, relative bioavailability, patient weight, severity of adverse side effects and preferred mode of administration.When used in the embodiments of the present invention, the effective amount of the LPA dsRNA agent or LPA antisense polynucleotide agent of the present invention can be the amount that produces the desired biological effect in cells when contacted with cells.

[0129] It should be appreciated that LPA gene silencing can be performed constitutively or by genomic engineering in any cell expressing LPA and can be determined by any suitable assay. In some embodiments of the present invention, LPA gene expression is reduced by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% by administration of the LPA dsRNA agent of the present invention. In some embodiments of the present invention, LPA gene expression is reduced by 5% to 10%, 5% to 25%, 10% to 50%, 10% to 75%, 25% to 75%, 25% to 100% or 50% to 100% by administration of the LPA dsRNA agent of the present invention.

[0130] Style The LPA dsRNA agent and LPA antisense polynucleotide agent are delivered in a pharmaceutical composition in a dosage sufficient to inhibit LPA gene expression. In certain embodiments of the invention, the dosage of the LPA dsRNA agent or LPA antisense polynucleotide agent is 0.01-200.0 mg per kg of subject's body weight per day, typically 1-50 mg / kg body weight, 5-40 mg / kg body weight, 10-30 mg / kg body weight, 1-20 mg / kg body weight, 1-10 mg / kg body weight, 4-15 mg / kg body weight (endpoints included) per day. For example, LPA The dsRNA agent or LPA antisense polynucleotide agent may be administered at a concentration of about 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 34 mg / kg, 35 mg / kg, 36 mg / kg, 37 mg / kg, 38 mg / kg, 39 mg / kg, 40 mg / kg, 41 mg / kg, 42 mg / kg, 43 mg / kg, 44 mg / kg, 45 mg / kg, 46 mg / kg, 47 mg / kg, 48 mg / kg, 49 mg / kg, 50 mg / kg, 51 mg / kg, 52 mg / kg, 53 mg g, 2.1mg / kg, 2.2mg / kg, 2.3mg / kg, 2.4mg / kg, 2.5mg / kg, 2.6mg / kg, 2.7mg / kg, 2.8mg / kg, 2.9mg / kg, 3.0mg / kg, 3 .1mg / kg, 3.2mg / kg, 3.3mg / kg, 3.4mg / kg, 3.5mg / kg, 3.6mg / kg, 3.7mg / kg, 3.8mg / kg, 3.9mg / kg, 4mg / kg, 4.1mg / k g, 4.2mg / kg, 4.3mg / kg, 4.4mg / kg, 4.5mg / kg, 4.6mg / kg, 4.7mg / kg, 4.8mg / kg, 4.9mg / kg, 5mg / kg, 5.1mg / kg, 5.2 mg / kg, 5.3mg / kg, 5.4mg / kg, 5.5mg / kg, 5.6mg / kg, 5.7mg / kg, 5.8mg / kg, 5.9mg / kg, 6mg / kg, 6.1mg / kg, 6.2mg / kg, 6.3mg / kg, 6.4mg / kg, 6.5mg / kg, 6.6mg / kg, 6.7mg / kg, 6.8mg / kg, 6.9mg / kg, 7mg / kg, 7.1mg / kg, 7.2mg / kg, 7.3mg / kg, 7.4mg / kg, 7.5mg / kg, 7.6mg / kg, 7.7mg / kg, 7.8mg / kg, 7.9mg / kg, 8mg / kg, 8.1mg / kg, 8.2mg / kg, 8.3mg / kg, 8.4mg / kg, 8.5mg / kg, 8.6mg / kg, 8.7mg / kg, 8.8mg / kg, 8.9mg / kg, 9mg / kg, 9.1mg / kg, 9.2mg / kg, 9.3mg / kg, 9.4mg / kg, 9.5mg / kg, 9.6mg / kg, 9.7mg / k g, 9.8mg / kg, 9.9mg / kg, 10mg / kg, 11mg / kg, 12mg / kg, 13mg / kg, 14mg / kg, 15mg / kg, 16mg / kg, 17mg / kg, 18mg / kg, 19mg / kg, 20mg / kg, 21mg / kg, 22mg / It may be administered in a single dose of 23mg / kg, 24mg / kg, 25mg / kg, 26mg / kg, 27mg / kg, 28mg / kg, 29mg / kg, 30mg / kg, 31mg / kg, 32mg / kg, 33mg / kg, 34mg / kg, 35mg / kg, 36mg / kg, 37mg / kg, 38mg / kg, 39mg / kg, 40mg / kg, 41mg / kg, 42mg / kg, 43mg / kg, 44mg / kg, 45mg / kg, 46mg / kg, 47mg / kg, 48mg / kg, 49mg / kg to 50mg / kg body weight.

[0131] Various factors can be considered when determining the delivery dose and time of the LPA dsRNA agent of the present invention.The absolute amount of the LPA dsRNA agent or LPA antisense polynucleotide agent to be delivered depends on various factors, including concurrent treatment, number of administrations, and individual subject parameters, including age, health condition, body size and weight.These factors are well known to those skilled in the art and can be analyzed by conventional experiments.In some embodiments, the maximum dose can be used, i.e. the highest safe dose based on reasonable medical judgment.

[0132] In some embodiments, the method of the present invention may include administering to a subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses of LPA dsRNA agent or LPA antisense polynucleotide agent. In some cases, the pharmaceutical compound (e.g., including LPA dsRNA agent or including LPA antisense polynucleotide agent) may be administered to a subject at a specific dose at least daily, every other day, weekly, biweekly, monthly, etc., and may be administered once a day or more than once a day, for example, 2, 3, 4, 5 or more times in a 24-hour period. The pharmaceutical composition of the present invention may be administered once a day; or the LPA dsRNA agent or LPA antisense polynucleotide agent may be administered in 2, 3 or more divided doses at appropriate intervals within a day, or may be delivered using continuous infusion or controlled release formulations. In some embodiments of the method of the present invention, the pharmaceutical composition of the present invention is administered to a subject at least once a day, at least once a week, at least once a month, or at least once a year.

[0133] In certain aspects, the methods of the present invention include administering a pharmaceutical compound alone or in combination with one or more other LPA dsRNA agents or LPA antisense polynucleotide agents and / or in combination with other medications or therapeutic activities or protocols administered to a subject with an LPA-related disease or condition. The pharmaceutical compound may be administered in the form of a pharmaceutical composition. The pharmaceutical composition used in the methods of the present invention may be sterile and contains an amount of an LPA dsRNA agent or an LPA antisense polynucleotide agent that reduces the level of an LPA polypeptide to a level sufficient to produce a desired response in a unit of weight or volume suitable for administration to a subject. The dose of a pharmaceutical composition containing an LPA dsRNA agent or an LPA antisense polynucleotide agent administered to a subject may be selected according to various parameters of reducing LPA protein levels, particularly the mode of administration used and the subject's condition. Other factors include the duration of treatment required. If the subject's response is insufficient with the initial dose, a higher dose may be used within the patient's tolerance (or the dose may be effectively increased by a different, more localized delivery route).

[0134] treatment As used herein, the terms "prevent" or "preventing", when used in reference to a disease, condition, or disorder that would benefit from a decrease in LPA gene expression, refer to a reduction in the likelihood that the subject will experience symptoms associated with such a disease, condition, or disorder, such as Berge's disease, peripheral artery disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic valve stenosis, aortic valve regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperapolipoprotein betalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease, and venous thrombosis, stroke, atherosclerosis, thrombosis, coronary heart disease, or aortic valve stenosis, and / or cardiovascular disease, including any other disease or condition associated with elevated levels of Lp(a)-containing particles. In such cases, the likelihood of such a disease occurring is reduced, e.g., when an individual has one or more cardiovascular disease risk factors but does not develop cardiovascular disease or develops only less severe cardiovascular disease, effective prevention is considered if the individual does not develop the relevant disease, condition or pathology, or has a reduced degree of onset of symptoms associated with such disease, condition or pathology (e.g., at least about a 10% reduction in clinical disease or condition measures), or has a delayed onset of symptoms (e.g., a delay of days, weeks, months or years), compared to a population having the same risk factors and not receiving the treatment described herein.

[0135] For LPA-related diseases and conditions, the reduction of LPA polypeptide level is effective in treating the disease or condition, the method and LPA dsRNA agent of the present invention can be used to treat and inhibit LPA expression.The examples of diseases and conditions that can be treated with the LPA dsRNA agent or LPA antisense polynucleotide agent of the present invention and the treatment method of the present invention include but are not limited to Berge's disease, peripheral artery disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic valve stenosis, aortic valve regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperapolipoprotein betalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease and venous thrombosis, stroke, atherosclerosis, thrombosis, coronary heart disease or aortic valve stenosis and / or any other disease or pathology associated with the elevated level of Lp(a)-containing particles. Such diseases and conditions may be referred to herein as "LPA-associated diseases and conditions" and "diseases and conditions caused and / or modulated by LPA."

[0136] In some aspects of the present invention, the LPA dsRNA agent or LPA antisense polynucleotide agent of the present invention can be administered to a subject at one or more times before or after diagnosis of an LPA-related disease or condition. In some aspects of the present invention, a subject is at risk of suffering from an LPA-related disease or condition or has developed an LPA-related disease or condition. A subject at risk of developing an LPA-related disease or condition is one who has an increased likelihood of developing an LPA-related disease or condition compared to a control who is at risk of developing an LPA-related disease or condition. In some embodiments of the present invention, the level of risk is statistically significant compared to the control level of risk. A subject at risk can include, for example, a subject with an underlying disease and / or genetic abnormality that is more susceptible to an LPA-related disease or condition than a control subject without the underlying disease or genetic abnormality; a subject with a family history and / or personal history of an LPA-related disease or condition; and a subject who has been previously treated for an LPA-related disease or condition or is a subject. It will be appreciated that the underlying disease and / or genetic abnormality that renders a subject more susceptible to an LPA-associated disease or condition may be a disease or genetic abnormality that, if present, has previously been determined to be associated with an increased likelihood of developing an LPA-associated disease or condition.

[0137] It will be appreciated that the LPA dsRNA agent or LPA antisense polynucleotide agent may be administered to a subject based on the medical condition of the individual subject. For example, medical care provided to a subject may assess the LPA level measured in a sample obtained from the subject and determine that it is desirable to reduce the subject's LPA level by administering an LPA dsRNA agent or LPA antisense polynucleotide agent of the present invention. In one non-limiting example, a biological sample such as a blood or serum sample may be obtained from the subject and the subject's LPA level may be determined in the sample. The LPA dsRNA agent or LPA antisense polynucleotide agent is administered to the subject, a blood or serum sample is obtained from the subject after administration, the LPA level is measured using the sample, and the results are compared to the results determined in the subject's sample before (previously) administration. A subsequent reduction in the LPA level in the subject's sample compared to the level before administration indicates the effectiveness of the administered LPA dsRNA agent or LPA antisense polynucleotide agent in reducing the subject's LPA level. In one non-limiting example, the level of Lp(a) in blood can be considered a physiological characteristic of an LPA-associated condition even if the subject has not been diagnosed with an LPA-associated condition such as those disclosed herein. A medical provider can monitor changes in the Lp(a) level in the subject's blood as a measure of the effectiveness of the administered LPA dsRNA or LPA antisense polynucleotide agent of the present invention.

[0138] Some embodiments of the method of the present invention include adjusting the treatment, which comprises administering a dsRNA agent or LPA antisense polynucleotide agent of the present invention to a subject based at least in part on an evaluation of the change in one or more physiological characteristics of the LPA-related disease or condition in the subject that is brought about by the treatment. For example, in some embodiments of the present invention, the effect of the dsRNA agent or LPA antisense polynucleotide agent of the present invention administered to the subject can be determined and used to help regulate the amount of the dsRNA agent or LPA antisense polynucleotide agent of the present invention subsequently administered to the subject. In one non-limiting example, a dsRNA agent or LPA antisense polynucleotide agent of the present invention is administered to a subject, and the Lp(a) level in the subject's blood is measured after administration; based at least in part on the determined level, it is determined whether a higher dose of the dsRNA agent or LPA antisense polynucleotide agent is required to improve the physiological effect of the administered agent, such as reducing or further reducing the Lp(a) level in the subject's blood. In another non-limiting example, a dsRNA agent or LPA antisense polynucleotide agent of the invention is administered to a subject, the level of Lp(a) in the subject's blood is determined following administration, and a smaller amount of the dsRNA agent or LPA antisense polynucleotide agent is expected to be administered to the subject based at least in part on the determined level.

[0139] Thus, some embodiments of the present invention include evaluating the changes in one or more physiological characteristics resulting from the subject's prior treatment to adjust the amount of the dsRNA agent or LPA antisense polynucleotide agent of the present invention that is subsequently administered to the subject. Some embodiments of the method of the present invention include measuring the physiological characteristics of the LPA-related disease or condition one, two, three, four, five, six or more times; evaluating and / or monitoring the effectiveness of the administered LPA dsRNA agent or LPA antisense polynucleotide agent of the present invention; and optionally using the measured results to adjust one or more of the following: the dosage, administration regimen and / or administration frequency of the dsRNA agent or LPA antisense polynucleotide agent of the present invention to treat the LPA-related disease or condition in the subject. In some embodiments of the method of the present invention, the desired result of administering an effective amount of a dsRNA agent or LPA antisense polynucleotide agent of the present invention to a subject is that the Lp(a) level in the subject's blood is reduced compared to the previous Lp(a) level in the blood determined for the subject; the Lp(a) level in the subject's blood is within the normal range.

[0140] As used herein, the terms "treat," "therapeutic," or "treated," when used in reference to an LPA-related disease or condition, may refer to prophylactic treatment that reduces the likelihood of developing an LPA-related disease or condition in a subject, may refer to treatment to eliminate or reduce the level of an LPA-related disease or condition after a subject has developed an LPA-related disease or condition, prevent an LPA-related disease or condition in a subject from becoming more severe and / or slow the progression of an LPA-related disease or condition compared to a subject in the absence of a therapy that reduces the level of an LPA polypeptide in the subject.

[0141] Certain embodiments of the agents, compositions and methods of the present invention can be used to inhibit LPA gene expression. As used herein, the terms "inhibit", "silence", "reduce", "downregulate" and "knock down" in reference to the expression of LPA gene refer to, for example, changing the expression of LPA gene by one or more of the following: the level of RNA transcribed by the gene, the level of LPA expressed, and the level of LPA polypeptide, protein or protein subunit translated from mRNA in a cell, cell population, tissue, organ or subject is reduced compared to the control level of RNA transcribed by LPA gene, the control level of LPA translated from mRNA, respectively, when the cell, cell population, tissue, organ or subject is contacted (e.g., treated) with the LPA dsRNA agent or LPA antisense polynucleotide agent of the present invention. In some embodiments, the control level is the level in a cell, tissue, organ or subject that is not contacted (e.g., not treated) with the LPA dsRNA agent or LPA antisense polynucleotide agent of the present invention.

[0142] How to apply Various routes of administration of LPA dsRNA agents or LPA antisense polynucleotide agents can be used in the methods of the present invention. The selection of a particular delivery mode depends at least in part on the particular condition being treated and the dosage required for therapeutic efficacy. In general, the methods of the present invention can be carried out using any medically acceptable mode of administration, meaning any mode that provides effective therapeutic levels for LPA-related diseases or conditions without causing clinically unacceptable side effects. In some embodiments of the present invention, LPA dsRNA agents or LPA antisense polynucleotide agents can be administered orally, enterally, transmucosally, subcutaneously, and / or parenterally. The term "parenteral" includes subcutaneous, intravenous, intrathecal, intramuscular, intraperitoneal, and intrasternal injection or infusion techniques. Other routes include, but are not limited to, nasal (e.g., through a nasogastric tube), transdermal, vaginal, rectal, sublingual, and inhalation. Delivery routes of the present invention can include intrathecal, intraventricular, or intracranial. In some embodiments of the invention, the LPA dsRNA agent or LPA antisense polynucleotide agent can be placed in a sustained release matrix and administered by placing the matrix in a subject. In some aspects of the invention, the LPA dsRNA agent or LPA antisense polynucleotide agent can be delivered to cells of a subject using nanoparticles coated with a delivery agent that targets a specific cell or organelle. A variety of delivery modes, methods, and agents are known in the art. Non-limiting examples of delivery methods and delivery agents are provided elsewhere herein. In some aspects of the invention, the term "delivery" with respect to an LPA dsRNA agent or LPA antisense polynucleotide agent can refer to administration of one or more "naked" LPA dsRNA agent or LPA antisense polynucleotide agent sequences to a cell or subject. In certain aspects of the invention, "delivery" refers to delivery to a cell or subject by transfection, delivery of a cell containing an LPA dsRNA agent or LPA antisense polynucleotide agent to a subject, delivery of a vector encoding an LPA dsRNA agent or LPA antisense polynucleotide agent to a cell and / or subject, and the like.Delivery of an LPA dsRNA agent or an LPA antisense polynucleotide agent using transfection methods can include administering a vector to a cell and / or a subject.

[0143] In some methods of the present invention, one or more LPA dsRNA agents or LPA antisense polynucleotide agents may be administered in a formulation or in a pharma- ceutically acceptable solution, which may generally contain pharma- ceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients. In some embodiments of the present invention, the LPA dsRNA agent or LPA antisense polynucleotide agent may be formulated with another therapeutic agent for co-administration. According to the methods of the present invention, the LPA dsRNA agent or LPA antisense polynucleotide agent may be administered in the form of a pharmaceutical composition. Typically, the pharmaceutical composition comprises an LPA dsRNA agent or LPA antisense polynucleotide agent and, optionally, a pharma- ceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those skilled in the art. As used herein, a pharma- ceutically acceptable vector refers to a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient (e.g., the ability of the LPA dsRNA agent or LPA antisense polynucleotide agent to inhibit LPA gene expression in a cell or subject). A variety of methods of administering and delivering dsRNA agents or LPA antisense polynucleotide agents for therapeutic use are known in the art and can be used in the methods of the invention.

[0144] Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers and other materials known in the art. Exemplary pharma-ceutically acceptable carriers are described in U.S. Pat. No. 5,211,657, while other carriers are known to those skilled in the art. Such formulations may generally contain salts, buffers, preservatives, compatible carriers and optionally other therapeutic agents. Although salts must be pharma-ceutically acceptable for use in medicine, pharma-ceutically unacceptable salts may be conveniently used to prepare pharma-ceutically acceptable salts thereof and are not excluded from the scope of the present invention. Such pharmacologically and pharma-ceutically acceptable salts include, but are not limited to, salts prepared from hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, and the like. Additionally, pharma-ceutically acceptable salts may be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts or calcium salts.

[0145] Some embodiments of the method of the present invention include administering one or more LPA dsRNA agents or LPA antisense polynucleotide agents directly to a tissue. In some embodiments, the tissue to which the compound is administered is a tissue in which an LPA-related disease or condition is present or likely to develop, non-limiting examples of tissues are the liver or kidney. Direct tissue administration can be achieved by direct injection or other means. Many orally delivered compounds naturally enter and pass through the liver and kidney, and some embodiments of the therapeutic method of the present invention include oral administration of one or more LPA dsRNA agents to a subject. The LPA dsRNA agent or LPA antisense polynucleotide agent can be administered once or multiple times, alone or in combination with other therapeutic agents. When administered multiple times, the LPA dsRNA agent or LPA antisense polynucleotide agent can be administered via different routes. For example, and not intended to be limiting, the first (or first few) administrations can be performed subcutaneously, and one or more additional administrations can be oral and / or systemic.

[0146] For the embodiment of the present invention in which it is desired to administer LPA dsRNA agent or LPA antisense polynucleotide agent systemically, LPA dsRNA agent or LPA antisense polynucleotide agent can be formulated for parenteral administration by injection, for example, by bolus or continuous infusion.Injectable formulations can be in unit dosage form, such as ampoules or multi-dose containers, with or without preservatives.LPA dsRNA agent formulations (also referred to as pharmaceutical compositions) can be in the form of suspension, solution or emulsion in oily or aqueous carriers, and can contain excipients such as suspending agents, stabilizing agents and / or dispersing agents.

[0147] Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's glucose solution, glucose and sodium chloride solution, lactated Ringer's solution or fixed oils. Intravenous vehicles include fluid and nutritional supplements, electrolyte supplements (such as those based on Ringer's glucose solution), and the like. Preservatives and other additives such as antibacterial agents, antioxidants, chelating agents, inert gases, and the like may also be present. Other forms of administration, such as intravenous administration, result in smaller doses. If the subject's response is inadequate with the initial dose, higher doses may be used within the tolerance of the patient (or the dose may be effectively increased by a different, more localized delivery route). Multiple doses per day can be used as necessary to achieve adequate systemic or local levels of one or more LPA dsRNA agents or LPA antisense polynucleotide agents and to achieve an appropriate reduction in LPA protein levels.

[0148] In other embodiments, the methods of the invention include the use of a delivery vehicle, such as a biocompatible microparticle, nanoparticle, or implant suitable for implantation into a recipient, such as a subject. Exemplary biodegradable implants that may be used according to the methods are described in PCT Publication WO 95 / 24929 (incorporated herein by reference), which describes biocompatible, biodegradable polymer matrices for incorporating biopolymers.

[0149] Both non-biodegradable and biodegradable polymer matrices can be used in the methods of the invention to deliver one or more LPA dsRNA agents or LPA antisense polynucleotide agents to a subject. In some embodiments, the matrix can be biodegradable. The matrix polymer can be a natural or synthetic polymer. The polymer can be selected based on the desired release period, typically on the order of a few hours to a year or more. Typically, release over a period of a few hours to 3 to 12 months can be used. The polymer is optionally in the form of a hydrogel that can absorb up to about 90% of its weight in water, and is optionally crosslinked with multivalent ions or other polymers.

[0150] In general, the LPA dsRNA agent or LPA antisense polynucleotide agent may be delivered using a biodegradable implant in some embodiments of the present invention, either by diffusion or by degradation of the polymer matrix. Exemplary synthetic polymers for this use are well known in the art. Using methods known in the art, biodegradable and non-biodegradable polymers may be used to deliver the LPA dsRNA agent or LPA antisense polynucleotide agent. Bioadhesive polymers such as biodegradable hydrogels (HSSawhney, CPPathak and JAHubell in Macromolecules, 1993, 26, 581-587) may also be used to deliver the LPA dsRNA agent or LPA antisense polynucleotide agent to treat LPA-related diseases or conditions. Other suitable delivery systems may include sustained, delayed or extended release delivery systems. Such systems avoid repeated administration of the LPA dsRNA agent or LPA antisense polynucleotide agent, thereby improving convenience for the subject and medical professional. Many types of release delivery systems are available and known to those skilled in the art. See, e.g., U.S. Patent Nos. 5,075,109, 4,452,775, 4,675,189, 5,736,152, 3,854,480, 5,133,974, and 5,407,686. In addition, pump-based hardware delivery systems can be used, some of which are also suitable for implantation.

[0151] The use of long-term sustained release implants can be adapted for prophylactic treatment of subjects and subjects at risk of developing recurrent LPA-related diseases or conditions.As used herein, long-term release refers to the construction and configuration of the implant to deliver therapeutic levels of LPA dsRNA agents or LPA antisense polynucleotide agents for at least up to 10 days, 20 days, 30 days, 60 days, 90 days, 6 months, 1 year or more.Long-term sustained release implants are well known to those skilled in the art and include some of the release systems mentioned above.

[0152] Therapeutic formulations of LPA dsRNA agents or LPA antisense polynucleotide agents are prepared by combining the molecules or compounds having the desired purity with optional pharma- ceutically acceptable carriers, excipients, or stabilizers in the form of a lyophilized formulation or an aqueous solution [Remington's Pharmaceutical Sciences 21 st edition, (2006)]. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol, or benzyl alcohol; parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin, gelatin, and the like. hydrophilic polymers (such as polyvinylpyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0153] Cells, Subjects and Controls The method of the present invention may be used in conjunction with cells, tissues, organs and / or subjects. In some aspects of the present invention, the subject is a vertebrate mammal, including but not limited to humans or primates such as dogs, cats, horses, cows, goats, mice, rats and monkeys. Thus, the present invention may be used to treat LPA-related diseases or conditions in both human and non-human subjects. In some aspects of the present invention, the subject may be a farm animal, a zoo animal, a domestic animal or a non-domestic animal, and the method of the present invention may be used in veterinary preventive and therapeutic regimens. In some embodiments of the present invention, the subject is a human being, and the method of the present invention may be used in preventive and therapeutic regimens for humans.

[0154] Non-limiting examples of subjects to which the present invention may be applied are subjects diagnosed with, suspected of, or at risk of developing a disease or condition associated with higher than expected LPA expression, also referred to as "elevated LPA expression levels". Non-limiting examples of diseases and conditions associated with higher than expected LPA expression are described elsewhere herein. The method of the present invention may be applied to subjects who have been diagnosed with a disease or condition at the time of treatment, subjects associated with higher than expected LPA expression, or subjects considered to be at risk of suffering from or developing a disease or condition associated with higher than expected LPA expression. In some aspects of the present invention, the disease or condition associated with higher than expected LPA expression levels is an acute disease or condition; in some aspects of the present invention, the disease or condition associated with higher than expected LPA expression levels is a chronic disease or condition.

[0155] In one non-limiting example, the LPA dsRNA agent of the present invention is administered to a subject diagnosed with cardiovascular disease, and cardiovascular disease includes Berge's disease, peripheral artery disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic stenosis, aortic regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperapolipoprotein betalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease and venous thrombosis, stroke, atherosclerosis, thrombosis, coronary heart disease or aortic stenosis and / or any other disease or condition associated with elevated levels of Lp(a)-containing particles.The method of the present invention can be applied to subjects who have been diagnosed with disease or condition at the time of treatment or subjects who are considered to be at risk of suffering from or developing disease or condition.

[0156] In another non-limiting example, the LPA dsRNA agent of the present invention is administered to treat the disease or disorder caused by or associated with renin-angiotensin-aldosterone system (RAAS) activation, or the disease or disorder whose symptoms or progression respond to RAAS inactivation.The term "LPA-related disease" includes the disease, disorder or condition that benefits from the reduction of LPA expression.These diseases are usually associated with hypertension. Non-limiting examples of LPA-associated diseases include cardiovascular diseases including Berge's disease, peripheral artery disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic stenosis, aortic regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperapolipoprotein betalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease, and venous thrombosis, stroke, atherosclerosis, thrombosis, coronary heart disease or aortic stenosis, and / or any other disease or condition associated with elevated levels of Lp(a)-containing particles.

[0157] The cells to which the methods of the invention can be applied include in vitro, in vivo and ex vivo cells. The cells can be in a subject, in culture and / or in suspension or any other suitable context or condition. The cells to which the methods of the invention can be applied can be liver cells, hepatic cells, cardiac cells, pancreatic cells, cardiovascular cells, renal cells or other types of vertebrate cells, including human and non-human mammalian cells. In some aspects of the invention, the cells to which the methods of the invention can be applied are healthy normal cells and are not known to be diseased cells. In some embodiments of the invention, the methods and compositions of the invention are applied to cells such as liver cells, hepatic cells, cardiac cells, pancreatic cells, cardiovascular cells and / or renal cells. It should also be understood that while in certain aspects of the invention, the control cells are normal cells, cells with a disease or condition can be used as control cells in certain cases, such as when comparing the results of a treated cell with a disease or condition to the results of an untreated cell with a disease or condition.

[0158] According to the method of the present invention, the LPA polypeptide level can be determined and compared to an LPA polypeptide control level. The control can be a predefined value that can take various forms. It can be a single cut-off, such as a median or mean value. It can be established based on comparison groups, for example, in a group with normal levels of LPA polypeptide and a group with increased levels of LPA polypeptide activity. Another non-limiting example of a comparison group can be a population with one or more symptoms or diagnoses of an LPA-related disease or condition versus a population without one or more symptoms or diagnoses of a disease or condition; a group of subjects with siRNA treatment of the present invention versus a group of subjects without siRNA treatment of the present invention. Typically, the control can be based on apparently healthy normal individuals or apparently healthy cells in an appropriate age group. In addition to a predefined value, it will be appreciated that the control according to the present invention can be a material sample that is tested in parallel with the experimental material. Examples include samples from a control population or a control sample generated by manufacturing for parallel testing with the experimental sample. In some embodiments of the invention, a control may include a cell or subject that is not contacted with or treated with an LPA dsRNA agent of the invention, in which case the control level of LPA polypeptide may be compared to the level of LPA polypeptide in a cell or subject that has been contacted with an LPA dsRNA agent or an LPA antisense polynucleotide agent of the invention.

[0159] In some embodiments of the invention, the control level may be an LPA polypeptide level determined for a subject, and LPA polypeptide levels determined for the same subject at different time points are compared to the control level. In one non-limiting example, the level of LPA is determined in a biological sample obtained from a subject who has not received LPA treatment according to the present invention. In some embodiments, the biological sample is a serum sample. The LPA polypeptide level measured in a sample obtained from a subject may serve as a baseline or control value for the subject. After one or more LPA dsRNA agents are administered to the subject according to the treatment method of the present invention, one or more additional serum samples may be obtained from the subject, and the LPA polypeptide level in the one or more subsequent samples may be compared to the subject's control / baseline level. Such a comparison may be used to assess the onset, progression, or regression of an LPA-related disease or condition in a subject. For example, the level of LPA polypeptide in a baseline sample obtained from a subject is higher than the level obtained from the same subject after an LPA dsRNA agent or an LPA antisense polynucleotide agent of the present invention has been administered to the subject, indicating regression of an LPA-related disease or condition and the effectiveness of the LPA dsRNA agent of the present invention administered in treating an LPA-related disease or condition.

[0160] In certain aspects of the present invention, one or more values ​​of LPA polypeptide level determined for a subject can be used as a control value and used to compare the LPA polypeptide level in the same subject later, thereby allowing evaluation of the change from the "baseline" LPA polypeptide level in the subject.Therefore, when an initial level is used as the control level of the subject, the initial LPA polypeptide level can be used to indicate and / or determine the level of LPA polypeptide in the subject that the method and compound of the present invention can reduce in the subject.

[0161] Using the method of the present invention, the LPA dsRNA agent and / or LPA antisense polynucleotide agent of the present invention can be administered to a subject. Such dsRNAi agents include, for example, the duplexes shown in Table 1. In some other embodiments, such dsRNAi agents include duplex variants such as those shown in Table 1. The effectiveness of the administration and treatment of the present invention can be evaluated as follows: after administration and treatment, the level of LPA polypeptide in serum samples obtained from the subject is reduced by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to the level of LPA polypeptide before administration in serum samples obtained from the subject at a previous time point or compared to the level of a non-contact control (e.g., the level of LPA polypeptide in a control serum sample). It will be recognized that the level of LPA polypeptide is all related to the level of LPA gene expression. Some embodiments and methods of the invention include administering to a subject an LPA dsRNA and / or an LPA antisense agent of the invention in an amount that effectively inhibits expression of the LPA gene, thereby reducing the level of LPA polypeptide in the subject.

[0162] Some embodiments of the present invention include determining the presence, absence and / or amount (also referred to herein as level) of LPA polypeptide from one or more biological samples obtained from one or more subjects. This determination can be used to evaluate the effectiveness of the treatment method of the present invention. For example, the methods and compositions of the present invention can be used to determine the level of LPA polypeptide in biological samples obtained from subjects previously treated with LPA dsRNA agents and / or LPA antisense agents of the present invention. After administration and treatment, the level of LPA polypeptide in serum samples obtained from the subject is reduced by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to the level of LPA polypeptide before administration in serum samples obtained from the subject at a previous time point or compared to the level of a non-contact control (e.g., the level of LPA polypeptide in a control serum sample), indicating the level of effectiveness of the treatment administered to the subject.

[0163] In some embodiments of the present invention, the physiological characteristics of an LPA-related disease or condition determined for a subject may serve as a control result, and the results of the determination of the physiological characteristics of the same subject are compared to the control result. In one non-limiting example, the Lp(a) level in blood (and / or the physiological characteristics of an LPA disease or condition) may be measured from a subject who has not been treated for LPA according to the present invention and used as a baseline or control value for the subject. After one or more LPA dsRNA agents are administered to the subject according to the treatment method of the present invention, the Lp(a) level in blood is measured and compared to the control / baseline level of the subject, respectively. Such a comparison may be used to evaluate the onset, progression or regression of an LPA-related disease or condition in a subject. For example, a baseline LPA level obtained from a subject is higher than the LPA level measured from the same subject after an LPA dsRNA agent or an LPA antisense polynucleotide agent of the present invention is administered to the subject, indicating regression of an LPA-related disease or condition and the efficacy of the LPA dsRNA agent of the present invention administered in treating an LPA-related disease or condition.

[0164] In some aspects of the present invention, the value of one or more physiological characteristics of an LPA-related disease or condition determined for a subject is used as a control value for subsequent comparison of the physiological characteristics of the same subject, thereby allowing evaluation of changes from the subject's "baseline" physiological characteristics. Thus, an initial physiological characteristic in an individual can be obtained, and the measurement value of the initial physiological characteristic is used as a control for the subject, and is used to demonstrate and / or determine the effectiveness that the methods and compounds of the present invention can be used to reduce the level of LPA polypeptide in the individual. Using the methods of the present invention, the LPA dsRNA agent and / or LPA antisense polynucleotide agent of the present invention can be administered to a subject in an effective amount for the treatment of an LPA disease or condition. The effectiveness of the administration and treatment of the present invention can be evaluated by determining changes in one or more physiological characteristics of an LPA disease or condition. In one non-limiting example, the Lp(a) level in the subject's blood is reduced compared to the Lp(a) level in the blood obtained from the subject at a previous time point or compared to the LPA level in a non-contact control, until the Lp(a) level in the subject's blood is within the normal range.

[0165] Some embodiments of the invention include determining the presence, absence, and / or changes in physiological characteristics of an LPA-associated disease or condition using methods such as, but not limited to, (1) measuring Lp(a) levels in the blood of a subject; (2) evaluating physiological characteristics of one or more biological samples obtained from one or more subjects; (3) or performing a physical examination on the subject. This determination can be used to evaluate the effectiveness of the treatment methods of the invention.

[0166] kit Also within the scope of the present invention is a kit comprising one or more LPA dsRNA agents and / or LPA antisense polynucleotide agents and instructions for their use in the methods of the present invention. The kit of the present invention may comprise one or more of LPA dsRNA agents, LPA sense polynucleotide agents and LPA antisense polynucleotide agents that can be used to treat LPA-related diseases or conditions. A kit comprising one or more LPA dsRNA agents, LPA sense polynucleotide agents and LPA antisense polynucleotide agents can be prepared for use in the treatment methods of the present invention. The components of the kit of the present invention can be packaged in aqueous media or lyophilized form. The kit of the present invention may comprise a support that is partitioned to contain one or more container devices or a series of container devices (e.g., test tubes, vials, flasks, bottles, syringes, etc.) sealed therein. A first container device or a series of container devices may comprise one or more compounds, such as LPA dsRNA agents and / or LPA sense or antisense polynucleotide agents. The second container device or series of container devices may contain targeting agents, labeling agents, delivery agents, etc., which may be included as part of the LPA dsRNA agent and / or LPA antisense polynucleotide administered in embodiments of the therapeutic methods of the invention.

[0167] The kits of the invention may also include instructions, which are typically written documents that provide guidance for carrying out the treatment embodied by the kit and for making decisions based on that treatment.

[0168] The following examples are provided to illustrate the practice of the present invention and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that the present invention can be applied to a variety of compositions and methods. EXAMPLES

[0169] Example 1. Synthesis of RNAi Agents The LPA RNAi agent duplexes shown in Tables 2-3 above were synthesized according to the following general procedure.

[0170] The sense and antisense strand sequences of siRNA were synthesized on an oligonucleotide synthesizer using a well-established solid-phase synthesis method based on phosphoramidite chemistry. Growth of the oligonucleotide strand is achieved through a four-step cycle: deprotection, condensation, capping, and an oxidation or sulfurization step for each nucleotide addition. Synthesis was performed on a solid support composed of controllable pore glass (CPG, 1000 Å). Monomeric phosphoramidites were purchased from commercial sources. Phosphoramidites bearing GalNAc ligand clusters (GLPA1 and GLPA2 as non-limiting examples) were synthesized according to the procedures in Examples 2-3 herein. For siRNAs used for in vitro screening (Table 2), synthesis was performed at a 2 μmol scale; for siRNAs used for in vivo testing (Tables 3, 4-5), the synthesis scale was 5 μmol or greater. When a GalNAc ligand (GLO-0 as a non-limiting example) was linked to the 3' end of the sense strand, a CPG solid support with an attached GalNAc ligand was used. When GalNAc ligands (GLS-1 or GLS-2 as non-limiting examples) were linked to the 5'-end of the sense strand, GalNAc phosphoramidites (GLPA1 or GLPA2 as non-limiting examples) were used for the final coupling reaction. Trichloroacetic acid (TCA) in 3% dichloromethane was used to deprotect the protecting group, i.e., 4,4'-dimethoxytriphenylmethyl (DMT). 5-Ethylthio-1H-tetrazole was used as the activating agent. I2 in THF / Py / H2O and phenylacetyl disulfide (PADS) in pyridine / MeCN were used for the oxidation and sulfurization reactions, respectively. After the final solid-phase synthesis step, the solid support-bound oligomers were cleaved and the protecting groups removed by treatment with 1:1 volume of 40 wt% aqueous methylamine and 28% ammonium hydroxide solutions. The crude mixture was concentrated to synthesize siRNA for in vitro screening. The remaining solid was dissolved in 1.0 M NaOAc and ice-cold EtOH was added to precipitate the single-stranded product as the sodium salt, which could be used for annealing without further purification.To synthesize siRNA for in vivo testing, the crude single-stranded product was further purified by ion-pair reversed-phase HPLC (IP-RP-HPLC). The purified single-stranded oligonucleotide products from IP-RP-HPLC were converted to sodium salts by dissolving them in 1.0M NaOAc and precipitating them by adding ice-cold EtOH. Annealing of the sense and antisense oligonucleotides with equimolar complementarity was carried out in water to form double-stranded siRNA products, which were lyophilized to obtain fluffy white solids.

[0171] Example 2. Preparation of Intermediate-A and Intermediate-B Intermediate-A was synthesized by treating commercially available galactosamine pentaacetate with trimethylsilyl trifluoromethanesulfonate (TMSOTf) in dichloromethane (DCM) as shown in Scheme 1 below. Glycosylation was then carried out with Cbz-protected 2-(2-aminoethoxy)ethan-1-ol to give compound II. The Cbz-protecting group was removed by hydrogenation to give Intermediate-A as a trifluoroacetic acid (TFA) salt. Intermediate-B was synthesized based on the same scheme, except for the use of Cbz-protected 2-(2-(2-aminomethoxy)ethoxy)ethan-1-ol as the feed material. [ka]

[0172] Scheme 1 TMSOTf (17.1 g, 77.2 mmol) was added to a solution of compound I (20.0 g, 51.4 mmol) in 100 mL of 1,2-dichloroethane (DCE). The resulting reaction mixture was stirred at 60° C. for 2 h, followed by 25° C. for 1 h; Cbz-protected 2-(2-aminoethoxy)ethan-1-ol (13.5 g, 56.5 mmol) was dried over 4 Å powdered molecular sieves (10 g) in DCE (100 mL) and added dropwise to the above reaction mixture at 0° C. under N2 atmosphere. The resulting reaction mixture was stirred at 25° C. for 16 h under N2 atmosphere. The reaction mixture was filtered and washed with saturated NaHCO3 (200 mL), water (200 mL) and saturated saline (200 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give the crude product, which was triturated with 2-methyltetrahydrofuran / heptane (5 / 3, v / v, 1.80 L) for 2 h. The resulting mixture was filtered and dried to give compound II (15.0 g, 50.3% yield) as a white solid.

[0173] 10% Pd / C (1.50 g) was carefully added to a dry, argon-purged hydrogenation bottle, followed by 10 mL of tetrahydrofuran (THF), followed by a solution of compound II (15.0 g, 26.4 mmol) in THF (300 mL) and TFA (trifluoroacetic acid, 3.00 g, 26.4 mmol). The resulting mixture was degassed and purged with H2 three times and stirred under H2 atmosphere (45 psi) at 25 °C for 3 h. Thin layer chromatography (TLC, solvent: DCM:MeOH = 10:1) showed that compound II was completely consumed. The reaction mixture was filtered and concentrated under reduced pressure. The residue was dissolved in anhydrous DCM (500 mL) and concentrated. This process was repeated three times to give intermediate-A (14.0 g, 96.5% yield) as a foamy white solid. 1H NMR (400MHz DMSO-d6): δ ppm 7.90(d,J=9.29Hz,1H),7.78(br s,3H),5.23(d,J=3.26Hz,1H),4.98(dd,J=11.29,3.26Hz,1H),4.56(d,J=8. 53Hz,1H),3.98-4.07(m,3H),3.79-3.93(m,2H),3.55-3.66(m,5H),2.98(br d,J=4.77Hz,2H),2.11(s,3H),2.00(s,3H),1.90(s,3H),1.76(s,3H).

[0174] Intermediate-B was synthesized using a procedure similar to that used for the synthesis of Intermediate-A. 1 H NMR(400MHz DMSO-d6):δ ppm 7.90(br d,J=9.03Hz,4H),5.21(d,J=3.51Hz,1H),4.97(dd,J=11.1Hz,1H),4.54(d,J=8.53Hz,1H), 3.98-4.06(m,3H),3.88(dt,J=10.9Hz,1H),3.76-3.83(m,1H),3.49-3.61(m,9H),2.97(br s,2H),2.10(s,3H),1.99(s,3H),1.88(s,3H),1.78(s,3H).Mass spectrometry calculation value C 20 H 34 N2O 11 : 478.22; Measured value: 479.3 (M+H + ).

[0175] Example 3. Synthesis of GalNAc Ligand Cluster Phosphoramidites GLPA1, GLPA2, and GLPA15 GLPA1 and GLPA2 were prepared according to Scheme 2 below. Starting with benzyl-protected propane-1,3-diamine, alkylation was carried out using tert-butyl 2-bromoacetate to give triester compound I. The benzyl protecting group was removed by hydrogenation to give secondary amine compound II. The amide was coupled with 6-hydroxycaproic acid to give compound III. The tert-butyl reactive group was then removed upon treatment with HCl in dioxane to form triacid compound IV. Amide coupling between triacid compound IV and intermediate-A or intermediate-B was carried out to give compound Va or Vb. Phosphoramidite GLPA1 or GLPA2 was synthesized by phosphitylation of compound Va or Vb with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite and a catalytic amount of 1H-tetrazole. [ka]

[0176] Scheme 2 2-tert-Butyl bromoacetate (23.7 g, 121 mmol) was added to a solution of N-benzyl-1,3-propanediamine (5.00 g, 30.4 mmol) in dimethylformamide (DMF, 100 mL); followed by dropwise addition of diisopropylethylamine (DIEA, 23.61 g, 182 mmol). The resulting reaction mixture was stirred at 25-30 °C for 16 h. LCMS showed that N-benzyl-1,3-propanediamine was completely consumed. The reaction mixture was diluted with H2O (500 mL) and extracted with EtOAc (500 mL × 2). The combined organics were washed with saturated brine (1 L), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography (gradient: petroleum ether: ethyl acetate 20:1 to 5:1). Compound I was obtained as a colorless oil (12.1 g, 78.4% yield). 1H NMR(400MHz,CDCl3):δ ppm 7.26-7.40(m,5H),3.79(s,2H),3.43(s,4H),3.21(s,2H),2.72(dt,J=16.9,7.34Hz,4H),1.70(quin,J=7.2Hz,2H),1.44-1.50(m,27H).

[0177] A dried hydrogenation bottle was purged with argon three times. Pd / C (200 mg, 10%) was added, followed by MeOH (5 mL), followed by a solution of compound I (1.00 g, 1.97 mmol) in MeOH (5 mL). The reaction mixture was degassed in vacuum and backfilled with H2. This process was repeated three times. The mixture was stirred at 25° C. under H2 atmosphere (15 psi) for 12 h. LCMS showed that compound I was completely consumed. The reaction mixture was filtered under reduced pressure under N2 atmosphere. The filtrate was concentrated under reduced pressure to give compound II (655 mg, 79.7% yield) as a yellow oil, which could be used in the next step without further purification. 1 H NMR (400MHz, CDCl3): δ ppm 3.44(s,4H),3.31(s,2H),2.78(t,J=7.1Hz,2H),2.68(t,J=6.9Hz,2H),1.88(br s,1H),1.69(quin,J=7.03Hz,2H),1.44-1.50(s,27H).

[0178] A mixture of compound II (655 mg, 1.57 mmol), 6-hydroxyhexanoic acid (249 mg, 1.89 mmol), DIEA (1.02 g, 7.86 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 904 mg, 4.72 mmol) and 1-hydroxybenzotriazole (HOBt, 637 mg, 4.72 mmol) in DMF (6 mL) was degassed and purged with N2 three times; then stirred at 25 °C under N2 atmosphere for 3 h. LCMS showed the desired product. The reaction mixture was diluted with H2O (10 mL) and extracted with 20 mL of EtOAc (10 mL x 2). The organics were combined, washed with saturated brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated to give the crude product, which was purified by silica gel column chromatography (gradient: petroleum ether:ethyl acetate 5:1 to 1:1) to give compound III (650 mg, 77.8% yield) as a yellow oil. 1 H NMR(400MHz,CDCl3):δ ppm 3.90-3.95(s,2H),3.63(t,J=6.40Hz,2H),3.38-3.45(m,6H),2.72(t,J=6.6 Mass spectrometry calculation value C 27 H 50 NO8: 530.36; Measured value: 531.3 (M+H) + .

[0179] A mixture of compound III (5.5 g, 10.3 mmol) in HCl / dioxane (2 M, 55 mL) was stirred at 25° C. for 3 h. LCMS showed complete consumption of compound III. The reaction mixture was filtered, washed with EtOAc (50 mL), and dried under reduced pressure to give the crude product. The crude product was dissolved in CH3CN (50 mL) and the volatiles were removed in vacuo. This process was repeated three times to give compound IV (2.05 g, 54.5% yield) as a white solid. 1H NMR(400MHz,D2O):δ ppm 4.21(s,1H),4.07(d,J=4.5Hz,4H),3.99(s,1H),3.45-3.52(m,3H),3. 42(t,J=6.5Hz,1H),3.32-3.38(m,1H),3.24-3.31(m,1H),2.37(t,J=7 .4Hz,1H),2.24(t,J=7.4Hz,1H),1.99(dt,J=15.5,7.53Hz,1H),1.85-1.94(m,1H),1.85-1.94(m,1H),1.39-1.56(m,4H),1.19-1.31(m,2H).

[0180] A mixture of compound IV (500 mg, 1.05 mmol), intermediate-A (2.02 g, 3.67 mmol), DIEA (813 mg, 6.30 mmol), EDCI (704 mg, 3.67 mmol) and HOBt (496 mg, 3.67 mmol) in DMF (10 mL) was degassed and purged with N2 three times, then the mixture was stirred under N2 atmosphere at 25 °C for 3 h. LCMS showed the desired product. The reaction mixture was quenched by adding H2O (10 mL) and extracted with DCM (10 mL x 2). The combined organics were extracted with 10% citric acid (20 mL). The aqueous phase was neutralized with saturated NaHCO3 solution and re-extracted with DCM (10 mL x 2). The organics were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give compound Va (570 mg, 0.281 mmol, 26.8% yield) as a white solid. 1 H NMR:(400MHz,CDCl3)ppm δ 7.84-8.12(m,3H),6.85-7.15(m,2H),6.66-6.81(m,1H),5.36(br d,J=2.7Hz,3H),5.11-5.27(m,3H),4.63-4.85(m,3H),3.90-4.25(m,18H),3.37-3.75(m,28H),3.15-3.28(m,4H),2.64(br d,J=6.53Hz,2H),2.30-2.46(m,2H),2.13-2.18(m,9H),2.05(s,9H),1.94-2.03(m,18H),1.68(br s,2H),1.45(br s,2H),1.12(br t,J=7.0Hz,2H).

[0181] Tetrazolediisopropylammonium (30.3 mg, 0.177 mmol) was added to a solution of compound Va (260 mg, 0.161 mmol) in anhydrous DCM (5 mL); followed by dropwise addition of 3-bis(diisopropylamino)phosphoryloxypropionitrile (194 mg, 0.645 mmol) at ambient temperature under N2. The reaction mixture was stirred at 20-25 °C for 2 h. LCMS showed that compound Va was completely consumed. After cooling to -20 °C, the reaction mixture was added to a stirred solution of brine / saturated NaHCO3 (1:1, 5 mL) at 0 °C. After stirring for 1 min, DCM (5 mL) was added. Layering occurred. The organic layer was washed with brine / saturated aqueous NaHCO3 (1:1, 5 mL), dried over Na2SO4, filtered, and concentrated to a volume of approximately 1 mL. The remaining solution was added dropwise to 20 mL of methyl tert-butyl ether (MTBE) under stirring. This resulted in the precipitation of a white solid. The mixture was centrifuged and the solid was collected. The solid was redissolved in 1 mL of DCM and precipitated by the addition of MTBE (20 mL). The solid was again isolated by centrifugation. The collected solid was dissolved in anhydrous CH3CN. The volatiles were removed. This process was repeated two more times to give the GalNAc ligand phosphoramidite compound GLPA1 (153 mg, 84.4 μmol) as a white solid. 1 H NMR(400MHz,CDCl3):ppm δ 7.71-8.06(m,2H),6.60-7.06(m,3H),5.37(br d,J=3.0Hz,3H),5.18-5.32(m,3H),4.70-4.86(m,3H),3.92-4.25(m,18H),3.42-3.85(m,30H),3.25(m ,4H),2.59-2.75(m,4H),2.27-2.44(m,2H),2.15-2.20(s,9H)2.07(s,9H),1.96-2.03(m,18H),1.65(br s,4H),1.44(br d,J=7.28Hz,2H),1.14-1.24(m,12H). 31 P NMR(CDCl3): ppm δ 147.15.

[0182] The GalNAc ligand phosphoramidite compound GLPA2 was synthesized using the same procedure, except using intermediate-B. 1 H NMR(400MHz,CDCl3):ppm δ 7.94-8.18(m,1H),7.69(br s,1H),6.66-7.10(m,3H),5.35(d,J=3.5Hz,3H),5.07-5.25(m,3H),4.76-4 .86(m,3H),4.01-4.31(m,10H),3.91-4.01(m,8H),3.74-3.86(m,4H),3.52- 3.71(m,30H),3.42-3.50(m,6H),3.15-3.25(m,4H),2.52-2.70(m,4H),2.2 2-2.45(m,2H),2.15-2.22(s,9H),2.06(s,9H),1.95-2.03(m,18H),1.77(br s,2H),1.58-1.66(m,4H),1.40(m,2H),1.08-1.24(m,12H). 31 P NMR(CDCl3): ppm δ 147.12.

[0183] GLPA15 was prepared according to Scheme 3 below. [ka]

[0184] Scheme 3 To a solution of intermediate compound II (275 g, 660 mmol, 1.00 eq.) in dichloromethane (2.75 L), triethylamine (133 g, 1.32 mol, 2.00 eq.) was added, followed by dropwise addition of Cbz-Cl (169 g, 990 mmol, 1.50 eq.). The reaction liquid was stirred at 25 °C for 2 h, and LCMS showed that compound II was completely converted. The reaction liquid was washed with saturated NaHCO3 solution (800 mL) and saturated aqueous sodium chloride solution (500 mL) in turn, and the organic phase was dried over anhydrous Na2SO4. After filtering to remove the drying agent, the filtrate was concentrated to dryness. The residue was subjected to column chromatography (SiO2, PE / EA = 100 / 1 to 5 / 1) to give compound 5 (290 g, 527 mmol, 75.7% yield) as a colorless oil.1 H NMR (400MHz in DMSO-d6): δ ppm 7.23-7.40(m,5H),5.00-5.12(m,2H),3.86-3.95(m,2H),3.23-3.39(m,6H ),2.55-2.67(m,2H),1.56-1.64(m,2H),1.31-1.46(m,27H).MS(ESI)[M+H] + m / z: 551.6.

[0185] HCOOH (2.9 L) was added to compound 5 (145 g, 263 mmol, 1.00 eq) and the solution was stirred at 60° C. for 12 h. LCMS showed that compound 5 was completely converted. 1.5 L of toluene and 1.5 L of acetonitrile were added to the reaction liquid and the mixture was concentrated under reduced pressure to about 500 mL. Then, toluene / acetonitrile (1:1, about 750 mL) was added and the mixture was concentrated to about 500 mL. Then, acetonitrile (about 1000 mL) was added and the mixture was concentrated to dryness. The crude product was triturated with 700 mL of acetonitrile at 60° C. for 2 h and filtered. The solid was collected and dried to give white solid compound 6 (105 g, quantitative). 1 H NMR (400MHz in DMSO-d6): δ ppm 7.26-7.40(m,5H),5.02-5.10(m,2H),3.89-4.00(m,2H),3.36-3.45(m,4H),3.24-3.34(m,2H),2.59-2.72(m,2H),1.40(s,2H).MS(ESI)[M+H] + m / z:383.0.

[0186] TBTU (327 g, 1.02 mol, 3.90 eq.) and triethylamine (212 g, 2.09 mol, 8.00 eq.) were added to a solution of compound 6 (100 g, 261 mmol.) and intermediate-A (502 g, 915 mmol, 3.50 eq.) in DMF (1.0 L), and the reaction was carried out at 25° C. for 1 h. LCMS showed that the conversion of compound 6 was complete. The reaction liquid was added to 4000 mL of water, extracted with methyl tert-butyl ether (2000 mL, two portions) to remove impurities, and the remaining aqueous phase was extracted with dichloromethane (3000 mL, two portions). The dichloromethane phase was washed successively with 10% aqueous citric acid (2000 mL, two portions), saturated NaHCO3 (2.0 L, two portions) and saturated brine (2.0 L) and dried over anhydrous Na2SO4. The filtrate was filtered and concentrated under reduced pressure to give compound 8 (260 g, 159 mmol, 60.9% yield) as a white solid. 1 H NMR (400MHz in DMSO-d6): δ ppm 7.99-8.08(m,2H),7.93(br d,J=5.50Hz,1H),7.79-7.86(m,3H),7.26-7.39(m,5H),5.22(d,J=3.13Hz,3H),4.95-5.08(m,5H),4.54(br d,J=8.38Hz,3H),4.03(s,9H),3.81-3.93(m,5H),3.76(br d,J=4.88Hz,3H),3.44-3.62(m,10H),3.34-3.43(m,6H),3.24(br d,J=6.13Hz,7H),3.02-3.09(m,4H),2.40-2.47(m,2H),2.10(s,9H),1.99(s,9H),1.89(s,9H),1.77(s,9H),1.57-1.68(m,2H).MS(ESI)[M+H] + m / z:816.4.

[0187] A 2L hydrogenation autoclave was inerted with argon and dry Pd / C (9 g) was carefully added. MeOH (50 mL) was added to the wet Pd / C, followed by the slow addition of compound 8 (90 g, 55.1 mmol, 1.00 eq.) in MeOH (850 mL) and trifluoroacetic acid (6.29 g, 55.1 mmol, 1.00 eq.) under argon atmosphere. The mixture was degassed / purged with H2 three times, placed under hydrogen atmosphere and stirred at 25° C. for 10 h. LCMS showed that compound 8 was completely converted. Pd / C was removed by filtration and the filtrate was concentrated under reduced pressure to give compound 9 (80 g, 90.2% yield). 1 H NMR (400MHz in DMSO-d6): δ ppm 9.12(br s,2H),8.50(br t,J=5.19Hz,1H),8.10(br t,J=5.50Hz,2H),7.85-7.91(m,3H),5.22(d,J=3.25Hz,3H),4.95-5.01(m,3H),4.52 -4.58(m,3H),4.03(s,9H),3.84-3.93(m,3H),3.75-3.83(m,3H),3.39-3.61(m,16H) ,3.23-3.32(m,6H),3.15-3.18(m,3H),2.97-3.05(m,2H),2.54-2.61(m,2H),2.10(s ,9H),2.00(s,9H),1.89(s,9H),1.77-1.80(m,9H),1.70-1.76(m,2H).MS(ESI)[M+H] + m / z:749.3.

[0188] Triethylamine (67.8 g, 672 mmol, 4.00 eq.) was added to a solution of compound 9 (270 g, 168 mmol, 1.00 eq.) and glutaric anhydride (28.6 g, 252 mmol, 1.50 eq.) in dichloromethane (2.7 L). The solution was stirred at 25° C. for 1 h. LCMS showed that compound 9 was completely converted to compound 11. 4-Hydroxypiperidine (42.4 g, 420 mmol, 2.50 eq.) and TBTU (107 g, 335 mmol, 2.00 eq.) were added to the reaction liquid and stirring was continued at 25° C. for 1 h. LCMS showed that the conversion of compound 11 was complete. The reaction was quenched by slow addition of saturated NH4Cl (3.0 L), the layers were separated, and the aqueous phase was extracted with dichloromethane (2 x 1000 mL) and combined with the previous organic phase. The combined organic phase was washed with a 1:1 mixture of saturated NaHCO3(aq) and saturated brine (3.0 L), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was dissolved in 1.5 L of dichloromethane, and the resulting solution was added dropwise to methyl tert-butyl ether (7.5 L). A translucent white precipitate formed gradually during the addition. The precipitate was filtered in vacuum, and the solid was collected and dried in vacuum to give compound 13 (207 g, 72.8% yield) as a white solid. 1 H NMR (400MHz in DMSO-d6): δ ppm 8.05(br d,J=2.00Hz,2H),7.82(br d,J=7.38Hz,3H),5.21(br s,3H),4.98(br d,J=10.26Hz,3H),4.72(br s,1H),4.54(br d,J=7.88Hz,3H),4.03(br s,9H),3.74-3.94(m,9H),3.45-3.71(m,12H),3.40(br s,6H),3.24(br s,7H),3.07(br d,J=14.13Hz,5H),2.91-3.01(m,1H),2.24-2.44(m,5H),2.20(br s,1H),2.10(s,9H),1.96-2.04(m,9H),1.89(br s,9H),1.74-1.81(m,9H),1.51-1.73(m,6H),1.07-1.36(m,3H).MS(ESI)[M+H] + m / z:848.0.

[0189] 3-Bis(diisopropylamino)phosphonyloxypropionitrile (53.3 g, 177 mmol, 1.50 eq.) was added to a solution of compound 13 (200 g, 118 mmol, 1.00 eq.) and tetrazole diisopropylammonium (8.08 g, 47.2 mmol, 0.40 eq.) in dichloromethane (2.0 L), and the reaction mixture was stirred at 40° C. for 2 h. LCMS showed that the conversion of compound 13 was complete. The reaction mixture was washed with a 1:1 mixture of saturated NaHCO3 and saturated aqueous sodium chloride (2.0 L), dried over anhydrous Na2SO4, the filtrate was concentrated, and the resulting crude product was dissolved in dichloromethane (1.2 L), and the resulting solution was added dropwise to stirred methyl tert-butyl ether (6.0 L). The suspension was filtered, the cake was rinsed with methyl tert-butyl ether, the solid was collected and dried in vacuum, the product was dissolved in dichloromethane (1.0 L) and concentrated to dryness, and the procedure was repeated four times to remove residual tert-butyl ether to give GLPA15 (164 g, 73.3% yield). 1 H NMR (400MHz in DMSO-d6): δ ppm 8.05(br d,J=6.50Hz,2H),7.81(br d,J=9.01Hz,3H),5.22(d,J=3.25Hz,3H),4.98(dd,J=11.26,3.25Hz,3H),4.55(br d,J=8.50Hz,3H),4.03(s,9H),3.64-3.97(m,12H),3.55-3.63(m,6H),3.50(br s,5H),3.40(br d,J=6.13Hz,6H),3.17-3.30(m,9H),3.07(br d,J=14.26Hz,4H),2.76(t,J=5.82Hz,2H),2.18-2.47(m,6H),2.10(s,9H),1.9 9(s,9H),1.89(s,9H),1.78(s,9H),1.52-1.74(m,6H),1.12-1.19(m,12H).31P NMR(DMSO-d6):ppm δ 145.25.MS(ESI)[M+H]+m / z:1895.7.

[0190] In particular studies, methods are provided for linking a GalNAc-containing targeting group (also referred to herein as a GalNAc delivery compound) to the 5' end of a sense strand, which includes using GalNAc phosphoramidite (GLPA1) in the final coupling step of solid phase synthesis and using a synthetic process such as that used during oligonucleotide chain extension (i.e., adding nucleotides to the 5' end of the sense strand) to link GLPA1 to the 5' end of the sense strand.

[0191] In some studies, the method of linking a GalNAc-containing targeting group to the 3'-end of the sense strand includes using a solid support (CPG) containing GLO-n. In some studies, the method of linking a GalNAc-containing targeting group to the 3'-end of the sense strand includes linking a GalNAc targeting group to a CPG solid support via an ester bond and using the CPG obtained with a GalNAc targeting group linked when the sense strand is synthesized, so that the GalNAc targeting group is linked to the 3'-end of the sense strand. Other GalNAc phosphoramidite compounds (GLPAn) can also be obtained by using the appropriate corresponding intermediates and using methods similar to those described herein or known in the art, and can be linked to the appropriate position of the siRNA duplex as a targeting group.

[0192] Example 4. Synthesis of isomannitol phosphoramidite (compound 2) [ka] 4,4'-Dimethoxytriphenylmethane chloride (DMTrCl, 232 g, 684 mmol, 1.0 eq.) in pyridine (400 ml) was added to a solution of compound A (isomannitol, 100 g, 684 mmol, 1.0 eq.) in pyridine (600 ml), and the mixture was stirred at 25 °C for 16 h. LC-MS showed that compound A was completely consumed and a major peak with the desired mass was detected. The resulting reaction mixture was diluted with water (500 mL), extracted with dichloromethane (500 mL*2), and the combined organic phase was washed with brine (500 mL), dried over Na2SO4, and concentrated in vacuum to give a residue. The residue was purified by column chromatography (DCM / MeOH=100 / 1 to 50 / 1, 0.1% Et3N) to give compound B (150 g, 48.9% yield) as a yellow solid. 1 H NMR:EC4783-404-P1B1_C(400MHz,DMSO-d6) δ ppm 7.46(br d,J=7.63Hz,2H)7.28-7.37(m,6H)7.19-7.25(m,1H)6.90(br d,J=7.88Hz,4H)4.70(d,J=6.50Hz,1H)3.99-4.09(m,6H)3.88-3.96(m,2H)3.83(br dd,J=7.82,6.94Hz,1H)3.74(s,6H)3.41(br t,J=8.13Hz,1H)3.05(t,J=8.44Hz,1H)2.85(br t,J=7.50Hz,1H).

[0193] 2H-Tetrazole (0.45M, 436mL, 1.1eq) was added dropwise to a solution of compound B (80.0g, 178mmol, 1.0eq) in dichloromethane (5.0mL) under N2 atmosphere at 25°C, followed by dropwise addition of a solution of compound C (2-cyanoethyldiisopropylchlorophosphoramidite, 80.6g, 267mmol, 85.0mL, 1.5eq) in dichloromethane (200mL); the reaction mixture was stirred at 25°C for 1.0 h; LC-MS showed that compound B was completely consumed and a major peak with the desired mass was detected. The resulting reaction mixture was cooled to -20°C, poured into ice-cold NaHCO3 (500mL), extracted with dichloromethane (500mL*3), and the combined organic layers were washed with NaHCO3 / brine=1:1 (300mL / 300mL), dried over Na2SO4, and concentrated in vacuo (35°C) to give a residue (100mL). The residue was purified by column chromatography (Al2O3, DCM / MeOH=100 / 1-50 / 1, 0.1% Et3N) to give isomannitol phosphoramidite compound 2 (77g, 119mmol, yield: 66.5%) as a white solid. 1 H NMR:EC4783-423-P1B1_C(400MHz,DMSO-d6) δ ppm 7.22(br d,J=7.50Hz,2H)7.05-7.14(m,6H)6.96-7.02(m,1H)6.67(br dd,J=8.82,1.81Hz,4H)3.95-4.07(m,2H)3.73-3.83(m,1H)3.62-3.72(m,2H)3.48-3.53(m ,6H)3.27-3.37(m,3H)3.11(s,6H)2.82(td,J=8.54,2.31Hz,1H)2.47-2.63(m,3H)2.28(br d,J=1.63Hz,3H)0.82-1.00(m,13H).

[0194] Example 5. Preparation of a solid support containing isomannitol monomer [ka] [ka] represents the macroporous amine methyl polyethylene resin support part. A 50 L glass kettle was placed under nitrogen protection, dichloromethane (19.50 kg) was added to the glass kettle, and stirring was started. The temperature was controlled at 20-30°C, DMTr-imann (1.47 kg) was added to the glass kettle, triethylamine (1.50 kg), 4-dimethylaminopyridine (0.164 kg) and succinic anhydride (1.34 kg) were added to the reaction kettle, and the system was kept warm at 20-30°C for 18 hours, followed by sampling and the reaction was terminated. Saturated sodium bicarbonate solution (22.50 kg) was added to the reaction mass and stirred for 10-20 minutes, followed by settling until layers were formed, the lower organic phase was decanted, and the upper aqueous phase was extracted twice with dichloromethane; the organic phases were combined and dried over anhydrous sodium sulfate, the filtrate was filtered, and then decanted for rotary evaporation and concentrated to zero fractions to form an off-white to off-white solid (1.83 kg).

[0195] N,N-Dimethylformamide (23.50 kg) was added to a 100 L glass kettle and stirred. The temperature was controlled at 20-30 °C. Under nitrogen protection, O-benzotriazole-tetramethyluronium hexafluorophosphate (0.33 kg), the product from the previous step, and N,N-diisopropylethylamine (0.13 kg) were added to the 100 L glass kettle through a solid feed funnel. After addition was complete, the mixture was stirred for 10-30 minutes and subsequently discharged into a 50 L galvanized bucket for reserve use. Macroporous amino-methyl resin (3.25 kg) (available from Tianjin Nankai Hecheng Technology Co., Ltd., batch number HA2X1209, loading capacity 0.48 mmol / g) was added to the above 100 L solid-phase synthesis kettle through a solid feed funnel, the temperature was controlled at 20-30 °C, N,N-dimethylformamide (21.00 kg + 21.00 kg) and the reaction liquid used in the galvanized bucket in the previous step were added to the solid-phase synthesis kettle. The system was reacted under temperature-controlled conditions and monitored until the solid loading ≥ 250 umol / g, the loading detection method was UV. The system was pressure filtered under nitrogen, and the filter cake was rinsed with N,N-dimethylformamide (26.00 kg + 26.10 kg + 26.00 kg) three times, and the filter cake was left in the kettle. CAP.A (4.40kg+4.42kg+4.30kg) and CAP.B (4.40kg+4.40kg+4.47kg) were added into an 80L glass kettle and stirred for 3-8 minutes. This operation was repeated three times for capping. Acetonitrile (18.00kg+18.00kg+18.00kg+17.50kg+17.50kg) was added into the solid phase synthesis kettle. Nitrogen was bubbled for 10-30 minutes before pressure filtration. This operation was repeated four times. The cake was purged with nitrogen in the solid phase synthesis kettle for 2-4 hours, and then transferred to a 50L pressure filtration tank. The temperature was controlled at 15-30°C and drying was continued. The yellow-white solid product after drying was weighed: 3.516kg.

[0196] Isosorbide residues (imann) can be added to the 5' or 3' end of the oligonucleotide chain by processes well known to those skilled in the art, such as the inverse abasic process (invab), to further add targeting groups.

[0197] Example 6. In vitro screening of LPA siRNA duplexes using Huh7 cells and dual fluorescent reporter gene vectors Huh7 cells were adjusted to an appropriate density and then seeded in 96-well plates. At the same time as seeding, the dual fluorescent reporter gene vector psciCHECK2 containing the target gene was co-transfected with siRNA into Huh7 cells using Lipofectamine RNAiMax (Invitrogen-13778-150) according to the manufacturer's recommendation. Cells were transfected with test siRNA or control siRNA. siRNA was tested in triplicate at two concentrations (0.1 nM and 1.0 nM), and 48 hours after transfection, Dual-Glo® Luciferase Assay Reagent was added to detect the fluorescence value. The ratio of Renilla luminescence to Firefly luminescence was calculated and normalized based on the ratio of control siRNA-treated sample to calculate knockdown efficiency. As a result, as shown in Table 4, the duplex AV# used is derived from the sequence corresponding to that shown in Table 2.

[0198] Table 4 provides experimental results from an in vitro study of inhibition of LPA expression using multiple LPA RNAi agents.

[0199] [Table 34]

[0200] [Table 35]

[0201] [Table 36]

[0202] [Table 37]

[0203] [Table 38]

[0204] Example 7. In vivo testing of LPA siRNA duplexes To evaluate the in vivo activity of LPA siRNA, mice infected with AAV encoding human LPA and luciferase genes were used (4 mice per group). Female C57BL / 6J mice were infected by intravenous injection of undiluted 2x10^11 viral particles of adeno-associated virus 8 (AAV8) vector encoding human LPA and luciferase genes 14 days before siRNA administration. On day 0, mice were subcutaneously injected with a single dose of 6 mg / kg LPA siRNA agent or PBS. Blood samples were collected on day 0, before siRNA administration, and at the end of day 7. Luciferase activity was measured. The percentage of knockdown was calculated by comparing the luciferase activity of blood samples from the siRNA-treated group before administration and the luciferase activity of blood samples collected at the end of day 7, and performing normalization based on the change in luciferase activity in serum samples from the PBS-treated group. As a result, as shown in Table 5, the duplexes AD# used are derived from sequences corresponding to those shown in Table 3.

[0205] Table 5 provides experimental results from an in vivo study of the inhibitory effects on LPA expression using multiple LPA_RNAi agents at a single dose of 6 mpk. On day 7, the remaining luciferase activity relative to day 0 was normalized to the change in the PBS treatment group (mean ± SD).

[0206] [Table 39]

[0207] Example 8. In vivo testing of LPA siRNA duplexes To evaluate the in vivo activity of LPA siRNA, mice infected with AAV encoding human LPA and luciferase genes were used (4 mice per group). Female C57BL / 6J mice were infected by intravenous injection of undiluted 2x10^11 viral particles of adeno-associated virus 8 (AAV8) vector encoding human LPA and luciferase genes 14 days prior to siRNA administration. On day 0, mice were subcutaneously injected with a single dose of 6mg / kg LPA siRNA agent or PBS. Blood samples were collected before siRNA administration on day 0, at the end of days 7 and 14. Luciferase activity was measured, and the percentage of knockdown was calculated by comparing the luciferase activity of blood samples from the siRNA-treated group before administration and blood samples collected on days 7 and 14, and performing normalization based on the change in luciferase activity in serum samples from the PBS-treated group. The percentage knockdown (retention) of human LPA mRNA levels (determined by qPCR) in the liver of mice on day 14 between the siRNA-treated and PBS-treated groups was compared, and the results are shown in Table 6. The duplex AD# used was derived from sequences corresponding to those shown in Table 3.

[0208] Table 6 provides experimental results from an in vivo study of inhibition of LPA expression using multiple LPA_RNAi agents at a single dose of 6mpk.

[0209] [Table 40]

[0210] Example 9. In vivo testing of LPA siRNA duplexes To evaluate the in vivo activity of LPA siRNA, mice infected with AAV encoding human LPA and luciferase genes were used (4 mice per group). Female C57BL / 6J mice were infected by intravenous injection of undiluted 2x10^11 viral particles of adeno-associated virus 8 (AAV8) vector encoding human LPA and luciferase genes 7 days prior to siRNA administration. On day 0, mice were subcutaneously injected with a single dose of 3mg / kg, 6mg / kg, and 10mg / kg LPA siRNA agent or PBS, respectively. Mice were sacrificed at termination. The knockdown (retention) percentage of human LPA mRNA levels (determined by qPCR) in the liver of mice on day 14 between siRNA-treated and PBS-treated groups was compared, and the results are shown in Table 7. The duplex AD# used was derived from the sequence corresponding to that shown in Table 3.

[0211] Table 7 provides experimental results from an in vivo study of inhibition of LPA expression using multiple LPA_RNAi agents at single doses of 3mpk, 6mpk and 10mpk, respectively.

[0212] [Table 41]

[0213] Example 10. In vivo testing of LPA siRNA duplexes To evaluate the in vivo activity of LPA siRNA, mice infected with AAV encoding human LPA and luciferase genes were used (4 mice per group). Female C57BL / 6J mice were infected by intravenous injection of undiluted 2x10^11 viral particles of adeno-associated virus 8 (AAV8) vector encoding human LPA and luciferase genes 7 days before siRNA administration. On day 0, mice were subcutaneously injected with a single dose of 2mg / kg or 6mg / kg LPA siRNA agent or PBS. Blood samples were collected on day 0, before siRNA administration, and at the end of days 7, 14, and 21, respectively. Luciferase activity was measured. The percentage of knockdown was calculated by comparing the luciferase activity of blood samples from the siRNA-treated group before administration and the luciferase activity of blood samples collected at the end of days 7, 14, and 21, and performing normalization based on the change in luciferase activity in serum samples from the PBS-treated group. As a result, as shown in Table 8, the duplexes AD# used are derived from sequences corresponding to those shown in Table 3.

[0214] Table 8 provides experimental results from an in vivo study of inhibition of LPA expression using multiple LPA_RNAi agents at single doses of 2 and 6 mpk on days 7, 14 and 21, with residual luciferase activity relative to day 0 normalized to the change in the PBS treatment group (mean ± SD).

[0215] [Table 42]

[0216] Example 11. In vivo testing of LPA siRNA duplexes To evaluate the in vivo activity of LPA siRNA, male cynomolgus monkeys (13-22 years old, weighing 7-9 kg) were employed in this study, with 3 animals in each group. Each animal was subcutaneously injected with 2 mg / kg of test article. The test articles used corresponded to the compounds shown in Table 3 (AD00377-1, AD00436-1, AD00480, AD00480-1, AD00480-2, and AD00474-2).

[0217] After an overnight fast, blood samples were taken on days -14 (pre-dose), -7 (pre-dose), 1 (pre-dose) and 8, 15, 22, 29, 43, 50, 57, 64, 71, 78, 85, 92 and 99 post-dose. Collected blood samples were left to clot at room temperature for at least 30 minutes, followed by centrifugation at 3500 rpm for 10 minutes at 4°C. Collected serum (approximately 1.0 mL) was transferred to two pre-labeled polypropylene screw-cap vials (0.5 mL / vial, one for ELISA assay and one for other spares) and stored in a refrigerator at -80°C until testing. The percentage of LPA remaining (normalized to the mean of days -14 (pre-dose), -7 (pre-dose) and 1 (pre-dose), pre-siRNA administration) is shown in Figure 1.

[0218] Example 12. In vivo testing of LPA siRNA duplexes To evaluate the in vivo activity of LPA siRNA, male cynomolgus monkeys (2-6 years old, weighing 2-6 kg) were employed in this study, with 4 animals in each group. Each animal was subcutaneously injected with saline or 2 mg / kg of AD00480-8 test product. The test product AD00480-8 used corresponds to the compound shown in Table 3. After an overnight fast, blood draws were performed on days -14 (pre-dose), 0 (pre-dose) and 7, 14 and 21 post-dose. The collected blood samples were left at room temperature for at least 30 minutes to clot, followed by centrifugation at 3500 rpm for 10 minutes at 4°C. The collected serum (approximately 1.0 mL) was transferred to two pre-labeled polypropylene screw-cap vials (0.5 ml / vial, one for ELISA assay and one for other spares) and stored in a refrigerator at -80°C until testing. The percentage of LPA remaining (normalized to the mean of day −14 (pre-dose) and day 0 (pre-dose), pre-siRNA administration) is shown in FIG. 2.

[0219] Example 13. In vitro screening of LPA siRNA duplexes using Huh7 cells and dual fluorescent reporter gene vectors Huh7 cells were adjusted to an appropriate density and then seeded in 96-well plates. At the same time as seeding, the dual fluorescent reporter gene vector psciCHECK2 containing the target gene was co-transfected with siRNA into Huh7 cells using Lipofectamine RNAiMax (Invitrogen-13778-150) according to the manufacturer's recommendation. Cells were transfected with test siRNA or control siRNA. siRNA was tested in triplicate at two concentrations (0.1 nM and 1.0 nM), and 48 hours after transfection, Dual-Glo® Luciferase Assay Reagent was added to detect the fluorescence value. The ratio of Renilla luminescence to Firefly luminescence was calculated and normalized based on the ratio of control siRNA-treated sample to calculate knockdown efficiency. As a result, as shown in Table 9, the duplex AV# used is derived from the sequence corresponding to that shown in Table 2.

[0220] Table 9 provides experimental results from an in vitro study of inhibition of LPA expression using multiple LPA RNAi agents.

[0221] [Table 43]

[0222] [Table 44]

[0223] Equivalent While several embodiments of the invention have been described and illustrated herein, it will be readily understood by those skilled in the art that various other means and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein, and each of these variations and / or modifications, are considered to be within the scope of the invention. More generally, it will be readily understood by those skilled in the art that all parameters, sizes, materials and configurations described herein are exemplary, and that the actual parameters, sizes, materials and / or configurations will depend on the particular application for which the teachings of the invention are used. Those skilled in the art will recognize or be able to determine, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Thus, the foregoing embodiments are merely illustrative, and it should be understood that within the scope of the appended claims and their equivalents, the invention may be practiced otherwise than as specifically described and claimed for protection. The invention is directed to each individual feature, system, article, material and / or method described herein. Moreover, any combination of two or more such features, systems, articles, materials and / or methods is also included within the scope of the invention, if such features, systems, articles, materials and / or methods are not mutually inconsistent.

[0224] All definitions defined and used herein shall be understood to refer to dictionary definitions, definitions in files incorporated by reference, and / or ordinary meaning of the defined terms.

[0225] When quantitative limitations are not used in the present specification and claims, they should be understood as "at least one" unless expressly stated to the contrary.

[0226] As used in this specification and claims, the term "and / or" should be understood to mean "either or both" of the elements so conjoined, i.e., such elements appear in combination in some cases and separately in other cases. In addition to the elements specifically identified by "and / or," other elements may optionally be present, whether or not associated with those specifically identified elements, unless expressly stated to the contrary.

[0227] All references, patents and patent applications and publications cited or referred to herein are hereby incorporated by reference in their entirety.

Claims

1. 1. A double-stranded RNA (dsRNA) agent that inhibits LPA gene expression, the dsRNA agent comprising a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 117 (5'-GAGAGUUAUCGAGGCACAUAA-3') and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 244 (5'-UUAUGUGCCUCGAUAACUCUC-3').

2. The sense strand comprises the sequence of SEQ ID NO: 889 ((GLS-15) * (Imann) * gagaguuaUcGaGgcacaua * a * (Imann)), and the antisense strand comprises the sequence of SEQ ID NO: 893 (u * U * auguGccucGaUaAcuc * u * c), wherein in each of SEQ ID NOs: 889 and 893, each nucleotide in lowercase is 2'-O-methyl (2'-OMe) modified, each nucleotide in uppercase is 2'-fluoro modified, each asterisk (*) indicates a phosphorothioate bond, and each Imann is 【Chemical 1】 and GLS-15 【Chemistry 2】 2. The dsRNA agent of claim 1, wherein:

3. A composition comprising the dsRNA agent of claim 1 and a pharmaceutically acceptable carrier.

4. 4. The composition of claim 3 packaged in a kit, container, packaging, dispenser, pre-filled syringe, or vial.

5. 4. The composition of claim 3, formulated for subcutaneous administration.

6. The composition of claim 3, wherein the pharmaceutically acceptable carrier comprises a sodium salt.

7. A method for producing a nucleic acid sequence comprising the steps of: (1) a nucleic acid sequence selected from the group consisting of: a ... 【Chemistry 3】 and GLS-15 【Chemistry 4】 The dsRNA agent of claim 2, wherein

8. A composition comprising the dsRNA agent of claim 7 and a pharmaceutically acceptable carrier.

9. The composition of claim 8, wherein the pharmaceutically acceptable carrier comprises a sodium salt.

10. The composition of claim 8 packaged in a kit, container, pack, dispenser, prefilled syringe or vial.

11. The composition of claim 8, formulated for subcutaneous administration.

12. 12. A pharmaceutical composition for use in treating an LPA-related disease or condition, said pharmaceutical composition comprising the dsRNA agent of any one of claims 1, 2 and 7 or the composition of any one of claims 3-6 and 8-11 in an amount effective to reduce serum Apo(a) levels, thereby treating the LPA-related disease or condition in a subject.

13. The pharmaceutical composition described in claim 12, wherein the LPA-related disease or condition is a cardiovascular disease.

14. The pharmaceutical composition of claim 13, wherein the cardiovascular disease is Berger's disease, peripheral artery disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic stenosis, aortic regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable angina, unstable angina, acute coronary syndrome, heterozygous familial hypercholesterolemia, homozygous familial hypercholesterolemia, hyperapolipoprotein betalipoproteinemia, cerebrovascular atherosclerosis, venous thrombosis, stroke, atherosclerosis, thrombosis, coronary heart disease, or a disease or condition associated with elevated levels of Apo(a)-containing particles.

15. The treatment of an LPA-related disease or condition, (i) using one or more physiological characteristics of an LPA-associated disease or condition in said subject; (ii) comparing the determined physiological characteristics to a baseline physiological characteristic before the treatment of the LPA-associated disease or condition and / or a control physiological characteristic of the LPA-associated disease or condition; It is evaluated by The pharmaceutical composition of claim 12, wherein the comparison indicates the presence or absence of inhibition of expression of the LPA gene in the subject, and optionally, the one or more physiological characteristics is Apo(a) protein levels in the blood, and a decrease in Apo(a) protein levels in the blood indicates a decrease in expression of the LPA gene in the subject.

16. i) inhibiting expression of the LPA gene in a subject. ii) reducing the level of Apo(a) protein in the subject compared to the baseline level of Apo(a) protein in the subject; or iii) altering physiological characteristics of the LPA-associated disease or condition in the subject compared to baseline physiological characteristics prior to treatment of the LPA-associated disease or condition in the subject; 12. Use of an effective amount of a dsRNA agent according to any one of claims 1, 2 and 7, or a composition according to any one of claims 3 to 6 and 8 to 11, for the manufacture of a medicament for:

17. The use described in claim 16, wherein the LPA-related disease or condition is cardiovascular disease.

18. The use of claim 17, wherein the cardiovascular disease is Berger's disease, peripheral artery disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic stenosis, aortic regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable angina, unstable angina, acute coronary syndrome, heterozygous familial hypercholesterolemia, homozygous familial hypercholesterolemia, hyperapolipoprotein betalipoproteinemia, cerebrovascular atherosclerosis, venous thrombosis, stroke, atherosclerosis, thrombosis, coronary heart disease, or a disease or condition associated with elevated levels of Apo(a)-containing particles.

19. The efficacy of the drug is (i) using one or more physiological characteristics of an LPA-associated disease or condition in said subject; (ii) comparing the one or more physiological characteristics to a baseline physiological characteristic before the treatment of the LPA-associated disease or condition and / or a control physiological characteristic of the LPA-associated disease or condition; It is evaluated by The comparison indicates the presence or absence of inhibition of expression of the LPA gene in the subject, and optionally, the one or more physiological characteristics is Apo(a) protein level in blood, and a decrease in Apo(a) protein level in blood indicates a decrease in expression of the LPA gene in the subject.

17. The use according to claim 16.

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