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 Art

[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), which is encoded by the LPA gene) linked to LDL-like particles via an ApoB polypeptide. Genetically defined high serum levels of Lp(a) particles are not affected by diet and exercise and are associated with an increased risk of developing cardiovascular disease through the associated potential for atherosclerosis (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 the serum of patients is an independently prevalent genetic risk factor for coronary heart disease and aortic 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 independent risk factors for other associated conditions including cardiovascular disease, stroke, and atherosclerotic stenosis. In addition, genome-wide association analysis has also identified LPA as a genetic risk factor for diseases such as atherosclerotic stenosis. A significant decrease in cardiovascular events is observed when both Lp(a) and LDL levels in hyperlipidemic patients are reduced using therapeutic lipoprotein apheresis. Therefore, there is a need for therapeutic agents and treatments related to these and other LPA-related diseases.

[0003] However, apart from indirect and standard general LDL reduction measures, there are currently no approved specific Lp(a) particle reduction therapies. Therefore, there is a need for effective treatment, prevention, and reduction of the risk of developing the following and related conditions: Berger's disease, peripheral artery disease, coronary artery disease, metabolic syndromes, 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 diseases or conditions associated with elevated levels of Lp(a) particles, and other related conditions, conditions or syndromes that have not yet been identified. This invention addresses this unmet medical need. [Overview of the Initiative]

[0004] According to aspects of the present invention, a double-stranded ribonucleic acid (dsRNA) agent that inhibits LPA(Apo(a)) expression is provided, the dsRNA agent comprising a sense strand and an antisense strand, and optionally comprising a targeted ligand. The region complementary to the LPA RNA transcript is located at nucleotide positions 2-18 in the antisense strand, and the complementary region comprises at least 15 consecutive 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 consecutive nucleotides that differ from one of the antisense sequences listed in Tables 1-3 by 3 or fewer 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 is 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 one of the sense strand sequences listed in Tables 1-3, the sense strand sequence being at least substantially complementary to the antisense strand sequence in the dsRNA agent. In certain embodiments, the dsRNA agent comprises one of the sense strand sequences listed in Tables 1-3, the sense strand sequence being fully complementary to the antisense strand sequence in the dsRNA agent. In some embodiments, the dsRNA agent comprises one of the antisense strand sequences listed in Tables 1-3. In some embodiments, the dsRNA agent comprises one of the sequences listed as double-stranded sequences in Tables 1-3. In some embodiments, the dsRNA agent contains a sense strand that differs from formula (A) by only 0, 1, 2, or 3 nucleotides: 5'-Z1GUUAUCGAGGCACAUAZ2-3' formula (A) (wherein Z1 is a nucleotide sequence containing 0 to 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 or absent 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 comprising 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 contains an antisense strand that differs from formula (B) by only 0, 1, 2, or 3 nucleotides: 5'-Z3UAUGUGCCUCGAUAACZ4-3' formula (B) (wherein Z3 is selected from one of A, U, C, and G or is absent, and Z4 is a nucleotide sequence containing 0 to 15 nucleotide motifs). In certain embodiments, Z3 is U. In some embodiments, the Z4 nucleotide sequence is selected from or absent 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. In some embodiments, Z4 is a nucleotide sequence comprising 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 containing nucleotide sequences described herein, each differing from formulas (A) and (B) by only 0, 1, 2, or 3 nucleotides, and optionally includes 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 to 23 nucleotides. In some embodiments, the complementary region is 19 to 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 containing at least 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the dsRNA agent contains a sense strand that differs from formula (C) by only 0, 1, 2, or 3 nucleotides: 5'-Z5CCAAGCUUGGUCAUCUZ6-3'formula (C) (wherein Z5 is a nucleotide sequence containing 0 to 15 nucleotide motifs, and Z6 is selected from one of A, U, C, and G or is absent). In certain embodiments, Z6 is A. In some embodiments, the Z5 nucleotide sequence is selected from one of the following motifs or is absent: 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 comprising one, two, three, or four 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 comprises an antisense strand differing from formula (D) by only 0, 1, 2, or 3 nucleotides: 5'-Z7AGAUGACCAAGCUUGGZ8-3'formula (D) (wherein Z7 is selected from one of A, U, C, and G or is absent, and Z8 is a nucleotide sequence comprising 0 to 15 nucleotide motifs).In certain embodiments, Z7 is U. In some embodiments, the Z8 nucleotide sequence is selected from or absent 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, CAAGUUCUUCCUG, CAAGUUCUUCCUGU, and CAAGUUCUUCCUGUG. In some embodiments, Z8 is a nucleotide sequence comprising 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, the dsRNA agent comprises a sense strand and an antisense strand containing nucleotide sequences described herein, each differing from formulas (C) and (D) by only 0, 1, 2, or 3 nucleotides, and optionally includes a targeted ligand. In certain embodiments, the length of each of the sense strand (C) and antisense strand (D) of the 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 complementary region is 16 to 23 nucleotides. In some embodiments, the complementary region is 19 to 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 containing at least 15, 16, 17, 18, or 19 nucleotides. In some embodiments, the dsRNA agent contains a sense strand different from formula (E) by only 0, 1, 2, or 3 nucleotides: 5'-Z9GACAGAGUUAUCGAGGZ. 10 -3' Equation (E) (wherein Z9 is a nucleotide sequence containing 0 to 15 nucleotide motifs, Z 10is selected from or absent among one of A, U, C, and G). In certain embodiments, Z 10 is A. In some embodiments, the Z9 nucleotide sequence is 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, or is absent. In some embodiments, Z9 is a nucleotide sequence comprising 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 comprises an antisense strand that differs from formula (F) by only 0, 1, 2, or 3 nucleotides: 5’-Z 11 CCUCGAUAACUCUGUCZ 12 -3’ formula (F) (where Z 11 is selected from or absent among one of A, U, C, and G, and Z 12 is a nucleotide sequence comprising a nucleotide motif of 0 to 15 nucleotides). In certain embodiments, Z 11 is U. In some embodiments, the Z 12 nucleotide sequence is one of 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, or is absent. In some embodiments, Z 12This is a nucleotide sequence comprising 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 comprises a nucleotide sequence described herein that differs from formulas (E) and (F) by only 0, 1, 2, or 3 nucleotides, respectively. The dsRNA agent comprises a sense strand and an antisense strand, and optionally includes a targeted ligand. In certain embodiments, the length of each of the sense strand (F) and antisense strand (F) of the dsRNA agent does not exceed 35 nucleotides. In certain embodiments, Z9 and Z 12 Nucleotide motifs are completely or partially complementary. In certain embodiments, Z 10 and Z 11 The nucleotide motifs are completely 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 to 23 nucleotides. In some embodiments, the complementary region is 19 to 21 nucleotides long. In some embodiments, the length of the sense strand is not greater than 35 nucleotides and includes a region complementary to the antisense strand containing at least 15, 16, 17, 18, or 19 nucleotides.

[0005] In some embodiments, the dsRNA agent includes at least one modified nucleotide. In certain embodiments, all or substantially all nucleotides of the antisense strand are modified nucleotides. In some embodiments, at least one modified nucleotide includes 2'-O-methylnucleotide, 2'-fluoronucleotide, 2'-deoxynucleotide, 2',3'-seconucleotide mimetic, locked nucleotide, unlocked nucleic acid (UNA) nucleotide, glycol nucleic acid nucleotide (GNA), 2'-F-arabinose nucleotide, 2'-methoxyethyl nucleotide, debasalized nucleotide, ribitol, reversed nucleotide, reversed debasalized nucleotide, reversed 2'-OMe nucleotide, reversed 2'-deoxynucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholino nucleotide and 3'-OMe nucleotide, nucleotides containing a 5'-phosphorothioate group or a cholesterol derivative or terminal nucleotide linked to a dodecanoic acid bisdecylamide group, 2'-amino modified nucleotide, phosphoramidate or nucleotides containing a non-natural base. In some embodiments, the antisense strand contains 15 or more modified nucleotides independently selected from 2'-O-methylnucleotides and 2'-fluoronucleotides, with fewer than 6 2'-fluoronucleotide modified nucleotides. In some embodiments, the antisense strand contains 3 or 5 2'-fluoronucleotides, preferably 5 2'-fluoronucleotides. In some embodiments, the sense strand contains 15 or more modified nucleotides independently selected from 2'-O-methylnucleotides and 2'-fluoronucleotides, with fewer than 4 2'-fluoronucleotide modified nucleotides. In a particular embodiment, the sense strand contains 3 2'-fluoronucleotides.In some embodiments, the antisense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methylnucleotides and 2'-fluoronucleotides, where at least 16 modified nucleotides are 2'-O-methylnucleotides, and positions 2, 7, 12, 14 and / or position 16 at the 5' end of the antisense strand are 2'-fluoronucleotide modified nucleotides (counting from the first pair-forming 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-methylnucleotides and 2'-fluoronucleotides, where at least 18 modified nucleotides are 2'-O-methylnucleotides, and positions 9, 11 and / or position 13 at the 3' end of the sense strand are 2'-fluoronucleotide modified nucleotides (counting from the first pair-forming nucleotide at the 3' end of the sense strand). In some embodiments, the antisense strand contains 2'-fluoromodified nucleotides at positions 2, 7, 12, 14, and 16 of the antisense strand, counting from the first paired nucleotide at the 5' end of the antisense strand towards the 3' end, with each other nucleotide in the antisense strand being independently unfluoromodified. In some embodiments, the antisense strand contains 2'-fluoromodified nucleotides at positions 2, 5, 12, 14, and 18 of the antisense strand, counting from the first paired nucleotide at the 5' end of the antisense strand towards the 3' end, with each other nucleotide in the antisense strand being independently unfluoromodified. In some embodiments, the sense strand contains 2'-fluoromodified nucleotides at positions 9, 11, and 13 of the sense strand, counting from the first paired nucleotide at the 3' end of the sense strand towards the 5' end, with each other nucleotide in the sense strand being independently unfluoromodified. In some embodiments, the dsRNA agent includes an E-vinylphosphonate nucleotide at the 5' end of the guide strand. In certain embodiments, the dsRNA agent includes at least one phosphorothioate nucleoside bond.In certain embodiments, the sense strand includes at least one phosphorothioate nucleoside linkage. In some embodiments, the antisense strand includes at least one phosphorothioate nucleoside linkage. In some embodiments, the sense strand includes 1, 2, 3, 4, 5, or 6 phosphorothioate nucleoside linkages. In some embodiments, the antisense strand includes 1, 2, 3, 4, 5, or 6 phosphorothioate nucleoside 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 long. 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 chain is 40 nucleotides or less long. In some embodiments, each chain is 30 nucleotides or less long. In some embodiments, each chain is 25 nucleotides or less long. In some embodiments, each chain is 23 nucleotides or less long. In certain embodiments, the dsRNA agent comprises at least one modified nucleotide and further comprises one or more targeting or linking groups. In some embodiments, one or more targeting or linking groups are conjugated to the sense strand. In some embodiments, the targeting 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 either 1 or 2)

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

[0007] In certain embodiments, the dsRNA agent includes a targeting group conjugated to the 5' end of the sense strand. In some embodiments, the dsRNA agent includes a targeting group conjugated to the 3' end of the sense strand. In some embodiments, the antisense strand includes one reverse debased residue at its 3' end. In certain embodiments, the sense strand includes one or two reverse debased residues at its 3' end and / or 5' end. In certain embodiments, the sense strand includes one or two isomannitol residues at its 3' end and / or 5' end. In certain embodiments, the sense strand independently includes one isomannitol residue at its 3' end and one at its 5' end, respectively. In some embodiments, the sense strand independently includes one isomannitol residue at its 3' end and one at its 5' end, and further includes a targeting group conjugated to its 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 includes 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 contain a region complementary to the LPA RNA transcript, the antisense strand is fully or partially complementary to the sense strand, the agent optionally contains a targeted ligand, each strand is 14-30 nucleotides long, and the sense strand sequence is given 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'-fluoromodified nucleotide; N' N1 , N' N2 , N' N3 and N' N4 Each of these independently represents a modified or unmodified nucleotide; each N' L n' can be represented by (independently, a modified or unmodified nucleotide, but not a 2'-fluoromodified nucleotide, where 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 Independently, each N' represents a modified or unmodified nucleotide, but does not represent a 2'-fluoromodified 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 The terms independently represent a modified or unmodified nucleotide, but not a 2'-fluoromodified 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'-fluoromodified nucleotides in formula (I).

[0009] In certain embodiments, the present invention relates to unlocked nucleic acid (UNA) oligomers for therapeutic use. Unlocked nucleic acid (UNA) is an acyclic analog of RNA in which the C2' and C3' atoms of the ribose ring are broken. The incorporation of UNA has been shown to be well tolerant of siRNA gene silencing activity and, in some cases, to further enhance 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-unstable modification, and ribonucleotide substitution by UNA reduces pairing strength and double-strand stability. Strategically placing UNA in the seed region of siRNA antisense strands reduces off-target activity through the microRNA (miRNA)-mediated gene silencing mechanism. miRNAs primarily identify target genes through base pairing between the antisense seed region (positions 2-8, starting from the 5' end) and the target mRNA for gene repression. Each miRNA potentially regulates a large number of genes. siRNA antisense strands loaded by RNA-induced silencing complexes (RISCs) can also potentially regulate a large number of unintended genes through miRNA-mediated mechanisms. Therefore, the incorporation of heat-unstable 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 complexes of RNA oligonucleotides 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 complexes of RNA oligonucleotides include, but are not limited to, the following: 1. Off-target activity is reduced. Adding UNA to the siRNA seed region reduces base pairing intensity in the seed region, thereby reducing potential off-target activity induced by the microRNA mechanism. 2. UNA demonstrated sufficient tolerance in terms of siRNA activity. In some cases, UNA can lead to enhanced activity.

[0012] Examples of UNA monomers that may be used in the technical solutions of the present invention include: [ka] These include, but are not limited to, the following:

[0013] According to one aspect of the present invention, a composition comprising any embodiment of the dsRNA agent described above is provided. In certain embodiments, the composition further comprises a pharmaceutically 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 targeted therapy, AGT targeted therapy, APOC3 targeted 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 for intravenous (IV) administration.

[0014] According to another aspect of the present invention, cells comprising any embodiment of the dsRNA agent described above are provided. In some embodiments, the cells are mammalian cells and, optionally, human cells.

[0015] According to another aspect of the present invention, a method is provided for inhibiting LPA gene expression in cells, the method comprising (i) preparing cells containing an effective amount of any embodiment of the dsRNA agent or composition of the present invention. In certain embodiments, the method further comprises (ii) obtaining degradation of the mRNA transcript of the LPA gene and maintaining the prepared cells for a time sufficient to inhibit LPA gene expression in the cells. In some embodiments, the cells are in a subject, and the dsRNA agent is administered subcutaneously to the subject. In some embodiments, the cells are 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 features of an LPA-related disease or condition in the subject, and (ii) comparing the identified physiological features with the baseline physiological features of the LPA-related disease or condition prior to treatment and / or the physiological features of a control of the LPA-related disease or condition, the comparison results indicating the presence or absence of inhibition of LPA gene expression in the subject. In some embodiments, the identified physiological feature is the Lp(a) level in the blood. A reduction in LPA levels in the blood indicates a reduction in LPA gene expression in the subject.

[0016] According to another aspect of the present invention, a method is provided for inhibiting LPA gene expression in a subject, the method comprising administering to the subject an effective amount of the above-described embodiment of the dsRNA or embodiment of the above-described composition. In some embodiments, the dsRNA agent is administered subcutaneously to the subject. 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 features of an LPA-related disease or condition in the subject, and (ii) comparing the identified physiological features with the baseline physiological features of the LPA-related disease or condition before treatment and / or the physiological features 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 feature is the Lp(a) level in the blood. A reduction in the LPA level in the blood indicates a reduction in LPA gene expression in the subject.

[0017] Another aspect of the present invention provides a method for treating a disease or condition related to the LPA protein, comprising administering to a subject an effective amount of any embodiment of the aforementioned dsRNA agent of the present invention or any embodiment of the aforementioned composition 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 includes 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 / 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 includes administering an additional therapeutic regimen to the subject. In some embodiments, the additional therapeutic regimen includes the treatment of an LPA-related disease or condition. In some embodiments, the additional therapeutic regimen includes 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 inducing 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 targeted therapy, APOC3 targeted therapy, and niacin or any combination thereof.

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

[0019] According to another aspect of the present invention, a method is provided for reducing LPA protein levels in a subject compared to baseline levels of LPA protein in the subject before treatment, the method comprising administering to the subject an effective amount of any embodiment of the aforementioned dsRNA agent or any embodiment of the aforementioned composition 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] Another aspect of the present invention provides a method for modifying the physiological characteristics of an LPA-related disease or condition in a subject compared to baseline physiological characteristics of the LPA-related disease or condition in the subject before treatment, the method comprising modifying the physiological characteristics of the LPA-related disease or condition in the subject by administering to the subject an effective amount of any embodiment of the aforementioned dsRNA agent or any embodiment of the aforementioned composition of the present invention. In some embodiments, the dsRNA agent is administered to the subject subcutaneously or via IV. In certain embodiments, the physiological characteristic is the Lp(a) level in the blood.

[0021] Array description The double-stranded AV00122~AD00484-1, AD00474-2, and AV01867~AV01968 are shown in Table 1, and their sense strand sequences are indicated.

[0022] The double-stranded AV00122~AD00484-1, AD00474-2, and AV01867~AV01968 are shown in Table 1, and their antisense strand sequences are indicated.

[0023] In the sequences shown in Table 2, chemical modifications are indicated by uppercase: 2'-fluoro; lowercase: 2'-OMe; phosphorothioate: *.

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

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

[0026] [Figure 1] A schematic diagram of serum LPA protein levels in monkeys is shown. [Figure 2] A schematic diagram of serum LPA protein levels of AD00480-8 at a dose of 2 mpk in monkeys is shown. [Modes for carrying out the invention]

[0027] Some embodiments of the present invention include RNAi agents that can inhibit LPA (Apo(a)) gene expression, such as, but are not limited to, double-stranded (ds) RNAi agents. Some embodiments of the present invention further include compositions comprising LPA RNAi agents and methods of using such compositions. The LPA RNAi agents disclosed herein may be conjugated to delivery compounds for delivery to cells, including delivery to hepatocytes. The pharmaceutical compositions of the present invention may comprise at least one dsRNA agent and a delivery compound. In some embodiments of the present invention, the delivery compound is a GalNAc-containing delivery compound. The LPA RNAi agent delivered to cells can inhibit LPA gene expression, thereby reducing the LPA protein product of the gene. LPA-related diseases and conditions can be treated using the dsRNAi agents of the present invention. Examples of such dsRNAi agents include the double-stranded AV00122~AD00484-1, AD00474-2, and AV01867~AV01968 shown in Table 1. In other embodiments, such dsRNAi agents include double-stranded variants such as AV00122~AD00484-1, AD00474-2, and AV01867~AV01968 variants.

[0028] In some embodiments of the present invention, diseases or conditions associated with LPA expression in cells or subjects are treated by reducing LPA expression in those cells or subjects. Non-limiting examples of diseases and conditions that can be treated by reducing LPA expression include cardiovascular diseases, including Berger's disease, peripheral artery disease, coronary artery disease, metabolic syndromes, 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 diseases or conditions associated with elevated levels of Lp(a)-containing particles.

[0029] Methods for preparing and using compositions comprising single-stranded (ssRNA) and double-stranded (dsRNA) agents of LPA 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 may be referred to as "siRNA".

[0030] As used herein, the term “RNAi” refers to an agent containing RNA that mediates targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. As is known in the art, an RNAi target region refers to a contiguous portion of the nucleotide sequence of an RNA molecule formed during gene transcription, including messenger RNA (mRNA), which is the processed product of the major transcript RNA. The target portion of this sequence will be long enough to be used as a substrate for cleavage directed to RNAi, at least in that portion or its vicinity. Target sequences may be 8–30 nucleotides (all), 10–30 nucleotides (all), 12–25 nucleotides (all), 15–23 nucleotides (all), 16–23 nucleotides (all), or 18–23 nucleotides (all), including all shorter lengths within each specified range. In some embodiments of the present 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 long. In certain embodiments, the length of the target sequence is 9 to 25 nucleotides (including all of them), encompassing all subranges and integers in between. For example, but not intended to be limiting, in some embodiments of the present 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 long and is fully or at least substantially complementary to at least a portion of the RNA transcript of the LPA gene. Some aspects of the present invention include a pharmaceutical composition comprising one or more LPA dsRNA agents and a pharmaceutically acceptable carrier. In some embodiments of the present invention, LPA RNAi as described herein inhibits the expression of LPA protein.

[0031] As used herein, “dsRNA agent” refers to a composition containing an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule capable of degrading a target mRNA transcript or inhibiting the translation of a target mRNA transcript. Without intending to limit ourselves to any particular theory, the dsRNA agents of the present invention may function by an RNA interference mechanism (i.e., by inducing the generation of RNA interference by interacting with the RNA interference pathway mechanism (RNA-induced silencing complex or RISC) in mammalian cells) or by 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), and their respective disclosures are incorporated herein by reference as a whole. Gene silencing methods known in the art may be used in combination with the disclosures provided herein to achieve inhibition of LPA expression.

[0032] The dsRNA agents disclosed herein consist of a sense strand and an antisense strand, and include, but are not limited to, small interfering RNAs (siRNAs), RNAi agents, microRNAs (miRNAs), small hairpin RNAs (shRNAs), and Dicer substrates. The antisense strand of the dsRNA agents described herein is at least partially complementary to the targeted mRNA, and it is understood in the art that target gene expression can be inhibited using dsRNA double-strand structures of various lengths. For example, dsRNAs having double-strand 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 double-strand structures can efficiently induce RNA interference. In certain embodiments of the present invention, the LPA dsRNA may comprise at least one strand of at least 21nt length, or the double helix may have a length of minus 1, 2, 3nt 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 present invention, the LPA dsRNA agents may have a partial sequence of at least 15, 16, 17, 18, 19, 20 or more consecutive 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 induced by the dsRNA containing the complete sequence (also referred herein as the “parent” sequence).

[0033] Specific embodiments of the compositions and methods of the present invention involve administering single-stranded RNA and / or single-stranded RNA in a composition to a subject. For example, antisense strands listed in any of Tables 1-3 may be used in or as a composition that reduces the expression of LPA polypeptide and / or LPA gene in a subject when administered to the subject. Tables 1-3 show the core elongation nucleotide sequences of antisense and sense strands of several LPA dsRNA agents. Single-stranded antisense molecules contained in specific compositions of the present invention and / or administered in specific methods of the present invention are referred to herein as “single-stranded antisense agents” or “antisense polynucleotide agents.” Single-stranded sense molecules contained in specific compositions and / or administered in specific methods of the present invention are referred herein as “single-stranded sense agents” or “sense polynucleotide agents.” The term “nucleotide 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 nucleotide sequences in Table 3; each chemical modification and delivery compound is shown in the corresponding sequence in Table 3. 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#"; and a double helix containing both a sense strand and an antisense strand may be identified by "double helix AD#".

[0034] Table 1 includes both sense and antisense strands and provides identification numbers for the duplexes formed by the sense and antisense strands in the same row of Table 1. In some embodiments of the present invention, the antisense sequence contains either nucleic acid base u or nucleic acid base a at its position 1. In some embodiments of the present invention, the antisense sequence contains nucleic acid base u at position 1 of the antisense sequence. As used herein, the term “matching position” in sense refers to a position in each strand that “pairs” with each other when the two strands form a duplex. For example, in a sense strand of 21 nucleic acid bases and an antisense strand of 21 nucleic acid bases, position 1 of the sense strand is the “matching position” with the nucleic acid base at position 21 of the antisense strand. In another non-limiting example, with respect to a sense strand of 23 nucleic acid bases and an antisense strand of 23 nucleic acid bases, the nucleic acid base at position 2 of the sense strand is the “matching position” with the nucleic acid base at position 22 of the antisense strand. In another non-limiting example, in an 18-base sense strand and an 18-base antisense strand, the base at position 1 on the sense strand corresponds to the base at position 18 on the antisense strand; the base at position 4 on the sense strand corresponds to the base at position 15 on the antisense strand. Technicians will understand how to identify the corresponding positions between the sense and antisense strands of double-stranded and paired strands.

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

[0036] In some embodiments of the methods of the present invention, an RNAi agent comprising the polynucleotide sequences shown in Table 1 is administered to a subject. In some embodiments of the present invention, the RNAi agent administered to the subject comprises a double helix containing at least one of the nucleotide sequences listed in Table 1 and including 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. In some embodiments of the methods of the present invention, the RNAi agent of the polynucleotide sequences shown in Table 1 is also conjugated to a delivery molecule of a non-limiting example, 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 specific chemically modified LPA RNAi agents of the present invention. In some embodiments of the methods of the present invention, the RNAi agent having the polynucleotide sequence shown in Table 2 is administered to cells and / or subjects. In some embodiments of the methods of the present invention, the RNAi agent administered to the subjects includes the double helix labeled in column 1 of Table 2 and sequence modifications in the sense and antisense strand sequences shown in columns 3 and 6 of the same row in Table 2, respectively. In some embodiments of the methods of the present invention, the sequences shown in Table 2 can be conjugated (also referred to herein as "conjugated") to compounds that can deliver the RNAi agent to the target cells and / or tissues. Non-limiting examples of delivery compounds that may be used in specific embodiments of the present invention are GalNAc-containing compounds. In Table 2, column 1 represents the double helix AD# of the nucleotide sequence corresponding to Table 1. The nucleotide sequence identified by the double-stranded AD# not only represents the nucleotide sequences contained in the sense and antisense strands, but also includes the specified chemical modifications shown in the same row of Table 2. For example, the first row of Table 1 shows the single-stranded sense and antisense nucleotide sequences, which together form a double-stranded sequence identified as double-stranded AV00122; double-stranded AV00122, listed in Table 2, includes the nucleotide sequences AV00122-SS and AV00122-AS, with chemical modifications to the sense and antisense sequences shown in columns 3 and 6, respectively. "Sense strand SS#" in column 2 of Table 2 is the specified identifier for the sense sequence (including modifications) shown in column 3 of the same row. "Antisense strand AS#" in column 5 of Table 2 is the specified identifier for 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 specific chemically modified LPA RNAi agents of the present invention. In some embodiments of the methods of the present invention, the RNAi agents shown in Table 3 are administered to cells and / or subjects. In some embodiments of the methods of the present invention, RNAi agents having the polynucleotide sequences shown in Table 3 are administered to subjects. In some embodiments of the present invention, the RNAi agent administered to a subject comprises a double helix identified in column 1 of Table 3 and includes sequence modifications and / or delivery compounds shown in the sense strand and antisense strand sequences in columns 3 and 6 of the same row in Table 3, respectively. This sequence is used in several in vivo experimental studies described elsewhere in this specification. In some embodiments of the methods of the present invention, the sequence shown in Table 3 can be conjugated (also referred to herein as "conjugated") to a delivery compound (a non-limiting example is a GalNAc-containing compound, i.e., having a delivery compound labeled "GLX-n" on the sense strand in column 3 of Table 3). As used herein and as shown in Table 3, "GLX-n" is used to indicate linked GalNAc-containing compounds, which are 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 these are provided elsewhere in this specification. The first column of Table 3 provides the double-strand AD# assigned to the double-strand of sense and antisense arrays in the row of the table. For example, double-strand AD00122 is a double-strand composed of a sense strand AD00122-SS and an antisense strand AD00122-AS. Each row in Table 3 provides a sense strand and an antisense strand and discloses the double-strand formed by the sense strand and antisense strand shown. The "sense strand SS#" in the second column of Table 3 is the specified identifier of the sense array (including modifications) shown in the third column of the same row.In the fifth column of Table 3, “Antisense Chain AS#” is a designated identifier for the antisense sequence (including modifications) shown in the sixth column. The specific identifier for a concatenated GalNAc-containing GLO compound is shown as GLO-0, and the other GLO-n or GLS-n compound may be substituted for the compound shown as GLO-0, and it should be understood that the resulting compound is also included in embodiments of the methods and / or compositions of the present invention.

[0068] Table 3 provides antisense and sense strand sequences of chemically modified LPA RNAi agents for in vivo testing. All sequences are shown from 5' to 3'. These sequences are used in several in vivo testing studies described elsewhere in this specification. The delivery molecule used in the in vivo study is indicated as "GLO-0" at the 3' end of each sense strand. The delivery molecule used in the in vivo study is indicated as "GLS-5" or "GLS-15" at the 5' end of each sense strand. Chemical modifications are indicated by uppercase: 2'-fluoro; lowercase: 2'-OMe; phosphorothioate: *; unlocked nucleic acid: UNA; invab = reverse debase; imann: when at the end of each strand: [ka] Or, if further coupling to the delivery molecule: [ka]

[0069] [Table 30]

[0070] [Table 31]

[0071] [Table 32]

[0072] [Table 33]

[0073] mismatch In particular, it is known to those skilled in the art that mismatches are tolerable with respect to the efficacy of dsRNA when they occur within the terminal region of the dsRNA. Some mismatches, such as those caused by fluctuating base pairs G:U and A:C, exhibit better tolerance and better tolerance with respect to efficacy (Du et el., 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 with respect to the LPA target sequence. In some embodiments, the LPA dsRNA agent of the present invention does not contain any mismatches. In certain embodiments, the LPA dsRNA agent of the present invention contains one or fewer mismatches. In some embodiments, the LPA dsRNA agent of the present invention contains two or fewer mismatches. In certain embodiments, the LPA dsRNA agent of the present invention contains three or fewer mismatches. In some embodiments of the present invention, the antisense strand of the LPA dsRNA agent contains a mismatch to an LPA target sequence that is not at the center of the complementary region. In some embodiments, the antisense strand of the LPA dsRNA agent contains one, two, three, four or more mismatches located within the last 5, 4, 3, 2, or 1 nucleotide of one or both of the 5' or 3' ends of the complementary region. Methods described herein and / or methods known in the art may be used to determine whether an LPA dsRNA agent containing mismatches to an LPA target sequence is effective in inhibiting LPA gene expression.

[0074] Complementarity As used herein, unless otherwise specified, the term “complementary” refers to the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence (e.g., LPA dsRNA agent sense strand or target LPA mRNA) and to form a double helix or double helix structure under specific conditions. Other conditions, such as physiologically appropriate conditions that may occur in a living organism, may also apply. Technicians may determine the optimal set of conditions for testing the complementarity of the two sequences based on the final application of the hybridized nucleotide. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs and include natural or modified nucleotides or nucleotide mimetic, insofar as they are at least as described above with respect to hybridization. Sequence identity or complementarity is independent of modification.

[0075] For example, complementary sequences within an LPA dsRNA as described herein involve base pairing between an oligonucleotide or polynucleotide containing a first nucleotide on the full length of one or two nucleotide sequences and an oligonucleotide or polynucleotide containing a second nucleotide sequence. Such sequences may be referred to herein as “fully complementary.” In embodiments where the 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 contain a 19-nucleotide oligonucleotide and another 20-nucleotide oligonucleotide, 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 the contiguous sequence of the first polynucleotide hybridize with the same number of bases in the contiguous sequence of the second polynucleotide. A contiguous sequence may contain all or part of the first or second nucleotide sequence.

[0076] As used herein, the term “substantially complementary” means that in a hybrid pair of nucleic acid sequences, at least about 85% (but not all) of the bases in the contiguous sequence of the first polynucleotide hybridize with the same number of bases in the contiguous sequence of the second polynucleotide. If the 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” may be used to mean that the first sequence forms a double helix 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 its ability to hybridize under conditions most appropriate for its final application, such as inhibition of LPA gene expression via the RISC pathway. The term “partially complementary” may be used herein to mean that, in a hybrid pair of nucleic acid base sequences, at least 75% (but not all) of the bases in the contiguous sequence of the first polynucleotide hybridize with the same number of bases in the contiguous sequence of the 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 the contiguous sequence of the second polynucleotide hybridize with the same number of bases in the contiguous sequence of the second polynucleotide.

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

[0078] As used herein, the terms “substantially identical” or “substantially identical” used to refer to nucleic acid sequences mean that a nucleic acid sequence contains at least 85% sequence identity, 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 compared to a reference sequence. The percentage of sequence identity is determined by comparing the optimal alignments of the two sequences on an alignment window. The percentage is calculated by determining the number of positions in which the same nucleic acid bases occur 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 inventions disclosed herein contain nucleotide sequences that are substantially identical to those disclosed herein (for example, in Tables 1-5). In some embodiments, the nucleotide sequence is either completely identical to the sequences disclosed herein (for example, in Tables 1-3) or has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0079] As used herein, the term “sequence-containing chain” refers to an oligonucleotide containing a nucleotide chain described by a sequence shown using standard nucleotide nomenclature. As used herein, the term “double-stranded RNA” or “dsRNA” refers to a sequence containing an RNA molecule or RNAi molecular complex having a hybrid double-stranded region containing two antiparallel and substantially or completely complementary nucleic acid strands, which are referred to as having “sense” and “antisense” directions with respect to the target LPA RNA, respectively. The double-stranded region may have any desired length that allows for the specific degradation of the target LPA RNA via the RISC pathway, but is typically 9 to 30 base pairs long, e.g., 15 to 30 base pairs long. Considering double helix The length can be any length within its arbitrary sub-range, 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 with dicers 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 substantially complementary to the region of the target LPA RNA. The two strands forming the double-stranded structure may originate from a single RNA molecule having at least one self-complementary region, or they may 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 herein to as a “hairpin loop”) formed by one strand of a single-stranded nucleotide at the 3' end and the corresponding other strand at the 5' end. In some embodiments of the present invention, the hairpin structure contains 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 two essentially complementary strands of an LPA dsRNA agent consist of individual RNA molecules, these molecules do not need to be covalently linked, but may be. When the two strands are covalently linked by means other than a hairpin loop, the linked structure is called a “linker”. The term “siRNA” is also used herein to refer to the dsRNA agent as described herein.

[0080] In some embodiments of the present invention, the LPA dsRNA agent may comprise sense and antisense sequences having unpaired nucleotides or nucleotide analogs at one or both ends of the dsRNA agent. The end without an unpaired nucleotide is called a “blunt end” and does not have a nucleotide overhang. When both ends of a dsRNA agent are blunt ends, the dsRNA is referred to as “blunt-terminated.” In some embodiments of the present invention, the first end of the dsRNA agent is blunt-terminated; in some embodiments, the second end of the dsRNA is blunt-terminated; and in certain embodiments of the present invention, both ends of the LPA dsRNA agent are blunt-terminated.

[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, and vice versa. The dsRNA may contain overhangs of at least one, two, three, four, five, six or more nucleotides. Nucleotide overhangs may contain or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. In some embodiments, it should be understood that nucleotide overhangs are present at the sense strand, the antisense strand, or both ends of the dsRNA agent, and the nucleotides in the overhang may be present at the 5' end, 3' end, or both ends of the antisense or 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 terms “antisense strand” or “guide strand” refer to a strand of the LPA dsRNA agent containing a region substantially complementary to the LPA target sequence. As used herein, the terms “sense strand” or “passenger strand” refer to a strand of the LPA dsRNA agent containing a region substantially complementary to 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 to obtain one or more beneficial properties. The nucleic acids in certain embodiments of the present invention may be synthesized and / or modified by methods well known in the art. For example, see “Current protocols in Nucleic Acid Chemistry,” Beaucage, S. Let al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, incorporated herein by reference. Modifications that may be present in specific embodiments of the LPA dsRNA agent of the present invention include (a) terminal modifications such as 5'-end modifications (phosphorylation, conjugation, reverse bonding, etc.), 3'-end modifications (conjugation, DNA nucleotides, reverse bonding, etc.); (b) base modifications, such as base substitution, base deletion (debasalized nucleotide), or base conjugation with respect to base pairs, stabilized bases, destabilized bases, or expansion of the partner pool; (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; and (d) skeletal modifications, including modifications or substitutions of phosphodiester bonds. Specific examples of RNA compounds usable in specific embodiments of the LPA dsRNA agent, LPA antisense polynucleotide, and LPA sense polynucleotide of the present invention include, but are not limited to, RNA containing a modified skeleton or RNA without natural internucleoside bonds. As a non-limiting example, RNA with skeletal modifications may not have a phosphorus atom in its skeleton. RNA without a phosphorus atom in its internucleoside skeleton may be called oligonucleosides. In some embodiments of the present invention, the modified RNA has a phosphorus atom in its internucleoside skeleton.

[0084] The terms “RNA molecule” or “RNA” or “ribonucleic acid molecule” should be understood to include not only RNA molecules expressed or found in nature, but also RNA analogs and derivatives containing 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 with nucleic acid base structures or ribose-phosphate backbone structures (e.g., as shown below), and molecules containing ribonucleoside analogs or derivatives must retain the ability to form double helixes. As a non-limiting example, RNA molecules may include, but are not limited to, at least one modified ribonucleoside, such as 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, a debased nucleoside, a 2'-deoxy-2'-fluoro modified nucleoside, a 2'-amino modified nucleoside, a 2'-alkyl modified nucleoside, a morpholino nucleoside, a phosphoramidate or nucleoside, or any combination thereof. In some embodiments of the present invention, the RNA molecule contains 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 ribonucleosides of the LPA dsRNA agent molecule. With respect to each of the multiple modified ribonucleosides in such an RNA molecule, the modifications do not need to be the same.

[0085] In some embodiments, the dsRNA agents, LPA antisense polynucleotides and / or LPA sense polynucleotides of the present invention may comprise one or more independently selected modified nucleotides and / or one or more independently selected non-phosphodiester bonds. The term “independently selected,” as used herein, means selected elements such as modified nucleotides and non-phosphodiester bonds, and is used to mean that two or more selected elements may, but do not need to, be identical to one another. As used herein, “nucleotide base,” “nucleotide,” or “nucleic acid base” refers to heterocyclic pyrimidine or purine compounds, which are standard building blocks of all nucleic acids, including nucleotide-forming bases: adenine (a), guanine (g), cytosine (c), thymine (t), and uracil (u). Nucleic acid bases may be further modified to include universal bases, hydrophobic bases, ambiguous bases, enlarged-size bases, and fluorinated bases, but are not intended to be limited thereto. The term “ribonucleotide” or “nucleotide” may be used herein to refer to an unmodified nucleotide, a modified nucleotide, or an alternative portion. Those skilled in the art will recognize that guanine, cytosine, adenine, and uracil can be replaced by other parts without significantly altering the base-pairing properties of oligonucleotides containing such substituted parts.

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

[0087] Modified RNA backbones may include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotryesters, aminoalkylphosphotryesters, methyl and other alkylphosphonates (including 3'-alkylenephosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkylphosphoramidates, thionoalkylphosphotryesters, and boranophosphates (having 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 the form of 3'-5'~5'-3' or 2'-5'~5'-2'). Various salts, mixed salts, and free acid forms are also included. Methods for preparing phosphorus-containing bonds are conventional methods in the art, and such methods may be used to prepare specific modified LPA dsRNA agents, specific modified LPA antisense polynucleotides and / or specific modified LPA sense polynucleotides of the present invention.

[0088] Modified RNA skeletons that do not contain phosphorus atoms have skeletons formed by short alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or one or more short heteroatoms or heterocyclic nucleoside bonds, including those having other parts mixed with N, O, S and CH2 components. Methods for preparing modified RNA skeletons that do not contain phosphorus atoms are conventional practice in the art, and such methods may be used to prepare 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, the RNA mimoid comprises LPA dsRNA, LPA antisense polynucleotide and / or LPA sense polynucleotide, where, for example, but not limited to, the sugar-nucleoside bond (i.e., the backbone) of the nucleotide unit is replaced with a new group. In such embodiments, the base unit is maintained for hybridization with a suitable LPA nucleic acid target compound. One such oligomeric compound that is an RNA mimoid that has been shown to have excellent hybridization properties is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleic acid bases are retained and bond directly or indirectly to the aza nitrogen atom of the amide portion of the backbone. Methods for preparing RNA mimoids are conventionally practiced in the art, and such methods may be used to prepare certain modified LPA dsRNA agents of the present invention.

[0090] Some embodiments of the present invention include RNA having a phosphorothioate backbone and oligonucleotides having heteroatom backbones, particularly -CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [referred to as the methylene (methylimino) or MMI backbone], --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 having a phosphorothioate backbone and oligonucleotides having heteroatom backbones are conventionally practiced in the art, and such methods may be used to prepare certain modified LPA dsRNA agents, certain LPA antisense polynucleotides and / or certain LPA sense polynucleotides of the present invention.

[0091] Modified RNA may also contain one or more substituted sugar moieties. The LPA dsRNA, LPA antisense polynucleotide and / or LPA sense polynucleotide of the present 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 (wherein alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1-C) 10 Alkyl or C2-C 10 (Possibly alkenyl and alkinyl). Exemplary preferred 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 One example is CH3)2 (wherein n and m are 1 to approximately 10). In other embodiments, the dsRNA contains one of the following at the 2' position: C1-C 10Lower alkyl groups, substituted lower alkyl groups, alkali groups, aralkyl groups, O-alkaryl groups or O-aralkyl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl groups, heterocycloalkaryl groups, aminoalkylamino groups, polyalkylamino groups; substituted silyl groups, RNA cleavage groups, reporter groups, insertants; groups used to improve the pharmacokinetic properties of LPA dsRNA agents; or groups used to improve LPA dsRNA agents, groups for improving the pharmacodynamic properties of LPA dsRNA agents, LPA antisense polynucleotides and / or LPA sense polynucleotides, and other substituents having similar properties. In some embodiments, the modification includes 2'-methoxyethoxy(2'-O-(2-methoxyethyl) or 2'-MOE, also known as 2'-O--CH2CH2OCH3)(Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., alkoxy-alkoxy. Other exemplary modifications are 2'-dimethylaminoethoxyethoxy, i.e., O(CH2)2ON(CH3)2, also referred to as 2'-DMAOE, as described in the examples below; and 2'-dimethylaminoethoxyethoxy(2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE, also known in the art), i.e., 2'-O--CH2-O--CH2--N(CH2)2. Methods for preparing those modified RNAs described are conventionally practiced in the art, and such methods may 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 may also be carried out in the LPA dsRNA agents of the present invention at other positions on the RNA of the LPA antisense polynucleotide, other positions on the LPA sense polynucleotide and / or LPA sense polynucleotide, particularly at the 3' position of the sugar on the 3' terminal nucleotide, or at the 5' position of the 2'-5' linked LPA dsRNA, LPA antisense polynucleotide, or LPA sense polynucleotide and the 5' position of the 5' terminal nucleotide. The LPA dsRNA agents, LPA antisense polynucleotides, and / or LPA sense polynucleotides may have, for example, a sugar mimetic instead of the cyclobutyl moiety of pentofuranose. For example, methods for preparing those modified RNAs described are conventionally practiced in the art, and such methods may 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, the LPA dsRNA agent, LPA antisense polynucleotide, and / or LPA sense polynucleotide may include modifications or substitutions of nucleic acid bases (generally referred to simply as “bases” in the art). As used herein, “unmodified” or “natural” nucleic acid bases include the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases 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, and 5-uracil (psoy Examples include 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; other synthetic and natural nucleic acid bases such as 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-azaguanine and 7-azaadenine, and 3-azaguanine and 3-azaadenine.Additional nucleic acid bases that may be included in specific embodiments of the LPA dsRNA agent of the present 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, pp. 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, pp. 289-302, Crooke, STand Lebleu, B., Ed., CRC Press, 1993. Methods for preparing dsRNAs, LPA antisense polynucleotides, and / or LPA sense polynucleotides (such as those described herein) including nucleic acid base modifications and / or substitutions are conventionally practiced in the art, and such methods may be used to prepare the specific modified LPA dsRNA agents, LPA sense polynucleotides, and / or LPA antisense polynucleotides of the present invention.

[0094] Certain embodiments of the LPA dsRNA agent, LPA antisense polynucleotide, and / or LPA sense polynucleotide of the present invention include RNA modified to contain one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having such a modified ribose moiety that contains additional crosslinks linking the 2' and 4' carbons. This structure effectively "locks" the ribose in the three-dimensional structure of the 3'-internal structure. The addition of locked nucleic acids to the LPA dsRNA agents, LPA antisense polynucleotides, and / or LPA sense polynucleotides 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 containing locked nucleic acids are conventionally practiced in the art, and such methods may be used to prepare the specific modified LPA dsRNA agents of the present invention. Specific embodiments of the LPA dsRNA compounds, sense polynucleotides and / or antisense polynucleotides of the present invention include at least one modified nucleotide, including 2'-O-methylnucleotide, 2'-fluoronucleotide, 2'-deoxynucleotide, 2',3'-seconucleotide mimetic, locked nucleotide, 2'-F-arabinose nucleotide, 2'-methoxyethyl nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholino nucleotide and 3'-Ome nucleotide, nucleotides containing a 5'-phosphorothioate group or a cholesterol derivative or a terminal nucleotide linked to a dodecanoic acid bisdecylamide group, 2'-amino-modified nucleotide, phosphoramidate or a nucleotide containing a non-natural base.In some embodiments, the LPA dsRNA compound contains an E-vinylphosphonate 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' and 5' ends of the sense polynucleotide and / or the 3' end of the antisense polynucleotide, and the at least one modified nucleotide includes a debasalized nucleotide, a ribitol, a reversed nucleotide, a reversed debasalized nucleotide, a reversed 2'-OMe nucleotide, and a reversed 2'-deoxynucleotide. It is known to those skilled in the art that the inclusion of a debasalized or reversed debasalized nucleotide at the terminus 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 contains one or two isomannitol residues at the 3' and 5' ends of the sense strand. In certain embodiments, the sense strand independently contains one isomannitol residue at the 3' and 5' ends, respectively. Examples of isomannitol residues are given below: [ka] (In the formula, each word "Olig" independently represents a polynucleotide portion.) Examples of isomannitol residues (imann) are as follows: [ka] Examples include, but are not limited to, isomannitol residues, their stereoisomers, and non-limiting examples: [ka] It can also be replaced with the following. In some embodiments, the sense chain independently contains one isomannitol residue (imann) at either the 3' or 5' end, and has a targeting group conjugated to the 5' end having the following exemplary structure, for example, the targeting group N-acetyl-galactosamine, preferably the above GLS-15: [ka] (In the formula, each term "Olig" independently represents a polynucleotide portion.)

[0097] In certain embodiments of the present invention, the LPA dsRNA compound and / or antisense polynucleotide comprises at least one modified nucleotide, the at least one modified nucleotide comprising an unlocked nucleic acid (UNA) nucleotide and / or a glycol nucleic acid nucleotide (GNA). UNA and GNA are known to those skilled in the art as thermally unstable chemical modifications that can significantly improve 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; Laurens et al., Utilization of unlocked nucleic acid (UNA) to enhance siRNA performance in vitro and in vivo. Mol BioSyst. 2010;6:862-70).

[0098] Other modifications may be included in the RNA of the LPA dsRNA agent, LPA antisense polynucleotide and / or LPA sense polynucleotide of a particular embodiment of the present invention, each comprising RNA that enhances one or more characteristics of the LPA dsRNA agent, one or more ligands for the LPA antisense polynucleotide and / or LPA sense polynucleotide, and partially or chemically linked conjugates. Non-limiting examples of characteristics that may be enhanced are the activity, cell distribution, delivery of the LPA dsRNA agent, pharmacokinetic properties of the LPA dsRNA agent and / or LPA sense polynucleotide. In some embodiments of the present invention, the LPA dsRNA agent comprises one or more targeting or linking groups conjugated to the sense strand in some embodiments of the LPA dsRNA agent of the present invention. Non-limiting examples of targeting groups are compounds containing N-acetyl-galactosamine (GalNAc). The terms “targeting group,” “targeting agent,” “conjugate,” “targeting compound,” and “targeting ligand” are used interchangeably herein. In some embodiments of the present invention, the LPA dsRNA agent comprises a targeting compound conjugated to the 5' end of the sense strand. In some embodiments of the present invention, the LPA dsRNA agent comprises a targeting compound conjugated to the 3' end of the sense strand. In some embodiments of the present invention, the LPA dsRNA agent comprises a targeting group containing GalNAc. In some embodiments of the present invention, the LPA dsRNA agent does not contain a targeting compound conjugated to either or both of the 3' and 5' ends of the sense strand. In some embodiments of the present invention, the LPA dsRNA agent does not contain a GalNAc-containing targeting compound conjugated to either or both of the 5' and 3' ends of the sense strand.

[0099] Furthermore, targeting agents and binders are well known in the art, and for example, targeting agents and binders that can be used in specific 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. NYAcad. Sci., 1992, 660:306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765-2770), and thiocholesterol (Oberhauser et al., Nucl. Acids 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 triethylammonium 1,2-di-O-hexadecyl-rac-glycerol-3-phosphonate (Manoharan et al.,Tetrahedron Lett.,1995,36:3651-3654; Shea et al.,Nucl.Acids Res., 1990, 18:3777-3783), polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmitoyl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or octadecylamine or hexaamino-carbonyloxycholesterol moiety (Crooke et al., J.Examples include, but are not limited to, those listed in Pharmacol. Exp. Ther., 1996, 277:923-937.

[0100] Certain embodiments of compositions comprising an LPA dsRNA agent, an LPA antisense polynucleotide, and / or an LPA sense polynucleotide may include ligands that modify properties of the LPA dsRNA agent, such as distribution and targeting. In some embodiments of the compositions comprising the LPA dsRNA agent of the present invention, the ligands increase the affinity for selected targets (molecules, cells or cell types, compartments, e.g., compartments of cells or organs, tissues, organs or regions of the body) compared to species in which such ligands are absent. 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 globulin), 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 include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-coglycolic 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 polyphosphatidine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptide-mimicking polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helix peptides.

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

[0102] Other examples of ligands include dyes, inserts (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphylline, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantane acetate, 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-(ole Oils) Lithocholic acid, O3-(oleoyl)cholic acid, dimethoxytrityl or phenoxazine and peptide conjugates (e.g., Antennapedia peptide, Tat peptide), alkylating agents, phosphates, amino acids, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole cluster, acridine-imidazole conjugate, Eu tetraaza macrocyclic molecules) 3+Examples include complexes, dinitrophenyl, HRP, or AP.

[0103] The ligands included in the compositions and / or methods of the present invention may be proteins such as glycoproteins or peptides, molecules having a specific affinity for coligands, or antibodies, such as antibodies that bind to specific cell types such as cancer cells, endothelial cells, cardiomyocytes, or osteocytes. Ligands useful in embodiments of the compositions and / or methods of the present invention may be hormones or hormone receptors. Ligands useful in embodiments of the compositions and / or methods of the present invention may be lipids, lectins, carbohydrates, vitamins, coenzymes, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, or polyvalent fucose. Ligands useful in embodiments of the compositions and / or methods of the present invention may be substances that increase the uptake of LPA dsRNA agents into cells by disrupting the cytoskeleton of cells (e.g., by disrupting microtubules, microfilaments, and / or intermediate filaments of cells). Non-exclusive examples of such drugs include taxone, vincristine, vinblastine, cytochalasin, nocodazole, jaspraquinolide, latruncrine A, phalloidin, swinford A, indanosine, and myoserbine.

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

[0105] LPA dsRNA formulation In some embodiments of the present invention, the LPA dsRNA agent is present in the composition. The composition of the present invention may include one or more LPA dsRNA agents and, optionally, one or more pharmaceutically acceptable carriers, delivery agents, targeting agents, detectable labels, etc. Non-limiting examples of targeting agents available according to some embodiments of the methods of the present invention are agents that deliver the LPA dsRNA agent of the present invention to the cells to be treated and / or into the cells. 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 cells. 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 hepatocytes. In some embodiments of the methods of the present invention, it will be recognized that the therapeutic agent includes an LPA dsRNA agent having only a delivery agent, such as a delivery agent containing N-acetylgalactosamine (GalNAc), without any additional linking elements. For example, in some aspects of the present invention, the LPA dsRNA agent may be administered to cells or subjects without any detectable label or targeting agent linked to the LPA dsRNA agent, contained in a composition comprising a delivery compound containing GalNAc and a pharmaceutically acceptable carrier.

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

[0107] Delivery of LPA dsRNA agents and LPA antisense polynucleotide agents Some embodiments of the methods of the present invention involve delivering an LPA dsRNA agent to cells. As used herein, the term “delivery” means promoting or influencing cellular uptake or absorption. Absorption or uptake of the LPA dsRNA agent may occur by independent diffusion or active cellular processes or by using a delivery agent, targeting agent, etc., that can associate with the LPA dsRNA agent of the present invention. Preferred delivery modes for the methods of the present invention include, but are not limited to, in vivo delivery, in which the LPA dsRNA agent is injected into a tissue site or administered incrementally. In some embodiments of the present invention, the LPA dsRNA agent is ligated to a delivery agent.

[0108] Non-limiting examples of methods that may be used to deliver LPA dsRNA agents to cells, tissues and / or targets 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 a variety of diseases and conditions, including but not limited to liver diseases, acute intermittent porphyria (AIP), hemophilia, and pulmonary fibrosis. Details of the multiple delivery methods can be found in papers such as Nikam, RR & KRGore (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, J. K. 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 involve the use of lipid nanoparticles (LNPs) for delivering 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 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 RNAi-mediated molecules.

[0110] Another non-limiting example of a delivery agent that may be used in embodiments of the present invention for delivering the LPA dsRNA agent of the present invention to cells, tissues and / or targets is a GalNAc-containing agent that is linked to the LPA dsRNA agent of the present invention for delivering the LPA dsRNA agent to cells, tissues and / or targets. Examples of specific other delivery agents containing GalNAc that may be used in specific embodiments of the methods and compositions of the present invention are disclosed in PCT International Publication No. 2020191183A1. Another non-limiting example of a GalNAc-targeting ligand that may be used in compositions and methods of the present invention for delivering the LPA dsRNA agent to cells is a targeted ligand cluster. Examples of targeted ligand clusters provided herein are GalNAc ligands having a phosphodiester linkage (GLO) and GalNAc ligands having a phosphorothioate linkage (GLS). The term "GLX-n" may be used herein to indicate that the GalNAC-containing compound to be linked is one 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 having the structure shown below). In the figure below, the linkage site of the GalNAc-targeting ligand to the RNAi agent of the present invention is at the right end of each targeting ligand. It will be recognized that any RNAi and dsRNA molecule of the present invention can be ligated 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.The following 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 may also be by beta-dextran delivery systems, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677 and U.S. Patent Application Publication 2005 / 0281781, which are incorporated herein by reference in whole. 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 methods of the present invention, LPA dsRNAs are delivered without a targeting agent. These RNAs may be delivered as “naked” RNA molecules. As a non-limiting example, the LPA dsRNA of the present invention may be administered to a subject in a pharmaceutical composition that includes an RNAi agent for treating LPA-related diseases or conditions in subjects such as cardiovascular diseases, but does not include a targeting agent (e.g., a GalNAc-targeting compound), and cardiovascular diseases include Berger's disease, peripheral artery disease, coronary artery disease, metabolic syndromes, 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 diseases or conditions associated with elevated levels of Lp(a)-containing particles.

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

[0113] The LPA dsRNA agents of the present invention may 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 methods of the present invention, one or more LPA dsRNA agents are administered to cells and / or subjects to treat a disease or condition associated with LPA expression. In some embodiments, the methods of the present invention include administering one or more LPA dsRNA agents to a subject that requires them to alleviate a disease or condition associated with LPA expression in the subject. The LPA dsRNA agents or LPA antisense polynucleotide agents of the present invention may be administered in vitro, ex vivo, and in vivo to reduce LPA expression in one or more cells.

[0114] In some embodiments of the present invention, the level of LPA polypeptide in cells is reduced by the delivery (e.g., introduction) of an LPA dsRNA agent or an LPA antisense polynucleotide agent to the cells. Targeting agents and methods may be used to facilitate the delivery of an LPA dsRNA agent or an LPA antisense polynucleotide agent to specific cell types, cell subtypes, organs, spatial regions and / or intracellular regions within cells in a subject. An LPA dsRNA agent may 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 the subject. In some embodiments of the present invention, an LPA dsRNA agent is administered to the subject in combination with one or more additional therapeutic regimens for treating an LPA-related disease or condition in order to treat an LPA-related disease or condition. Other non-limiting examples of therapeutic regimens include the administration of one or more LPA antisense polynucleotides of the present invention, the administration of non-LPA dsRNA therapeutic agents and behavioral modifications. Additional treatment regimens may be administered at one or more of the following time points: before, concurrently with, and after administration of the LPA dsRNA agent of the present invention. It will be recognized that, as used herein, “concurrent” means within the 5th minute, 10th minute, 30th minute, 45th minute, and 60th minute of zero (where “zero” 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 targeted therapy, APOC3 targeted therapy, and niacin or any combination thereof. Non-limiting examples of behavioral modifications are dietary regimens, counseling, and exercise regimens. These and other therapeutic agents and behavioral modifiers are known in the art and may be used to treat a target LPA disease or condition, and may be combined with one or more LPA dsRNA agents of the present invention administered to a subject to treat an LPA disease or condition.The LPA dsRNA agent of the present invention, administered to cells or subjects to treat LPA-related diseases or conditions, may act synergistically with one or more other therapeutic agents or active ingredients to increase the efficacy of one or more therapeutic agents or active ingredients and / or increase the efficacy of the LPA dsRNA agent in treating LPA-related diseases or conditions.

[0115] The therapeutic method of the present invention includes the administration of an LPA dsRNA agent which can be used when an LPA-related disease or condition is present, including before the onset of the disease or condition and / or at all time points before and after any of these stages, including the early, middle, and late stages of the disease or condition. The method of the present invention may also treat a subject that has been previously treated with one or more other therapeutic agents and / or therapeutically effective components for an LPA-related disease or condition, wherein the one or more other therapeutic agents and / or therapeutically effective components have been unsuccessful, minimally successful, and / or no longer successful in treating the subject's LPA-related disease or condition.

[0116] dsRNA encoded in the vector In some embodiments of the present invention, LPA dsRNA agents can be delivered into cells using a vector. The LPA dsRNA agent transcription unit may be contained in a DNA or RNA vector. The preparation and use of vectors encoding such transgenes for the delivery of sequences into cells and / or subjects is well known in the art. For example, a vector resulting in transient expression of LPA dsRNA for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or longer may be used in the methods of the present invention. The length of transient expression may be determined using conventional methods based on factors such as a selected specific vector construct and target cells and / or tissues, but is not limited. Such transgenes may be introduced as linear constructs, circular plasmids or viral vectors, which may be embedded or non-embedded vectors. The introduced gene 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. If two individual strands are expressed, for example, to produce dsRNA, the two individual expression vectors can be co-introduced into cells, for example, by 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 a reverse repeat polynucleotide linked by a linker polynucleotide sequence such that the LPA dsRNA agent has a stem-loop structure.

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

[0119] Examples of viral vector systems that may be included in embodiments of the method include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentivirus vectors and Moloney's mouse leukemia virus; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) poliomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) poxvirus vectors such as orthopoxvirus vectors, e.g., vaccinia virus vectors or tripoxvirus vectors, e.g., canary poxvirus or fowlpox virus vectors; and (j) helper-dependent or gutless adenovirus vectors. The construct for recombinant expression of LPA dsRNA agents may include regulatory elements such as promoters and enhancers, which can be selected to result in constitutive or regulatory / inducible expression. The use of viral vector systems and promoters and enhancers is customary in the art and may be used in combination with the methods and compositions described herein.

[0120] Some embodiments of the present invention involve delivering an LPA dsRNA agent into cells using a viral vector. Many adenovirus-based delivery systems are conventionally used in the art for delivery to, for example, the lungs, liver, central nervous system, endothelial cells, and muscles. Non-limiting examples of viral vectors that may be used in the methods of the present invention are AAV vectors, poxviruses such as vaccinia virus, modified Ankara virus (MVA), NYVAC, tripoxviruses such as tripoxvirus or canary poxvirus.

[0121] Certain embodiments of the present invention include a method for delivering an LPA dsRNA agent into cells using a vector, such vector may reside in a pharmaceutically acceptable carrier that does not necessarily contain a sustained-release matrix into which the gene delivery vector is incorporated. In some embodiments, the vector for delivering LPA dsRNA may be generated from recombinant cells, and the pharmaceutical composition of the present invention may comprise one or more cells that generate an LPA dsRNA delivery system.

[0122] Pharmaceutical composition containing LPA dsRNA or ssRNA agent Specific embodiments of the present invention involve the use of a pharmaceutical composition comprising an LPA dsRNA agent or an LPA antisense polynucleotide agent and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising an LPA dsRNA agent or an LPA antisense polynucleotide agent may be used in the manner of the present invention to reduce LPA gene expression in cells and may be used in the treatment of LPA-related diseases or conditions. Such pharmaceutical compositions may be formulated based on a delivery mode. Non-limiting examples of formulations for delivery modes include compositions formulated for subcutaneous delivery, compositions formulated for systemic administration by parenteral delivery, compositions formulated for intravenous (IV) delivery, compositions formulated for intrathecal delivery, and compositions formulated for direct delivery to the brain, etc. The pharmaceutical compositions of the present invention may 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 a transdermal patch); lungs, such as by inhalation or blowing of a powder or aerosol, including via a sprayer; intra-airway, intranasal, epithelial and transdermal, oral or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; for example, subepidermal via an implantable device; or intracranial via a parenchymal device; or 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 kidneys, etc. Naturally, "delivery of LPA dsRNA agents" or "delivery of LPA antisense polynucleotide agents" into cells includes, respectively, delivery of LPA dsRNA agents or LPA antisense polynucleotide agents, direct expression of LPA dsRNA agents in cells, expression of LPA dsRNA agents from a coding vector delivered into cells, or any preferred mode of making LPA dsRNA or LPA antisense polynucleotide agents appear in cells. The preparation and use of formulations and means for delivering inhibitory RNA are well known and conventionally used in the art.

[0123] As used herein, “pharmaceutical composition” comprises a pharmacologically effective amount of the LPA dsRNA agent or 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 solution, buffered saline solution, glucose, water, glycerin, ethanol, and combinations thereof. This term expressly excludes cell culture media. With respect to orally administered drugs, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients, such as inactive diluents, disintegrants, binders, lubricants, sweeteners, flavorings, colorants, and preservatives. Preferred inactive diluents include sodium carbonate and calcium carbonate, sodium phosphate and calcium phosphate, and lactose, while corn starch and arginate are preferred disintegrants. Binders may include starch and gelatin, while lubricants, if present, are usually magnesium stearate, stearic acid, or talc. If necessary, tablets may be coated with a substance such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract. Reagents contained in the pharmaceutical formulation are further described below. Terms used herein, such as “pharmacologically effective amount,” “therapeutic 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 prophylactic outcome. 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 therapeutic effective amount of a drug to treat a disease or condition is the amount required to reduce that parameter by at least 10%. For example, a therapeutic effective amount of the LPA dsRNA agent or LPA antisense polynucleotide agent may reduce LPA polypeptide levels by at least 10%. The pharmaceutical composition may include, for example, a dsRNAi agent containing a double helix as shown in Table 1. In some other embodiments, such a dsRNAi agent includes a double helix variant in Table 1.

[0124] Effective amount In some embodiments, the method of the present invention involves contacting cells with an effective amount of LPA dsRNA or LPA antisense polynucleotide to reduce LPA gene expression in the contacted cells. Some embodiments of the method of the present invention involve administering the LPA dsRNA or LPA antisense polynucleotide to a target in an amount that effectively reduces the expression of the target LPA gene and effectively treats the LPA-related disease or condition. The “effective amount” used to reduce LPA expression and / or treat the LPA-related disease or condition is the amount necessary or sufficient to achieve the desired biological effect. For example, an effective amount of LPA dsRNA or LPA antisense polynucleotide for the treatment of the LPA-related disease or condition may be (i) the amount necessary to slow or stop the progression of the disease or condition; or (ii) the amount that can reverse, reduce or eliminate one or more symptoms of the disease or condition. In some embodiments of the present invention, the effective dose is the amount of the LPA dsRNA agent or LPA antisense polynucleotide agent that, when administered to a subject requiring treatment for an LPA-related disease or condition, produces a therapeutic response to prevent and / or treat the disease or condition. According to some embodiments of the present invention, the effective dose is the amount of the LPA dsRNA agent or LPA antisense polynucleotide agent of the present invention that, when administered in combination with or concurrently with another therapeutic treatment for an LPA-related disease or condition, produces a therapeutic response to prevent and / or treat the disease or condition. In some embodiments of the present invention, the biological effect of treating a subject with the LPA dsRNA agent or LPA antisense polynucleotide agent of the present invention may be improvement and / or complete elimination of symptoms caused by an LPA-related disease or condition. In some embodiments of the present invention, the biological effect is the complete elimination of an LPA-related disease or condition, as demonstrated, for example, 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 after administration of the agent of the present invention. The effects of the agent and / or method of the present invention on LPA-related diseases or conditions can be determined using other methods known in the art for evaluating the status of LPA-related diseases or conditions.

[0125] The effective dose of an LPA dsRNA agent or LPA antisense polynucleotide agent for reducing LPA polypeptide to a level for the treatment of an LPA-related disease or condition is generally determined in a clinical trial that establishes an effective dose for a test population and a control population in a blinded trial. In some embodiments, the effective dose is the amount that elicits a desired response, such as the amount that reduces the LPA-related disease or condition in cells, tissues and / or subjects having the disease or condition. Thus, the effective dose of an LPA dsRNA agent or LPA antisense polynucleotide agent for the treatment of an LPA-related disease or condition that can be treated by reducing LPA polypeptide may be the amount that, when administered, reduces the amount of LPA polypeptide in the subject to less than the amount present in cells, tissues and / or subjects without administration of the LPA dsRNA agent or LPA antisense polynucleotide agent. In some embodiments of the present invention, the level of LPA polypeptide and / or LPA gene expression present in cells, tissues and / or subjects that have not been contacted with or administered the LPA dsRNA agent or LPA antisense polynucleotide agent of the present invention is referred to as the “control” dose. In some embodiments of the method of the present invention, the control dose for a subject is the dose for the subject before treatment; in other words, the level for a subject before administration of the LPA agent may be a control level for the subject and may be used for comparison with the LPA polypeptide and / or LPA gene expression levels after administration of siRNA to the subject. In the case of treatment for LPA-related diseases or conditions, the desired response may be a reduction or elimination of one or more symptoms of the disease or condition in cells, tissues and / or the subject. The reduction or elimination may be temporary or persistent. It will be recognized that the status of the LPA-related disease or condition may be monitored using methods such as determining LPA polypeptide and LPA gene expression, symptom assessment, clinical tests, etc. In some embodiments of the present invention, the desired response to the 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 dose of a compound that reduces LPA polypeptides may also be determined by evaluating the physiological effects of administering the LPA dsRNA agent or LPA antisense polynucleotide agent to cells or subjects, such as a reduction in LPA-related disease or condition after administration. Subject assays and / or symptom monitoring may be used to determine the efficacy of the LPA dsRNA agent or LPA antisense polynucleotide agent of the present invention that may be administered with the pharmaceutical compound of the present invention, and to determine whether a response to treatment is present. 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 may be used to determine the status of LPA-related disease or condition in a subject before and after treatment with the LPA dsRNA agent of the present invention. Another non-limiting example is that one or more cholesterol accumulation tests in the liver known in the art may be used to determine the status of LPA-related disease in a subject. In this embodiment, the disease includes cholesterol accumulation, and this test is used to determine cholesterol levels in the subject before and after treatment with the LPA dsRNA agent of the present invention.

[0127] Some embodiments of the present invention include methods for determining the efficacy 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 evaluating and / or monitoring one or more “physiological features” of the subject. Non-limiting examples of physiological features of an LPA-related disease or condition include 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, the subject’s liver fat, and physical symptoms. Standard methods for determining such physiological features are known in the art and include, but are not limited to, blood tests, imaging tests, and physical examinations.

[0128] It will be recognized that the amount of LPA dsRNA or LPA antisense polynucleotide administered to a subject may be modified, at least in part, based on the determination of the subject's disease and / or condition and / or physiological characteristics. The amount of therapeutic agent may be modified, for example, by increasing or decreasing the amount of LPA dsRNA or LPA antisense polynucleotide by changing the composition in which the LPA dsRNA or LPA antisense polynucleotide is administered, by changing the route of administration, or by changing the time of administration. The effective dose of LPA dsRNA or LPA antisense polynucleotide will vary depending on the specific condition being treated, the age and health status of the subject being treated, the severity of the condition, the duration of treatment, the nature of concurrent treatment (if any), the specific route of administration, and other factors within the knowledge and expertise of the healthcare professional. For example, the effective dose may depend on the level of LPA polypeptide and / or the desired level of LPA gene expression that is effective in treating LPA-related diseases or conditions. Those skilled in the art can empirically determine an effective amount of a particular LPA dsRNA agent or LPA antisense polynucleotide agent for use in the methods of the present invention without excessive experimentation. Combined with the teachings provided herein, an effective prophylactic or therapeutic regimen may be planned to effectively treat a particular target 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 embodiments of the present invention, the effective amount of the LPA dsRNA agent or LPA antisense polynucleotide agent of the present invention may be an amount that produces a desired biological effect in the cell when in contact with the cell.

[0129] It should be recognized that LPA gene silencing can be performed constitutively or by genomic manipulation in any cells 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 LPA dsRNA agents and LPA antisense polynucleotide agents are delivered in a pharmaceutical composition at a dose sufficient to inhibit LPA gene expression. In certain embodiments of the present invention, the dose of the LPA dsRNA agent or LPA antisense polynucleotide agent is 0.01 to 200.0 mg per kg of body weight of the subject per day, generally 1 to 50 mg / kg body weight, 5 to 40 mg / kg body weight, 10 to 30 mg / kg body weight, 1 to 20 mg / kg body weight, 1 to 10 mg / kg body weight, and 4 to 15 mg / kg body weight (including endpoint values) per day. For example, LPA dsRNA agents or LPA antisense polynucleotide agents are available in approximately 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, and 2 mg / kg. 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 can be administered as a single dose based on body weight in kg, with doses of 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, and 49 mg / kg to 50 mg / kg.

[0131] Various factors may be considered when determining the delivery dose and timing of the LPA dsRNA agent of the present invention. The absolute amount of LPA dsRNA agent or LPA antisense polynucleotide agent delivered depends on various factors, including concurrent treatment, number of administrations, and parameters of the individual subject, including age, health status, 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, i.e., the safest dose based on reasonable medical judgment, may be used.

[0132] In some embodiments, the method of the present invention may involve administering to a subject one, two, three, four, five, six, seven, eight, nine, ten or more doses of an LPA dsRNA agent or an LPA antisense polynucleotide agent. In some cases, the pharmaceutical compound (e.g., containing an LPA dsRNA agent or an LPA antisense polynucleotide agent) may be administered to the subject in specific doses, such as at least daily, every other day, weekly, bi-weekly, or monthly, and may be administered once a day or more than twice a day, for example, two, three, four, five times or more times in a 24-hour cycle. 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 two, three or more divided doses at appropriate intervals of one day or less, or may be delivered using continuous infusion or a controlled-release formulation. In some embodiments of the method of the present invention, the pharmaceutical composition of the method of the present invention is administered to the subject once or more times a day, once or more times a week, once or more times a month, or once or more times a year.

[0133] In certain embodiments, 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 pharmacotherapy or therapeutic activities or protocols administered to a subject having 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 a certain amount of an LPA dsRNA agent or LPA antisense polynucleotide agent that reduces the level of LPA polypeptide to a level sufficient to produce a desired response in units of weight or volume suitable for administration to the subject. The dose of the pharmaceutical composition containing the LPA dsRNA agent or LPA antisense polynucleotide agent administered to the subject may be selected according to various parameters for reducing LPA protein levels, in particular the method of administration used and the subject's condition. Other factors include the required duration of treatment. 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 “prevent” mean, when used in reference to a disease, condition, or disorder that benefits from reduced LPA gene expression, a reduction in the likelihood of experiencing symptoms associated with such diseases, conditions, or disorders, including cardiovascular diseases such as Berger's disease, peripheral artery disease, coronary artery disease, metabolic syndromes, 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 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 such cases, the likelihood of such disease occurring is reduced, and if, for example, an individual has one or more cardiovascular disease risk factors but does not develop cardiovascular disease or develops only cardiovascular disease of low severity, then effective prevention is considered to occur if, compared to a population with the same risk factors but who has not received the treatments described herein, the individual does not develop the associated disease, condition or pathology, or has a reduced degree of onset of symptoms associated with such disease, condition or pathology (e.g., a reduction of at least about 10% on a clinically measurable measure of disease or condition), or the onset of symptoms is delayed (e.g., a delay of several days, weeks, months or years).

[0135] For LPA-related diseases and conditions where reducing LPA polypeptide levels is effective in treating the disease or condition, the methods and LPA dsRNA agents of the present invention may be used for therapeutic purposes that inhibit LPA expression. Examples of diseases and conditions that can be treated with the LPA dsRNA agents or LPA antisense polynucleotide agents of the present invention and the therapeutic methods of the present invention include, but are not limited to, Berger's disease, peripheral artery disease, coronary artery disease, metabolic syndromes, 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 diseases or conditions associated with elevated levels of Lp(a)-containing particles. Such diseases and conditions may be referred to herein as “LPA-related diseases and conditions” and “diseases and conditions caused and / or regulated by LPA.”

[0136] In some embodiments of the present invention, the LPA dsRNA agent or LPA antisense polynucleotide agent of the present invention may be administered to a subject at one or more time points before or after the diagnosis of an LPA-related disease or condition. In some embodiments of the present invention, a subject is at risk of developing an LPA-related disease or condition or has already developed an LPA-related disease or condition. A subject at risk of developing an LPA-related disease or condition is one in which the likelihood of developing an LPA-related disease or condition is increased compared to a control 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. Subjects at risk may include, for example, subjects with underlying diseases and / or genetic abnormalities that make them more susceptible to LPA-related diseases or conditions than a control subject without underlying diseases or genetic abnormalities; subjects with a family and / or personal history of LPA-related diseases or conditions; and subjects who have been previously treated for or are subject to treatment for an LPA-related disease or condition. It will be recognized that any underlying diseases and / or genetic abnormalities that make a subject more susceptible to LPA-related diseases or conditions may be those previously determined to be associated with an increased likelihood of developing LPA-related diseases or conditions.

[0137] It will be recognized that LPA dsRNA agents or LPA antisense polynucleotide agents may be administered to subjects based on their individual medical condition. For example, the medical treatment 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 the 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, the blood or serum sample is obtained from the subject after administration, the LPA level is measured using the sample, and the result is compared to the result determined in a sample of the subject before administration. The subsequent reduction in the LPA level in the subject's sample compared to the pre-administration level demonstrates the effectiveness of the LPA dsRNA agent or LPA antisense polynucleotide agent administered in reducing the subject's LPA level. In one non-limiting example, blood levels of Lp(a) may be considered a physiological feature of an LPA-related condition even if the subject has not been diagnosed with such a condition, such as those disclosed herein. Healthcare providers may monitor changes in blood levels of Lp(a) in a subject as a measure of the effectiveness of the LPA dsRNA agent or LPA antisense polynucleotide agent of the present invention administered.

[0138] Some embodiments of the methods of the present invention include modulating a treatment, the treatment of which includes administering the dsRNA agent or LPA antisense polynucleotide agent of the present invention to a subject, at least in part, on the basis of evaluating changes in one or more physiological features of an LPA-related disease or condition in the subject to be 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 a subject may be determined and may be used to help modulate the amount of the dsRNA agent or LPA antisense polynucleotide agent of the present invention administered to the subject. In one non-limiting example, the 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; at least in part, on the level determined, it is determined whether a higher dose of the dsRNA agent or LPA antisense polynucleotide agent is necessary 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, the dsRNA agent or LPA antisense polynucleotide agent of the present invention is administered to a subject, the level of Lp(a) in the subject's blood is determined after 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 partially on the determined level.

[0139] Accordingly, some embodiments of the present invention include evaluating changes in one or more physiological characteristics brought about by prior treatment of a subject in order to adjust the amount of the dsRNA agent or LPA antisense polynucleotide agent of the present invention administered to the subject. Some embodiments of the methods of the present invention include measuring physiological characteristics of an LPA-related disease or condition one, two, three, four, five, six or more times; evaluating and / or monitoring the effectiveness of the LPA dsRNA agent or LPA antisense polynucleotide agent of the present invention administered; and optionally using the measured results to adjust one or more of the following: the dose, administration regime and / or frequency of administration of the dsRNA agent or LPA antisense polynucleotide agent of the present invention for treating an LPA-related disease or condition in the subject. In some embodiments of the methods of the present invention, the desired outcome of administering an effective amount of the dsRNA agent or LPA antisense polynucleotide agent of the present invention to the subject is that the Lp(a) level in the subject's blood is reduced compared to a previous Lp(a) level in the blood determined with respect to the subject; or the Lp(a) level in the subject's blood was within the normal range.

[0140] As used herein, the terms “treat,” “therapeutic,” or “treated” may, when used in relation to LPA-related diseases or conditions, refer to prophylactic treatment that reduces the likelihood of developing an LPA-related disease or condition in a subject, treatment that prevents the LPA-related disease or condition from becoming more severe in the subject and / or slows its progression compared to the subject in the absence of a therapy that reduces the levels of LPA polypeptides in the subject, or treatment that eliminates or reduces the level of an LPA-related disease or condition after it has developed in the subject.

[0141] Certain embodiments of the agents, compositions, and methods of the present invention may be used to inhibit LPA gene expression. As used herein, with respect to LPA gene expression, the terms “inhibit,” “silence,” “reduce,” “downregulate,” and “knockdown” mean, for example, altering LPA gene expression 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 cells, cell populations, tissues, organs, or subjects, when the cells, cell populations, tissues, organs, or subjects are contacted with (e.g., treated with) the LPA dsRNA agent or LPA antisense polynucleotide agent of the present invention, compared to control levels of RNA transcribed by the LPA gene and LPA translated from mRNA, respectively. In some embodiments, the control level is the level in cells, tissues, organs, or subjects that are not contacted with (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 may be used in the methods of the present invention. The choice of a particular delivery method depends at least in part on the specific condition being treated and the dose required for therapeutic efficacy. In general, the methods of the present invention may be carried out using any medically acceptable delivery method, meaning any method that provides an effective therapeutic level for an LPA-related disease or condition without causing clinically unacceptable side effects. In some embodiments of the present invention, LPA dsRNA agents or LPA antisense polynucleotide agents may 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., via a nasogastric tube), percutaneous, vaginal, rectal, sublingual, and inhalation. The delivery routes of the present invention may include intrathecal, ventricular, or intracranial. In some embodiments of the present invention, the LPA dsRNA agent or LPA antisense polynucleotide agent may be placed in a sustained-release matrix and administered by placing the matrix on a target. In some aspects of the present invention, the LPA dsRNA agent or LPA antisense polynucleotide agent may be delivered to target cells using nanoparticles coated with a delivery agent that targets specific cells or organelles. Various delivery modes, methods, and agents are known in the art. Non-limiting examples of delivery methods and delivery agents are provided elsewhere in this specification. In some aspects of the present invention, the term “delivery” with respect to the LPA dsRNA agent or LPA antisense polynucleotide agent may mean the administration of one or more “naked” LPA dsRNA agent or LPA antisense polynucleotide agent sequences to cells or targets. In certain aspects of the present invention, “delivery” means delivery to cells or targets by transfection, delivery of cells containing the LPA dsRNA agent or LPA antisense polynucleotide agent to targets, delivery of vectors encoding the LPA dsRNA agent or LPA antisense polynucleotide agent to cells and / or targets, etc.Delivery of LPA dsRNA agents or LPA antisense polynucleotide agents using a transfection method may involve administering a vector to cells and / or subjects.

[0143] In some methods of the present invention, one or more LPA dsRNA agents or LPA antisense polynucleotide agents may be administered in formulation form or in a pharmaceutically acceptable solution, which may generally contain salts, buffers, preservatives, compatible carriers, adjuvants, and optionally other therapeutic components in pharmaceutically acceptable concentrations. 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 the LPA dsRNA agent or LPA antisense polynucleotide agent and optionally a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those skilled in the art. As used herein, a pharmaceutically acceptable vector refers to a non-toxic material that does not impair the efficacy 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 cells or subjects). Various methods for administering and delivering dsRNA agents or LPA antisense polynucleotide agents for therapeutic use are known in the art and may be used in the methods of the present invention.

[0144] Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials known in the art. Exemplary pharmaceutically acceptable carriers are described in U.S. Patent No. 5,211,657, while other carriers are known to those skilled in the art. Such formulations generally contain salts, buffers, preservatives, compatible carriers, and, optionally, other therapeutic agents. Salts must be pharmaceutically acceptable for use in pharmaceuticals; however, unpharmaceutically acceptable salts can be conveniently used to prepare such pharmacochemically acceptable salts and are not excluded from the scope of the invention. Examples of such pharmacochemically and pharmaceutically 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 others. Furthermore, pharmacochemically 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 methods of the present invention involve the direct administration of one or more LPA dsRNA agents or LPA antisense polynucleotide agents 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 exists or is likely to develop, and non-limiting examples of tissues include the liver or kidneys. Direct tissue administration can be achieved by direct injection or by other means. Many orally delivered compounds spontaneously enter and pass through the liver and kidneys, and some embodiments of the therapeutic methods of the present invention involve the oral administration of one or more LPA dsRNA agents to a subject. The LPA dsRNA agents or LPA antisense polynucleotide agents may be administered once or multiple times, alone or in combination with other therapeutic agents. If administered multiple times, the LPA dsRNA agents or LPA antisense polynucleotide agents may be administered via different routes. For example, for the first (or first few) doses may be administered subcutaneously, and one or more additional doses may be oral and / or systemic.

[0146] For embodiments of the present invention in which systemic administration of an LPA dsRNA agent or LPA antisense polynucleotide agent is desirable, the LPA dsRNA agent or LPA antisense polynucleotide agent may be formulated for parenteral administration by injection, for example, by bolus or serial infusion. Injectable formulations may exist in unit dosage forms such as ampoules or multi-dose containers with or without preservatives. LPA dsRNA agent formulations (also referred to as pharmaceutical compositions) may exist in the form of suspensions, solutions or emulsions in oily or aqueous carriers and may contain excipients such as suspending agents, stabilizers and / or dispersants.

[0147] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions, including physiological saline and buffer media. Parenteral carriers include sodium chloride solution, Ringer's glucose solution, glucose and sodium chloride solution, Ringer's lactate solution, or fixative oils. Intravenous excipients include liquids and nutritional supplements, electrolyte supplements (such as those based on Ringer's glucose solution), etc. Antimicrobial agents, antioxidants, chelating agents, preservatives such as inert gases, and other additives may also be present. Other forms of administration, such as intravenous administration, result in lower doses. If the subject's response is insufficient with the initial dose, higher doses may be used within the patient's tolerance (or the dose may be effectively increased by a different, more localized delivery route). Multiple daily doses may be used as needed to achieve adequate systemic or local levels of one or more LPA dsRNA agents or LPA antisense polynucleotide agents, and to achieve adequate reduction of LPA protein levels.

[0148] In other embodiments, the method of the present invention includes the use of a delivery carrier such as an implantable tablet suitable for implantation into a recipient such as biocompatible microparticles, nanoparticles, or other objects. An exemplary biodegradable implantable tablet that may be used according to this method is described in PCT International Publication No. 95 / 24929 (incorporated herein by reference), which describes a biocompatible, biodegradable polymer matrix for incorporating biomacromolecules.

[0149] Both non-biodegradable and biodegradable polymer matrices can be used in the methods of the present invention to deliver one or more LPA dsRNA agents or LPA antisense polynucleotide agents to a target. In some embodiments, the matrix may be biodegradable. The matrix polymer may be natural or synthetic polymer. The polymer may typically be selected based on a desired release period ranging from several hours to over a year. Typically, releases over a period of several hours to 3 to 12 months may be used. The polymer may optionally exist in the form of a hydrogel capable of absorbing up to about 90% of the weight of water, and may also optionally be crosslinked with polyvalent ions or other polymers.

[0150] In general, LPA dsRNA agents or LPA antisense polynucleotide agents may be delivered in some embodiments of the present invention using biodegradable implantable tablets by diffusion or by degradation of a polymer matrix. Exemplary synthetic polymers for this use are well known in the art. LPA dsRNA agents or LPA antisense polynucleotide agents can be delivered using biodegradable and non-biodegradable polymers using methods known in the art. LPA dsRNA agents or LPA antisense polynucleotide agents can also be delivered to treat LPA-related diseases or conditions using bioadhesive polymers such as biodegradable hydrogels (HSSawhney, CPPathak and JAHubell in Macromolecules, 1993, 26, 581-587). Other suitable delivery systems may include sustained-release, delayed-release, or prolonged-release delivery systems. Such systems avoid repeated administration of LPA dsRNA agents or LPA antisense polynucleotide agents, thereby improving convenience for subjects and healthcare professionals. Many types of release delivery systems are available and known to those skilled in the art. See, for example, 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 planting.

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

[0152] Therapeutic formulations of LPA dsRNA agents or LPA antisense polynucleotide agents are prepared by providing molecules or compounds of desired purity to an optional pharmaceutically acceptable carrier, excipient, or stabilizer in the form of a lyophilized formulation or aqueous solution. [Remington's Pharmaceutical Sciences 21] st It can be prepared for storage by mixing with [edition, (2006)]. Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the doses and concentrations used, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (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, etc.); low molecular weight (less than about 10 residues) polypeptides; serum albumin, gelatin This includes proteins such as benzoyl sulfate or immunoglobulins; 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 dextrin; 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 nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0153] Cells, subjects, and controls The methods of the present invention may be used in combination with cells, tissues, organs and / or subjects. In some embodiments of the present invention, the subjects are vertebrate mammals, including but not limited to humans or primates such as dogs, cats, horses, cattle, goats, mice, rats and monkeys. Thus, the present invention can be used to treat LPA-related diseases or conditions in both human and non-human subjects. In some embodiments of the present invention, the subjects may be farm animals, zoo animals, livestock or non-livestock, and the methods of the present invention may be used in prophylactic and therapeutic regimens in veterinary medicine. In some embodiments of the present invention, the subjects are humans, and the methods of the present invention may be used in prophylactic and therapeutic regimens for humans.

[0154] Non-limiting examples of subjects to whom the present invention may be applied are subjects diagnosed with, suspected of having, or at risk of having, 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 in this specification. The methods of the present invention may be applied to subjects diagnosed with a disease or condition at the time of treatment, subjects associated with higher-than-expected LPA expression, or subjects considered to have or be at risk of developing a disease or condition associated with higher-than-expected LPA expression. In some embodiments of the present invention, the disease or condition associated with higher-than-expected LPA expression levels is an acute disease or condition; in some embodiments 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, which includes 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 / 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 may be applied to a subject diagnosed with a disease or condition at the time of treatment, or a subject considered to be at risk of developing or suffering from a disease or condition.

[0156] In another non-limiting example, the LPA dsRNA agent of the present invention is administered to treat diseases or disorders caused by or associated with renin-angiotensin-aldosterone system (RAAS) activation, or diseases or disorders whose symptoms or progression respond to RAAS inactivation. The term “LPA-related disease” includes diseases, disorders, or conditions that benefit from reduced LPA expression. These diseases are typically associated with hypertension. Non-exclusive examples of LPA-related diseases include cardiovascular diseases, including Berger's disease, peripheral artery disease, coronary artery disease, metabolic syndromes, 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 diseases or conditions associated with elevated levels of Lp(a)-containing particles.

[0157] The cells to which the methods of the present invention may be applied include in vitro, in vivo, and ex vivo cells. The cells may exist as subjects, cultures and / or suspensions or in any other preferred circumstances or conditions. The cells to which the methods of the present invention may be applied may be liver cells, hepatocytes, cardiac cells, pancreatic cells, cardiovascular cells, kidney cells, or other types of vertebrate cells, including human and non-human mammalian cells. In some embodiments of the present invention, the cells to which the methods of the present invention may be applied are healthy, normal cells and are not known to be diseased cells. In some embodiments of the present invention, the methods and compositions of the present invention are applied to cells such as liver cells, hepatocytes, cardiac cells, pancreatic cells, cardiovascular cells and / or kidney cells. In certain embodiments of the present invention, while the control cells are normal cells, it should also be understood that cells with disease or a condition may be used as control cells in certain cases, such as when comparing the results of treated cells with disease or a condition to the results of untreated cells with disease or a condition.

[0158] According to the method of the present invention, LPA polypeptide levels can be determined and compared to LPA polypeptide control levels. The control may be a predetermined value that can take various forms. It may be a single cutoff, such as the median or mean. It may be established based on a comparison group in which there are, for example, 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 may be a group without one or more symptoms or diagnoses of an LPA-related disease or condition compared to a group with one or more symptoms or diagnoses of an LPA-related disease or condition; or a group of subjects not treated with the siRNA therapy of the present invention compared to a group of subjects treated with the siRNA therapy of the present invention. Typically, the control may be based on obviously healthy normal individuals or obviously healthy cells in an appropriate age group. In addition to predetermined values, it will be recognized that controls according to the present invention may be material samples tested in parallel with experimental materials. Examples include samples from a control population or control samples produced by manufacturing for parallel testing with experimental samples. In some embodiments of the present invention, the control may include cells or subjects that have not been contacted with or treated with the LPA dsRNA agent of the present invention, in which case the control level of LPA polypeptide may be compared to the level of LPA polypeptide in cells or subjects that have been contacted with the LPA dsRNA agent or LPA antisense polynucleotide agent of the present invention.

[0159] In some embodiments of the present 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 LPA level is determined in a biological sample obtained from a subject that has never 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 have been administered to a subject according to the therapeutic method of the present invention, one or more additional serum samples may be obtained from the subject, and the LPA polypeptide levels in one or more subsequent samples may be compared to the control / baseline level of the subject. Such comparisons may be used to assess the onset, progression, or regression of LPA-related disease or condition in the subject. For example, the level of LPA polypeptide in baseline samples obtained from subjects is higher than the level obtained from the same subjects after administration of the LPA dsRNA agent or LPA antisense polynucleotide agent of the present invention, which demonstrates the regression of LPA-related diseases or conditions and the efficacy of the LPA dsRNA agent of the present invention when administered in the treatment of LPA-related diseases or conditions.

[0160] In certain embodiments of the present invention, one or more values ​​of the LPA polypeptide level determined with respect to a subject may be used as a control value to later compare the LPA polypeptide level in the same subject, thereby enabling an evaluation of the change from the “baseline” LPA polypeptide level in the subject. Thus, when the initial level is used as the control level for the subject, the initial LPA polypeptide level may be used to indicate and / or determine the level of LPA polypeptide in the subject that the method and compounds 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 double strands shown in Table 1. In some other embodiments, such dsRNAi agents include double-strand 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 the LPA polypeptide in the serum sample obtained from the subject is at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more reduced compared to the pre-administration level of the LPA polypeptide in the serum sample obtained from the subject at a previous time point or compared to the level of a non-contact control (e.g., the level of the LPA polypeptide in a control serum sample). It will be recognized that all levels of the LPA polypeptide are associated with the level of LPA gene expression. Some embodiments and methods of the present invention include administering the LPA dsRNA and / or LPA antisense agent of the present invention to a subject in an amount effective to inhibit the expression of the LPA gene, thereby reducing the level of the LPA polypeptide in the subject.

[0162] Some embodiments of the present invention involve determining the presence, absence, and / or amount (also referred to herein as level) of LPA polypeptides derived from one or more biological samples obtained from one or more subjects. This determination can be used to evaluate the effectiveness of the therapeutic methods of the present invention. For example, the methods and compositions of the present invention can be used to determine the level of LPA polypeptides in biological samples obtained from subjects previously treated with the LPA dsRNA agent and / or LPA antisense agent of the present invention. After administration and treatment, the level of LPA polypeptides in serum samples obtained from subjects 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 polypeptides before administration in serum samples obtained from subjects at a previous point in time, or compared to the level of a non-contact control (e.g., the level of LPA polypeptides 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 determination of the physiological characteristics of the same subject may be compared with the control result. In one non-limiting example, blood Lp(a) levels (and / or physiological characteristics of an LPA disease or condition) may be measured from a subject that has never received LPA treatment according to the present invention and may be used as a baseline or control value for the subject. After one or more LPA dsRNA agents are administered to a subject according to the treatment method of the present invention, blood Lp(a) levels are measured and compared, each to the subject's control / baseline level. Such comparisons may be used to assess the onset, progression, or regression of an LPA-related disease or condition in the subject. For example, a baseline LPA level obtained from a subject may be higher than the LPA level measured from the same subject after the subject has been administered the LPA dsRNA agent or LPA antisense polynucleotide agent of the present invention, indicating regression of an LPA-related disease or condition and the effectiveness of the LPA dsRNA agent of the present invention administered when treating an LPA-related disease or condition.

[0164] In some embodiments of the 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 enabling assessment of changes from the "baseline" physiological characteristics of the subject. Thus, an initial physiological characteristic of an individual can be obtained, the measured value of the initial physiological characteristic is used as a control for the subject, and the methods and compounds of the invention are shown to be effective and / or used to determine the effect of reducing the level of LPA polypeptide in an individual. Using the methods of the invention, the LPA dsRNA agents and / or LPA antisense polynucleotide agents of the 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 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 blood of a subject 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 blood of the subject is within the normal range.

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

[0166] Kit A kit comprising one or more LPA dsRNA agents and / or LPA antisense polynucleotide agents, along with instructions for their use in the methods of the present invention, is also within the scope of the present invention. The kit of the present invention may comprise one or more 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 may be prepared for use in the therapeutic methods of the present invention. The components of the kit of the present invention may be packaged in an aqueous medium or in a lyophilized form. The kit of the present invention may comprise a support partitioned to seal and contain one or more container devices or a series of container devices (e.g., test tubes, vials, flasks, bottles, syringes, etc.) therein. The first container device or 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 include a targeting agent, a labeling agent, a delivery agent, etc., which may be included as part of the LPA dsRNA agent and / or LPA antisense polynucleotide administered in an embodiment of the therapeutic method of the present invention.

[0167] The kit of the present invention may also include instructions. Instructions are typically a document that provides guidance for performing the treatment embodied by the kit and for making decisions based on that treatment.

[0168] The following examples are provided to illustrate concrete examples of the practical application of the present invention and are not intended to limit the scope of the 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 double-stranded reagents 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 chemical reactions. Oligonucleotide chain growth is achieved through a four-step cycle: deprotection, condensation, capping, and oxidation or sulfidation for the addition of each nucleotide. Synthesis was carried out on a solid support composed of controllable porous glass (CPG, 1000 Å). Monomeric phosphoramidites were purchased from commercial suppliers. Phosphoramidites with GalNAc ligand clusters (GLPA1 and GLPA2 as non-limiting examples) were synthesized according to the procedures of Examples 2-3 herein. For siRNAs used for in vitro screening (Table 2), synthesis was carried out on a 2 μmol scale; for siRNAs used for in vivo testing (Tables 3, 4-5), the synthesis scale was 5 μmol or more. When a GalNAc ligand (GLO-0 as a non-limiting example) was ligated to the 3' end of the sense strand, a CPG solid support with the ligated GalNAc ligand was used. When a GalNAc ligand (non-limiting example: GLS-1 or GLS-2) was ligated to the 5' end of the sense chain, a GalNAc phosphoramidite (non-limiting example: GLPA1 or GLPA2) was 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 an activator. I2 in THF / Py / H2O and phenylacetyl disulfide (PADS) in pyridine / MeCN were used for oxidation and sulfidation reactions, respectively. After the final solid-phase synthesis step, the solid support-bound oligomers were cleaved by treatment with 40 wt% aqueous methylamine solution and 28% ammonium hydroxide solution in a 1:1 volume ratio, and the protecting group was removed. 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-chain product as a sodium salt. This product could then 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 reverse-phase HPLC (IP-RP-HPLC). The single-stranded oligonucleotide products purified by IP-RP-HPLC were converted to sodium salts by dissolving them in 1.0 M NaOAc and precipitating them by adding ice-cold EtOH. Equimolar complementarity annealing of sense and antisense oligonucleotides was performed in water to form double-stranded siRNA products, which were freeze-dried to obtain a fluffy white solid.

[0171] Example 2. Preparation of intermediates A and B Intermediate A was synthesized by treating commercially available galactosamine pentaacetate with trimethylsilyltrifluoromethanesulfonate (TMSOTf) in dichloromethane (DCM), as shown in Scheme 1 below. Next, glycosylation was carried out with Cbz-protected 2-(2-aminoethoxy)ethane-1-ol to obtain compound II. The Cbz protecting group was removed by hydrogenation to obtain intermediate A as a trifluoroacetic acid (TFA) salt. Intermediate B was synthesized according to the same scheme, except that Cbz-protected 2-(2-(2-aminomethoxy)ethoxy)ethane-1-ol was used as the feedstock. [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 hours, followed by 25°C for 1 hour; Cbz-protected 2-(2-aminoethoxy)ethane-1-ol (13.5 g, 56.5 mmol) was dried over a 4 Å powder molecular sieve (10 g) in DCE (100 mL) and added dropwise to the reaction mixture at 0°C under an N2 atmosphere. The resulting reaction mixture was stirred at 25°C for 16 hours under an N2 atmosphere. The reaction mixture was filtered and washed with saturated NaHCO3 (200 mL), water (200 mL), and saturated physiological saline (200 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product, which was triturated with 2-methyltetrahydrofuran / heptane (5 / 3, v / v, 1.80 L) for 2 hours. The resulting mixture was filtered and dried to obtain compound II (15.0 g, yield 50.3%) as a white solid.

[0173] 10% Pd / C (1.50 g) was carefully added to a hydrogenation bottle purged with dry argon, followed by the addition of 10 mL of tetrahydrofuran (THF), and then a solution of compound II (15.0 g, 26.4 mmol) in TFA (trifluoroacetic acid, 3.00 g, 26.4 mmol) was added. The resulting mixture was degassed, purged three times with H2, and stirred at 25°C for 3 hours under an H2 atmosphere (45 psi). 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 obtain intermediate-A as a foamy white solid (14.0 g, yield 96.5%). 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 obtain triester compound I. The benzyl protecting group was removed by hydrogenation to obtain secondary amine compound II. The amide was coupled with 6-hydroxycaproic acid to obtain compound III. Next, the tert-butyl reactive group was removed by treatment with HCl in dioxane to form tribasic acid compound IV. Amide coupling was carried out between tribasic acid compound IV and intermediate A or intermediate B to obtain compound Va or Vb. Phosphoramidite GLPA1 or GLPA2 was synthesized by phosphytylation of compound Va or Vb with 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite 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 hours. LC-MS showed that N-benzyl-1,3-propanediamine had been completely consumed. The reaction mixture was diluted with H2O (500 mL) and extracted with ethyl acetate (500 mL x 2). The combined organic matter was washed with saturated brine (1 L), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product, which was purified by silica gel column chromatography (gradient: petroleum ether:ethyl acetate 20:1-5:1). Compound I was obtained as a colorless oil (12.1 g, yield 78.4%). 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] The dried hydrogenation bottle was purged three times with argon. After adding Pd / C (200 mg, 10%), MeOH (5 mL) was added, followed by the addition of a solution of compound I (1.00 g, 1.97 mmol) in MeOH (5 mL). The reaction mixture was degassed under vacuum and refilled with H2. This process was repeated three times. The mixture was stirred at 25°C for 12 hours under an H2 atmosphere (15 psi). LCMS showed that compound I was completely consumed. The reaction mixture was filtered under reduced pressure under an N2 atmosphere. The filtrate was concentrated under reduced pressure to obtain compound II (655 mg, yield 79.7%) 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 three times with N2; subsequently, it was stirred at 25 °C for 3 hours under a N2 atmosphere. LCMS indicated the desired product. The reaction mixture was diluted with H2O (10 mL) and extracted with 20 mL (10 mL×2) of EtOAc. The organics were combined, washed with saturated brine (20 mL), dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography (gradient: petroleum ether:ethyl acetate 5:1~1:1) to give Compound III (650 mg, yield 77.8%) as a yellow oil. 1 1H NMR (400 MHz, CDCl3): δ ppm 3.90-3.95 (s, 2H), 3.63 (t, J = 6.40 Hz, 2H), 3.38-3.45 (m, 6H), 2.72 (t, J = 6.65 Hz, 2H), 2.40 (t, J = 7.28 Hz, 2H), 1.55-1.75 (m, 8H), 1.44 (s, 27H). Mass spectrometry calculated value for C 27 1H 50 1N2O8: 530.36; measured value: 531.3 (M+H) + .

[0179] A mixture of Compound III (5.5 g, 10.3 mmol) in HCl / dioxane (2M, 55 mL) was stirred at 25 °C for 3 hours. LCMS indicated the complete consumption of Compound III. The reaction mixture was filtered, washed with EtOAc (50 mL), and dried under reduced pressure to obtain a crude product. The crude product was dissolved in CH3CN (50 mL), and the volatile substances were removed in vacuo. This process was repeated three times to give Compound IV (2.05 g, yield 54.5%) 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, purged three times with N2, and then the mixture was stirred at 25°C for 3 hours under an N2 atmosphere. LC-MS showed the desired product. The reaction mixture was quenched by adding H2O (10 mL) and extracted with DCM (10 mL x 2). The combined organic matter was extracted with 10% citric acid (20 mL). The aqueous phase was neutralized with a saturated NaHCO3 solution and re-extracted with DCM (10 mL x 2). The organic matter was dried with sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound Va (570 mg, 0.281 mmol, yield 26.8%) 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] Tetrazole diisopropylammonium (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 the addition of 3-bis(diisopropylamino)phosphoryloxypropionitrile (194 mg, 0.645 mmol) dropwise at ambient temperature under N2. The reaction mixture was stirred at 20-25°C for 2 hours. LC-MS showed that compound Va had been completely consumed. After cooling to -20°C, the reaction mixture was added at 0°C to a stirred brine / saturated NaHCO3 (1:1, 5 mL) solution. After stirring for 1 minute, DCM (5 mL) was added. Stratification occurred. The organic layer was washed with brine / saturated NaHCO3 aqueous solution (1:1, 5 mL), dried over Na2SO4, filtered, and concentrated to approximately 1 mL. The residual solution was added dropwise to 20 mL of methyl tert-butyl ether (MTBE) under stirring. This resulted in a white solid precipitate. The mixture was centrifuged and the solid was recovered. The solid was redissolved in 1 mL of DCM and precipitated by adding MTBE (20 mL). The solid was separated again by centrifugation. The recovered solid was dissolved in anhydrous CH3CN. Volatile substances were removed. This process was repeated two more times to obtain 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 1P NMR (CDCl3): ppm δ 147.15.

[0182] The galNAc ligand phosphoramidite compound GLPA2 was synthesized using the same procedure, except that intermediate B was used. 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 1P 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 mixture was stirred at 25°C for 2 hours, and LC-MS showed that compound II had been completely converted. The reaction mixture was washed sequentially with saturated NaHCO3 solution (800 mL) and saturated sodium chloride aqueous solution (500 mL), 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 obtain compound 5 (290 g, 527 mmol, yield 75.7%) 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 hours. LC-MS 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 approximately 500 mL. Next, toluene / acetonitrile (1:1, approximately 750 mL) was added, and the mixture was concentrated to approximately 500 mL. Next, acetonitrile (approximately 1000 mL) was added, and the mixture was concentrated to dryness. The crude product was pulverized with 700 mL of acetonitrile at 60°C for 2 hours and filtered. The solid was recovered and dried to obtain 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 hour. LC-MS showed that the conversion of compound 6 was complete. The reaction liquid was added to 4000 mL of water and extracted with methyl tert-butyl ether (2000 mL, 2 parts) to remove impurities, and the remaining aqueous phase was extracted with dichloromethane (3000 mL, 2 parts). The dichloromethane phase was successively washed with 10% citric acid aqueous solution (2000 mL, 2 parts), saturated NaHCO3 (2.0 L, 2 parts), and saturated brine (2.0 L), and dried over anhydrous Na2SO4. The filtrate was filtered and concentrated under reduced pressure to obtain compound 8 (260 g, 159 mmol, yield 60.9%) 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 2 L hydrogenated autoclave was inactivated 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.) and trifluoroacetic acid (6.29 g, 55.1 mmol, 1.00 eq.) in MeOH (850 mL) under an argon atmosphere. The mixture was degassed and subjected to three H2 substitutions, then stirred under a hydrogen atmosphere at 25°C for 10 hours. LC-MS showed that compound 8 had been completely converted. Pd / C was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain 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 hour. LC-MS 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 mixture, and stirring was continued at 25°C for 1 hour. LC-MS showed that the conversion of compound 11 was complete. The reaction mixture was quenched by slowly adding saturated NH4Cl (3.0 L), the layers were separated, and the aqueous phase was extracted with dichloromethane (2 × 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 gradually formed during the addition. The precipitate was filtered under vacuum, the solid was collected, and dried under vacuum to obtain compound 13 (207 g, yield 72.8%) 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 tetraazolium 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 indicated 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, 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 filter cake was rinsed with methyl tert-butyl ether, the solid was recovered, dried in vacuo, the product was dissolved in dichloromethane (1.0 L), concentrated to dryness, and the operation was repeated 4 times to remove the remaining tert-butyl ether, to give GLPA15 (164 g, yield 73.3%). 1 H NMR (400 MHz in DMSO-d6): δ ppm 8.05 (br d, J = 6.50 Hz, 2H), 7.81 (br d, J = 9.01 Hz, 3H), 5.22 (d, J = 3.25 Hz, 3H), 4.98 (dd, J = 11.26, 3.25 Hz, 3H), 4.55 (br d, J = 8.50 Hz, 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 = 6.13 Hz, 6H), 3.17 - 3.30 (m, 9H), 3.07 (br d, J = 14.26 Hz, 4H), 2.76 (t, J = 5.82 Hz, 2H), 2.18 - 2.47 (m, 6H), 2.10 (s, 9H), 1.99 (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 a particular study, a method is 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, the method comprising using a synthetic process such as using GalNAc phosphoramidite (GLPA1) in the final coupling step of a solid-phase synthesis and using a synthetic process used in oligonucleotide chain extension (i.e., adding a nucleotide to the 5' end of the sense strand) to link GLPA1 to the 5' end of the sense strand.

[0191] In some studies, a method for ligating a GalNAc-containing targeting group to the 3' end of a sense chain involves using a solid support (CPG) containing GLO-n. In some studies, a method for ligating a GalNAc-containing targeting group to the 3' end of a sense chain involves ligating the GalNAc targeting group to a CPG solid support via an ester bond and using the resulting CPG together with the ligated GalNAc targeting group when the sense chain is synthesized, thereby ligating the GalNAc targeting group to the 3' end of the sense chain. Other GalNAc phosphoramidite compounds (GLPAn) can also be obtained using appropriate corresponding intermediates and by methods similar to those described herein or known in the art, and can be ligated as a targeting group at a suitable position on an siRNA double helix.

[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 hours. 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 x 2), washed with brine (500 mL), dried over Na2SO4, and concentrated under vacuum to obtain the residue. The residue was purified by column chromatography (DCM / MeOH = 100 / 1~50 / 1, 0.1% Et3N) to obtain compound B (150 g, yield 48.9%) 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 (0.45 M, 436 mL, 1.1 eq) was added dropwise to a solution of compound B (80.0 g, 178 mmol, 1.0 eq) in dichloromethane (5.0 mL) under an N2 atmosphere at 25°C, followed by the dropwise addition of a solution of compound C (2-cyanoethyldiisopropyl chlorophosphoramidite, 80.6 g, 267 mmol, 85.0 mL, 1.5 eq) in dichloromethane (200 mL); the reaction mixture was stirred at 25°C for 1.0 hour; 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 (500 mL), extracted with dichloromethane (500 mL x 3), washed with NaHCO3 / salt water = 1:1 (300 mL / 300 mL), dried over Na2SO4, and concentrated under vacuum (35°C) to obtain a residue (100 mL). The residue was purified by column chromatography (Al2O3, DCM / MeOH = 100 / 1~50 / 1, 0.1% Et3N) to obtain isomannitol phosphoramidite compound 2 (77 g, 119 mmol, 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] The ∇ represents the macroporous amine-methyl polyethylene resin support portion. A 50 L glass kettle was placed under nitrogen protection, and dichloromethane (19.50 kg) was added to the glass kettle and stirring was started. The temperature was controlled at 20-30°C, and DMTr-imann (1.47 kg) was added to the glass kettle, followed by triethylamine (1.50 kg), 4-dimethylaminopyridine (0.164 kg), and succinic anhydride (1.34 kg) in the reaction kettle. The system was kept warm at 20-30°C for 18 hours, after which a sample was taken, and the reaction was completed. A saturated sodium bicarbonate solution (22.50 kg) was added to the reaction system and stirred for 10-20 minutes, then allowed to stand until layers formed. The organic phase in the lower layer was separated, and the aqueous phase in the upper layer was extracted twice with dichloromethane; the organic phases were combined, dried over anhydrous sodium sulfate, the filtrate was filtered, then transferred for rotary evaporation, concentrated to no fraction, and a gray to apparent white solid (1.83 kg) was formed.

[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) and N,N-diisopropylethylamine (0.13 kg), products from the previous step, were added to the 100 L glass kettle through a solid feed funnel. After the addition was complete, the mixture was stirred for 10-30 minutes and then drained 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, load capacity 0.48 mmol / g) was added to the aforementioned 100 L solid-phase synthesis kettle through a solid-feed funnel. The temperature was controlled at 20-30°C, and N,N-dimethylformamide (21.00 kg + 21.00 kg) and the reaction liquid used in the previously galvanized bucket were added to the solid-phase synthesis kettle. The system was reacted under temperature-controlled conditions, and the solid load was monitored until it reached ≥250 umol / g, with UV detection used for load detection. The system was pressure filtered under nitrogen, and the filtrate was rinsed three times with N,N-dimethylformamide (26.00 kg + 26.10 kg + 26.00 kg), leaving the filtrate in the kettle. CAP.A (4.40kg + 4.42kg + 4.30kg) and CAP.B (4.40kg + 4.40kg + 4.47kg) were added to an 80L glass kettle and stirred for 3-8 minutes. This procedure was repeated three times for capping. Acetonitrile (18.00kg + 18.00kg + 18.00kg + 17.50kg + 17.50kg) was added to the solid-phase synthesis kettle. Nitrogen was bubbling for 10-30 minutes before pressure filtration. This procedure was repeated four times. The filtrate was purged with nitrogen in the solid-phase synthesis kettle for 2-4 hours, and then transferred to a 50L pressure filtration tank. Drying was continued while controlling the temperature at 15-30°C. The yellow-to-white solid product after drying was weighed: 3.516kg.

[0196] Isosorbide residue (imann) can be added to the 5' or 3' end of the oligonucleotide chain by a process well known to those skilled in the art, such as a reverse debase process (invab), to further add a targeting group.

[0197] Example 6. In vitro screening of LPA siRNA double helix using Huh7 cells and a dual fluorescent reporter gene vector. Huh7 cells were adjusted to an appropriate density and subsequently seeded in 96-well plates. According to the manufacturer's recommendations, simultaneously with seeding, the double-stranded fluorescent reporter gene vector psciCHECK2, containing the target gene, was co-transfected into Huh7 cells with siRNA using lipofectamine RNAiMax (Invitrogen-13778-150). Cells were then transfected with either test siRNA or control siRNA. siRNA was tested in triplicates at two concentrations (0.1 nM and 1.0 nM), and 48 hours after transfection, the fluorescence values ​​were detected by adding Dual-Glo® luciferase assay reagent. The ratio of sea urchin luminescence to firefly luminescence was calculated and standardized based on the ratio of the control siRNA-treated samples to calculate the knockdown efficiency. As a result, the double-stranded AV# used, as shown in Table 4, originated from sequences corresponding to those shown in Table 2.

[0198] Table 4 presents experimental results from in vitro studies of LPA expression inhibition 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 study of LPA siRNA double-stranded 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 14 days prior to siRNA administration by intravenous injection of undiluted 2 × 10^11 viral particles of an adeno-associated virus 8 (AAV8) vector encoding human LPA and luciferase genes. On day 0, mice were subcutaneously injected with a single dose of either 6 mg / kg of LPA siRNA or PBS. Blood samples were collected on day 0, before siRNA administration, and at the end of day 7. Luciferase activity was measured. The knockdown percentage was calculated by comparing the luciferase activity of blood samples from the pre-administration siRNA treatment group with the luciferase activity of blood samples collected at the end of day 7, and performing standardization based on the change in luciferase activity in serum samples from the PBS treatment group. As a result, as shown in Table 5, the double-stranded AD# used is derived from the sequence corresponding to that shown in Table 3.

[0205] Table 5 presents experimental results from an in vivo study of the inhibitory effect on LPA expression using multiple LPA RNAi agents at a single dose of 6 mpk. On day 7, residual luciferase activity compared to day 0 was standardized (mean ± SD) for the changes in the PBS treatment group.

[0206] [Table 39]

[0207] Example 8. In vivo study of LPA siRNA double-stranded 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 14 days prior to siRNA administration by intravenous injection of undiluted 2 × 10^11 viral particles of adeno-associated virus 8 (AAV8) vector encoding human LPA and luciferase genes. On day 0, mice were subcutaneously injected with a single dose of either 6 mg / kg of LPA siRNA or PBS. Blood samples were collected before siRNA administration on day 0, and at the end of days 7 and 14. Luciferase activity was measured, and the knockdown percentage was calculated by comparing the luciferase activity of blood samples from the pre-administration siRNA treatment group with that of blood samples collected on days 7 and 14, and performing standardization based on the change in luciferase activity in serum samples from the PBS treatment group. The knockdown (retention) percentage of human LPA mRNA levels (determined by qPCR) in the liver of mice at day 14 was compared between the siRNA treatment group and the PBS treatment group, and the results are shown in Table 6. The double-stranded AD# used were derived from sequences corresponding to those shown in Table 3.

[0208] Table 6 presents experimental results from in vivo studies of LPA expression inhibition using multiple LPA RNAi agents at a single dose of 6 mpk.

[0209] [Table 40]

[0210] Example 9. In vivo study of LPA siRNA double-stranded 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 intravenously with a stock solution of 2 × 10^11 viral particles from an 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 single doses of LPA siRNA or PBS at 3 mg / kg, 6 mg / kg, and 10 mg / kg, respectively. Mice were sacrificed at the end of the study. The knockdown (retention) percentage of human LPA mRNA levels (determined by qPCR) in the liver of mice at day 14 was compared between the siRNA-treated and PBS-treated groups, and the results are shown in Table 7. The double-stranded AD# used were derived from sequences corresponding to those shown in Table 3.

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

[0212] [Table 41]

[0213] Example 10. In vivo study of LPA siRNA double-stranded 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 intravenously with a stock solution of 2 × 10^11 viral particles from an 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 either 2 mg / kg or 6 mg / kg of LPA siRNA or PBS. Blood samples were collected on day 0, before siRNA administration, and at the end of days 7, 14, and 21. Luciferase activity was measured. The knockdown percentage was calculated by comparing the luciferase activity of blood samples from the pre-administration siRNA treatment group with the luciferase activity of blood samples collected at the end of days 7, 14, and 21, and performing standardization based on the change in luciferase activity in serum samples from the PBS treatment group. As a result, as shown in Table 8, the double-stranded AD# used is derived from the sequence corresponding to that shown in Table 3.

[0214] Table 8 presents experimental results from in vivo studies of LPA expression inhibition 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 (mean ± SD) for changes in the PBS treatment group.

[0215] [Table 42]

[0216] Example 11. In vivo study of LPA siRNA double-stranded To evaluate the in vivo activity of LPA siRNA, male cynomolgus monkeys (13–22 years old, weighing 7–9 kg) were selected for this study, with three animals in each group. Each animal was subcutaneously injected with 2 mg / kg of the test substance. The test substances 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 collected on day -14 (pre-administration), day -7 (pre-administration), day 1 (pre-administration), and on days 8, 15, 22, 29, 43, 50, 57, 64, 71, 78, 85, 92, and 99 post-administration. The collected blood samples were allowed to coagulate at room temperature for at least 30 minutes, followed by centrifugation at 3500 rpm at 4°C for 10 minutes. The collected serum (approximately 1.0 mL) was transferred to two pre-labeled polypropylene screw-cap vials (0.5 ml / vial, one for the ELISA assay and one for other backups) and stored in a refrigerator at -80°C until testing. The residual percentage of LPA (standardized against the mean of day -14 (pre-administration), day -7 (pre-administration), and day 1 (pre-administration), pre-administration of siRNA) is shown in Figure 1.

[0218] Example 12. In vivo study of LPA siRNA double-stranded To evaluate the in vivo activity of LPA siRNA, male cynomolgus monkeys (2–6 years old, weighing 2–6 kg) were used in this study, with four animals in each group. Each animal was subcutaneously injected with either physiological saline or 2 mg / kg of the AD00480-8 test product. The AD00480-8 test product used corresponds to the compounds shown in Table 3. After overnight fasting, blood collection was performed on day -14 (pre-administration), day 0 (pre-administration), and days 7, 14, and 21 post-administration. The collected blood samples were allowed to coagulate at room temperature for at least 30 minutes, followed by centrifugation at 3500 rpm at 4°C for 10 minutes. The collected serum (approximately 1.0 mL) was transferred to two pre-labeled polypropylene screw-cap vials (0.5 ml / vial, one for the ELISA assay and one for other reserves) and stored in a refrigerator at -80°C until testing. The percentage of LPA remaining (standardized against the average of day 14 (pre-administration) and day 0 (pre-administration), after siRNA pre-administration) is shown in Figure 2.

[0219] Example 13. In vitro screening of LPA siRNA double helix using Huh7 cells and a dual fluorescent reporter gene vector. Huh7 cells were adjusted to an appropriate density and subsequently seeded in 96-well plates. According to the manufacturer's recommendations, simultaneously with seeding, the double-stranded fluorescent reporter gene vector psciCHECK2, containing the target gene, was co-transfected into Huh7 cells with siRNA using lipofectamine RNAiMax (Invitrogen-13778-150). Cells were then transfected with either test siRNA or control siRNA. siRNA was tested in triplicates at two concentrations (0.1 nM and 1.0 nM), and 48 hours after transfection, the fluorescence values ​​were detected by adding Dual-Glo® luciferase assay reagent. The ratio of sea urchin luminescence to firefly luminescence was calculated and standardized based on the ratio of the control siRNA-treated samples to calculate the knockdown efficiency. As a result, the double-stranded AV# used, as shown in Table 9, originated from sequences corresponding to those shown in Table 2.

[0220] Table 9 presents experimental results from in vitro studies of LPA expression inhibition using multiple LPA RNAi agents.

[0221] [Table 43]

[0222] [Table 44]

[0223] Equal parts While several embodiments of the present invention are described and shown herein, it will be readily apparent to those skilled in the art that various other means and / or structures, as well as each of their variations and / or modifications, for carrying out the functions described herein and / or obtaining the results and / or one or more advantages, are considered to be within the scope of the present invention. More generally, it will be readily apparent to those skilled in the art that all parameters, sizes, materials and configurations described herein are illustrative, and the actual parameters, sizes, materials and / or configurations will depend on the specific application in which the teachings of the present invention are used. Those skilled in the art will be able to recognize or determine many equivalents of the specific embodiments of the present invention described herein by means of conventional experimentation alone. Thus, it should be understood that the embodiments described herein are merely illustrative and fall within the scope of the appended claims and their equivalents, and that the present invention may be carried out in a manner different from those specifically described and claimed for protection. The present invention covers each of the individual features, systems, articles, materials and / or methods described herein. Furthermore, any combination of two or more such features, systems, articles, materials and / or methods is also within the scope of the present invention, provided that such features, systems, articles, materials and / or methods are not contradictory to each other.

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

[0225] Where quantitative limitations are not used herein or in the claims, they should be understood as “at least one” unless expressly stated to the contrary.

[0226] As used herein and in the claims, the phrase “and / or” should be understood to mean “one or both” of the elements thus combined, that is, such elements appear together in some cases and separately in others. In addition to the elements specifically identified by “and / or,” other elements may be present, at their discretion, whether related to those specifically identified elements or not, unless explicitly stated otherwise.

[0227] All references, patents, patent applications, and publications cited or referenced herein are incorporated herein by reference as a whole. The inventions described in the original claims of this application are listed below. [Invention 1] A double-stranded ribonucleic acid (dsRNA) agent that inhibits LPA(Apo(a)) expression, comprising a sense strand and an antisense strand, and optionally comprising a targeted ligand; wherein a region complementary to the LPA RNA transcript is located at nucleotide positions 2-18 in the antisense strand, and the complementary region comprises at least 15 consecutive nucleotides that differ by 0, 1, 2, or 3 nucleotides from one of the antisense sequences listed in one of Tables 1-3. [Invention 2] The dsRNA agent according to Invention 1, wherein the region complementary to the LPA RNA transcript comprises at least 15, 16, 17, 18, or 19 consecutive nucleotides that differ by three or fewer nucleotides from one of the antisense sequences listed in one of Tables 1 to 3. [Invention 3] The dsRNA agent according to invention 1 or 2, wherein the antisense strand of the dsRNA is at least substantially complementary to any target region in the mRNA of the human LPA gene, and is provided in any one of Tables 1 to 3. [Invention 4] The dsRNA agent according to Invention 3, wherein the antisense strand of the dsRNA is completely complementary to any target region in the mRNA of the human LPA gene, and is provided in any one of Tables 1 to 3. [Invention 5] The dsRNA agent according to Invention 1, comprising one sense strand sequence from Tables 1 to 3, wherein the sense strand sequence is at least substantially complementary to the antisense strand sequence in the dsRNA agent. [Invention 6] The dsRNA agent according to Invention 1, comprising one sense strand sequence from Tables 1 to 3, wherein the sense strand sequence is completely complementary to the antisense strand sequence in the dsRNA agent. [Invention 7] The dsRNA agent according to Invention 1, comprising the antisense strand sequence listed in any one of Tables 1 to 3. [Invention 8] The dsRNA agent according to Invention 1, comprising a sequence listed as a double-stranded sequence in any one of Tables 1 to 3. [Invention 9] A dsRNA agent according to Invention 1, comprising at least one modified nucleotide. [Invention 10] The dsRNA agent according to Invention 1, wherein all or substantially all nucleotides in the antisense strand are modified nucleotides. [Invention 11] The dsRNA agent according to Invention 5 or 6, wherein the at least one modified nucleotide includes a 2'-O-methylnucleotide, a 2'-fluoronucleotide, a 2'-deoxynucleotide, a 2',3'-seconucleotide mimetic, a locked nucleotide, an unlocked nucleic acid (UNA) nucleotide, a glycol nucleic acid nucleotide (GNA), a 2'-F-arabinose nucleotide, a 2'-methoxyethyl nucleotide, a debasalized nucleotide, a ribitol, a reversed nucleotide, a reversed debasalized nucleotide, a reversed 2'-OMe nucleotide, a reversed 2'-deoxynucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide and a 3'-OMe nucleotide, a nucleotide containing a 5'-phosphorothioate group, or a cholesterol derivative or a terminal nucleotide linked to a dodecanoic acid bisdecylamide group, a 2'-amino modified nucleotide, a phosphoramidate or a nucleotide containing a non-natural base. [Invention 12] E-vinylphosphonate nucleotide is included in the 5' end of the guide strand, as described in invention 9 or 10 of the dsRNA agent. [Invention 13] A dsRNA agent according to Invention 1, comprising at least one phosphorothioate nucleoside interbinding. [Invention 14] The sense strand comprises at least one phosphorothioate nucleoside interbonding, as described in Invention 1, for the dsRNA agent. [Invention 15] The dsRNA agent according to Invention 1, wherein the antisense strand comprises at least one phosphorothioate nucleoside bond. [Invention 16] The dsRNA agent according to Invention 1, wherein the sense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate nucleoside interbonds. [Invention 17] The dsRNA agent according to Invention 1, wherein the antisense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate nucleoside interbonds. [Invention 18] The dsRNA agent according to Invention 1, wherein all or substantially all nucleotides of the sense strand and the antisense strand are modified nucleotides. [Invention 19] The dsRNA agent according to Invention 1, wherein the modified sense strand is one of the modified sense strand sequences listed in Tables 2-3. [Invention 20] The dsRNA agent according to Invention 1, wherein the modified antisense strand is one of the modified antisense strand sequences listed in Tables 2-3. [Invention 21] The dsRNA agent according to Invention 1, wherein the sense strand is complementary or substantially complementary to the antisense strand, and the complementary region is 16 to 23 nucleotides long. [Invention 22] The dsRNA agent according to Invention 21, wherein the complementary region is 19 to 21 nucleotides long. [Invention 23] The dsRNA agent according to Invention 1, wherein each chain has a length of 30 nucleotides or less. [Invention 24] The dsRNA agent according to Invention 1, wherein each strand is 25 nucleotides or less in length. [Invention 25] The dsRNA agent according to Invention 1, wherein each chain has a length of 23 nucleotides or less. [Invention 26] The dsRNA agent according to Invention 1, comprising at least one modified nucleotide and further comprising one or more targeting groups or linking groups. [Discussion 27] The dsRNA agent according to invention 26, wherein one or more targeting groups or linking groups are conjugated to the sense strand. [Invention 28] The targeting group or linking group comprises N-acetyl-galactosamine (GalNAc), as described in invention 26 or 27, which is a dsRNA agent. [Invention 29] The aforementioned targeting group is

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Claims

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

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

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

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

5. The composition according to claim 3, formulated for subcutaneous administration.

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

7. The aforementioned sense strand is sequence (5'-(GLS-15) of sequence number 889 * (Imann) * gagaguuaUcGaGgcacaua * a * (Imann)-3') is included, and the antisense strand is the sequence (5'-u) of sequence number 893. * U * auguGccucGaUaAcuc * u * It consists of c-3'), and in the sequences of sequence numbers 889 and 893, each lowercase nucleotide is modified with 2'-O-methyl (2'-OMe), and each uppercase nucleotide is modified with 2'-fluoro, and each asterisk ( * ) shows a phosphorothioate bond, and each Imann is 【Transformation 3】 And, GLS-15 is 【Chemistry 4】 The dsRNA agent according to claim 2.

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

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

10. The composition according to claim 8, packaged in a kit, container, pack, dispenser, pre-filled syringe or vial.

11. The composition according to claim 8, formulated for subcutaneous administration.

12. A pharmaceutical composition used for the treatment of LPA-related diseases or conditions, wherein the pharmaceutical composition contains 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 in an effective amount to lower serum Apo(a) levels, thereby treating the target LPA-related disease or condition.

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

14. The pharmaceutical composition according to 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 pectoris, unstable angina pectoris, 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 an increase in the level of Apo(a)-containing particles.

15. i) Inhibit the expression of the LPA gene in the target, ii) Reduce the level of Apo(a) protein in the subject compared to the baseline level of Apo(a) protein in the subject, or iii) Alter the physiological characteristics of LPA-related disease or condition in the subjects compared to the baseline physiological characteristics of LPA-related disease or condition in the subjects before treatment. Use of an effective amount of the dsRNA agent according to any one of claims 1, 2, and 7, or the composition according to any one of claims 3 to 6 and 8 to 11, for the manufacture of a drug for the purpose of

16. The use according to claim 15, wherein the LPA-related disease or condition is a cardiovascular disease.

17. The use according to claim 16, 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 pectoris, unstable angina pectoris, 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 an increase in the level of Apo(a)-containing particles.

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