Nucleic acid conjugate having dual-target-gene inhibitory effect and use thereof

Abstract

The present invention relates to a nucleic acid conjugate having a dual-target-gene inhibitory effect and the use thereof. Provided is a liver-targeting double-stranded nucleic acid conjugate capable of simultaneously inhibiting PCSK9 mRNA and LPA mRNA, which nucleic acid conjugate comprises a first nucleic acid single strand and a second nucleic acid single strand that are partially or completely complementary, each containing a region at least partially complementary to a sequence of PCSK9 mRNA or LPA mRNA. The double-stranded nucleic acid conjugate can be cleaved to form two independent siRNA conjugates, which respectively inhibit two target genes. The nucleic acid conjugate of the present invention has a therapeutic effect on hypercholesterolemia.

Description

Nucleic acid conjugate with dual-target gene inhibition and use thereof

[0001] Cross-reference to Related Applications

[0002] This application claims priority to Chinese Patent Application No. 2024110199180, filed on July 29, 2024, Chinese Patent Application No. 2024116690975, filed on November 21, 2024, Chinese Patent Application No. 2025102443144, filed on March 03, 2025, and Chinese Patent Application No. 2025104541264, filed on April 11, 2025, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application belongs to the field of biochemistry, and provides a nucleic acid double-stranded conjugate capable of inhibiting PCSK9 and LPA target genes, which has good stability, excellent PCSK9 and LPA gene inhibition activity, satisfactory cytotoxicity and immunostimulatory activity. BACKGROUND

[0004] Cardiovascular disease (CVD) is the most important chronic non-communicable disease that threatens human life and health worldwide. It is estimated that by 2050, the global CVD patient population will rise to 1.14 billion, which may lead to 35.6 million deaths (European Journal of Preventive Cardiology (2024) 00, 1-15). Atherosclerotic cardiovascular disease (ASCVD) is the main CVD (such as ischemic heart disease and ischemic stroke, etc.), which is the first cause of death among urban and rural residents in China, accounting for more than 40% of the cause of death. Epidemiological, genetic, and clinical intervention research evidence has fully confirmed that low-density lipoprotein cholesterol (LDL-C) is a key risk factor for ASCVD, and the lipid management guidelines in most countries or regions recommend LDL-C as the primary target for lipid-lowering therapy. Other apolipoprotein B (ApoB) containing lipoproteins, including triglyceride-rich lipoproteins (TRLs) and their remnants, as well as lipoprotein (a) [Lp(a)], also participate in the pathophysiological process of ASCVD, among which Lp(a) is an independent risk factor for ASCVD, regardless of age, gender, smoking status, and serum LDL-C levels.

[0005] Statins are currently the first choice for treating elevated LDL-C levels, but they have many shortcomings in clinical application: some patients have adverse reactions, LDL-C levels are not reduced to the target, and Lp(a) levels are increased. PCSK9 inhibitors can reduce LDL-C by about 50%-70%(China Lipid Management Guidelines, 2023), among which monoclonal antibodies and siRNA have been approved for marketing, and PCSK9 monoclonal antibodies are recommended as the recommended drug for controlling LDL-C in many national guidelines. In clinical application, PCSK9 inhibitors have weak effects on Lp(a)(reduction of about 18%-25%), and there is still room for improvement.

[0006] Because Lp(a) levels are less affected by acquired factors, lifestyle interventions such as healthy diet and exercise cannot directly reduce Lp(a) levels, and there is currently no lipid-lowering therapy targeting Lp(a) specifically. The existing traditional lipid-lowering therapy cannot achieve a satisfactory reduction in Lp(a), and the targeted Lp(a) lipid-lowering therapy has become one of the important breakthroughs in the prevention and treatment of cardiovascular diseases. Apo(a) protein (gene name LPA) is the main component of Lp(a). Existing animal and clinical tests have shown that inhibiting the expression of Apo(a) protein by siRNA can effectively reduce Lp(a) levels by more than 95%, but the effect on LDL-C is not obvious (less than 20%). There is currently a lack of a drug that can simultaneously reduce LDL-C and Lp(a).

[0007] The present application provides a small interfering RNA (siRNA) preparation that simultaneously targets PCSK9 and LPA, which can bind to the mRNA of PCSK9 and LPA specifically, destroy the normal translation template function of PCSK9 and LPA mRNA, and thus prevent the translation of PCSK9 and Apo(a) protein, thereby inhibiting the relevant protein pathway from the source, for treating / preventing diseases caused by the relevant protein pathway. SUMMARY

[0008] The present disclosure provides a nucleic acid double-stranded conjugate structure with a double-target gene inhibition effect, which comprises two partially or completely complementary nucleic acid single strands, and the nucleic acid double-stranded conjugate can be specifically recognized and cleaved by an endonuclease in vivo, forming two independent siRNA conjugates and inhibiting two target genes respectively. The nucleic acid double-stranded conjugate can form 0, 1 or more than 1 Bubble structure at the site specifically recognized and cleaved by the endonuclease.

[0009] The present disclosure first provides a double-stranded nucleic acid conjugate with a double-target gene inhibition effect, comprising:

[0010] a first nucleic acid single strand and a second nucleic acid single strand, which are partially or fully complementary; the two nucleic acid single strands are capable of forming a stable double-stranded structure; the first nucleic acid single strand or the second nucleic acid single strand has a length of 30-80 nt, and comprises a region that is at least partially complementary to a mRNA sequence of a target gene, respectively; each nucleic acid single strand comprises at least 2 unmodified RNA nucleotides or DNA nucleotides;

[0011] each nucleic acid single strand is conjugated with at least one carrier group comprising an ASGPR ligand, or a carrier group comprising a long fatty chain, or a carrier group comprising a long fatty acid chain, or a carrier group comprising a polypeptide, or a carrier group comprising a monoclonal antibody, or a carrier group comprising a targeting small molecule; wherein the group is conjugated to each nucleic acid single strand.

[0012] each nucleic acid single strand is represented by formula (I):

[0013] wherein each N is independently an unmodified or chemically modified nucleotide; each L is an independent carrier group, which is present or absent; (N) represents consecutive unmodified nucleotides; the sum of X1, X2, X3, X4, and X5 is an integer of 30-80.

[0014] In some preferred embodiments of the present disclosure, at least one of the first nucleic acid single strand and the second nucleic acid single strand has a structure as shown in formula (II):

[0015] In some preferred embodiments of the present disclosure, both the first nucleic acid single strand and the second nucleic acid single strand have a structure as shown in formula (II).

[0016] In some preferred embodiments of the present disclosure, the first nucleic acid single strand or the second nucleic acid single strand has a structure as shown in formula (III):

[0017] In some preferred embodiments of the present disclosure, the first nucleic acid single strand has a structure as shown in formula (II); and the second nucleic acid single strand has a structure as shown in formula (III).

[0018] In some preferred embodiments of the present disclosure, the first nucleic acid single strand has a structure as shown in formula (III); and the second nucleic acid single strand has a structure as shown in formula (II).

[0019] In some embodiments of the disclosure, the nucleic acid conjugate can be cleaved into two independent siRNA conjugates that inhibit mRNA of a target gene selected from the group consisting of: PCSK9, LPA, C3, C5, ANGPTL3, ANGPTL4, AGT, CFB, FXI, FXII, ANG7, ANG8, PLIN2, PNPLA3, HSD17B13, APOC3, DGAT2, MARC1, ALDH2, GRB14, GPR146, APOB, DPP4, PLG, INHBE, PD-L1, etc.

[0020] In some embodiments of the disclosure, the nucleic acid conjugate can be cleaved into two independent siRNA conjugates that inhibit mRNA of a target gene, which can also be selected from the group consisting of ADRB1, AGTR1, CACNA1C, SCN2A, HCN1, HCN4, HCN3, KCNAS, KCNJ3, KCNJ4, PLN, CAMK2D, PDE1, DEGS1, LEP, FLCN, ZFP423, CDK6, RPTOR, mTOR, FOXP1, PDE3B, ACVR1C, MSTN, CHRNA1, CHRNB1, CHRND, CHRNE, CHRNG, COL13A1, DOK7, LRP4, MUSK, RAPSN, SCN4A, DUX4, etc.

[0021] In some embodiments of the disclosure, the nucleic acid conjugate can be cleaved into two independent siRNA conjugates that inhibit mRNA of a target gene, which can also be selected from the group consisting of SOD1, FUS, C9orf72, MAPT, APP, SMN2, SCN9A, SCN10A, HTT, p21, UTRN, DUX4, SNCA, ATXN1, ATXN2, ATXN3, SCA1, SCA7, SCA8, UCP1, VEGFA, MeCP2, PRNP, DMPK, TARDBP, TTR, etc.

[0022] In some embodiments of the disclosure, the two independent siRNA conjugates inhibit the same gene or different genes, and both are selected from one or two members of the above-mentioned gene list.

[0023] In some embodiments of the disclosure, the two independent siRNA conjugates inhibit different genes or the same gene in the liver tissue.

[0024] In some embodiments of the disclosure, the two independent siRNA conjugates inhibit different genes or the same gene in the adipose tissue.

[0025] In some embodiments of the disclosure, the two independent siRNA conjugates inhibit different genes in CNS tissue.

[0026] In some embodiments of the disclosure, the two independent siRNA conjugates inhibit different genes or the same gene in muscle tissue (quadriceps).

[0027] In some embodiments of the disclosure, the two independent siRNA conjugates inhibit different genes or the same gene in muscle tissue (heart).

[0028] In some embodiments of the disclosure, the two independent siRNA conjugates inhibit different genes or the same gene in lung tissue.

[0029] In some embodiments of the disclosure, the two independent siRNA conjugates inhibit different genes or the same gene in kidney tissue.

[0030] In some embodiments of the disclosure, the target gene combination includes, but is not limited to, the following combinations:

[0031] (1) PCSK9 and LPA;

[0032] (2) C3 and C5;

[0033] (3) PCSK9 and ANGPTL3;

[0034] (4) ANGPTL4 and ANGPTL3;

[0035] (5) CTNNB1 and SOD1;

[0036] (6) ALK7 and SOD1;

[0037] (7) PCSK9 and AGT;

[0038] (8) PCSK9 and APOC3;

[0039] (9) AGT and CFB;

[0040] (10) PLIN2 and PNPLA3;

[0041] (11) PNPLA3 and HSD17B13;

[0042] (12) PNPLA3 and DGAT2;

[0043] (13) PNPLA3 and MARC1;

[0044] (14) Tau and APP;

[0045] (15) SCN9A and SCN10A;

[0046] (16) INHBE and DPP4.

[0047] Further, the nucleic acid conjugate is capable of forming 0, 1 or more than 1 Bubble structure at the position of 15-30 nt from the 5'-end of the nucleic acid single strand 1 or the nucleic acid single strand 2.

[0048] In some embodiments of the present disclosure, X1 is an integer from 14-29; the nucleic acid conjugate is capable of forming 0, 1 or more than 1 Bubble structure at the position of (N).

[0049] In some embodiments of the present disclosure, the region complementary to at least part of the mRNA sequence of the target gene is located at the position of (N) or 5' side of the Bubble structure.

[0050] Further, the position of (N) or the Bubble structure comprises at least 2 unmodified RNA nucleotides or DNA nucleotides.

[0051] In some embodiments of the present disclosure, each single strand of the nucleic acid conjugate has the same or different nucleotide sequence at the position of (N).

[0052] In some preferred embodiments of the present disclosure, the position of (N) or the Bubble structure comprises unmodified RNA nucleotides (rN) or DNA nucleotides (dN).

[0053] In some embodiments of the present disclosure, X3 is selected from an integer from 1-5 or 6-7.

[0054] In some embodiments of the present disclosure, X3 is selected from an integer from 1-4.

[0055] In some preferred embodiments of the present disclosure, X2 is selected from 2 or 3.

[0056] In some embodiments of the present disclosure, the position of (N) or the Bubble structure is capable of being recognized and cleaved by an endonuclease, cleaving into two independent siRNA conjugates.

[0057] Further, the two independent siRNA conjugates after cleavage are capable of independently inducing the inhibition of the mRNA of the two target genes through the RNAi-induced silencing complex process, respectively.

[0058] In some embodiments of the disclosure, the two independent siRNA conjugates each comprise a sequence complementary to the mRNA of two target genes.

[0059] In some embodiments of the disclosure, the nucleotide sequence at the (N) position on the first nucleic acid single strand or the second nucleic acid single strand in the nucleic acid conjugate is dNrN or dNrNdN.

[0060] In some preferred embodiments of the disclosure, dN is dA or dT.

[0061] In some preferred embodiments of the disclosure, rN is rU or rC.

[0062] In some more preferred embodiments of the disclosure, the nucleotide sequence at the (N) position is selected from dArU, or dTrCdT or dAdTrUdT.

[0063] In some embodiments of the disclosure, the nucleotide sequence at the (N) position on the first nucleic acid single strand is dTrCdT, and the nucleotide sequence at the (N) position on the second nucleic acid single strand is dTrCdT.

[0064] In some embodiments of the disclosure, the nucleotide sequence at the (N) position on the first nucleic acid single strand is dArU, and the nucleotide sequence at the (N) position on the second nucleic acid single strand is dTrCdT.

[0065] In some embodiments of the disclosure, the nucleotide sequence at the (N) position on the first nucleic acid single strand is dArU, and the nucleotide sequence at the (N) position on the second nucleic acid single strand is dAdTrUdT.

[0066] In some preferred embodiments of the disclosure, the carrier group comprising an ASGPR ligand, the targeting ligand is GalNAc (L96) or Ser (GN), or GalNAc (Ser1), GalNAc (Ser2), GalNAc (Ser3), GalNAc (Ser4), or LP-GalNAc, XY-GalNAc, GalNAc (A1GN), GalNAc (A1dGN), GalNAc (A3GN), GalNAc (A3dGN), GalNAc (A5GN), GAlNAc (NAG25), GAlNAc (NAG37),

[0067] In some preferred embodiments of the disclosure, the carrier group comprising an ASGPR ligand, the targeting ligand is GalNAc (L96) or Ser (GN), or GalNAc (Ser1), GalNAc (Ser2), GalNAc (Ser3), GalNAc (Ser4), or LP-GalNAc, XY-GalNAc, GalNAc (A1GN), GalNAc (A1dGN), GalNAc (A3GN), GalNAc (A3dGN), GalNAc (A5GN), GAlNAc (NAG25), GAlNAc (NAG37),

[0068] In some preferred embodiments of the disclosure, the carrier group comprising an ASGPR ligand, the targeting ligand is GalNAc (L96) or Ser (GN), or GalNAc (Ser1), GalNAc (Ser2), GalNAc (Ser3), GalNAc (Ser4), or LP-GalNAc, XY-GalNAc, GalNAc (A1GN), GalNAc (A1dGN), GalNAc (A3GN), GalNAc (A3dGN), GalNAc (A5GN), GAlNAc (NAG25), GAlNAc (NAG37), In some preferred embodiments of the disclosure, the carrier group comprising an ASGPR ligand, the targeting ligand is GalNAc (L96) or Ser (GN), or GalNAc (Ser1), GalNAc (Ser2), GalNAc (Ser3), GalNAc (Ser4), or LP-GalNAc, XY-GalNAc, GalNAc (A1GN), GalNAc (A1dGN), GalNAc (A3GN), GalNAc (A3dGN), GalNAc (A5GN), GAlNAc (NAG25), GAlNAc (NAG37),

[0069] GalNAc(Ser1), GalNAc(Ser2), GalNAc(Ser3), GalNAc(Ser4) have the following structures, respectively:

[0070] LP-GalNAc has the following structure:

[0071] XY-GalNAc has the following structure:

[0072] GalNAc(A1GN), GalNAc(A1dGN), GalNAc(A3GN), GalNAc(A3dGN), GalNAc(A5GN), GAlNAc(NAG25), GAlNAc(NAG37) have the following structures, respectively:

[0073] In some embodiments of the disclosure, the fatty chain in the carrier group comprising a long fatty chain comprises a C8-C30 alkyl, a substituted C8-C30 alkyl, a C8-C30 alkoxy, a substituted C8-C30 alkoxy, a C8-C30 alkenyl, a substituted C8-C30 alkenyl, a C8-C30 alkynyl, or a substituted C8-C30 alkynyl.

[0074] In some embodiments of the disclosure, the long fatty acid chain in the carrier group comprising a long fatty acid chain has the structure wherein B is independently a covalent bond linker, A is independently a substituted or unsubstituted alkylene, n is an integer from 1 to 5, and m is an integer from 1 to 30; wherein B can be independently selected from the group consisting of a bond -N(R 1 )-, -O-, -S-, -C(O)-, -N(R 1 )C(O)-, -C(O)N(R 2 )-, -N(R 1 )C(O)N(R 2 )-, -C(O)O-, -OC(O)-, -N(R 1 )C(O)O-, -OC(O)N(R 2 )-, -OPO2-O-, -O-P(O)(S)-O-, -O-P(O)(R 3 )-O-, -O-P(S)(R 3 )-O-, -O-P(O)(NR 1 R 2 )-N-, -O-P(S)(NR 1 R 2)-N-, -0-P(0)(NR 1 R 2 )-0-, -0-P(S)(NR 1 R 2 )-0-, -P(0)(NR 1 R 2 )-N-, -P(S)(NR 1 R 2 )-N-, -P(0)(NR 1 R 2 )-0-, -P(S)(NR 1 R 2 )-0-, -S-S-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; and each R 1 , R 2 and R 3 is independently hydrogen or unsubstituted C1-C6alkyl.

[0075] In some embodiments of the present disclosure, the polypeptide in the carrier group comprising a polypeptide is selected from a cell-penetrating peptide, a targeting peptide; the small molecule in the carrier group comprising a targeting small molecule is selected from folate, biotin, cholesterol.

[0076] In some embodiments of the present disclosure, the nucleic acid conjugate has liver targeting, extrahepatic targeting; preferably, the extrahepatic targeting is selected from targeting CNS tissue, fat, muscle, lung, kidney, eye, etc.

[0077] In some embodiments of the present disclosure, the long fatty chain, long fatty chain acid, polypeptide and other small molecules with targeting in the carrier group containing long fatty chain, long fatty chain acid, polypeptide and other small molecules with targeting have the following structure:

[0078] wherein R6is hydrogen or C1-C6alkyl, and R7is H or an amino protecting group.

[0079] In addition to the above described nucleic acid conjugate for silencing two different target gene mRNAs, the present disclosure provides some embodiments comprising two partially or fully complementary nucleic acid single strands, which can be specifically recognized and cleaved by an endonuclease in vivo, forming two independent siRNA conjugates and simultaneously inhibiting the same / different transcripts of the same target gene, improving the silencing efficiency and long-term effect of siRNA on the target gene. The nucleic acid double-stranded conjugate can form 0, 1 or more Bubble structures at the site specifically recognized and cleaved by the endonuclease.

[0080] The present disclosure also provides a double-stranded nucleic acid conjugate for inhibiting different target regions of the same transcript or different transcripts of the same target gene, comprising:

[0081] a partially or fully complementary first nucleic acid single strand and a second nucleic acid single strand; the two nucleic acid single strands can form a stable double-stranded structure; the first nucleic acid single strand or the second nucleic acid single strand is 30-80 nt in length and comprises a region at least partially complementary to the mRNA sequence of a target gene; each nucleic acid single strand comprises at least 2 unmodified RNA nucleotides or DNA nucleotides;

[0082] each nucleic acid single strand is conjugated to at least one carrier group comprising an ASGPR ligand, or a carrier group comprising a long aliphatic chain, or a carrier group comprising a long aliphatic acid chain, or a carrier group comprising a polypeptide, or a carrier group comprising a monoclonal antibody, or a carrier group comprising a targeting small molecule; wherein the group is conjugated to the middle or end of each nucleic acid single strand.

[0083] Further, the nucleic acid conjugate can form 0, 1 or more Bubble structures at a position of 15-30 nt from the 5'-end of the first nucleic acid single strand or the second nucleic acid single strand.

[0084] Further, the position of (N) or the Bubble structure comprises at least 2 unmodified RNA nucleotides or DNA nucleotides.

[0085] In some embodiments of the present disclosure, the Bubble structure comprises a nucleotide sequence at the position of (N) as described above.

[0086] In some embodiments of the present disclosure, the position of (N) or the Bubble structure can be recognized and cleaved by an endonuclease, cleaving into two independent siRNA conjugates.

[0087] Further, the two independent siRNA conjugates after cleavage can independently inhibit the mRNA of the same transcript of the same target gene, different target regions or different transcripts through the RNAi-induced silencing complex process.

[0088] In some embodiments of the present disclosure, the two independent siRNA conjugates respectively comprise sequences complementary to different transcripts of the same target mRNA.

[0089] In some embodiments of the present disclosure, the two independent siRNA conjugates respectively comprise sequences complementary to different target regions of the same target mRNA.

[0090] In some preferred embodiments of the present disclosure, the carrier group comprising ASGPR ligand, the targeting ligand is GalNAc(L96) or Ser(GN), or GalNAc(Ser1), GalNAc(Ser2), GalNAc(Ser3), GalNAc(Ser4), or LP-GalNAc, XY-GalNAc, GalNAc(A1GN), GalNAc(A1dGN), GalNAc(A3GN), GalNAc(A3dGN), GalNAc(A5GN), GAlNAc(NAG25), GAlNAc(NAG37),

[0091] GalNAc(L96) has the following structure:

[0092] Ser(GN) can have the following structure:

[0093] GalNAc(Ser1), GalNAc(Ser2), GalNAc(Ser3), GalNAc(Ser4) respectively have the following structure:

[0094] LP-GalNAc has the following structure:

[0095] XY-GalNAc has the following structure:

[0096] or

[0097] GalNAc(A1GN), GalNAc(A1dGN), GalNAc(A3GN), GalNAc(A3dGN), GalNAc(A5GN), GAlNAc(NAG25), GAlNAc(NAG37) respectively have the following structure:

[0098] In some embodiments of the disclosure, the fatty chain in the carrier group comprising a long fatty chain comprises a C8-C30alkyl, a substituted C8-C30alkyl, a C8-C30alkoxy, a substituted C8-C30alkoxy, a C8-C30alkenyl, a substituted C8-C30alkenyl, a C8-C30alkynyl, or a substituted C8-C30alkynyl.

[0099] In some embodiments of the disclosure, the long fatty acid chain in the carrier group comprising a long fatty acid chain has the structure where B is independently a covalent bond linker, A is independently a substituted or unsubstituted alkylene, n is an integer from 1 to 5, and m is an integer from 1 to 30; wherein B can be independently selected from the group consisting of a bond -N(R 1 )-, -O-, -S-, -C(O)-, -N(R 1 )C(O)-, -C(O)N(R 2 )-, -N(R 1 )C(O)N(R 2 )-, -C(O)O-, -OC(O)-, -N(R 1 )C(O)O-, -OC(O)N(R 2 )-, -OPO2-O-, -O-P(O)(S)-O-, -O-P(O)(R 3 )-O-, -O-P(S)(R 3 )-O-, -O-P(O)(NR 1 R 2 )-N-, -O-P(S)(NR 1 R 2 )-N-, -O-P(O)(NR 1 R 2 )-O-, -O-P(S)(NR 1 R 2 )-O-, -P(O)(NR 1 R 2 )-N-, -P(S)(NR 1 R 2 )-N-, -P(O)(NR 1 R 2 )-O-, -P(S)(NR 1 R 2 )-O-, -S-S-, a substituted or unsubstituted alkylene, a substituted or unsubstituted heteroalkylene, a substituted or unsubstituted cycloalkylene, a substituted or unsubstituted heterocycloalkylene, a substituted or unsubstituted arylene, or a substituted or unsubstituted heteroarylene; and each R 1 , R 2 , and R 3independently hydrogen or unsubstituted C1-C6alkyl.

[0100] In some embodiments of the present disclosure, the polypeptide in the carrier group comprising polypeptide is selected from a transmembrane peptide, a targeting peptide; the small molecule in the carrier group comprising targeting small molecule is selected from folate, biotin, cholesterol, etc.

[0101] In some embodiments of the present disclosure, the nucleic acid conjugate has liver targeting, extrahepatic targeting; preferably, the extrahepatic targeting is selected from targeting CNS tissue, fat, muscle, lung, kidney, eye, etc.

[0102] In some preferred embodiments of the present disclosure, the nucleic acid conjugate has liver targeting; the carrier group comprising ASGPR ligand has the following structure:

[0103] The present disclosure also provides a double-stranded nucleic acid conjugate with liver targeting, capable of simultaneously inhibiting PCSK9 mRNA and LPA mRNA, comprising:

[0104] a. a first nucleic acid single strand and a second nucleic acid single strand that are partially complementary or fully complementary; the two nucleic acid single strands can form a stable double-stranded structure; the first nucleic acid single strand or the second nucleic acid single strand is 30-80 nt in length and comprises a region that is at least partially complementary to the sequence of PCSK9 mRNA or LPA mRNA, respectively; each nucleic acid single strand comprises at least 2 unmodified RNA nucleotides or DNA nucleotides;

[0105] b. each nucleic acid single strand is conjugated to at least one carrier group comprising an ASGPR ligand; the group is conjugated to the end of each nucleic acid single strand;

[0106] wherein each nucleic acid single strand is represented by formula (I):

[0107] wherein each N is independently an unmodified or chemically modified nucleotide; each L is an independent carrier group, with or without; (N) represents consecutive unmodified nucleotides; the sum of X1, X2, X3, X4 and X5 is an integer of 30-80.

[0108] In some embodiments of the present disclosure, in the nucleic acid conjugate, X1 and / or X5 in the first nucleic acid single strand and / or the second nucleic acid single strand is / are 0.

[0109] In some preferred embodiments of the present disclosure, at least one of the first nucleic acid single strand and the second nucleic acid single strand has a structure as shown in formula (II):

[0110] In some preferred embodiments of the present disclosure, the first nucleic acid single strand and the second nucleic acid single strand each has a structure as shown in Formula (II).

[0111] In some preferred embodiments of the present disclosure, the first nucleic acid single strand has a structure as shown in Formula (II); and the second nucleic acid single strand has a structure as shown in Formula (III).

[0112] In some preferred embodiments of the present disclosure, the first nucleic acid single strand has a structure as shown in Formula (III); and the second nucleic acid single strand has a structure as shown in Formula (II).

[0113] Further, the position of 15-30 nt from the 5'-end of the nucleic acid conjugate nucleic acid single strand 1 or nucleic acid single strand 2 can serve as a recognition and / or cleavage site for an endonuclease, cleaving into two independent siRNA conjugates; the two independent siRNA conjugates after cleavage respectively inhibit PCSK9 mRNA or LPA mRNA through the process of RNAi silencing complex induction. The nucleic acid double-stranded conjugate can form 0, 1 or more than 1 Bubble structure at the site specifically recognized and cleaved by the endonuclease.

[0114] In some embodiments of the present disclosure, the sum of X1 and X2 in the nucleic acid conjugate is an integer of 14-29; the nucleic acid conjugate can form 0, 1 or more than 1 Bubble structure at the position of (N).

[0115] In some embodiments of the present disclosure, the region complementary to at least part of the sequence of PCSK9 mRNA or LPA mRNA is located at the position of (N) or the 5' side of the Bubble structure.

[0116] In some embodiments of the present disclosure, the nucleotide sequence of the first nucleic acid single strand or the second nucleic acid single strand is selected from the unmodified nucleotide sequences shown in Table 0a:

[0117] Table 0a. Unmodified nucleotide sequences

[0118] In some embodiments of the present disclosure, the first nucleic acid single strand or the second nucleic acid single strand comprises a nucleotide sequence as shown in SEQ ID NOs. 1001-1114.

[0119] In some embodiments of the present disclosure, the nucleotide sequence of the first nucleic acid single strand or the second nucleic acid single strand is selected from the following combinations:

[0120] (1) SEQ ID NO. 1001 and 1002;

[0121] (2) SEQ ID NO. 1003 and 1002;

[0122] (3) SEQ ID NO. 1004 and 1005;

[0123] (4) SEQ ID NO. 1006 and 1007;

[0124] (5) SEQ ID NO. 1006 and 1008;

[0125] (6) SEQ ID NO. 1009 and 1008;

[0126] (7) SEQ ID NO. 1010 and 1002;

[0127] (8) SEQ ID NO. 1011 and 1002;

[0128] (9) SEQ ID NO. 1012 and 1002;

[0129] (10) SEQ ID NO. 1013 and 1002;

[0130] (11) SEQ ID NO. 1014 and 1005;

[0131] (12) SEQ ID NO. 1015 and 1007;

[0132] (13) SEQ ID NO. 1016 and 1008;

[0133] (14) SEQ ID NO. 1017 and 1008;

[0134] (15) SEQ ID NO. 1018 and 1008;

[0135] (16) SEQ ID NO. 1019 and 1008;

[0136] (17) SEQ ID NO. 1020 and 1021;

[0137] (18) SEQ ID NO. 1022 and 1021;

[0138] (19) SEQ ID NO. 1023 and 1024;

[0139] (20) SEQ ID NO. 1025 and 1026;

[0140] (21) SEQ ID NO. 1025 and 1027;

[0141] (22) SEQ ID NO. 1028 and 1027;

[0142] (23) SEQ ID NO. 1001 and 1029;

[0143] (24) SEQ ID NO. 1030 and 1008;

[0144] (25) SEQ ID NO. 1031 and 1032;

[0145] (26) SEQ ID NO. 1033 and 1032;

[0146] (27) SEQ ID NO. 1034 and 1035;

[0147] (28) SEQ ID NO. 1034 and 1036;

[0148] (29) SEQ ID NO. 1037 and 1036;

[0149] (30) SEQ ID NO. 1031 and 1038;

[0150] (31) SEQ ID NO. 1039 and 1040;

[0151] (32) SEQ ID NO. 1041 and 1036;

[0152] (33) SEQ ID NO. 1042 and 1043;

[0153] (34) SEQ ID NO. 1044 and 1045;

[0154] (35) SEQ ID NO. 1046 and 1045;

[0155] (36) SEQ ID NO. 1047 and 1048;

[0156] (37) SEQ ID NO. 1049 and 1048;

[0157] (38) SEQ ID NO. 1050 and 1051;

[0158] (39) SEQ ID NO. 1050 and 1052;

[0159] (40) SEQ ID NO. 1053 and 1052;

[0160] (41) SEQ ID NO. 1047 and 1054;

[0161] (42) SEQ ID NO. 1055 and 1056;

[0162] (43) SEQ ID NO. 1057 and 1052;

[0163] (44) SEQ ID NO. 1058 and 1059;

[0164] (45) SEQ ID NO. 1060 and 1059;

[0165] (46) SEQ ID NO. 1061 and 1062;

[0166] (47) SEQ ID NO. 1061 and 1063;

[0167] (48) SEQ ID NO. 1064 and 1063;

[0168] (49) SEQ ID NO. 1058 and 1065;

[0169] (50) SEQ ID NO. 1066 and 1067;

[0170] (51) SEQ ID NO. 1068 and 1063;

[0171] (52) SEQ ID NO. 1069 and 1070;

[0172] (53) SEQ ID NO. 1071 and 1048;

[0173] (54) SEQ ID NO. 1072 and 1073;

[0174] (55) SEQ ID NO. 1072 and 1074;

[0175] (56) SEQ ID NO. 1075 and 1076;

[0176] (57) SEQ ID NO. 1077 and 1078;

[0177] (58) SEQ ID NO. 1079 and 1080;

[0178] (59) SEQ ID NO. 1081 and 1082;

[0179] (60) SEQ ID NO. 1083 and 1084;

[0180] (61) SEQ ID NO. 1085 and 1086;

[0181] (62) SEQ ID NO. 1087 and 1088;

[0182] (63) SEQ ID NO. 1089 and 1090;

[0183] (64) SEQ ID NO. 1091 and 1092;

[0184] (65) SEQ ID NO. 1093 and 1094;

[0185] (66) SEQ ID NO. 1095 and 1096;

[0186] (67) SEQ ID NO. 1097 and 1098;

[0187] (68) SEQ ID NO. 1099 and 1100;

[0188] (69) SEQ ID NO. 1101 and 1102;

[0189] (70) SEQ ID NO. 1103 and 1104;

[0190] (71) SEQ ID NO. 1105 and 1106;

[0191] (72) SEQ ID NO. 1107 and 1108;

[0192] (73) SEQ ID NO. 1020 and 1109;

[0193] (74) SEQ ID NO. 1071 and 1113;

[0194] (75) SEQ ID NO. 1071 and 1114.

[0195] In some embodiments of the present disclosure, the first nucleic acid single strand or the second nucleic acid single strand comprises a modified nucleotide.

[0196] In some embodiments of the disclosure, the modified nucleotides are selected from 2'-methoxy nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-cyclic nucleotide analogs, 2'-fluoro-arabinonucleotides, 2'-methoxyethyl nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 3'-methoxy nucleotides, 2'-allyl modified nucleotides, nucleotides comprising phosphorothioate groups, nucleotides comprising methylphosphonate groups, nucleotides comprising 5'-phosphates, nucleotides comprising 5'-phosphate mimics, glycol modified nucleotides, abasic nucleotides, morpholino nucleotides, locked nucleotides, unlocked nucleotides, threose nucleotides, or glycerol nucleotides.

[0197] In some embodiments of the disclosure, the first nucleic acid single strand or the second nucleic acid single strand can adopt the following chemical modification patterns:

[0198] wherein B1, B2, B3, B4, B5, B6are each independently 2'-OMe, 2'-F, LNA, 2'-OMOE modified nucleotides;

[0199] T1, T2, T3, T4are each independently 2'-F, 2'-OMe, DNA modified nucleotides;

[0200] Z1, Z2, Z3are each independently 2'-F, 2'-OMe, 2'-DNA, RNA (2'-OH modified) nucleotides;

[0201] q 1 ~q 13 are each independently an integer from 0 to 10.

[0202] In some embodiments of the disclosure, the first nucleic acid single strand comprises the modified nucleotide sequence of nucleic acid single strand 1 of modified nucleic acid duplexes 1-272 in Table 0b.

[0203] In some preferred embodiments of the disclosure, the second nucleic acid single strand comprises the modified nucleotide sequence of nucleic acid single strand 2 of modified nucleic acid duplexes 1-272 in Table 0b.

[0204] In some embodiments of the disclosure, the first nucleic acid single strand or the second nucleic acid single strand further comprises a 5'-(E)-vinylphosphate modified nucleotide at its 5' end.

[0205] In some preferred embodiments of the disclosure, the first nucleic acid single strand comprises the modified nucleotide sequence of nucleic acid single strand 1 of modified nucleic acid duplexes 1, 233, 234, 235, 236, 263, 264, 265, 266, 267, 267, 271, 272 in Table 0b.

[0206] In some embodiments of the disclosure, the second nucleic acid single strand comprises the modified nucleotide sequence of the nucleic acid single strand 2 of modified nucleic acid duplex 1, 233, 234, 235, 236, 263, 264, 265, 266, 267, 267, 271, 272 in Table 0b.

[0207] In some embodiments of the disclosure, the nucleotide sequence combination of the first nucleic acid single strand or the second nucleic acid single strand is selected from:

[0208] (1) the modified nucleotide sequences of the nucleic acid single strand 1 and the nucleic acid single strand 2 of modified nucleic acid duplex 1 in Table 0b;

[0209] (2) the modified nucleotide sequences of the nucleic acid single strand 1 and the nucleic acid single strand 2 of modified nucleic acid duplex 233 in Table 0b;

[0210] (3) the modified nucleotide sequences of the nucleic acid single strand 1 and the nucleic acid single strand 2 of modified nucleic acid duplex 234 in Table 0b;

[0211] (4) the modified nucleotide sequences of the nucleic acid single strand 1 and the nucleic acid single strand 2 of modified nucleic acid duplex 235 in Table 0b;

[0212] (5) the modified nucleotide sequences of the nucleic acid single strand 1 and the nucleic acid single strand 2 of modified nucleic acid duplex 236 in Table 0b;

[0213] (6) the modified nucleotide sequences of the nucleic acid single strand 1 and the nucleic acid single strand 2 of modified nucleic acid duplex 263 in Table 0b;

[0214] (7) the modified nucleotide sequences of the nucleic acid single strand 1 and the nucleic acid single strand 2 of modified nucleic acid duplex 264 in Table 0b;

[0215] (8) the modified nucleotide sequences of the nucleic acid single strand 1 and the nucleic acid single strand 2 of modified nucleic acid duplex 265 in Table 0b;

[0216] (9) the modified nucleotide sequences of the nucleic acid single strand 1 and the nucleic acid single strand 2 of modified nucleic acid duplex 266 in Table 0b;

[0217] (10) the modified nucleotide sequences of the nucleic acid single strand 1 and the nucleic acid single strand 2 of modified nucleic acid duplex 267 in Table 0b;

[0218] (11) the modified nucleotide sequences of the nucleic acid single strand 1 and the nucleic acid single strand 2 of modified nucleic acid duplex 270 in Table 0b;

[0219] (12) the modified nucleotide sequences of the nucleic acid single strand 1 and the nucleic acid single strand 2 of modified nucleic acid duplex 271 in Table 0b;

[0220] (13) Modified nucleotides sequences of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 272 in Table 0b.

[0221] In some embodiments of the present disclosure, the modified nucleic acid duplexes with PCSK9 and LPA dual-target gene silencing are shown in Table 0b:

[0222] Table 0b. Modified nucleic acid duplexes with PCSK9 and LPA target mRNA inhibition:

[0223] In some embodiments of the present disclosure, the nucleic acid conjugates with PCSK9 and LPA dual-target gene inhibition are selected from the group consisting of Conjugate 1 to Conjugate 314 and Conjugate 316 to Conjugate 318 in Table la.

[0224] In some preferred embodiments of the present disclosure, the nucleic acid conjugate is Conjugate 1, Conjugate 278, Conjugate 279, Conjugate 280, Conjugate 281, Conjugate 308, Conjugate 309, Conjugate 310, Conjugate 311, Conjugate 312, Conjugate 316, Conjugate 317 or Conjugate 318.

[0225] In another aspect, the present disclosure also provides a pharmaceutical composition comprising the aforementioned nucleic acid conjugate alone or in combination with a pharmaceutically acceptable carrier or excipient.

[0226] In some embodiments of the present disclosure, the pharmaceutical composition is an injection formulation or a subcutaneous administration formulation.

[0227] In some preferred embodiments of the present disclosure, the pharmaceutical composition is a subcutaneous injection formulation.

[0228] In some embodiments of the present disclosure, the pharmaceutical composition further comprises an excipient, which includes a buffer solution or a non-buffer solution.

[0229] In another aspect, the present disclosure also provides a use of the aforementioned nucleic acid conjugate or pharmaceutical composition in the preparation of a medicament for treating a disease mediated by dysregulation of PCSK9 and / or LPA expression in a subject in need thereof.

[0230] In another aspect, the present disclosure also provides a method of treating a disease mediated by dysregulation of PCSK9 and / or LPA expression, wherein the method comprises administering to a subject in need thereof the aforementioned nucleic acid conjugate or pharmaceutical composition.

[0231] In another aspect, the present disclosure also provides a use of the aforementioned nucleic acid conjugate or pharmaceutical composition for treating a disease mediated by dysregulation of PCSK9 and / or LPA expression in a subject in need thereof.

[0232] In another aspect, the present disclosure also provides a use of the aforementioned nucleic acid conjugate or pharmaceutical composition for treating a disease mediated by dysregulation of PCSK9 and / or LPA expression in a subject in need thereof.

[0233] In some preferred embodiments of the present disclosure, the aforementioned disease mediated by dysregulation of PCSK9 and / or LPA expression comprises hyperlipidemia.

[0234] In some preferred embodiments of the present disclosure, the aforementioned hyperlipidemia comprises hypercholesterolemia.

[0235] In some embodiments of the present disclosure, the aforementioned double-stranded conjugate or pharmaceutical composition is administered by injection or subcutaneous administration.

[0236] In some preferred embodiments of the present disclosure, the aforementioned double-stranded conjugate or pharmaceutical composition is administered by subcutaneous injection.

[0237] In some embodiments of the present disclosure, the aforementioned double-stranded conjugate or pharmaceutical composition is administered with an excipient, which comprises a buffered solution or a non-buffered solution.

[0238] In some preferred embodiments of the present disclosure, the aforementioned buffered solution comprises acetate, citrate, prolamine, carbonate or phosphate or phosphate buffered saline or any combination thereof.

[0239] In some preferred embodiments of the present disclosure, the aforementioned non-buffered solution is saline or water.

[0240] In some preferred embodiments of the present disclosure, the aforementioned subject in need thereof is a human.

[0241] In some embodiments of the present disclosure, the aforementioned double-stranded conjugate or pharmaceutical composition is administered at a dose of 1-100 mg / Kg.

[0242] In some more preferred embodiments of the present disclosure, the double-stranded conjugate is administered at a dose of 1-20 mg / Kg.

[0243] In some more preferred embodiments of the present disclosure, the double-stranded conjugate is administered at a dose of 1-6 mg / Kg.

[0244] In some more preferred embodiments of the present disclosure, the double-stranded conjugate is administered at a dose of 6-20 mg / Kg.

[0245] In some more preferred embodiments of the present disclosure, the double-stranded conjugate is administered at a dose of 6.0 mg / Kg.

[0246] In order to achieve the targeting of the nucleic acid double-stranded conjugate, the middle or end of each nucleic acid single strand is conjugated with a carrier group containing an ASGPR ligand, or a carrier group containing a long fatty chain, or a carrier group containing a long fatty acid chain, or a carrier group containing a polypeptide, or a carrier group containing a monoclonal antibody, or a carrier group containing a small molecule targeting, to achieve liver targeting, CNS targeting, lung targeting, kidney targeting, fat muscle targeting, eye targeting, etc.

[0247] In the nucleic acid double-stranded conjugate of the present disclosure, at least two specific base nucleotides (unmodified DNA or RNA) are introduced into each nucleic acid single strand, forming a site that can be specifically recognized and cut by an endonuclease in vivo into two independent siRNA conjugates, respectively inhibiting two target genes through the RNAi-induced silencing complex process. The nucleic acid double-stranded conjugate can form 0, 1 or more Bubble structures at the site specifically recognized and cut by the endonuclease.

[0248] Based on the above characteristics, the double-stranded nucleic acid conjugate capable of simultaneously inhibiting two target genes involved in the present disclosure has the structure shown in Figures 1-3 (A-U), where the protruding part is the Bubble design of the nucleic acid double-stranded conjugate, and L represents the carrier fragment of the compound with targeting ability, which can be conjugated at the end or middle of each nucleic acid single strand.

[0249] Meanwhile, the present disclosure utilizes the design of the nucleic acid double-stranded structure to design a nucleic acid double-stranded conjugate for simultaneously inhibiting the expression of PCSK9 and LPA genes, which comprises two partially or completely complementary nucleic acid single strands, and the nucleic acid double-stranded conjugate forms a site in the sequence that can be specifically recognized and cleaved by an endonuclease in vivo. The nucleic acid double-stranded conjugate provided by the present disclosure has good stability, excellent PCSK9 and LPA target mRNA inhibitory activity, satisfactory cytotoxicity and immunostimulatory activity, and a therapeutic effect on hypercholesterolemia diseases. The nucleic acid double-stranded conjugate can form 0, 1 or more Bubble structures at the site that can be specifically recognized and cleaved by an endonuclease.

[0250] The nucleic acid double-stranded conjugate capable of inhibiting PCSK9 and LPA target genes provided by the present disclosure includes but is not limited to those having the structures (A to U) shown in FIGS. 1 to 3, wherein L represents a GalNAc carrier fragment having liver targeting property, which can be conjugated at the end or in the middle of the two nucleic acid single strands; the protruding part in the schematic diagram is the Bubble part sensitive to endonuclease.

[0251] According to the above structure schematic, the designed nucleic acid conjugate capable of simultaneously inhibiting PCSK9 and LPA target mRNA is shown in Table 1a.

[0252] The modification scheme used in Table 1a is as follows:

[0253] wherein the capital letters A, C, G, U, I, T, 5mC represent adenosine-3'-phosphate, cytidine-3'-phosphate, guanosine-3'-phosphate, uridine-3'-phosphate, inosine-3'-phosphate, 5-methyluracil-3'-phosphate, 5-methylcytosine-3'-phosphate, respectively; the lowercase letter m represents that the nucleotide adjacent to the right of the letter m is a 2'-methoxy modified nucleotide; the lowercase letter f represents that the nucleotide adjacent to the right is a 2'-fluorinated modified nucleotide; the lowercase letter d represents that the nucleotide adjacent to the right is DNA; the lowercase letter r represents that the nucleotide adjacent to the right is a 2'-hydroxyl nucleotide; the "*" between the two nucleotides adjacent to the left is a phosphorothioate group; eVP represents a 5'-(E)-vinyl phosphate modified nucleotide; wherein the liver-targeting molecule (L96, GN11, GN12, etc.) is as described above; ib represents an inverted abasic nucleotide; invdA represents an inverted deoxyribonucleotide;

[0254] Table 1a. Nucleic acid double-stranded conjugate structure with PCSK9 and LPA target mRNA inhibitory effect:

[0255] Structure characterization method and results of conjugates, see Table 1b:

[0256] LC-MS representative test method: when the test sample is detected by denaturing IP RP-LC, the complementary paired double strands are separated into single strands (positive and negative strands), and then the positive / negative strand parent ions are gas-phase fragmented by tandem mass spectrometry. The software CONFIRM Sequence is used to analyze all detected fragment ions and resolve them. The sequence of the test sample is consistent with the theoretical sequence, that is, the actual molecular weight (MW) deviates from the theoretical molecular weight (MW) by less than 5 parts per million. The results are shown in Table 1b.

[0257] Table 1b. Molecular weight (MW) of nucleic acid double-stranded conjugates

[0258] Definitions of terms used in the present disclosure: unless otherwise specified, the initial definition of a group or term provided herein applies throughout the specification for that group or term; for terms not specifically defined herein, the meaning given to them by those skilled in the art should be given in the light of the disclosure and context.

[0259] In the present disclosure, "nucleic acid drug" refers to an RNA or RNA-like (e.g. chemically modified RNA) oligonucleotide molecule that is capable of reducing or inhibiting the translation of messenger RNA (mRNA) in a sequence-specific manner.

[0260] A nucleic acid molecule can function through an RNA interference mechanism (e.g., by interacting with the mRNA interference pathway machinery of a mammalian cell (the RNA-induced silencing complex, RISC) to induce mRNA degradation), or through any other mechanism or pathway. While it is believed that the nucleic acid drug agents used in the present disclosure function through an RNA interference mechanism, the nucleic acid drug agents are not limited or restricted to any particular mechanism or pathway of action. Nucleic acid drug molecule types include, but are not limited to, single-stranded antisense oligonucleotides, short interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs), and dicer substrates. The nucleic acid drug agents described in the present disclosure are composed of an oligonucleotide strand that is at least partially complementary to an mRNA that is a target. In some embodiments, the nucleic acid drug agents or conjugates described in the present disclosure are double-stranded and composed of an antisense strand and a sense strand that is at least partially complementary to the antisense strand.

[0261] The terms "silence," "reduce," "inhibit," "down-regulate," or "knockdown" refer to a reduction or decrease in the level of expression of a given gene when a cell, tissue, organ, or animal is treated with a nucleic acid drug molecule described in the present disclosure, as compared to the level of expression of the gene when the cell, tissue, organ, or animal is not so treated.

[0262] The term "sequence" or "nucleotide sequence" refers to the order or succession of nucleobases or nucleotides, represented in alphabetical order using standard nucleotide nomenclature.

[0263] The term "complementary" when used to describe the relationship between a first nucleotide sequence (e.g., a nucleic acid drug sense strand or a target mRNA) and a second nucleotide sequence (e.g., a single-stranded antisense oligonucleotide or a double-stranded nucleic acid drug antisense strand) refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize (under physiological conditions in a mammal or similar in vitro conditions) to an oligonucleotide or polynucleotide comprising the second nucleotide sequence to form base pairs and form a duplex or double helix structure. Complementary sequences include Watson-Crick base pairing or non-Watson-Crick base pairing, and include natural or modified nucleotides or nucleotide analogs to the extent necessary to satisfy the above-mentioned hybridization requirements. For example, monomers mA and fA are complementary to U (or T) and are equivalent to A for purposes of determining identity or complementarity.

[0264] In the present disclosure, "fully complementary" means that all (100%) of the bases in a contiguous sequence of a first oligonucleotide will hybridize to the same number of bases in a contiguous sequence of a second oligonucleotide in a pair of hybridizing nucleobase or nucleotide sequence molecules. The contiguous sequence can comprise all or a portion of the first or second nucleotide sequence.

[0265] In the present disclosure, "partially complementary" means that at least 70% (but not all) of the bases in a contiguous sequence of a first oligonucleotide will hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide, in a pair of hybridized nucleobase or nucleotide sequence molecules. The contiguous sequence can comprise all or part of the first or second nucleotide sequence.

[0266] In the present disclosure, the terms "complementary", "fully complementary", "partially complementary" are used in relation to nucleobase or nucleotide matching between nucleic acid single strand 1 and nucleic acid single strand 2 of an RNAi agent, or between the antisense strand of an RNAi agent and the sequence of a target mRNA.

[0267] In the present disclosure, "unmodified RNA" is ribonucleotides (2'-hydroxyl nucleotides); "unmodified DNA" is deoxyribonucleotides (2'-deoxy nucleotides).

[0268] In the present disclosure, "covalent bond linker" is a piece of molecular structure or functional group that connects two molecules or groups of atoms together through a covalent bond, such as -N(R 1 )-, -O-, -S-, -C(O)-, -N(R 1 )C(O)-, -C(O)N(R 2 )-, -N(R 1 )C(O)N(R 2 )-, -C(O)O-, -OC(O)-, -N(R 1 )C(O)O-, -OC(O)N(R 2 )-, -OPO2-O-, -O-P(O)(S)-O-, -O-P(O)(R 3 )-O-, -O-P(S)(R 3 )-O-, -O-P(O)(NR 1 R 2 )-N-, -O-P(S)(NR 1 R 2 )-N-, -O-P(O)(NR 1 R 2 )-O-, -O-P(S)(NR 1 R 2 )-O-, -P(O)(NR 1 R 2 )-N-, -P(S)(NR 1 R 2 )-N-, -P(O)(NR 1 R 2 )-O-, -P(S)(NR 1 R 2)-O-, -S-S-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; and each R 1 , R 2 , and R 3 are independently hydrogen or unsubstituted C1-C6alkyl.

[0269] In the present disclosure, "Bubble structure" refers to that, during the annealing pairing process of two nucleic acid single strands, due to the incomplete matching or mismatching of bases, the nucleic acid double strand cannot form a standard double-stranded RNA structure, and a bulge part, i.e., a circular or bubble-shaped region, is formed in the incomplete matching or mismatching region (unpaired region).

[0270] In the present disclosure, "PCSK9 and / or LPA expression disorder mediated disease" refers to a group of metabolic cardiovascular diseases with low-density lipoprotein cholesterol (LDL-C) clearance disorder and lipoprotein (a) mediated vascular inflammation / thrombosis as the core pathological mechanism, caused by the pathological increase of protein products of proprotein convertase subtilisin 9 (PCSK9) and lipoprotein (a) (LPA) genes due to their abnormal high expression, which covers typical diseases including but not limited to hyperlipidemia, hypercholesterolemia, atherosclerotic cardiovascular disease, coronary heart disease, ischemic stroke, peripheral arterial disease, calcified aortic valve stenosis, premature cardiovascular disease, etc. BRIEF DESCRIPTION OF DRAWINGS

[0271] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present specification and serve to explain the principles of the present specification, together with the description.

[0272] Figure 1 shows exemplary structures A-G of the nucleic acid double-stranded conjugate with dual target gene inhibition effect provided by the present disclosure.

[0273] Figure 2 shows exemplary structures H-N of the nucleic acid double-stranded conjugate with dual target gene inhibition effect provided by the present disclosure.

[0274] Figure 3 shows exemplary structures O-U of the nucleic acid double-stranded conjugate with dual target gene inhibition effect provided by the present disclosure. DETAILED DESCRIPTION

[0275] It is known to those skilled in the art that the nucleic acid double-stranded conjugate described in the present disclosure can be obtained by the conventional siRNA preparation method in the art (for example, solid-phase synthesis and liquid-phase synthesis), both of which have commercial customization services. It is also clear to those skilled in the art that the modified nucleotide group can be introduced into the nucleic acid double-stranded conjugate described in the present disclosure by using nucleotide monomers with corresponding modifications. The method for preparing nucleotide monomers with corresponding modifications is well known to those skilled in the art, and there are commercial monomer suppliers on the market.

[0276] Example 1: Synthesis of nucleic acid double-stranded conjugate

[0277] For the nucleic acid single strand 1 and the nucleic acid single strand 2 of the nucleic acid double-stranded conjugate sequence of the present disclosure, and the modified nucleic acid single strand 1 and the nucleic acid single strand 2 of the duplex, CPG is used as a solid-phase carrier; GalNAc-modified CPG is used as the starting cycle for the synthesis of the nucleic acid single strand 1 and the nucleic acid single strand 2.

[0278] Using YB-192S synthesizer, using phosphoramidite triester solid-phase synthesis method, starting with a solid-phase carrier, sequentially connecting nucleoside monomers in the 3'-5' direction, sequence synthesis with a scale of 0.2 umol is carried out.

[0279] The connection of phosphoramidite monomers is carried out by continuous cycles of four-step chemical reactions of deprotection, coupling, oxidation / sulfuration, and capping. The phosphoramidite monomers are prepared into a 0.05M acetonitrile solution, the activator is 0.3M BTT acetonitrile, the deprotection reagent is 3% trichloroacetic acid / dichloromethane solution, the oxidation reagent is 0.05M iodine / pyridine / tetrahydrofuran / water solution (v / v / v=2 / 1 / 7), the capping reagent A is acetic anhydride / acetonitrile solution (v / v=2 / 8), the capping reagent B is pyridine / azimethylimidazole / tetrahydrofuran (v / v / v=10 / 16 / 74), and the thio reagent is 0.05M DTTT pyridine / acetonitrile solution (v / v=4 / 6).

[0280] After the completion of solid-phase synthesis, the carrier is transferred to a 2 mL centrifuge tube, 0.8 ml of concentrated ammonia is added, and the reaction is carried out at 55°C for 16 h. After cooling to room temperature, the solution is transferred to a 2 mL centrifuge tube and concentrated to dryness. 0.2 mL of anhydrous DMSO is added for dissolution, and then 0.25 mL of triethylamine trifluoride is added, and the reaction is carried out at 65°C for 2 h. After the reaction is completed, it is cooled to room temperature, and the crude sequence is obtained by ethanol precipitation.

[0281] The crude product is purified by reverse-phase HPLC, and the fractions are collected and lyophilized. Ethanol precipitation is carried out by adding 1M sodium acetate solution 0.3 mL and ethanol 0.9 mL, and the sequence is replaced with sodium salt. Then, desalting is carried out using a 3KD ultrafiltration tube to remove excess free salt.

[0282] The nucleic acid single strand 1 and the nucleic acid single strand 2 are mixed into an aqueous solution at a certain concentration, the nucleic acid single strand 1 / nucleic acid single strand 2 are mixed at a molar ratio of (1:1.05), incubated at 95°C for 5 minutes, then naturally cooled to room temperature, freeze-dried and the target product is obtained. The compounds are as described in Table 1a.

[0283] Example 2: Immunogenicity test of nucleic acid double-stranded conjugates

[0284] The purchased hPBMC cells (Shanghai Sailai Biotechnology Co., Ltd.) were centrifuged and resuspended with RPMI-1640 cell culture medium (containing 10% FBS and 1% Penicillin-streptomycin solution), mixed and cultured overnight in a 5% CO2, 37°C incubator. The test compound was diluted to 20x working solution, and Opti-MEM were mixed at a ratio of 1.5:23.5 to prepare a mixed solution, which was used after vortexing. The prepared 20x working solution and mixed solution were mixed at a ratio of 1:1 in a 96-well V-bottom plate. The overnight culture medium was discarded and the hPBMC cells were resuspended with RPMI-1640 cell culture medium (containing 10% FBS and 1% Penicillin-streptomycin solution), counted and diluted to the required density for transfection, and then added to the 96-well plate. The final cell density in the cell plate was 2.0x10 5 cells / well, and the total volume was 200 μL. The cell plate was continued to be cultured in a 5% CO2, 37°C incubator for 24 h.

[0285] After 24 h of transfection of the hPBMC cells, the cell supernatant was taken and the content of IFN alpha, IL-6 and TNF alpha in the hPBMC supernatant was detected using the ProcartaPlex Mix&Match 3-plex Kit. The fold change of each factor of the test compound was calculated, wherein the fold change of the test siRNA, nake siRNA and ployIC = the detected factor concentration / the factor concentration in the transfection reagent well; the fold change of GS9688 = the detected factor concentration / the factor concentration in the DMSO well.

[0286] Table 2. Results of in vitro PBMC immunogenicity test of different conjugates

[0287] Conclusion: Through immunogenicity detection, the modified conjugates conjugate 1, conjugate 2, conjugate 61 and conjugate 307 do not cause immune activation.

[0288] Example 3: In vivo activity detection of nucleic acid double-stranded conjugates

[0289] 6-8 weeks old male hPCSK9 humanized mice were randomly grouped according to body weight, 3-5 mice per group. Day 1, subcutaneous administration of different doses, injection of equal volume of PBS as control. At Day 7, all mice were injected with 8% of their body weight volume of hLPA plasmid DNA solution through the tail vein within 5 seconds. At Day 8 (24 hours after LPA plasmid injection), all mice were euthanized by CO2 inhalation, and the livers were collected. The hPCSK9 and hLPA mRNA levels in the livers were detected by QPCR method to evaluate the knockdown effect of different conjugates on target genes. The test results are shown in Table 3

[0290] 6-8 weeks old male hPCSK9 or hLPA humanized mice (provided by BioSens or Jiangsu Jicui) were randomly grouped according to body weight. Before grouping and administration, the mice were fasted, blood was collected to separate serum for ELISA detection of hPCSK9 (R&D, #DPC900) and hApo(a) (abcam, #ab212165). Day 1, subcutaneous injection of PBS, positive drug mix (PC mix, i.e. Inclisiran and Olpasiran, administration dose 3mpk+mpk), conjugate 1, or PBS, conjugate 308, conjugate 277, conjugate 278, conjugate 279, conjugate 280, conjugate 281, conjugate 283, drug dose 6.0mpk (mg / Kg). After administration on day 8, day 15, day 22, day 29, blood was collected to separate serum for ELISA detection of hPCSK9 and hApo(a), respectively. The serum protein concentration of each group at each time point was compared with the protein concentration of the corresponding mice at day 0. The test results are shown in Tables 4-5.

[0291] The positive drug mix (PC mix) is a mixture of Inclisiran and Olpasiran, wherein the structures of Inclisiran and Olpasiran are as follows:

[0292] Inclisiran:

[0293] Sense strand: mC*mU*mAmGmAmCfCmUfGmUdTmUmUmGmCmUmUmUmUmGmUL96

[0294] Antisense strand: mA*fC*mAfAfAfAmGfCmAfAmAmAmCfAmGfGmUfCmUmAmG*mA*mA

[0295] Olpasiran:

[0296] Sense: NAG25*mC*mAmGmCmCmCmCmUfUfAfUmUmGmUmUmAmUmAmCmG*invdA

[0297] Antisense: mU*fC*mGfUmAfUmAmAmCmAmAfUmAfAmGfGmGfGmC*fU*mG

[0298] Table 3. Inhibition of liver hPCSK9 / hLPA mRNA by conjugates in LPA-HDI test in hPCSK9 mice

[0299] Conclusion: Conjugate 1, conjugate 2, conjugate 61, conjugate 307 exhibit better activity than conjugate 315 in the inhibition of the target of PCSK9 and LPA; at the same time, the silencing activity of the conjugate molecules is comparable to that of PC mix.

[0300] Table 4. Changes in the expression of serum hPCSK9 protein in mice before and after administration of different conjugates

[0301] Table 5. Changes in the expression of serum hApo(a) protein in mice before and after administration of different conjugates

[0302] Example 4: In vivo activity detection of nucleic acid double-stranded conjugates

[0303] In this experiment, cynomolgus monkeys (Macaca fascicularis, LDL-C greater than 1.0 mmol / L, and fluctuation less than 15% during the screening period) were used, three in each group. Serum samples were taken after overnight fasting on day -7 and day 0 (before administration). On the first day of the study, the test conjugate molecules were injected subcutaneously into the cynomolgus monkeys. Then, serum was collected after overnight fasting on days 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, etc. every week. The TC, HDL-C, LDL-C, Lp(a), TG, ApoB in the serum of cynomolgus monkeys before and after administration were detected using a blood biochemical detector, non HDL-C was calculated, and the content of PCSK9 protein in the serum sample was determined using an ELISA kit (R&D, #DPC900). The changes in each index before and after administration were calculated by comparing the blood lipids and protein content before administration (day 0), and the detection results are shown in Tables 6-21.

[0304] Table 6. Inhibition effect of conjugate 1, 6mpk on blood lipids and proteins before and after administration NA: not detected.

[0305] Table 7. Inhibition of serum LDL-C levels after dosing of conjugate 308 and positive drug Inclisiran

[0306] Table 8. Inhibition of serum Lp(a) levels after dosing of conjugate 308 and positive drug Inclisiran

[0307] Table 9. Inhibition of serum ApoB levels after dosing of conjugate 308 and positive drug Inclisiran

[0308] [Rule 91 correction 06.01.2026] Table 10. Inhibition of serum nonHDL-C levels after dosing of conjugate 308 and positive drug Inclisiran

[0309] Table 11. Inhibition of serum PCSK9 levels after dosing of conjugate 308 and positive drug Inclisiran

[0310] Table 12. Inhibition of serum LDL-C levels after dosing of conjugate and positive drugs Inclisiran, Olpasiran

[0311] Table 13. Inhibition of serum Lp(a) levels after dosing of conjugate and positive drugs Inclisiran, Olpasiran

[0312] Table 14. Inhibition of serum nonHDL-C levels after dosing of conjugate and positive drugs Inclisiran, Olpasiran

[0313] [Rule 91 correction 06.01.2026] Table 15. Inhibition of serum ApoB levels after dosing of conjugate and positive drugs Inclisiran, Olpasiran

[0314] [Rule 91 correction 06.01.2026] Table 16. Inhibition of serum PCSK9 levels after dosing of conjugate and positive drugs Inclisiran, Olpasiran

[0315] [Rule 91 correction 06.01.2026] NA: not detected.

[0316] [Corr. R91 06.01.2026] Table 17. Inhibition effect on serum LDL-C content after administration of conjugate 312 and positive drug Inclisiran, Olpasiran

[0317] [Corr. R91 06.01.2026] Table 18. Inhibition effect on serum Lp(a) content after administration of conjugate 312 and positive drug Inclisiran, Olpasiran

[0318] [Corr. R91 06.01.2026] Table 19. Inhibition effect on serum ApoB content after administration of conjugate 312 and positive drug Inclisiran, Olpasiran

[0319] [Corr. R91 06.01.2026] Table 20. Inhibition effect on serum nonHDL-C content after administration of conjugate 312 and positive drug Inclisiran, Olpasiran

[0320] [Corr. R91 06.01.2026] Table 20a. Inhibition effect on serum TC content after administration of conjugate 312 and positive drug Inclisiran, Olpasiran [0320.1][Corr. R91 06.01.2026] Table 21. Inhibition effect on serum PCSK9 content after administration of conjugate 312 and positive drug Inclisiran, Olpasiran

[0321] Conclusion: Conjugate 1, conjugate 279, conjugate 278, conjugate 281, conjugate 308, conjugate 312, etc. conjugate molecules exhibit excellent activity in cynomolgus monkeys, and the inhibition effect on double targets (PCSK9 and LPA) is maintained or better than PC mix; at the same time, the inhibition effect on LDL-C, Lp(a), ApoB, non HDL-C is better than Inclisiran.

Claims

1. A double-stranded nucleic acid conjugate with liver targeting, capable of simultaneously inhibiting PCSK9 mRNA and LPA mRNA, comprising: a. a first nucleic acid single strand and a second nucleic acid single strand which are partially or completely complementary; the first nucleic acid single strand or the second nucleic acid single strand has a length of 30-80 nt, respectively comprises a region which is at least partially complementary to the sequence of PCSK9 mRNA or LPA mRNA, and comprises at least 2 unmodified RNA nucleotides or DNA nucleotides; b. each nucleic acid single strand is conjugated with at least one carrier group comprising an ASGPR ligand; the group is conjugated on each nucleic acid single strand; each N is independently an unmodified or chemically modified nucleotide; each L is an independent carrier group, with or without; (N) represents consecutive unmodified nucleotides; the sum of X1, X2, X3, X4 and X5 is an integer of 30-80. The X1 and / or X5 on the first nucleic acid single strand and / or the second nucleic acid single strand is / are 0; Preferably, at least one of the first nucleic acid single strand and the second nucleic acid single strand has a structure as shown in formula (II): each nucleic acid single strand is represented by formula (I): wherein The first nucleic acid single strand or the second nucleic acid single strand has a structure as shown in formula (III):

2. The nucleic acid conjugate of claim 1, wherein, The sum of X1 and X2 is an integer of 14-29; the nucleic acid conjugate at the position of (N) is capable of forming 0, 1 or more than 1 Bubble structure; the region which is at least partially complementary to the sequence of PCSK9 mRNA or LPA mRNA is located at the 5' side of the position of (N). The first nucleic acid single strand or the second nucleic acid single strand comprises a nucleotide sequence as shown in SEQ ID NOs. 1001-1114. 5' N X2 -(N) X3 -N X4 -L 3' (II); and / or The nucleotide sequences of the first nucleic acid single strand and the second nucleic acid single strand are selected from the following combinations: 5' N X2 -(N) X3 -N X4 3' (III).

3. The nucleic acid conjugate of claim 1 or 2, characterized in that: (1) SEQ ID NO. 1001 and 1002; 4. The nucleic acid conjugate of any one of claims 1-3, wherein: (2) SEQ ID NO. 1003 and 1002; 5. The nucleic acid conjugate of any one of claims 1-4, characterized in that: (3) SEQ ID NO. 1004 and 1005; (4) SEQ ID NO. 1006 and 1007; (5) SEQ ID NO. 1006 and 1008; (6) SEQ ID NO. 1009 and 1008; (7) SEQ ID NO. 1010 and 1002; (8) SEQ ID NO. 1011 and 1002; (9) SEQ ID NO. 1012 and 1002; (10) SEQ ID NO. 1013 and 1002; (11) SEQ ID NO. 1014 and 1005; (12) SEQ ID NO. 1015 and 1007; (13) SEQ ID NO. 1016 and 1008; (14) SEQ ID NO. 1017 and 1008; (15) SEQ ID NO. 1018 and 1008; (16) SEQ ID NO. 1019 and 1008; (17) SEQ ID NO. 1020 and 1021; (18) SEQ ID NO. 1022 and 1021; ​ ​ ​ (19) SEQ ID NO. 1023 and 1024; (20) SEQ ID NO. 1025 and 1026; (21) SEQ ID NO. 1025 and 1027; (22) SEQ ID NO. 1028 and 1027; (23) SEQ ID NO. 1001 and 1029; (24) SEQ ID NO. 1030 and 1008; (25) SEQ ID NO. 1031 and 1032; (26) SEQ ID NO. 1033 and 1032; (27) SEQ ID NO. 1034 and 1035; (28) SEQ ID NO. 1034 and 1036; (29) SEQ ID NO. 1037 and 1036; (30) SEQ ID NO. 1031 and 1038; (31) SEQ ID NO. 1039 and 1040; (32) SEQ ID NO. 1041 and 1036; (33) SEQ ID NO. 1042 and 1043; (34) SEQ ID NO. 1044 and 1045; (35) SEQ ID NO. 1046 and 1045; (36) SEQ ID NO. 1047 and 1048; (37) SEQ ID NO. 1049 and 1048; (38) SEQ ID NO. 1050 and 1051; (39) SEQ ID NO. 1050 and 1052; (40) SEQ ID NO. 1053 and 1052; (41) SEQ ID NO. 1047 and 1054; (42) SEQ ID NO. 1055 and 1056; (43) SEQ ID NO. 1057 and 1052; (44) SEQ ID NO. 1058 and 1059; (45) SEQ ID NO. 1060 and 1059; (46) SEQ ID NO. 1061 and 1062; (47) SEQ ID NO. 1061 and 1063; (48) SEQ ID NO. 1064 and 1063; (49) SEQ ID NO. 1058 and 1065; (50) SEQ ID NO. 1066 and 1067; (51) SEQ ID NO. 1068 and 1063; (52) SEQ ID NO. 1069 and 1070; (53) SEQ ID NO. 1071 and 1048; (54) SEQ ID NO. 1072 and 1073; (55) SEQ ID NO. 1072 and 1074; (56) SEQ ID NO. 1075 and 1076; (57) SEQ ID NO. 1077 and 1078; (58) SEQ ID NO. 1079 and 1080; (59) SEQ ID NO. 1081 and 1082; (60) SEQ ID NO. 1083 and 1084; (61) SEQ ID NO. 1085 and 1086; (62) SEQ ID NO. 1087 and 1088; (63) SEQ ID NO. 1089 and 1090; (64) SEQ ID NO. 1091 and 1092; (65) SEQ ID NO. 1093 and 1094; (66) SEQ ID NO. 1095 and 1096; (67) SEQ ID NO. 1097 and 1098; (68) SEQ ID NO. 1099 and 1100; (69) SEQ ID NO. 1101 and 1102; (70) SEQ ID NO. 1103 and 1104; (71) SEQ ID NO. 1105 and 1106; (72) SEQ ID NO. 1107 and 1108; (73) SEQ ID NO. 1020 and 1109; (74) SEQ ID NO. 1071 and 1113; (75) SEQ ID NO. 1071 and 1114.

6. The nucleic acid conjugate of any one of claims 1-5, wherein: the first nucleic acid single strand or the second nucleic acid single strand comprises a modified nucleotide selected from the group consisting of 2'-methoxy nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-seco nucleotide analogs, 2'-fluoro-arabinonucleotides, 2'-methoxyethyl nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 3'-methoxy nucleotides, 2'-allyl modified nucleotides, nucleotides comprising phosphorothioate groups, nucleotides comprising methylphosphonate groups, nucleotides comprising 5'-phosphates, nucleotides comprising 5'-phosphate mimics, glycol modified nucleotides, abasic nucleotides, morpholino nucleotides, locked nucleotides, unlocked nucleotides, threose nucleotides, or glycerol nucleotides.

7. The nucleic acid conjugate of any one of claims 1-6, wherein: the first nucleic acid single strand comprises the modified nucleotide sequence of nucleic acid single strand 1 of modified nucleic acid duplexes 1 to 272 in Table 0b; and / or the second nucleic acid single strand comprises the modified nucleotide sequence of nucleic acid single strand 2 of modified nucleic acid duplexes 1 to 272 in Table 0b; and / or the first nucleic acid single strand or the second nucleic acid single strand further comprises at its 5' end a 5'-(E)-vinylphosphate modified nucleotide; preferably the first nucleic acid single strand comprises the modified nucleotide sequence of nucleic acid single strand 1 of modified nucleic acid duplexes 1, 233, 234, 235, 236, 263, 264, 265, 266, 267, 267, 271, 272 in Table 0b; preferably the second nucleic acid single strand comprises the modified nucleotide sequence of nucleic acid single strand 2 of modified nucleic acid duplexes 1, 233, 234, 235, 236, 263, 264, 265, 266, 267, 267, 271, 272 in Table 0b.

8. The nucleic acid conjugate of any one of claims 1-7, wherein: the modified nucleotide sequence combination of the first nucleic acid single strand or the second nucleic acid single strand is selected from the modified nucleotide sequence combinations of modified nucleic acid duplexes 1 to 272 in Table 0b; Preferably, the nucleotide sequence combination of the first nucleic acid single strand or the second nucleic acid single strand is selected from: (1) the modified nucleotide sequence of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 1 in Table 0b; (2) the modified nucleotide sequence of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 233 in Table 0b; (3) the modified nucleotide sequence of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 234 in Table 0b; (4) the modified nucleotide sequence of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 235 in Table 0b; (5) the modified nucleotide sequence of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 236 in Table 0b; (6) the modified nucleotide sequence of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 263 in Table 0b; (7) the modified nucleotide sequence of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 264 in Table 0b; (8) the modified nucleotide sequence of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 265 in Table 0b; (9) the modified nucleotide sequence of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 266 in Table 0b; (10) the modified nucleotide sequence of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 267 in Table 0b; (11) the modified nucleotide sequence of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 270 in Table 0b; (12) the modified nucleotide sequence of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 271 in Table 0b; (13) the modified nucleotide sequence of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 272 in Table 0b.

9. The nucleic acid conjugate of any one of claims 1-8, wherein: the nucleic acid conjugate has liver targeting; Preferably, the carrier group comprising an ASGPR ligand is GalNAc(L96) or Ser(GN), or GalNAc(Ser1), GalNAc(Ser2), GalNAc(Ser3), GalNAc(Ser4), or LP-GalNAc, XY-GalNAc, GalNAc(A1GN), GalNAc(A1dGN), GalNAc(A3GN), GalNAc(A3dGN), GalNAc(A5GN), GAlNAc(NAG25), GAlNAc(NAG37), wherein GalNAc(L96) has the following structure: Ser(GN) has the following structure: GalNAc(Ser1), GalNAc(Ser2), GalNAc(Ser3), GalNAc(Ser4) have the following structures, respectively: LP-GalNAc has the following structure: XY-GalNAc has the following structure: GalNAc(A1GN), GalNAc(A1dGN), GalNAc(A3GN), GalNAc(A3dGN), GalNAc(A5GN), GAlNAc(NAG25), GAlNAc(NAG37) have the following structures, respectively:

10. The nucleic acid conjugate of any one of claims 1-9, wherein: The nucleic acid conjugate has liver targeting; the carrier group comprising ASGPR ligand has the following structure:

11. The nucleic acid conjugate of any one of claims 1-10, wherein: the nucleic acid conjugate is selected from conjugate 1 to conjugate 314 and conjugate 316 to conjugate 318 in Table 1a; preferably, the nucleic acid conjugate is conjugate 1, conjugate 278, conjugate 279, conjugate 280, conjugate 281, conjugate 308, conjugate 309, conjugate 310, conjugate 311, conjugate 312, conjugate 316, conjugate 317 or conjugate 318.

12. A pharmaceutical composition comprising the nucleic acid conjugate according to any one of claims 1-11, alone or in combination with a pharmaceutically acceptable carrier or excipient.

13. The pharmaceutical composition of claim 12, wherein, The pharmaceutical composition is an injection formulation or a subcutaneous administration formulation; preferably, the pharmaceutical composition is a subcutaneous injection formulation.

14. The pharmaceutical composition of claim 13, wherein, The pharmaceutical composition further comprises an excipient, which includes a buffered solution or a non-buffered solution; Preferably, the buffered solution includes acetate, citrate, zeath, carbonate or phosphate or phosphate buffered saline or any combination thereof, or the non-buffered solution is saline or water.

15. Use of the nucleic acid conjugate according to any one of claims 1-11 or the pharmaceutical composition according to any one of claims 12-14 in the manufacture of a medicament for treating a disease mediated by dysregulation of PCSK9 and / or LPA expression in a subject in need thereof; preferably, the disease mediated by dysregulation of PCSK9 and / or LPA expression comprises hyperlipidemia; the hyperlipidemia comprises hypercholesterolemia. Preferably, the subject in need is a human.

16. A method of treating a disease mediated by dysregulation of PCSK9 and / or LPA expression, wherein, The method comprises administering to a subject in need the nucleic acid conjugate according to any one of claims 1-11 or the pharmaceutical composition according to any one of claims 12-14; preferably, the disease mediated by dysregulation of PCSK9 and / or LPA expression comprises hyperlipidemia; the hyperlipidemia comprises hypercholesterolemia.

17. A nucleic acid conjugate or a pharmaceutical composition for use in the treatment of a disease mediated by dysregulation of PCSK9 and / or LPA expression; wherein, The nucleic acid conjugate is defined in any one of claims 1-11 and the pharmaceutical composition is defined in any one of claims 12-14; preferably, the disease mediated by dysregulation of PCSK9 and / or LPA expression comprises hyperlipidemia; the hyperlipidemia comprises hypercholesterolemia.

18. Use of a nucleic acid conjugate or a pharmaceutical composition for treating a disease mediated by dysregulation of PCSK9 and / or LPA expression in a subject in need thereof; wherein, The nucleic acid conjugate is defined in any one of claims 1-11 and the pharmaceutical composition is defined in any one of claims 12-14; preferably, the disease mediated by dysregulation of PCSK9 and / or LPA expression comprises hyperlipidemia; the hyperlipidemia comprises hypercholesterolemia.

19. The method of claim 16, wherein the double stranded conjugate or pharmaceutical composition is administered by injection or subcutaneous administration; preferably, the double stranded conjugate or pharmaceutical composition is administered by subcutaneous injection.

20. The method of claim 16, wherein the double stranded conjugate or pharmaceutical composition is administered with an excipient, which includes a buffered solution or a non-buffered solution; Preferably, the non-buffered solution is saline or water, or the buffered solution includes acetate, citrate, zeath, carbonate or phosphate or phosphate buffered saline or any combination thereof.

21. The method of claim 16, wherein the subject in need is a human.

22. The method of claim 16, wherein the double stranded conjugate or pharmaceutical composition is administered at a dose of 1-100 mg / Kg; preferably, the double stranded conjugate is administered at a dose of 1-20 mg / Kg; more preferably, the double stranded conjugate is administered at a dose of 1-6 mg / Kg or 6-20 mg / Kg.

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