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

Abstract

The present invention provides a nucleic acid conjugate having a double-target gene inhibition effect and a use thereof. The nucleic acid conjugate has a first nucleic acid single strand and second nucleic acid single strand that are partially or completely complementary to each other, and can be cleaved to form two independent siRNA conjugates that respectively inhibit two target genes. The nucleic acid conjugate provided by the present invention exhibits good stability, excellent dual-target gene inhibition activity, and satisfactory cytotoxicity and immunogenicity in the process of inhibiting PCSK9 and LPA target genes. The dual-target modification mode of the present invention can also be applied to other targets and to various forms of tissue targeting.

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 3, 2025, Chinese Patent Application No. 2025104541264, filed on April 11, 2025, Chinese Patent Application No. 2024116584266, filed on November 20, 2024, Chinese Patent Application No. 2025102443110, filed on March 3, 2025, Chinese Patent Application No. 2025105499724, filed on April 29, 2025, Chinese Patent Application No. 2025100102287, filed on January 3, 2025, Chinese Patent Application No. 202510417184X, filed on April 3, 2025, Chinese Patent Application No. 2024114502736, filed on October 17, 2024, and Chinese Patent Application No. 2024117971069, filed on December 9, 2024, 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 for simultaneously inhibiting the expression of two target genes, which comprises two partially or completely complementary nucleic acid single strands, and the nucleic acid double strand comprises a site that can be specifically recognized and cleaved by an endonuclease in vivo, can form two independent siRNA conjugates, and can inhibit two target genes respectively. BACKGROUND

[0004] Cardiovascular disease (CVD) is the most important chronic non-communicable disease that threatens human life and health worldwide. It is estimated that the global CVD patients will rise to 1.14 billion by 2050, 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 in 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 LDL-C is recommended as the primary target for lipid-lowering therapy in most countries or regions. Other apolipoprotein B (ApoB) containing lipoproteins, including triglyceride-rich lipoproteins (TRL) and their remnants, and 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 have many shortcomings in clinical application: some patients have adverse reactions, LDL-C reduction is not up to standard, and Lp(a) levels are elevated. PCSK9 inhibitors reduce LDL-C by about 50%-70% (Chinese Lipid Management Guidelines, 2023), among which monoclonal antibodies and siRNA have been approved for marketing, especially PCSK9 monoclonal antibodies are recommended as recommended drugs for controlling LDL-C by multiple 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] Since 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) alone. The existing traditional lipid-lowering therapies cannot achieve a satisfactory reduction in Lp(a) levels, and targeting 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 no drug that can simultaneously reduce LDL-C and Lp(a).

[0007] The present application provides a small interfering RNA (siRNA) preparation targeting PCSK9 and LPA simultaneously, 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 related protein pathway from the source for the treatment / prevention of diseases caused by the related protein pathway.

[0008] On this basis, since the double-target siRNA silences two pathogenic genes simultaneously through a single molecule, its core advantage lies in the multi-level synergistic effect. This double-target modification mode can also be applied to other targets and tissues (such as liver, CNS, fat, muscle, kidney, lung, etc.). In terms of treatment mechanism, double-target design can block upstream and downstream genes in the same disease pathway, or simultaneously regulate complementary pathological pathways, thereby inhibiting the compensatory escape phenomenon from the source, i.e. the attenuation of therapeutic effect caused by the compensatory activation of other pathways after a single target is inhibited. This multi-pathway synergistic effect not only significantly improves the treatment intensity, but also expands the coverage of indications, such as simultaneously regulating blood lipid and blood pressure related genes in metabolic diseases. SUMMARY

[0009] The present disclosure provides a nucleic acid double-stranded conjugate structure with double-target gene inhibition, 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.

[0010] The present disclosure first provides a double-stranded nucleic acid conjugate with double-target gene inhibition, comprising:

[0011] 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 which 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;

[0012] 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.

[0013] each nucleic acid single strand is represented by formula (I):

[0014] wherein each N is independently an unmodified or chemically modified nucleotide; each L is an independent carrier group, present or absent; (N) represents consecutive unmodified nucleotides; the sum of X1, X2, X3, X4, and X5 is an integer of 30-80.

[0015] 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):

[0016] 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).

[0017] 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):

[0018] 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).

[0019] 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).

[0020] In some embodiments of the disclosure, the nucleic acid conjugate can be cleaved into two independent siRNA conjugates that inhibit the 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.

[0021] In some embodiments of the disclosure, the nucleic acid conjugate can be cleaved into two independent siRNA conjugates that inhibit the 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.

[0022] In some embodiments of the disclosure, the nucleic acid conjugate can be cleaved into two independent siRNA conjugates that inhibit the 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.

[0023] 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.

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

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

[0026] In some embodiments of the disclosure, the two independent siRNA conjugates inhibit different genes in CNS tissue.

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

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

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

[0030] In some embodiments of the disclosure, the two independent siRNA conjugates inhibit different genes or the same gene in kidney tissue.

[0031] In some embodiments of the disclosure, the target gene combination includes, but is not limited to, the following combinations:

[0032] (1) PCSK9 and LPA;

[0033] (2) C3 and C5;

[0034] (3) PCSK9 and ANGPTL3;

[0035] (4) ANGPTL4 and ANGPTL3;

[0036] (5) CTNNB1 and SOD1;

[0037] (6) ALK7 and SOD1;

[0038] (7) PCSK9 and AGT;

[0039] (8) PCSK9 and APOC3;

[0040] (9) AGT and CFB;

[0041] (10) PLIN2 and PNPLA3;

[0042] (11) PNPLA3 and HSD17B13;

[0043] (12) PNPLA3 and DGAT2;

[0044] (13) PNPLA3 and MARC1;

[0045] (14) Tau and APP;

[0046] (15) SCN9A and SCN10A;

[0047] (16) INHBE and DPP4.

[0048] 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.

[0049] 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).

[0050] In some embodiments of the present disclosure, the region that is at least partially complementary to the mRNA sequence of the target gene is located at the position of (N) or 5' side of the Bubble structure.

[0051] Further, the position of (N) or the Bubble structure comprises at least 2 unmodified RNA nucleotides or DNA nucleotides.

[0052] 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).

[0053] 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).

[0054] In some embodiments of the present disclosure, X3 is selected from an integer from 1-5 or 6-7.

[0055] In some embodiments of the present disclosure, X3 is selected from an integer from 1-4.

[0056] In some preferred embodiments of the present disclosure, X2 is selected from 2 or 3.

[0057] 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.

[0058] Further, the two independent siRNA conjugates after cleavage are capable of independently inhibiting the mRNA of the two target genes through the RNAi-induced silencing complex process, respectively.

[0059] In some embodiments of the disclosure, the two independent siRNA conjugates each comprise a sequence complementary to the mRNA of two target genes.

[0060] 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.

[0061] In some preferred embodiments of the disclosure, dN is dA or dT.

[0062] In some preferred embodiments of the disclosure, rN is rU or rC.

[0063] In some more preferred embodiments of the disclosure, the nucleotide sequence at the (N) position is selected from dArU, dTrCdT or dAdTrUdT.

[0064] 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.

[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 dTrCdT.

[0066] 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.

[0067] In some preferred embodiments of the disclosure, the carrier group comprising an ASGPR ligand includes, but is not limited to, the following structures:

[0068] In some embodiments of the disclosure, the fatty chain in the carrier group comprising a long fatty chain includes 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.

[0069] 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 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-, 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.

[0070] In some embodiments of the 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.

[0071] In some embodiments of the disclosure, the nucleic acid conjugate has a liver targeting, an extrahepatic targeting.

[0072] In some embodiments of the disclosure, the extrahepatic targeting is selected from targeting CNS tissue, fat, muscle, lung, kidney, eye.

[0073] 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.

[0074] 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:

[0075] 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;

[0076] 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.

[0077] 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.

[0078] Further, the position of (N) or the Bubble structure comprises at least 2 unmodified RNA nucleotides or DNA nucleotides.

[0079] In some embodiments of the present disclosure, the Bubble structure comprises a nucleotide sequence at the position of (N) as described above.

[0080] 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.

[0081] 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.

[0082] In some embodiments of the present disclosure, the two independent siRNA conjugates each comprise a sequence complementary to a different transcript of the same target mRNA.

[0083] In some embodiments of the present disclosure, the two independent siRNA conjugates each comprise a sequence complementary to a different target region of the same target mRNA.

[0084] In some preferred embodiments of the present disclosure, the carrier group comprising 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),

[0085] GalNAc(L96) has the following structure:

[0086] Ser(GN) can have the following structure: GalNAc(Ser1), GalNAc(Ser2), GalNAc(Ser3), GalNAc(Ser4) respectively have the following structures:

[0087] LP-GalNAc has the following structure:

[0088] XY-GalNAc has the following structure:

[0089] or

[0090] GalNAc(A1GN), GalNAc(A1dGN), GalNAc(A3GN), GalNAc(A3dGN), GalNAc(A5GN), GAlNAc(NAG25), GAlNAc(NAG37) respectively have the following structures:

[0091] 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.

[0092] 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-, -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.

[0093] 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.

[0094] 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.

[0095] In some embodiments of the present disclosure, the long fatty chain, long fatty chain acid, polypeptide and other targeting small molecules in the carrier group containing long fatty chain, long fatty chain acid, polypeptide and other targeting small molecules have the following structure:

[0096] wherein R6 is hydrogen or C1-C6 alkyl, and R7 is H or an amino protecting group.

[0097] In some preferred embodiments of the present disclosure, the nucleic acid conjugate has liver targeting; the carrier group comprising an ASGPR ligand has the following structure:

[0098] The present disclosure also provides a double-stranded nucleic acid conjugate with liver targeting, capable of simultaneously inhibiting PCSK9 mRNA and LPA mRNA, comprising:

[0099] 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;

[0100] 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;

[0101] wherein each nucleic acid single strand is represented by formula (I):

[0102] 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.

[0103] In some embodiments of the present disclosure, in the nucleic acid conjugate, X1and / or X5on the first nucleic acid single strand and / or the second nucleic acid single strand is 0.

[0104] 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):

[0105] 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).

[0106] 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).

[0107] 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).

[0108] Further, the position of 15-30 nt from the 5'-end of the nucleic acid single strand 1 or the nucleic acid single strand 2 of the nucleic acid conjugate 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 the PCSK9 mRNA or the 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.

[0109] 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:

[0110] Table 0a. Unmodified nucleotide sequences

[0111] 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.

[0112] 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:

[0113] (1) SEQ ID NO. 1001 and 1002;

[0114] (2) SEQ ID NO. 1020 and 1021;

[0115] (3) SEQ ID NO. 1069 and 1070;

[0116] (4) SEQ ID NO. 1071 and 1048;

[0117] (5) SEQ ID NO. 1071 and 1113;

[0118] (6) SEQ ID NO. 1071 and 1114.

[0119] In some embodiments of the present disclosure, the first nucleic acid single strand or the second nucleic acid single strand comprises modified nucleotides.

[0120] In some embodiments of the present 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'-phosphate, nucleotides comprising 5'-phosphate mimics, glycol modified nucleotides, abasic nucleotides, morpholino nucleotides, locked nucleotides, unlocked nucleotides, threose nucleotides, or glycerol nucleotides.

[0121] In some embodiments of the present disclosure, the first nucleic acid single strand or the second nucleic acid single strand can be chemically modified in the following ways:

[0122] wherein B1, B2, B3, B4, B5, B6 are each independently 2'-OMe, 2'-F, LNA, 2'-OMOE modified nucleotides;

[0123] T1, T2, T3, T4 are each independently 2'-F, 2'-OMe, DNA modified nucleotides;

[0124] Z1, Z2, Z3 are each independently 2'-F, 2'-OMe, 2'-DNA, RNA (2'-OH modified) nucleotides;

[0125] q 1 ~q 13 are each independently an integer from 0 to 10;

[0126] In some embodiments of the disclosure, the first nucleic acid single strand comprises the nucleotide sequence of nucleic acid single strand 1 of modified nucleic acid duplex 1, 233, 234, 235, 236, 263, 264, 265, 266, 267, 267, 271, 272 in Table 0b.

[0127] In some embodiments of the disclosure, the second nucleic acid single strand comprises the nucleotide sequence of 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.

[0128] 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.

[0129] 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:

[0130] (1) the nucleotide sequences of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 1 in Table 0b;

[0131] (2) the nucleotide sequences of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 233 in Table 0b;

[0132] (3) the nucleotide sequences of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 234 in Table 0b;

[0133] (4) the nucleotide sequences of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 235 in Table 0b;

[0134] (5) the nucleotide sequences of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 236 in Table 0b;

[0135] (6) the nucleotide sequences of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 263 in Table 0b;

[0136] (7) the nucleotide sequences of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 264 in Table 0b;

[0137] (8) the nucleotide sequences of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 265 in Table 0b;

[0138] (9) the nucleotide sequences of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 266 in Table 0b;

[0139] (10) the nucleotide sequences of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 267 in Table 0b;

[0140] (11) the nucleotide sequences of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 270 in Table Ob;

[0141] (12) the nucleotide sequences of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 271 in Table Ob;

[0142] (13) the nucleotide sequences of nucleic acid single strand 1 and nucleic acid single strand 2 of modified nucleic acid duplex 272 in Table Ob.

[0143] In some embodiments of the disclosure, the modified nucleic acid duplexes with PCSK9 and LPA dual-target gene silencing are shown in Table 0:

[0144] Table Ob. Modified nucleic acid duplexes with PCSK9 and LPA target mRNA inhibition:

[0145] In some embodiments of the disclosure, the nucleic acid conjugates with PCSK9 and LPA dual-target gene inhibition are selected from all the conjugates in Table la, such as Conjugate 1 to Conjugate 318.

[0146] In some embodiments of the disclosure, the nucleic acid conjugates with ANGPTL4 and ANGPTL3 dual-target gene inhibition are shown in Conjugate 402 to Conjugate 410 in Table 22.

[0147] In some embodiments of the disclosure, the nucleic acid conjugates with CNS tissue dual-target gene inhibition are shown in Conjugate 504 in Table 29.

[0148] In some embodiments of the disclosure, the nucleic acid conjugates with adipose tissue dual-target gene inhibition are shown in Conjugate 603 in Table 33.

[0149] 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.

[0150] In some embodiments of the present disclosure, the pharmaceutical composition further comprises a delivery formulation.

[0151] In some preferred embodiments of the present disclosure, the delivery formulation comprises a lipid nanoparticle (LNP), a lipopolyplex (LPP), a polymer nanoparticle (PNP), an inorganic nanoparticle (INP), a cationic nanoemulsion (CNE), an exosome, a biological microvesicle.

[0152] In another aspect, the present disclosure also provides use of the aforementioned nucleic acid conjugate or pharmaceutical composition in the preparation of a medicament for modulating gene expression.

[0153] In another aspect, the present disclosure also provides a method for modulating gene expression, wherein the method comprises administering the aforementioned nucleic acid conjugate or pharmaceutical composition to a subject in need thereof.

[0154] In another aspect, the present disclosure also provides the aforementioned nucleic acid conjugate or pharmaceutical composition for use in modulating gene expression.

[0155] In another aspect, the present disclosure also provides use of the aforementioned nucleic acid conjugate or pharmaceutical composition for modulating gene expression in a subject in need thereof.

[0156] In order to achieve the targeting of the nucleic acid double-stranded conjugate, the middle or terminal of each nucleic acid single strand is conjugated with a 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 small molecule targeting agent, so as to achieve liver targeting, CNS targeting, lung targeting, kidney targeting, fat muscle targeting, eye targeting, etc. of the nucleic acid double-stranded conjugate.

[0157] The nucleic acid double-stranded conjugate of the present disclosure introduces at least two specific base nucleotides (unmodified DNA or RNA) in each nucleic acid single strand, forming a site that can be specifically recognized and cleaved 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 that can be specifically recognized and cleaved by an endonuclease.

[0158] Based on the above features, the double-stranded nucleic acid conjugate capable of simultaneously inhibiting two target genes of the present disclosure has the structure shown in Figures 1-3 (A-U), wherein the raised part is the Bubble design of the nucleic acid double-stranded conjugate, and L represents a carrier fragment of a compound with targeting ability, which can be conjugated to the end or middle of each nucleic acid single strand.

[0159] Meanwhile, the present disclosure uses 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 inhibition activity, satisfactory cytotoxicity and immunostimulatory activity, and therapeutic effect in 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.

[0160] 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 the structure shown in Figures 1-3 (A-U), wherein L represents a GalNAc carrier fragment with liver targeting ability, which can be conjugated to the end or middle of the two nucleic acid single strands; and the raised part in the schematic diagram is the Bubble part sensitive to endonuclease.

[0161] Based on the good inhibition activity of the above double-target conjugate on double-target genes (PCSK9 and LPA) in mice and cynomolgus monkeys, we applied this double-target modification mode to other target and tissue (such as CNS) targeting.

[0162] According to the above structure schematic, the designed nucleic acid conjugate with partial double-target gene inhibition effect is shown in Table 1a.

[0163] The modification scheme used in Table 1a is as follows:

[0164] 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 lower-case letter m represents that the nucleotide adjacent to the right of the letter m is a 2'-methoxy-modified nucleotide; the lower-case letter f represents that the nucleotide adjacent to the right is a 2'-fluoro-modified nucleotide; the lower-case letter d represents that the nucleotide adjacent to the right is a DNA; the lower-case letter r represents that the nucleotide adjacent to the right is a 2'-hydroxyl nucleotide; the “*” represents that the two nucleotides adjacent to the left and right are connected by a phosphorothioate group; eVP represents a 5'-(E)-vinylphosphate-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;

[0165] Table 1a. Nucleic acid double-stranded conjugate structures with PCSK9 and LPA target mRNA inhibition effect:

[0166] Structure characterization method and results of the conjugates, see Table 1b in detail:

[0167] LC-MS representative test method: When the test article is subjected to denaturing IP RP-LC, the complementary paired double strands are unzipped into single strands (sense and antisense strands), and then the parent ions of the sense / antisense strands are subjected to gas-phase fragmentation by tandem mass spectrometry. The software CONFIRM Sequence is used to analyze all detected fragment ions and to resolve the sequence of the test article. The sequence of the test article is consistent with the theoretical sequence, i.e., 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.

[0168] Table 1b. Molecular weight (MW) of nucleic acid duplex conjugates

[0169] Definitions of terms used in connection with the present disclosure: Unless otherwise indicated, the initial definition of a group or term provided herein applies to that group or term throughout the specification; for terms not specifically defined herein, the meaning attributable to them should be given in light of the disclosure and context, as would be given to those skilled in the art.

[0170] 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.

[0171] The 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 term nucleic acid drug as used in the present disclosure functions through an RNA interference mechanism, the nucleic acid drug is not limited or restricted to any particular mechanism or pathway of action. The types of nucleic acid drug molecules include, but are not limited to, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer substrates. The nucleic acid drugs described in the present disclosure are composed of an oligonucleotide strand that is at least partially complementary to the mRNA that is the target. In some embodiments, the nucleic acid drug or conjugate described in the present disclosure is double-stranded and is composed of an antisense strand and a sense strand that is at least partially complementary to the antisense strand.

[0172] 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 administered without such treatment.

[0173] The term "sequence" or "nucleotide sequence" refers to the order or succession of nucleobases or nucleotides, denoted in alphabetical order using standard nucleotide nomenclature.

[0174] 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 mammalian physiological conditions or analogous 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 comprise Watson-Crick base pairing or non-Watson-Crick base pairing and comprise 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.

[0175] In the present disclosure, "fully complementary" means that in a pair of hybridized nucleobase or nucleotide sequence molecules, 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. The contiguous sequence can comprise all or a portion of the first or second nucleotide sequence.

[0176] In the present disclosure, "partially complementary" means that in a pair of hybridized nucleobase or nucleotide sequence molecules, at least 70% (but not all) 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. The contiguous sequence can comprise all or a portion of the first or second nucleotide sequence.

[0177] 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.

[0178] In the present disclosure, "unmodified RNA" is ribonucleotides (2'-hydroxyl nucleotides); "unmodified DNA" is deoxyribonucleotides (2'-deoxy nucleotides).

[0179] In the present disclosure, a "covalent bond linker" is a section of molecular structure or functional group that links 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 is independently hydrogen or unsubstituted C1-C6alkyl.

[0180] In the present disclosure, “Bubble structure” refers to that, during the annealing pairing process, 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).

[0181] In the present disclosure, “Regulating gene expression” refers to the process that small interfering RNA (siRNA) specifically silences the expression of target genes at the post-transcriptional level through the mechanism of RNA interference (RNAi). BRIEF DESCRIPTION OF DRAWINGS

[0182] 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, in which:

[0183] 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.

[0184] 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.

[0185] 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

[0186] 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, such as 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.

[0187] Example 1: Synthesis of nucleic acid double-stranded conjugate

[0188] 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.

[0189] 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, and synthesizing a sequence with a synthesis scale of 0.2 umol.

[0190] 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).

[0191] After the solid phase synthesis is completed, the support is transferred to a 2 mL centrifuge tube, 0.8 mL of concentrated ammonia is added, and the reaction is sealed and incubated 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 0.25 mL of triethylamine trifluoride is added, and the reaction is incubated 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.

[0192] The crude product is purified by reverse-phase HPLC, the fractions are collected and lyophilized. Ethanol precipitation is performed by adding 0.3 mL of a 1 M sodium acetate solution and 0.9 mL of ethanol, and the sequence is converted to a sodium salt. Then, desalting is performed using a 3 KD ultrafiltration tube to remove excess free salt.

[0193] Nucleic acid single strand 1 and nucleic acid single strand 2 are mixed in a certain concentration aqueous solution, and 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 min, then naturally cooled to room temperature, lyophilized and obtained as the target product. The compounds are as described in Table 1a.

[0194] Example 2: Immunogenicity test of nucleic acid double-stranded conjugate

[0195] The purchased hPBMC cells (Shanghai Sailai Biotechnology Co., Ltd.) are 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 is diluted to 20x working solution, and Opti-MEM is mixed at a ratio of 1.5:23.5 to prepare a mixed solution, which is used after vortexing. The prepared 20x working solution and mixed solution are mixed at a ratio of 1:1 in a 96-well V-bottom plate. The overnight culture medium is discarded, and the hPBMC cells are resuspended with RPMI-1640 cell culture medium (containing 10% FBS and 1% Penicillin-streptomycin solution), counted, diluted to the required density for transfection, and added to the 96-well plate. The final cell density in the plate is 2.0x10 5 cells / well, and the total volume is 200 μL. The cell plate is continued to be cultured in a 5% CO2, 37°C incubator for 24 h.

[0196] After 24h transfection, the supernatant of hPBMC cells was collected, and the content of IFN alpha, IL-6 and TNF alpha in the supernatant of hPBMC was detected using ProcartaPlex Mix&Match 3-plex Kit. The fold change of each factor of the test compound was calculated, wherein the fold change of siRNA, nake siRNA and ployIC to be tested = the detected factor concentration / the factor concentration of the transfection reagent well; the fold change of GS9688 = the detected factor concentration / the factor concentration of the DMSO well.

[0197] Table 2. Results of in vitro PBMC immunogenicity test of different conjugates

[0198] Conclusion: Through immunogenicity test, the modified conjugates of conjugate 1, conjugate 2, conjugate 61 and conjugate 307 do not cause immune activation.

[0199] Example 3: In vivo activity detection of nucleic acid double-stranded conjugate

[0200] 6-8 week old male hPCSK9 humanized mice were randomly divided according to body weight, 3-5 mice per group. On Day 1, subcutaneous administration was performed, and the administration dose was 6mpk. An equal volume of PBS was injected as a control. On Day 7, all mice were injected with a solution of hLPA plasmid DNA with a volume of 8% of their body weight through the tail vein within 5 seconds. On Day 8 (24h after LPA plasmid injection), all mice were euthanized by CO2 inhalation, and the liver was collected. The hPCSK9 and hLPA mRNA levels in the liver were detected by QPCR method to evaluate the knockdown effect of different conjugates on target genes. The test results are shown in Table 3.

[0201] 6-8 week old male hPCSK9 or hLPA humanized mice (provided by BioSens or Jiangsu Jiequ) were randomized according to body weight. Before grouping, mice were fasted, blood was collected to isolate serum for ELISA detection of hPCSK9 (R&D, #DPC900) and hApo(a) (abcam, #ab212165). On Day 1, mice were subcutaneously injected with PBS, positive drug mix (PC mix, i.e. Inclisiran and Olpasiran, at a dose of 3 mpk+mpk), Conjugate 1, or PBS, Conjugate 308, Conjugate 277, Conjugate 278, Conjugate 279, Conjugate 280, Conjugate 281, Conjugate 283, at a dose of 6.0 mpk, respectively. On Day 8, Day 15, Day 22, Day 29, blood was collected to isolate serum for ELISA detection of hPCSK9 and hApo(a), respectively. The serum protein concentration of each time point was compared with the corresponding mouse protein concentration on Day 0. The test results are shown in Tables 4-5.

[0202] The positive drug mix (PC mix) is a mixture of Inclisiran and Olpasiran, wherein the structures of Inclisiran and Olpasiran are as follows:

[0203] Inclisiran:

[0204] Sense strand: mC*mU*mAmGmAmCfCmUfGmUdTmUmUmGmCmUmUmUmUmGmUL96

[0205] Antisense strand: mA*fC*mAfAfAfAmGfCmAfAmAmAmCfAmGfGmUfCmUmAmG*mA*mA

[0206] Olpasiran:

[0207] Sense strand: NAG25*mC*mAmGmCmCmCmCmUfUfAfUmUmGmUmUmAmUmAmCmG*invdA

[0208] Antisense strand: mU*fC*mGfUmAfUmAmAmCmAmAfUmAfAmGfGmGfGmC*fU*mG

[0209] Table 3. Inhibition of liver hPCSK9 / hLPA mRNA by Conjugates in LPA-HDI test in hPCSK9 mice

[0210] Conclusion: Conjugate 1, Conjugate 2, Conjugate 61, Conjugate 307 exhibited better activity in the target inhibition of PCSK9 and LPA than Conjugate 315; at the same time, the silencing activity of the conjugate molecules was comparable to that of PC mix.

[0211] Table 4. Changes in the expression of hPCSK9 protein in the serum of mice before and after administration of different conjugates

[0212] Table 5. Changes in the expression of hApo(a) protein in the serum of mice before and after administration of different conjugates

[0213] Example 4: In vivo activity detection of nucleic acid double-stranded conjugate

[0214] 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 cynomolgus monkeys were subcutaneously injected with the test conjugate molecules. 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 PCSK9 protein content in the serum samples 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.

[0215] Table 6. Inhibition effect of conjugate 1, 6mpk on blood lipids and proteins before and after administration of conjugate

[0216] NA: indicates not detected.

[0217] Table 7. Inhibition effect of conjugate 308 and positive drug Inclisiran on serum LDL-C content after administration

[0218] Table 8. Inhibition effect of conjugate 308 and positive drug Inclisiran on serum Lp(a) content after administration

[0219] Table 9. Inhibition effect of conjugate 308 and positive drug Inclisiran on serum ApoB content after administration

[0220] Table 10. Inhibition of serum nonHDL-C levels after dosing of conjugate 308 and positive drug Inclisiran

[0221] Table 11. Inhibition of serum PCSK9 levels after dosing of conjugate 308 and positive drug Inclisiran

[0222] Table 12. Inhibition of serum LDL-C levels after dosing of conjugate and positive drugs Inclisiran, Olpasiran

[0223] Table 13. Inhibition of serum Lp(a) levels after dosing of conjugate and positive drugs Inclisiran, Olpasiran

[0224] Table 14. Inhibition of serum nonHDL-C levels after dosing of conjugate and positive drugs Inclisiran, Olpasiran

[0225] Table 15. Inhibition of serum ApoB levels after dosing of conjugate and positive drugs Inclisiran, Olpasiran

[0226] Table 16. Inhibition of serum PCSK9 levels after dosing of conjugate and positive drugs Inclisiran, Olpasiran

[0227] NA: Not Assayable.

[0228] Table 17. Inhibition of serum LDL-C levels after dosing of conjugate 312 and positive drugs Inclisiran, Olpasiran

[0229] Table 18. Inhibition of serum Lp(a) levels after dosing of conjugate 312 and positive drugs Inclisiran, Olpasiran

[0230] Table 19. Inhibition of serum ApoB levels after dosing of conjugate 312 and positive drugs Inclisiran, Olpasiran

[0231] Table 20. Inhibition effect of serum nonHDL-C content after administration of conjugate 312 and positive drugs Inclisiran, Olpasiran

[0232] Table 20a. Inhibition effect of serum TC content after administration of conjugate 312 and positive drugs Inclisiran, Olpasiran

[0233] Table 21. Inhibition effect of serum PCSK9 content after administration of conjugate 312 and positive drugs Inclisiran, Olpasiran

[0234] Conclusion: Conjugate 1, conjugate 279, conjugate 278, conjugate 281, conjugate 308, conjugate 312 exhibit excellent activity in cynomolgus monkeys, and the inhibition of double targets (PCSK9 and LPA) is maintained or better than PC mix; at the same time, they exhibit better inhibition of LDL-C, Lp(a), ApoB, non HDL-C than Inclisiran.

[0235] Example 5: Other double-target inhibition nucleic acid conjugates

[0236] Based on the above double-target conjugates exhibiting good inhibition activity of double-target genes (PCSK9 and LPA) in mice and cynomolgus monkeys, we applied this double-target modification mode to other targets, and the specific conjugates are shown in Table 22 below.

[0237] Table 22. Nucleic acid conjugates with ANGPTL4, ANGPTL3 single / double target inhibition

[0238] Example 6: In vivo activity detection of nucleic acid double-stranded conjugates

[0239] 6-8 week old C57BL / 6J mice were randomly divided into groups according to body weight, 6 mice in each group. The mice in each group were calculated for drug dosage according to body weight, and a single dose was administered by abdominal subcutaneous injection on Day 1, with a dose of 6 mpk, and an equal volume of PBS was injected as a control. The day of administration was recorded as day 1 (D1). On D8 and D29, three mice were euthanized in each group, and the mouse livers were collected. The expression of ANGPTL4 and ANGPTL3 mRNA in the liver at different times was detected by QPCR method to evaluate the knockdown effect of different conjugates on target genes. The detection results are shown in Tables 23, 24, 25, 26, 27, and 28.

[0240] Table 23. Residual expression of ANGPTL3 mRNA in liver after administration of conjugates and siRNA mixtures

[0241] Table 24. Residual expression of ANGPTL4 mRNA in liver after administration of conjugates and siRNA mixtures

[0242] Table 25. Residual expression of ANGPTL3 mRNA in liver after administration of conjugates and siRNA mixtures

[0243] Table 26. Residual expression of ANGPTL4 mRNA in liver after administration of conjugates and siRNA mixtures

[0244] Table 27. Residual expression of ANGPTL3 mRNA in liver after administration of conjugates and siRNA mixtures

[0245] Table 28. Residual expression of ANGPTL4 mRNA in liver after administration of conjugates and siRNA mixtures

[0246] Example 7: Nucleic acid conjugates with dual-target inhibition of SOD1 and CTNNB1

[0247] Based on the above liver-targeted dual-target conjugates exhibiting good inhibitory activity on dual-target genes in mice and cynomolgus monkeys, we applied this dual-target modification mode to nucleic acid double-stranded conjugates with CNS targeting, and the specific conjugates are shown in Table 29 below.

[0248] Table 29. Nucleic acid conjugates with single / dual-target inhibition of SOD1 and CTNNB1

[0249] Example 8: In vivo activity evaluation of nucleic acid conjugates with dual-target inhibition of SOD1 and CTNNB1

[0250] 6-8 week old C57BL / 6J mice were randomly divided into groups according to body weight, with 4-6 mice in each group. The mice in each group were calculated for drug dosage according to body weight, and ICV administration (intracerebroventricular administration) was performed according to Table 30. The first day of administration was Day 1. The mice were euthanized on D8 and D22 after administration, and the mouse brain tissues were isolated, and the hippocampus, frontal cortex, hypothalamus, remaining other brain tissues and heart were taken, and QPCR was used to detect the changes in SOD1 mRNA and CTNNB1 mRNA expression in each tissue, and the detection results are shown in Tables 31-32 below.

[0251] Table 30. Dosing route, dose and schedule in animal model

[0252] Table 31. Residual expression of SOD1 mRNA in various tissues on the eighth day after administration

[0253] Table 32. Residual expression of CTNNB1 mRNA in various tissues

[0254] Example 9: Nucleic acid conjugate with SOD1 and ALK7 dual-target inhibition

[0255] Based on the above liver-targeted dual-target conjugate showing good inhibition activity on dual-target genes in mice and cynomolgus monkeys, we applied this dual-target modification mode to a nucleic acid double-stranded conjugate with fat targeting, and the specific conjugate is shown in Table 33 below.

[0256] Table 33. Nucleic acid conjugate with SOD1 and ALK7 single / dual-target inhibition

[0257] Example 10: In vivo activity evaluation of nucleic acid conjugate with SOD1 and ALK7 dual-target inhibition

[0258] 6-8 week old C57BL / 6J mice were randomly divided into groups according to body weight, with 3 mice in each group. The mice in each group were calculated for drug dose according to body weight, and administered according to Table 34. Day 1 was administered by abdominal subcutaneous injection, with a single dose of 6mpk, and an equal volume of PBS was injected as a control. The day of administration was recorded as day 1 (D1). On D8 and D29, three mice were euthanized each time, and different fats (inguinal subcutaneous fat, perigonadal fat, scapula brown fat, perirenal fat, etc.) were collected, and the expression of SOD1 and ALK7 mRNA in different tissues was detected by QPCR method to evaluate the knockdown effect of different conjugates on target genes. The test results are shown in Table 34 below.

[0259] Table 34. Dosing route, dose and schedule in animal model

[0260] Conjugate 603 can achieve efficient knockdown of SOD1 and ALK7 target genes in adipose tissue (inguinal subcutaneous fat, perigonadal fat, scapula brown fat, perirenal fat, etc.), achieving mRNA silencing effect comparable to or slightly better than the mixture composition.

[0261] Summary: The application adopts a novel double-target dsRNA design method, which can realize efficient silencing of double-target genes by a single molecule, such as efficient silencing of double-target in liver, double-target silencing in CNS tissue, and double-target silencing in adipose tissue.

Claims

1. A double-stranded nucleic acid conjugate having dual-target gene inhibition, comprising: a. a first nucleic acid single strand and a second nucleic acid single strand that are partially or fully 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 that is at least partially complementary to a mRNA sequence of a target gene, and comprises at least 2 unmodified RNA nucleotides or DNA nucleotides; b. a moiety conjugated to each nucleic acid single strand; wherein each N is independently an unmodified or chemically modified nucleotide; each L is an independent carrier moiety, with or without; (N) represents consecutive unmodified nucleotides; the sum of X1, X2, X3, X4 and X5 is an integer of 30-80; c. wherein X1 and / or X5 on the first nucleic acid single strand and / or the second nucleic acid single strand is / are 0; d. 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): wherein each N is independently an unmodified or chemically modified nucleotide; each L is an independent carrier moiety, with or without; (N) represents consecutive unmodified nucleotides; the sum of X1, X2, X3, X4 and X5 is an integer of 30-80; e. preferably, the first nucleic acid single strand or the second nucleic acid single strand has a structure as shown in formula (III): wherein each N is independently an unmodified or chemically modified nucleotide; each L is an independent carrier moiety, with or without; (N) represents consecutive unmodified nucleotides; the sum of X1, X2, X3, X4 and X5 is an integer of 30-80; f. the nucleic acid conjugate can be cleaved into two independent siRNA conjugates that inhibit the 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. or 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. or 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.; preferably, the target gene is selected from the following combinations of genes: (1) PCSK9 and LPA; (2) C3 and C5; (3) PCSK9 and ANGPTL3; (4) ANGPTL4 and ANGPTL3; (5) CTNNB1 and SOD1; (6) ALK7 and SOD1; (7) PCSK9 and AGT; ​ b. each nucleic acid single strand is conjugated to 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 mAb, or a carrier group comprising a small molecule targeting agent; wherein, ​ each nucleic acid single strand is represented by formula (I): ​ 2. The nucleic acid conjugate of claim 1, wherein, ​ ​ 5' N X2 -(N) X3 -N X4 -L 3' (II); and / or ​ 5' N X2 -(N) X3 -N X4 3' (III).

3. The nucleic acid conjugate of claim 1 or 2, characterized in that: ​ ​ ​ ​ ​ ​ ​ ​ (8) PCSK9 and APOC3; (9) AGT and CFB; (10) PLIN2 and PNPLA3; (11) PNPLA3 and HSD17B13; (12) PNPLA3 and DGAT2; (13) PNPLA3 and MARC1; (14) Tau and APP; (15) SCN9A and SCN10A; (16) INHBE and DPP4.

4. The nucleic acid conjugate of any one of claims 1-3, wherein: The sum of X1 and X2 is an integer from 14 to 29; the nucleic acid conjugate is capable of forming 0, 1 or more than 1 Bubble structure at the position of (N); the region complementary to at least part of the mRNA sequence of the target gene is located 5' to the position of (N); Each single strand in the nucleic acid conjugate has the same or different nucleotide sequence at the position of (N); Preferably, the Bubble structure comprises unmodified RNA nucleotides (rN) or DNA nucleotides (dN).

5. The nucleic acid conjugate of any one of claims 1-4, characterized in that: X3 is selected from an integer from 1 to 5 or 6 to 7.

6. The nucleic acid conjugate of any one of claims 1-5, wherein: X3 is selected from an integer from 1 to 4; preferably, X3 is selected from 2 or 3.

7. The nucleic acid conjugate of claim 3, wherein: The two independent siRNA conjugates respectively comprise sequences complementary to the mRNA of two target genes; or The two independent siRNA conjugates respectively comprise sequences complementary to different transcripts of the mRNA of the same target; or The two independent siRNA conjugates respectively comprise sequences complementary to different target regions of the mRNA of the same target.

8. The nucleic acid conjugate of any one of claims 1-7, wherein: The nucleotide sequence at the position of (N) on the first or second nucleic acid single strand is dNrN or dNrNdN or dNdNrNdN; wherein dN is preferably dA or dT; rN is preferably rU or rC; More preferably, the nucleotide sequence at the position of (N) is selected from dArU, dTrCdT or dAdTrUdT.

9. The nucleic acid conjugate according to any one of claims 1-8, characterized in that: The nucleotide sequence at the position of (N) on the first nucleic acid single strand is dTrCdT, and the nucleotide sequence at the position of (N) on the second nucleic acid single strand is dTrCdT; or The nucleotide sequence at the position of (N) on the first nucleic acid single strand is dArU, and the nucleotide sequence at the position of (N) on the second nucleic acid single strand is dTrCdT; or The nucleotide sequence at the position of (N) on the first nucleic acid single strand is dArU, and the nucleotide sequence at the position of (N) on the second nucleic acid single strand is dAdTrUdT.

10. The nucleic acid conjugate of any one of claims 1-9, wherein: The nucleic acid conjugate has liver targeting or extrahepatic targeting; preferably, the extrahepatic targeting is selected from targeting CNS tissue, fat, muscle, lung, kidney, eye; 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) can have 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: or GalNAc(A1GN), GalNAc(A1dGN), GalNAc(A3GN), GalNAc(A3dGN), GalNAc(A5GN), GAlNAc(NAG25), GAlNAc(NAG37) have the following structures, respectively: Preferably, 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; Preferably, 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; Preferably, 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.

11. The nucleic acid conjugate of any one of claims 1-10, wherein: The nucleic acid conjugate is selected from conjugate 1 to conjugate 318 in Table 1.

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 further comprises a delivery formulation; preferably, the delivery formulation comprises a lipid nanoparticle (LNP), a lipid polyplex (LPP), a polymeric nanoparticle (PNP), an inorganic nanoparticle (INP), a cationic nanoemulsion (CNE), an exosome, a biological microvesicle. The pharmaceutical composition further comprises a delivery formulation; preferably, the delivery formulation comprises a lipid nanoparticle (LNP), a lipid polyplex (LPP), a polymeric nanoparticle (PNP), an inorganic nanoparticle (INP), a cationic nanoemulsion (CNE), an exosome, a biological microvesicle.

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