Polypeptide-oligonucleotide complexes and uses thereof

Abstract

Provided are polypeptide-oligonucleotide multimeric complexes and uses thereof. Specifically, provided is a multimeric complex carrying at least one (a1) polypeptide element and at least one (b1) oligonucleotide pharmacophore, linked by a reciprocal nucleic acid backbone complex structure.

Description

Polypeptide-oligonucleotide complex and its use Technical Field

[0001] The present invention relates to the field of biotechnology drugs, in particular to polypeptide-oligonucleotide complexes and applications thereof. Background Art

[0002] Since oligonucleotides have the potential to precisely regulate gene expression to treat diseases, oligonucleotide-based gene therapy has become increasingly popular. The length of oligonucleotides is generally between 20-60bps and can be in single-stranded or double-stranded form. The forms of nucleic acid monomers include ribonucleotides, deoxyribonucleotides and other nucleic acid derivatives. The types of oligonucleotide drugs currently under development include antisense oligonucleotides (AON), small interfering RNA (siRNA), microRNA (miRNA), nucleic acid aptamers, etc. Oligonucleotide drugs pair with DNA, mRNA or pre-mRNA through the principle of complementary base pairing to achieve precise regulation of gene expression. Oligonucleotide drugs further broaden the means and boundaries of disease treatment and have great potential for drug development.

[0003] However, oligonucleotide drugs also have some problems, including difficulty penetrating cell membranes and the blood-brain barrier, and lack of targeting. Therefore, antibody-oligonucleotide conjugates (AOCs) with targeted characteristics have emerged. AOCs are a new type of biomacromolecule formed by coupling antibodies and oligonucleotides. Oligonucleotides have the ability to regulate gene expression and are developed as gene therapy drugs. The conjugated antibodies provide targeting for oligonucleotides, resulting in precise targeting and better pharmacokinetic properties than oligonucleotides.

[0004] AOC drug candidates are primarily in the preclinical and early clinical stages, with no marketed AOCs currently available. These candidates cover indications for a range of diseases, including muscle disorders, central nervous system diseases, and tumors. Due to the relatively new concept of AOCs and the complex and demanding preparation process, only a handful of companies possess AOC technology platforms, creating significant technical barriers.

[0005] Therefore, there is an urgent need to develop new AOC drugs that can improve the targeting of oligonucleotide drugs and break through the blood-brain barrier to enhance the efficacy of the drugs. Summary of the Invention

[0006] The purpose of the present invention is to provide an oligonucleotide drug with precise targeting based on a mutually compatible nucleic acid backbone composite structure and its use.

[0007] In a first aspect of the present invention, a polypeptide-oligonucleotide polymer complex is provided, wherein the complex is a polymer formed by the complexation of n monomers having mutually compatible nucleic acid backbones, where n is a positive integer of 2-8;

[0008] Wherein, the monomers include:

[0009] (a) a polypeptide monomer comprising: (a1) a polypeptide element and (a2) a first backbone nucleic acid single strand; wherein the polypeptide element is covalently linked to the first backbone nucleic acid single strand;

[0010] (b) an oligonucleotide-based monomer comprising: (b1) an oligonucleotide pharmacodynamic element and (b2) a second backbone nucleic acid single strand; wherein the oligonucleotide pharmacodynamic element is covalently linked to the second backbone nucleic acid single strand; and

[0011] (c) an optional pure backbone monomer, wherein the pure backbone monomer comprises a third backbone nucleic acid single strand, and the third backbone nucleic acid single strand is not covalently linked to a targeting element (such as an antibody) or a pharmacodynamic element (such as an oligonucleotide drug);

[0012] Furthermore, in the polymer, the backbone nucleic acid single strand of each monomer forms a mutually compatible double strand with the backbone nucleic acid single strand of other monomers through base complementarity, thereby forming a mutually compatible nucleic acid backbone composite structure;

[0013] And a complex carries at least one (a1) polypeptide element and at least one (b1) oligonucleotide pharmacodynamic element.

[0014] In another preferred embodiment, the polypeptide element is selected from the following group: a polypeptide element with targeting function, a polypeptide element that prolongs half-life, or a combination thereof.

[0015] In another preferred embodiment, n is a positive integer of 2-6; more preferably, n is a positive integer of 3-6.

[0016] In another preferred embodiment, n is 2, 3, 4, 5, 6, 7 or 8.

[0017] In another preferred embodiment, the polypeptide element with targeting function is selected from the following group: antibodies or active fragments thereof, ligands or active fragments thereof, or a combination thereof.

[0018] In another preferred embodiment, the antibody or its binding fragment includes a humanized antibody or its binding fragment, a chimeric antibody or its binding fragment, a monoclonal antibody or its binding fragment, a monovalent Fab', a divalent Fab2, a single-chain variable fragment (scFv), a tandem scFv, a diabody, a triabody, a minibody, a TriBi minibody, a single domain antibody (sdAb), a tandem single domain antibody (sdAb), IgG-scFv, BiTE, DART, TandAb, scDiabody, scDiabody-Fc, scDiabody-CH3, scFv-CH3 KIH, Fab-scFv-Fc, Fab-scFv-Fc KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2-scFv2, scFv-KIH, a tetravalent HCAb, an intracellular antibody or a camelid antibody or its binding fragment.

[0019] In another preferred embodiment, the polypeptide element with targeting function targets a target selected from the group consisting of TfR1, BCMA, CD73, GPC3, HER2, PMSA, 4-1BB, OX40, GLP-1, Trop2, FGL1, LFA-3, 2B4, 5T4, α-4 integrin, α-V integrin, α4β7 integrin, α4β7 integrin, α-SMA, AGR2, Apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9, carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, and CD47. , CD51, CD52, CD56, CD64, CD69, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD107a, CD117, CD123 , CD125, CD132(IL-2Rg), CD133, CD137, CD138, CD160, CD166, CD172A, CD248, CEACAM5(CEA), CEA CAM6 (NCA-90), CLAUDIN-3, CLAUDIN-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), FSP-1, GAL3ST1, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSFR, GPIlb / IIIa receptor, Gpl30, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, hGH, HLA-DR, HVEM, hyaluronidase, ICOS, IgE, IgE receptor (FceRI), IGF1R, IL1B, IL1R, IL12p40, IL-12R, IL-12Rβ1, IL13R, IL13Ra2, IL23R, IL27 / IL27R(wsxl), IL29, IL-31R, IL31 / IL31R, IL2R, IL4R, IL6R, IL1 receptor accessory protein (IL1RAP), insulin receptor, Jagged ligand, Jagged 1, Jagged2, KISS1-R, KLRG1, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, Nicastrin, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, Siglec15, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TfR1, TGFβ, TIG IT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, WISP-3, or a combination thereof.

[0020] In another preferred embodiment, the polypeptide element with targeting function is selected from the following group: single domain antibody (VHH), single chain antibody, natural ligand, or a combination thereof.

[0021] In another preferred embodiment, the polypeptide element with targeting function includes an antibody or a binding fragment thereof targeting TfR1.

[0022] In another preferred embodiment, the polypeptide element with targeting function includes a single domain antibody targeting TfR1.

[0023] In another preferred embodiment, the polypeptide element with targeting function has a sequence as shown in SEQ ID NOs: 361.

[0024] In another preferred embodiment, the half-life-extending polypeptide element is selected from the following group: natural albumin, recombinant albumin, anti-albumin antibodies (including single domain antibodies, single chain antibodies, Fab, monoclonal antibodies), proteins that directly bind to FcRn, any protein with a long half-life, or a combination thereof.

[0025] In another preferred embodiment, the polypeptide element that prolongs the half-life comprises an anti-albumin single domain antibody.

[0026] In another preferred embodiment, the polypeptide element that extends half-life has a sequence as shown in SEQ ID NOs: 362.

[0027] In another preferred embodiment, the complex comprises 1, 2 or 3 different types of polypeptide monomers.

[0028] In another preferred embodiment, the different types are distinguished based only on polypeptide elements.

[0029] In another preferred embodiment, the complex includes both a polypeptide element with targeting function and a polypeptide element with extended half-life.

[0030] In another preferred embodiment, the complex includes both a single-domain antibody targeting TfR1 and a single-domain antibody against albumin.

[0031] In another preferred embodiment, the oligonucleotide pharmacodynamic element is selected from the group consisting of antisense oligonucleotides (ASOs), short interfering nucleic acids (siNAs), short interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs), PMOs or mRNAs, or a combination thereof.

[0032] The present invention also provides a polypeptide-oligonucleotide polymer complex, wherein the complex carries at least one (a1) polypeptide element and at least one (b1) oligonucleotide pharmacodynamic element, and the (a1) polypeptide element and the (b1) oligonucleotide pharmacodynamic element are coupled together via a mutually compatible nucleic acid backbone composite structure;

[0033] The mutually compatible nucleic acid backbone composite structure is formed by the composite of n mutually compatible nucleic acid backbone monomers having mutually compatible nucleic acid backbone pairing regions, wherein n is a positive integer of 2-8;

[0034] Furthermore, the nucleic acid single strand of each mutually compatible nucleic acid backbone monomer forms a mutually compatible double strand with the nucleic acid single strand of other mutually compatible nucleic acid backbone monomers through base complementarity, thereby forming a mutually compatible nucleic acid backbone composite structure.

[0035] In another preferred embodiment, the complex comprises at least one polypeptide element having a targeting function and at least one oligonucleotide pharmacodynamic element having a pharmacodynamic function.

[0036] In another preferred embodiment, the polypeptide element can specifically bind to a cell surface receptor and be internalized into the cell.

[0037] In another preferred embodiment, the polypeptide element optionally further comprises a half-life extending portion.

[0038] In another preferred embodiment, the polypeptide monomer is not covalently linked to the oligonucleotide pharmacodynamic element.

[0039] In another preferred embodiment, the polypeptide monomer is covalently linked to the oligonucleotide pharmacodynamic element.

[0040] In another preferred embodiment, the polypeptide monomer has a structure of formula I: Z1-W1 (I)

[0041] Where,

[0042] Z1 is a polypeptide moiety;

[0043] W1 is the first backbone nucleic acid single strand;

[0044] “-” is a linker or a bond.

[0045] In another preferred embodiment, the W1 comprises a pairing region of a compatible nucleic acid backbone.

[0046] In another preferred embodiment, the oligonucleotide-type monomer has a structure of formula II: Y1-W2-Y2 (II)

[0047] Where,

[0048] Y1 is none or the first oligonucleotide pharmacodynamic element;

[0049] Y2 is none or a second oligonucleotide pharmacodynamic element;

[0050] W2 is the second backbone nucleic acid single strand;

[0051] “-” is a connector or a bond;

[0052] Wherein, Y1 and Y2 are not both absent, and the first oligonucleotide pharmacodynamic element and the second oligonucleotide pharmacodynamic element are the same or different.

[0053] In another preferred embodiment, the W2 comprises a pairing region of a mutually compatible nucleic acid backbone.

[0054] In another preferred embodiment, the pure skeleton monomer has a structure of formula III: V1-W3-V2 (III)

[0055] Where,

[0056] V1 is none or the first detectable labeling group;

[0057] V2 is none or a second detectable labeling group;

[0058] W3 is the third backbone nucleic acid single strand;

[0059] “-” is a linker or a bond.

[0060] In another preferred embodiment, V1 and V2 are both absent.

[0061] In another preferred embodiment, the first detectable labeling group and the second detectable labeling group are the same or different.

[0062] In another preferred embodiment, the single-stranded backbone nucleic acid is selected from the group consisting of left-handed nucleic acid, peptide nucleic acid, locked nucleic acid, thio-modified nucleic acid, 2'-fluorine-modified nucleic acid, 5-hydroxymethylcytosine nucleic acid, phosphorodiamidate morpholino nucleic acid, phosphorodiamidate morpholino nucleic acid, or a combination thereof.

[0063] In another preferred embodiment, the complex comprises a structure shown in formula i: (AY-)m-PNSC-(-ZR)ni

[0064] in,

[0065] A comprises a polypeptide element with targeting function;

[0066] R comprises an oligonucleotide pharmacodynamic element;

[0067] PNSC is a mutually compatible nucleic acid backbone complex structure;

[0068] Y is a bond or the first linker;

[0069] Z is a bond or a second linker; and

[0070] m and n are each independently a positive integer ≥1.

[0071] In another preferred embodiment, n≥2; preferably, n≥3; more preferably, n≥4.

[0072] In another preferred embodiment, when m≥2, the polypeptide elements in the complex may be the same or different.

[0073] In another preferred embodiment, m is 2, and the oligonucleotide complex comprises A1 and A2, A1 is an antibody targeting TfR1, and A2 is an anti-albumin antibody.

[0074] In another preferred embodiment, when n≥2, the oligonucleotide pharmacodynamic elements in the complex may be the same or different.

[0075] In another preferred embodiment, the ratio of n to m is 10:1, preferably 4:1, and more preferably 2:1.

[0076] In another preferred embodiment, the first linker is selected from: a cleavable linker and a non-cleavable linker; preferably, the first linker is a non-cleavable linker.

[0077] In another preferred embodiment, the second linker is selected from: a cleavable linker and a non-cleavable linker; preferably, the second linker is a cleavable linker.

[0078] In another preferred embodiment, the cleavable linker is selected from the group consisting of MC-VC-PAB, hydrazone linker, disulfide (selenide) linker, boronate ester, thioketal, azoketone, polypeptide substrate, enzyme-cleavable linker, or a combination thereof.

[0079] In another preferred embodiment, the non-cleavable linker is selected from the group consisting of SMCC, amino-amino coupling bifunctional non-cleavable linker, amino-sulfhydryl coupling bifunctional non-cleavable linker, and sulfhydryl-sulfhydryl coupling bifunctional non-cleavable linker.

[0080] In another preferred embodiment, the oligonucleotide pharmacodynamic element comprises at least about 10 to about 30 nucleotides in length.

[0081] In another preferred embodiment, the oligonucleotide pharmacodynamic element comprises modified nucleotides.

[0082] In another preferred embodiment, the oligonucleotide pharmacodynamic element comprises at least one 2'-modified nucleotide and at least one modified internucleotide linkage.

[0083] In another preferred embodiment, the 2'-modified nucleotides include morpholino, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-ODMAP), T-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) or 2'-O-N-methylacetamido (2'-O-NMA) modified nucleotides.

[0084] In another preferred embodiment, the 2'-modified nucleotide comprises locked nucleic acid (LNA), ethylene nucleic acid (ENA) or peptide nucleic acid (PNA).

[0085] In another preferred embodiment, the oligonucleotide pharmacodynamic element comprises one or more morpholino-modified nucleotides.

[0086] In another preferred embodiment, the oligonucleotide pharmacodynamic element comprises one or more phosphorodiamidate morpholino oligonucleotides.

[0087] In another preferred embodiment, the oligonucleotide pharmacodynamic element comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorodiamidate morpholino oligonucleotides.

[0088] In another preferred embodiment, the complex comprises an oligonucleotide pharmacodynamic element comprising the nucleotide sequence shown in SEQ ID NOs: 363, and / or an oligonucleotide pharmacodynamic element comprising the nucleotide sequence shown in SEQ ID NOs: 364.

[0089] In another preferred embodiment, the complex contains both an oligonucleotide pharmacodynamic element comprising the nucleotide sequence shown in SEQ ID NOs: 363 and an oligonucleotide pharmacodynamic element comprising the nucleotide sequence shown in SEQ ID NOs: 364.

[0090] In another preferred embodiment, the nucleotide sequence comprises one or more nucleotides modified with phosphorodiamidate morpholino.

[0091] In another preferred embodiment, the nucleotide sequence comprises at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% phosphorodiamidate morpholino-modified nucleotides.

[0092] In another preferred embodiment, the PNSC comprises a structure shown in Formula 1: C1-R1-C2-R2-C3 (1)

[0093] in,

[0094] R1 is base complementary pairing region 1;

[0095] R2 is base complementary pairing region 2;

[0096] C1, C2, and C3 are each independently a null or redundant nucleic acid;

[0097] “-” is a key.

[0098] In another preferred embodiment, the lengths of R1 and R2 are independently 10-20 bases, preferably 14-16 bases.

[0099] In another preferred embodiment, the length of C1 is 0-5 bases.

[0100] In another preferred embodiment, the length of C3 is 0-5 bases.

[0101] In another preferred embodiment, the length of C2 is 0-3 bases.

[0102] In another preferred embodiment, the sequence of C2 is selected from the group consisting of A, AA, AGA or AAA.

[0103] In another preferred embodiment, R1 of each monomer forms a base complementary pairing structure with R2 of the left adjacent (or left side) monomer; and R2 forms a base complementary pairing structure with R1 of the right adjacent (or right side) monomer.

[0104] In another preferred embodiment, the PNSC is a trimer, a tetramer, a pentamer or a hexamer; preferably, the structure of the PNSC is as shown in FIG25 .

[0105] In another preferred embodiment, the polymer has a backbone nucleic acid single-stranded sequence set selected from Table A or Table B:

[0106] Table A

[0107] Table B

[0108] The second invention of the present invention provides a monomer library, comprising:

[0109] (a) a first monomer, wherein the first monomer is a polypeptide-type monomer, comprising: (a1) a polypeptide element and (a2) a first backbone nucleic acid single strand; wherein the polypeptide element is covalently linked to the first backbone nucleic acid single strand;

[0110] (b) a second monomer, wherein the second monomer is an oligonucleotide-type monomer, comprising: (b1) an oligonucleotide pharmacodynamic element and (b2) a second backbone nucleic acid single strand; wherein the oligonucleotide pharmacodynamic element is covalently linked to the second backbone nucleic acid single strand; and

[0111] (c) a third monomer, wherein the third monomer is a pure backbone-type monomer, which includes a third backbone nucleic acid single strand, and the third backbone nucleic acid single strand is not covalently linked to a targeting element (such as an antibody) or a pharmacodynamic element (such as an oligonucleotide drug).

[0112] In another preferred embodiment, the polypeptide element is selected from the following group: a polypeptide element with targeting function, a polypeptide element that prolongs half-life, or a combination thereof.

[0113] In another preferred embodiment, the polypeptide element with targeting function is selected from the following group: antibodies or active fragments thereof, ligands or active fragments thereof, or a combination thereof.

[0114] In another preferred embodiment, the antibody or binding fragment thereof includes a humanized antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a divalent Fab2, a single-chain variable fragment (scFv), a tandem scFv, a tandem scFv, a diabody, a triabody, a minibody, a minibody, a TriBi minibody, a tandem single domain antibody, a single domain antibody (sdAb), an IgG-scFv, a BiTE, a DART, a TandAb, a scDiabody, a scDiabody-Fc, a scDiabody-CH3, a scFv-CH3 KIH, a Fab-scFv-Fc, a Fab-scFv-Fc KIH, a Fab-scFv, a scFv-CH-CL-scFv, a F(ab')2-scFv2, a scFv-KIH, a tetravalent HCAb, an intracellular antibody, a diabody or a camelid antibody or binding fragment thereof.

[0115] In another preferred embodiment, the polypeptide element with targeting function targets a target selected from the group consisting of TfR1, BCMA, CD73, GPC3, HER2, PMSA, 4-1BB, OX40, GLP-1, Trop2, FGL1, LFA-3, 2B4, 5T4, α-4 integrin, α-V integrin, α4β7 integrin, α4β7 integrin, α-SMA, AGR2, Apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9, carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, and CD47. , CD51, CD52, CD56, CD64, CD69, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD107a, CD117, CD123 , CD125, CD132(IL-2Rg), CD133, CD137, CD138, CD160, CD166, CD172A, CD248, CEACAM5(CEA), CEA CAM6 (NCA-90), CLAUDIN-3, CLAUDIN-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), FSP-1, GAL3ST1, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSFR, GPIlb / IIIa receptor, Gpl30, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, hGH, HLA-DR, HVEM, hyaluronidase, ICOS, IgE, IgE receptor (FceRI), IGF1R, IL1B, IL1R, IL12p40, IL-12R, IL-12Rβ1, IL13R, IL13Ra2, IL23R, IL27 / IL27R(wsxl), IL29, IL-31R, IL31 / IL31R, IL2R, IL4R, IL6R, IL1 receptor accessory protein (IL1RAP), insulin receptor, Jagged ligand, Jagged 1, Jagged2, KISS1-R, KLRG1, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, Nicastrin, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, Siglec15, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TfR1, TGFβ, TIG IT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, WISP-3, or a combination thereof.

[0116] In another preferred embodiment, the polypeptide element with targeting function is selected from the following group: single domain antibody (VHH), single chain antibody, or a combination thereof.

[0117] In another preferred embodiment, the polypeptide element with targeting function includes an antibody or a binding fragment thereof targeting TfR1.

[0118] In another preferred embodiment, the polypeptide element with targeting function includes a single domain antibody targeting TfR1.

[0119] In another preferred embodiment, the half-life-extending polypeptide element is selected from the following group: natural albumin, recombinant albumin, anti-albumin antibodies (including single domain antibodies, single chain antibodies, Fab, monoclonal antibodies), proteins that directly bind to FcRn, any protein with a long half-life, or a combination thereof.

[0120] In another preferred embodiment, the oligonucleotide pharmacodynamic element is selected from the group consisting of antisense oligonucleotides (ASOs), short interfering nucleic acids (siNAs), short interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs), PMOs or mRNAs, or a combination thereof.

[0121] In another preferred embodiment, the backbone nucleic acid single strand is selected from the group consisting of L-nucleic acid, peptide nucleic acid, locked nucleic acid, thio-modified nucleic acid, 2'-fluorine-modified nucleic acid, 5-hydroxymethylcytosine nucleic acid, morpholine phosphate nucleic acid, L-morpholine phosphate nucleic acid or a combination thereof.

[0122] The third aspect of the present invention provides a pharmaceutical composition, comprising:

[0123] (i) the polypeptide-oligonucleotide multimer complex according to the first aspect of the present invention as an active ingredient; and

[0124] (ii) a pharmaceutically acceptable carrier.

[0125] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: injection and lyophilized preparation.

[0126] The fourth aspect of the present invention provides the use of the polypeptide-oligonucleotide multimer complex according to the first aspect of the present invention for preparing a drug for treating diseases caused by abnormal gene expression.

[0127] In another preferred embodiment, the polypeptide-oligonucleotide polymer complex is used to inhibit the expression of abnormal genes.

[0128] In another preferred embodiment, the disease caused by abnormal gene expression is selected from the group consisting of cancer, cardiovascular disease, inflammatory disease, neuromuscular disease, genetic disease, metabolic disease, and preventable infectious disease.

[0129] In another preferred embodiment, the disease caused by abnormal gene expression is Duchenne muscular dystrophy.

[0130] The fifth aspect of the present invention provides a method for treating diseases caused by abnormal gene expression, comprising the steps of administering a therapeutically effective amount of the polypeptide-oligonucleotide multimer complex of the first aspect of the present invention or the pharmaceutical composition of the second aspect of the present invention to a subject in need.

[0131] In another preferred embodiment, the subject is a mammal, such as a human.

[0132] In the sixth aspect of the present invention, a set of single-stranded nucleic acid sequences for forming a hexameric intermatched nucleic acid backbone complex structure is provided; the set of single-stranded nucleic acid sequences is selected from the backbone nucleic acid single-stranded sequence sets numbered 6-1 to 6-20 in Table B.

[0133] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0134] Figure 1 shows a schematic diagram of the MAOC drug molecule (modular AOC, modular antibody oligonucleotide conjugate).

[0135] Figure 2 shows the coupling and purification of the targeting module. (a) SDS-PAGE verification of the coupling efficiency of the anti-TfR1 single-domain antibody (anti-TfR1 nanobody) and L-DNA1. Lane 1: standard protein; Lane 2: anti-TfR1 single-domain antibody; Lane 3: mixture after the anti-TfR1 single-domain antibody and L-DNA1 reacted at room temperature for 2 hours. (b) Anion exchange chromatography was used to separate and remove free anti-TfR1 single-domain antibody from the coupling product. (c) The sample buffer was replaced with molecular sieves to obtain anti-TfR1 single-domain antibody-L-DNA1 with a purity greater than 95%.

[0136] Figure 3 shows the conjugation and purification of the half-life extension module. (a) SDS-PAGE verification of the conjugation efficiency of the anti-HSA single-domain antibody (anti-HSA nanobody) and L-DNA2. Lane 1: Standard protein; Lane 2: Anti-HSA single-domain antibody; Lane 3: The mixture of the anti-HSA single-domain antibody and L-DNA2 after a 2-hour reaction at room temperature. (b) Anion exchange chromatography was used to separate and remove free anti-HSA single-domain antibody from the reaction mixture. (c) The sample buffer was replaced with molecular sieves to obtain anti-HSA single-domain antibody-L-DNA2 with a purity greater than 95%.

[0137] FIG4 shows the molecular structure of the antisense nucleic acid PMO.

[0138] FIG5 shows agarose gel verification of in vitro DMD exon 22 and 23 skipping.

[0139] Figure 6 shows agarose gel analysis of the coupling efficiency of the antisense nucleic acid PMO with the nucleic acid backbone L-DNA. Lanes 1 and 4: Molecular weight standards; Lane 2: L-DNA3; Lane 3: The mixture of L-DNA3 and EXON22 PMO after 2-4 hours of reaction. Lane 5: L-DNA4; Lane 6: The mixture of L-DNA4 and EXON23 PMO after 2-4 hours of reaction.

[0140] Figure 7 shows the purification of PMO-(L-DNA)-PMO. (a) Free PMO was removed from the reaction mixture by anion exchange chromatography. (b) PMO-(L-DNA) coupled to a PMO molecule was removed from the reaction mixture by hydrophobic interaction chromatography. (c) The purity of PMO-(L-DNA)-PMO was determined by agarose gel analysis. Lane 1: EXON22-(L-DNA3)-EXON22; Lane 2: EXON23-(L-DNA4)-EXON23; Lane 3: EXON23-(L-DNA3)-EXON23;

[0141] Figure 8 shows schematic diagrams of MAOC molecules: (a) MAOC-001; (b) MAOC-002.

[0142] Figure 9 shows gel electrophoresis analysis of the purity of MAOC assembly samples. (a) MAOC-001 demonstrated greater than 90% purity by SDS-PAGE. (b) MAOC-002 demonstrated greater than 90% purity by SDS-PAGE. (c) 2% TAE agarose gel analysis of the purity of MAOC-002 samples before and after assembly. Lane 1: Molecular weight standard; Lane 2: Anti-TfR1 single-domain antibody-L-DNA1; Lane 3: Anti-HSA single-domain antibody-L-DNA2; Lane 4: EXON22-(L-DNA3)-EXON22; Lane 5: EXON23-(L-DNA4)-EXON23; Lane 6: MAOC-002.

[0143] FIG10 shows a schematic diagram of the ELISA experimental protocol.

[0144] FIG11 shows the ELISA analysis of the binding activity of assembled MAOC-001 with human TfR1 protein and nucleic acid probe.

[0145] FIG12 shows a curve of body weight changes in hTfR1 humanized mice administered with MAOC-001.

[0146] Figure 13 shows the development of an ELISA method for detecting PMO concentrations in serum and tissue samples. (a) Schematic diagram of the ELISA protocol. (b) Double logarithmic linear fit of the standard curve for the EXON23 PMO sample. (c) Double logarithmic linear fit of the standard curve for the MAOC-001 sample.

[0147] FIG14 shows the concentration-time curve of EXON23 PMO in the serum of mice administered with MAOC-001.

[0148] FIG15 shows the content of EXON23 PMO in various muscle tissues of mice administered with MAOC-001.

[0149] FIG16 shows the agarose gel analysis of DMD exon 23 skipping in various muscle tissues of MAOC-001-administered mice.

[0150] FIG17 is a bar graph showing the DMD exon 23 skipping status in various muscle tissues of MAOC-001-administered mice.

[0151] FIG18 shows 3% TAE agarose gel analysis of in vitro DMD exon 22 and 23 simultaneous skipping PCR product fragments.

[0152] FIG19 shows the sequencing results of DMD exon 22 and 23 skipping PCR product fragments.

[0153] FIG20 shows the changes in body weight of hTfR1 humanized mice administered MAOC-002.

[0154] FIG21 shows the content of EXON22 PMO in various muscle tissues of mice administered with MAOC-002.

[0155] FIG22 shows the content of EXON23 PMO in various muscle tissues of mice administered with MAOC-002.

[0156] FIG23 shows the agarose gel analysis of DMD exon 22 and exon 23 skipping in muscle tissues of MAOC-002-administered mice.

[0157] FIG24 is a bar graph showing the skipping of exons 22 and 23 of DMD in muscle tissues of mice treated with MAOC-002.

[0158] FIG25 shows a schematic diagram of the composite structure of the intercompatible nucleic acid backbone. DETAILED DESCRIPTION

[0159] After extensive and in-depth research, the inventors have developed for the first time a polypeptide-oligonucleotide complex and its applications. Based on a complementary nucleic acid backbone composite structure, the present invention connects a targeting polypeptide moiety with a pharmacologically active oligonucleotide, resulting in a multifunctional complex. This complex exhibits precise targeting and excellent pharmacokinetic properties, while also possessing a high oligonucleotide-to-antibody ratio, enabling precise and efficient gene therapy. This is the basis for the present invention.

[0160] The complex of the present invention is simple and convenient to prepare. Based on the preparation of polypeptide monomers, oligonucleotide monomers and pure backbone monomers containing backbone nucleic acid single chains, flexible assembly can be performed through the mutual matching structure of the backbone nucleic acid single chains to obtain a multifunctional complex containing the desired oligonucleotide / antibody ratio.

[0161] the term

[0162] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0163] As used herein, the terms "mutually compatible nucleic acid backbone complex structure", "mutually compatible nucleic acid backbone structure", and "PNSC (paired nucleotide scaffold complex structure)" are used interchangeably and refer to a composite paired structure formed by two or more (such as 2, 3, 4, 5, 6) mutually compatible nucleic acid backbone monomers, wherein the backbone nucleic acid single strand of each monomer forms a mutually compatible double strand with the backbone nucleic acid single strand of another monomer through base complementarity, thereby forming a mutually compatible nucleic acid backbone complex structure. The mutually compatible nucleic acid backbone complex structure in the present invention can be a dimer, trimer, tetramer, pentamer, hexamer, heptamer or octamer. Representative structures include, but are not limited to, the structure shown in Figure 25.

[0164] As used herein, the terms "monomer having a compatible nucleic acid backbone" and "compatible nucleic acid backbone monomer" are used interchangeably to refer to a monomer having a single nucleic acid strand containing a backbone nucleic acid pairing region for forming a compatible nucleic acid backbone.

[0165] As used herein, the term "backbone nucleic acid single strand" refers to a nucleic acid single strand having a backbone nucleic acid pairing region. The backbone nucleic acid single strand can exist in polypeptide-type monomers, oligonucleotide-type monomers, and pure backbone-type monomers.

[0166] Pharmacodynamic components

[0167] In the present invention, the pharmacodynamic component is an oligonucleotide drug, which typically includes but is not limited to antisense oligonucleotides (AON), small interfering RNA (siRNA), and microRNA.

[0168] In some embodiments, the pharmacodynamic element can be a natural nucleotide and / or a chemically modified nucleotide. Chemical modification is one of the most effective methods for enhancing the efficacy of oligonucleotide drugs. Chemical modification helps antisense oligonucleotides resist degradation by endogenous nucleases, enhances the stability of oligonucleotides in plasma, tissues, and cells, and can also enhance the affinity of oligonucleotides for target nucleic acids, achieving higher antisense activity.

[0169] Chemical modifications include modifications to the nucleic acid backbone, modifications to the bases, modifications to the ribose sugar groups, and the use of nucleic acid analogs. The nucleic acid analogs refer to groups that can replace natural nucleotides in nucleic acids, including but not limited to bridged nucleotides (BNAs), acyclic nucleotides, or isonucleotides. BNAs are a type of nucleotide in which the ribose is restricted in a 3′-endo conformation by a bridge between the 2′ and 4′ carbon atoms. BNAs include locked nucleic acids (LNAs), ENAs, cETs, or other types of bicyclic nucleotides. BNAs increase the stability of oligonucleotides to nucleases and their affinity for target RNA. Acyclic nucleotides include unlocked nucleic acids (UNA) or glycerol nucleic acids (GNAs). Isonucleotides include peptide nucleic acids (PNAs) or phosphorodiamidate morpholinolines (PMOs).

[0170] L-nucleic acid

[0171] Left-handed nucleic acids exist as mirror images of naturally occurring right-handed nucleic acids (D-nucleic acids). They can be divided into left-handed DNA (L-DNA) and left-handed RNA (L-RNA). Left-handed chiral centers are primarily found in the deoxyribose or ribose sugar moieties of nucleic acids, forming a mirror image. Therefore, left-handed nucleic acids are resistant to degradation by ubiquitous nucleases (such as exonucleases and endonucleases) in plasma.

[0172] polypeptide element

[0173] In the present invention, the polypeptide element portion can specifically target cell surface molecules and induce endocytosis. Typically, the polypeptide element comprises a humanized antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a divalent Fab2, a single-chain variable fragment (scFv), a diabody, a minibody, a single domain antibody, a single domain antibody (sdAb), or a camelid antibody or binding fragment thereof.

[0174] In another preferred embodiment of the present invention, the polypeptide element further comprises a protein or polypeptide that extends half-life (a polypeptide element that extends half-life). Representative examples include, but are not limited to, anti-human serum albumin antibodies (anti-HSA antibodies), Fc elements, or a combination thereof.

[0175] In a preferred embodiment, the half-life extension module adopts an anti-HSA single-domain antibody, which can extend the half-life of the macromolecular drug by binding to the HSA protein in the serum through the FcRn-mediated protein circulation mechanism.

[0176] Targeting peptide element

[0177] As used herein, the terms "targeting polypeptide element" and "targeting polypeptide element" are used interchangeably. Representative targeting polypeptide elements include (but are not limited to): antibodies, ligands, nucleic acid aptamers, or combinations thereof targeting cell surface receptors.

[0178] A preferred targeting polypeptide element is an antibody or an active fragment thereof, a ligand or an active fragment thereof, or a combination thereof.

[0179] transferrin receptor 1

[0180] Transferrin receptor protein 1 (TfR1) is a membrane protein widely expressed in almost all cell and tissue types in the human body, including muscle tissue and cells. TFR1 is widely expressed in the heart, liver, kidney, muscle, etc.

[0181] Pharmaceutical composition

[0182] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the above-mentioned antibody or active fragment thereof or fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): oral, respiratory, intratumoral, intraperitoneal, intravenous, or topical administration.

[0183] The pharmaceutical composition of the present invention can be used directly for treatment (eg, anti-tumor treatment), and thus can be used to prolong the half-life of the drug. In addition, other therapeutic agents can also be used simultaneously.

[0184] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned monoclonal antibody of the present invention (or its conjugate) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 μg / kg body weight to about 10 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used in conjunction with other therapeutic agents.

[0185] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight, and in most cases does not exceed about 8 mg / kg body weight. Preferably, the dose is about 10 μg / kg body weight to about 1 mg / kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0186] Duchenne muscular dystrophy

[0187] Duchenne muscular dystrophy (DMD) is a hereditary muscle wasting disease. It is caused by a mutation in the Dystrophin gene, resulting in a loss of the Dystrophin protein, and is primarily characterized by progressive muscle wasting and weakness. Patients begin to experience muscle weakness around the age of 2 to 5, becoming unable to walk between the ages of 9 and 12. Eventually, all smooth muscles, as well as the heart and respiratory muscles, are affected, leading to respiratory failure or heart dysfunction and death. The average life expectancy for patients with the disease is approximately 30 years. Current treatments include glucocorticoid injections and gene therapy, but these methods can only delay progression and cannot cure DMD.

[0188] The main advantages of the present invention are:

[0189] (1) Compared with traditional oligonucleotide drugs, the polypeptide-oligonucleotide polymer complex of the present invention has precise targeting.

[0190] (2) Compared with traditional oligonucleotide drugs, the polypeptide-oligonucleotide polymer complex of the present invention can penetrate the blood-brain barrier and has better pharmacokinetic properties.

[0191] (3) Compared with conventional AOC drugs, the polypeptide-oligonucleotide polymer complex of the present invention can achieve the loading of multiple oligonucleotide pharmacodynamic elements through the mutually compatible nucleic acid backbone composite structure in an extremely simple and quick connection manner, that is, it has a higher oligonucleotide / antibody ratio.

[0192] (4) The polypeptide-oligonucleotide polymer complex of the present invention is simple and flexible to assemble and can simultaneously load different types of oligonucleotide pharmacodynamic elements.

[0193] (5) The polypeptide-oligonucleotide polymer complex of the present invention is simple and flexible to assemble, can target multiple targets simultaneously, and can achieve more precise positioning.

[0194] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0195] Example 1: Nucleic acid backbone and MAOC molecular design

[0196] The present invention uses a tetrameric L-DNA nucleic acid backbone, and the sequences of the four nucleic acid single strands are as follows (from 5' to 3'):

[0197] Chain 1 (L-DNA1): SEQ ID NO: 121

[0198] 5'-AGGCGATCACAATCCAAATGAGCGTGTTACGG-3'

[0199] Chain 2 (L-DNA2): SEQ ID NO: 122

[0200] 5'-ACCGTAACACGCTCAAAACCGAAGTGCCAATT-3'

[0201] Chain 3 (L-DNA3): SEQ ID NO: 123

[0202] 5'-AAATTGGCACTTCGGAAAACTATGCGGCTGCT-3'

[0203] Chain 4 (L-DNA4): SEQ ID NO: 124

[0204] 5'-AAGCAGCCGCATAGTAAAGGATTGTGATCGCC-3'

[0205] The 5' ends of chains 1 and 2 are modified with NH2 groups, and the 5' and 3' ends of chains 3 and 4 are modified with disulfide bonds, respectively.

[0206] The MAOC molecule is mainly composed of a targeting module consisting of a single-domain antibody-nucleic acid backbone conjugate and a pharmacodynamic module consisting of an oligonucleotide-nucleic acid backbone-oligonucleotide conjugate, supplemented by a half-life extension module. A schematic diagram of some molecules is shown in Figure 1.

[0207] Example 2: Targeting Module: Preparation of Anti-TfR1 Single Domain Antibody-L-DNA1

[0208] A cysteine ​​mutation was introduced at the carboxyl terminus of the single-domain antibody for nucleic acid backbone coupling. The gene sequence of the anti-TfR1 single-domain antibody was optimized to yeast-preferred codons and then subcloned into the pPICZ alpha A plasmid. The amino acid sequence of the anti-TfR1 single-domain antibody is SEQ ID NO: 361. To facilitate purification, a His tag was added to the N-terminus of the single-domain antibody.

[0209] SEQ ID NO: 361, amino acid sequence of anti-TfR1 single domain antibody mutant:

[0210] After linearization, the plasmid was electroporated into Pichia pastoris strain X33, and strains with high target gene copies were screened using Zeocin concentration gradient YPD agar plates. Monoclonal strains were cultured using GMGY medium at 30∑C, 250 rpm. Once sufficient cells were obtained, secretory expression of the target single-domain antibody was induced using GMMY medium at 20∑C, 250 rpm, supplemented with 1% methanol every 24 hours. Strains that have undergone high-copy selection can produce single-domain antibody yields of 40-200 mg / L in laboratory-grade glass flasks. The single-domain antibody in the culture supernatant was purified using a His-tag affinity column.

[0211] The 5'-terminal NH2 of L-DNA1 was modified with an SMCC linker to impart a maleimide-reactive group to the 5' end of L-DNA1. The cysteine ​​residue at the carboxyl terminus of the single-domain antibody was reduced with a reducing agent. The reduced single-domain antibody was then mixed with a 1- to 2-fold molar excess of SMCC-L-DNA1 single chain. The reaction was allowed to proceed at room temperature for 2 hours, and the coupling efficiency was verified by SDS-PAGE (Figure 2(a)).

[0212] The unreacted SMCC-L-DNA1 single chain was removed using a his-tag affinity column, and the single-domain antibody and the single-domain antibody-L-DNA mixture were collected. The buffer was replaced with the loading buffer of the anion exchange column. The single-domain antibody-L-DNA was further separated and purified using HiTrap Q HP (Cytiva) to remove a very small amount of unreacted single-domain antibody (Figure 2(b)). The separation process was achieved by gradient elution. The loading buffer was 20mM Tris-HCl, 15mM NaCl, pH 7.4, and the elution buffer was 20mM Tris-HCl, 1M NaCl, pH 7.4. The elution buffer was 0-100% for gradient elution. The unreacted single-domain antibody and single-domain antibody-L-DNA peaked one after another. The single-domain antibody-L-DNA was collected and concentrated using Superdex TM The buffer was replaced with PBS buffer using 75increase 10 / 300GL (Cytiva), and the anti-TfR1 single-domain antibody-L-DNA1 with a purity greater than 95% was finally obtained ( Figure 2( c )).

[0213] Example 3: Preparation of half-life extension module: anti-HSA single domain antibody-L-DNA2

[0214] The single-domain antibody (SDA) used in the half-life extension module is an anti-HSA SDA. By binding to HSA protein in serum, this antibody can extend the half-life of macromolecular drugs through an FcRn-mediated protein recycling mechanism. The amino acid sequence of the anti-HSA SDA is SEQ ID NO: 362. To facilitate purification, a His tag is added to the N-terminus of the SDA.

[0215] SEQ ID NO: 362, amino acid sequence of an anti-HSA single domain antibody mutant:

[0216] The expression and purification of the anti-HSA single domain antibody and the conjugation purification with L-DNA2 were the same as in Example 2. After the conjugation purification step, the anti-HSA single domain antibody-L-DNA2 with a purity greater than 95% was obtained ( FIG. 3 ).

[0217] Example 4: Synthesis of antisense oligonucleotide PMO sequences

[0218] The present invention uses two PMO sequences: EXON22 can jump over exon 22 on mouse DMD, and its sequence is SEQ ID NO: 363; EXON23 can jump over exon 23 on mouse DMD, and its sequence is SEQ ID NO: 364.

[0219] EXON22: SEQ ID NO: 363

[0220] 5'-ATGTCCACAGACCTGTAATT-3'

[0221] EXON23: SEQ ID NO: 364

[0222] 5'-GGCCAAACCTCGGCTTACCTGAAAT-3'

[0223] The 3' ends of the two PMOs were modified with MC-VC-PAB linkers for L-DNA coupling. The molecular formula is shown in Figure 4.

[0224] Example 5: PMO induces DMD exon skipping in mouse myoblasts C2C12

[0225] Mouse myoblast C2C12 cells were seeded at 100,000 cells / well in a 12-well plate and incubated overnight in a 37°C, 5% CO2 incubator in DMEM + 10% FBS. The next day, the medium was replaced with differentiation medium (DMEM + 2% Horse Serum (Procell, Cat#164215) + 1 μM insulin (Procell, Cat#PB180432). The plate was incubated in the incubator for 3-5 days, with the medium refreshed every two days. A 10-fold concentration of EXON22, EXON23 PMO, and a control oligo were prepared, 100 μl added to 900 μl of fresh differentiation medium, and the cells were incubated. After 24 hours of drug exposure, the medium was replaced with drug-free differentiation medium. The medium was then replaced daily until 72 hours after drug exposure, after which the cells were harvested. Cells were lysed using TRIzol reagent (Thermo, Cat#15596018), and total RNA was isolated using the phenol / chloroform method. The RNA was then reverse-transcribed into cDNA using the M-MuLV First-Strand cDNA Synthesis Kit (Sangon, Cat#B532435). A primary PCR reaction was performed using 300 ng of cDNA using Taq PCR Mix (Sangon, Cat#B639293). The primers used were Ex20F: 5'-CAGAATTCTGCCAATTGCTGAG-3' (SEQ ID NO: 365) and Ex26R: 5'-TTCTTCAGCTTGTGTCATCC-3' (SEQ ID NO: 366). The reaction scheme is shown in Table 1.

[0226] Table 1 Primary PCR protocol

[0227] 1 μl of the primary PCR product was used for a 50 μl nested PCR reaction. The primers used were Ex20F2: 5'-ACCCAGTCTACCACCCTATC-3' (SEQ ID NO: 367) and Ex25R: 5'-CTCTTTATCTTCTGCCCACCTT-3' (SEQ ID NO: 368). The reaction scheme is shown in Table 2.

[0228] Table 2 Nested PCR protocol

[0229] PCR products were analyzed on a 1% TAE agarose gel. The wild-type (WT) DMD product was 788 bp, the exon 22-skipping DMD product (skip exon 22) was 642 bp, and the exon 23-skipping DMD product (skip exon 23) was 575 bp. Exon skipping did not occur in the no-drug control (Vehicle Ctrl) or the oligo control (Oligo Ctrl) (Figure 5).

[0230] Example 6: Preparation of pharmacodynamic module: PMO-(L-DNA3 / 4)-PMO

[0231] The pharmacodynamic module PMO-(L-DNA)-PMO was prepared by coupling two PMOs to the 3' and 5' ends of L-DNA, respectively. First, the disulfide bonds at the 3' and 5' ends of the L-DNA were reduced and modified, exposing a thiol group at each end. Next, a 5-fold molar amount of PMO modified with a 3'-end MC-VC-PAB linker was added, mixed rapidly, and incubated at 60°C for 2-4 hours. After the reaction, the coupling efficiency was assessed using a 2% TAE agarose gel. The experimental results showed that the proportion of EXON22 and EXON23 simultaneously coupled to both the 3' and 5' ends of L-DNA1 and L-DNA2 was as high as over 95%, with only a small amount of L-DNA attached to only one PMO, and virtually no free L-DNA remaining (Figure 6).

[0232] The purification of PMO-(L-DNA)-PMO involved two main steps: 1) removing free PMO using HiTrap Q HP (Cytiva), leveraging its uncharged nature (Figure 7(a)); 2) separating PMO-(L-DNA)-PMO and PMO-(L-DNA) using HiCap Butyl 4FF, leveraging their different hydrophobic properties (Figure 7(b)). Analysis on a 2% TAE agarose gel revealed that the purity of the resulting PMO-(L-DNA)-PMO samples was greater than 98% (Figure 7(c)).

[0233] Example 7: Self-assembly of MAOC drugs

[0234] This example uses two MAOC molecules, MAOC-001 and MAOC-002, as examples to illustrate the self-assembly of MAOC molecules. MAOC-001 is conjugated to four EXON23 PMOs (Figure 8(a)), enabling a single antibody to simultaneously mediate the delivery of four oligonucleotides. MAOC-002, on the other hand, is conjugated to two PMOs (Figure 8(b)), enabling dual exon skipping.

[0235] The self-assembly process of MAOC-001 drug is as follows:

[0236] The concentrations of anti-TfR1 single-domain antibody-L-DNA1, anti-HSA single-domain antibody-L-DNA2, EXON23-(L-DNA3)-EXON23, and EXON23-(L-DNA4)-EXON23 were measured using a NanoDrop One (Thermo). Appropriate amounts of these components were preheated at 37°C for 5 minutes, then mixed in a 1:1 molar ratio at 37°C and incubated for 1 minute to complete the self-assembly of the MAOC-001 drug. The assembled samples were analyzed for purity using SDS-PAGE, which demonstrated a purity exceeding 90% (Figure 9(a)).

[0237] The self-assembly process of MAOC-002 drug is as follows:

[0238] The concentrations of anti-TfR1 single-domain antibody-L-DNA1, anti-HSA single-domain antibody-L-DNA2, EXON22-(L-DNA3)-EXON22, and EXON23-(L-DNA4)-EXON23 were measured using a NanoDrop One (Thermo). Appropriate amounts of the above components were preheated at 37°C for 5 minutes, then mixed in a 1:1 molar ratio at 37°C and incubated for 1 minute to complete the self-assembly of the MAOC-002 drug. The assembled samples were analyzed for purity using SDS-PAGE and 2% TAE agarose gel, respectively. The results demonstrated that the assembled samples were greater than 90% pure (Figures 9(b, c)).

[0239] Example 8: Targeted Activity Verification of MAOC Drugs

[0240] The binding activity of assembled MAOC-001 and MAOC-002 to human TfR1 protein and nucleic acid probes, respectively, was analyzed by enzyme-linked immunosorbent assay (ELISA). Two PTO / DNA probes complementary to EXON22 PMO and EXON23 PMO, respectively, were synthesized. The probes were modified with digoxigenin at the 5' end and biotin at the 3' end.

[0241] EXON22 PTO / DNA probe: SEQ ID NO: 369

[0242] 5'-digoxigenin-AATTACAGGTCTGTGGACAT-biotin-3'

[0243] EXON23 PTO / DNA probe: SEQ ID NO: 370

[0244] 5'-digoxigenin-ATTTCAGGTAAGCCGAGGTTTGGCC-biotin-3'

[0245] Among them, the nucleotides marked in bold are phosphorothioate-modified.

[0246] The schematic diagram of the ELISA experimental design is shown in Figure 10. MAOC molecules are captured by binding of human TfR1 protein to anti-TfR1 single-domain antibodies on MAOC. Then, a PTO / DNA probe complementary to PMO is used to detect the PMO on the MAOC molecule to verify the activity of the targeting module and pharmacodynamic module on the MAOC molecule and the integrity of the MAOC molecule. The experimental steps are as follows:

[0247] A human TfR1-His (ACRO, Cat#CD1-H5243) antigen solution was prepared in PBS to a final concentration of 1 μg / mL. 25 μl was added to each well of a 384-well microtiter plate and coated overnight at 4°C. The plates were washed three times with PBST (PBS + 0.05% Tween 20). 50 μl / well of blocking buffer (PBST + 3% BSA) was added and blocked for 1 hour at room temperature. Washed once with PBST. MAOC-001 or MAOC-002 was serially diluted in sample dilution buffer (10 mM Tris, 1 mM EDTA, 0.1% (v / v) Triton X-100, pH 8.0). EXON22 PTO / DNA or EXON23 PTO / DNA capture probes were serially diluted in probe dilution buffer (10 mM Tris, 1 mM EDTA, 1 M NaCl, 0.1% (v / v) Triton X-100, pH 8.0). Mix equal volumes of the diluted MAOC drug and capture probe and incubate at 37°C for 30 minutes. Transfer the mixture to a 384-well microtiter plate pre-coated with hTfR1 at a dilution of 25 μl / well in duplicate and incubate at 37°C for 30 minutes. Prepare a 1:5000 dilution of Micrococcal Nuclease (CST, Cat#10011) solution in nuclease reaction buffer (50 mM Tris, 5 mM CaCl2, 200 mM NaCl, 0.1 mg / ml BSA, pH 8.2) and add 25 μl / well to the microtiter plate. Incubate at 37°C for 1 hour to allow the Micrococcal Nuclease to cleave any unbound PMO probe. Wash three times with PBST and add 25 μl / well of a 1:5000 dilution of horseradish peroxidase-conjugated anti-digoxigenin antibody (R&D Systems, Cat#HAM7520) and incubate at 37°C for 30 minutes. Wash 3 times with PBST, pat dry, add 25ul / well of chromogenic substrate TMB solution (Beyotime, Cat#P0209), develop color at room temperature for 5 to 30 minutes, and then add 25ul / well of chromogenic stop solution (Beyotime, Cat#P0215). The absorbance at 450 of each well was measured using a multifunctional microplate reader (Molecular Devices, SpectraMax i3x). The binding EC50 value was calculated using the S-shaped curve 4-parameter equation of GraphPad Prism 9 software. The experimental results showed a standard S-shaped curve, as shown in Figure 11, with an EC50 value of 0.15nM, indicating that MAOC-001 can simultaneously bind to human TfR1 protein and PTO / DNA probe, has a complete molecular structure, and has strong binding ability.

[0248] Example 9: Serum pharmacokinetics analysis of MAOC-001 in hTfR1 humanized mice In vivo efficacy and pharmacokinetic experiments in mice hTfR1 humanized C57BL / 6 mice weighing 16-24g and aged 6-8 weeks were selected. A single injection of 20mg / kg PMO dose of non-modified EXON23 PMO and MAOC-001 (the molar amount of administration is 1 / 4 of EXON23 PMO) was given, and the PBS group was used as a control. Serum and tissue samples at each time point were collected to further analyze the pharmacokinetics of the drug in mice, the tissue distribution of the drug, and the exon skipping in vivo. 17 mice were grouped according to body weight and randomly divided into 3 groups, with 5-6 mice in each group. Detailed dosing information is shown in Table 3:

[0249] Table 3 Grouping and dosing Note: a: Single dose refers to administration once on the day of grouping.

[0250] All mice underwent clinical observation daily, including but not limited to their condition and diet. Body weight was measured twice weekly, and monitoring data showed that all mice's body weights fluctuated within the normal range during the experiment, indicating that the mice tolerated the drug well at the doses tested (Figure 12). At the end of the experiment or at a humane endpoint, animals were euthanized using an overdose of CO2.

[0251] Blood samples were collected from the drug-treated group at 5 minutes, 2 hours, 4 hours, 8 hours, 1 day, 4 days, 7 days, and 14 days after administration. Blood samples were collected from the non-drug-treated group as a control. Serum was rapidly separated and stored at -80°C.

[0252] The ELISA method was used to detect the PMO concentration in the serum sample. The ELISA method is shown in the schematic diagram (Figure 13 (a)). By coating Streptavidin (ACRO, Cat#STN-N5116), the biotin-labeled PTO / DNA probe was captured, and the probe then captured the PMO in the serum through base complementary pairing. Micrococcal nuclease was then used to cut the probe that was not bound to PMO. After washing to remove free digoxigenin, a 1:5000 diluted anti-digoxigenin antibody (R&D Systems, Cat#HAM7520) was used to detect the PMO content in the serum. The standard curve used a 2-fold gradient dilution method, starting from 2000pM, and 11 concentration gradients were set. All samples were set in duplicate and a secondary antibody control group and a blank mouse blood sample control group were set. Net OD450 = T OD -C OD ,T OD is the actual OD450 measurement value of each serum sample, C ODThe double logarithmic analysis method was used to linearly fit the standard curve, and the detection range of EXON23 samples was 10pM-313pM. 2 ≥0.99 (Figure 13(b)); the detection range of MAOC-001 sample was 20pM-313pM, R 2 ≥0.99 (Figure 13(c)).

[0253] Each serum sample was diluted to a concentration within the assay range, and the corresponding Net OD450 was measured. The corresponding serum drug concentration was then calculated based on the standard curve, and a drug concentration-time curve was plotted (Figure 14). The experimental results showed that, at equal PMO dosages, serum PMO concentrations in the MAOC-001 group were higher than those in the EXON23 PMO group at all blood collection time points, indicating that MAOC-001 has higher bioavailability than EXON23 PMO.

[0254] Example 10: Analysis of oligonucleotide delivery efficiency of MAOC-001 in hTfR1 humanized mouse muscle tissue

[0255] On days 7 and 14 after administration, two to three mice were collected from each group. After anesthesia, they underwent cardiac perfusion with pre-chilled PBS. Following perfusion, tissue samples from the heart, diaphragm, quadriceps femoris, gastrocnemius, soleus, and tibialis anterior muscles were quickly collected. Each tissue sample was divided into two aliquots: the first aliquot was weighed, snap-frozen in liquid nitrogen, and stored at -80°C for PMO content analysis; the second aliquot was weighed, immersed in 10 volumes of RNAsolid (Servicebio, Cat#G3019) solution, immersed overnight at 4°C, and then frozen at -80°C for total RNA extraction and exon skipping analysis.

[0256] After grinding the tissue samples, PMO concentrations in the tissues were measured using an ELISA method similar to that used in Example 9. A bar graph (Figure 15) was plotted based on the experimental data. The results showed that the EXON23 PMO content in the MAOC-001-treated group (G3) was significantly higher than that in the EXON23 PMO-treated group (G2) across all tissue samples, including the heart, diaphragm, quadriceps femoris, gastrocnemius, and tibialis anterior. Therefore, the delivery efficiency of MAOC-001 oligonucleotides in mice was significantly higher than that of the oligonucleotides themselves.

[0257] Example 11: Analysis of the efficacy of MAOC-001 in exon skipping of DMD in hTfR1 humanized mice. Tissues were ground and then subjected to total RNA extraction and exon skipping analysis. The ground tissue samples were lysed using TRIzol reagent (Thermo, Cat#15596018), and total RNA was isolated using the phenol / chloroform method. 10-200 ng of total RNA was used for simultaneous reverse transcription and primary PCR reactions using the HiScript II One Step RT-PCR Kit (Vazyme, Cat#P611). Primers used were Ex20F2: 5'-ACCCAGTCTACCACCCTATC-3' and Ex25R: 5'-CTCTTTATCTTCTGCCCACCTT-3'. The reaction scheme is shown in Table 4.

[0258] Table 4 Reverse transcription and primary PCR protocols

[0259] 1 μl of the primary PCR product was used for a 50 μl nested PCR reaction. The primers used were Ex22F: 5′-TGAGTAGCATCAGGACGTGG-3′ (SEQ ID NO: 371) and Ex24R: 5′-GCAGGCCATTCCTCTTTCAG-3′ (SEQ ID NO: 372). The reaction scheme is shown in Table 5.

[0260] Table 5 Nested PCR protocol

[0261] PCR products were analyzed on a 3% TAE agarose gel. The wild-type (WT) DMD product was 388 bp, and the exon 23-skipped DMD product was 175 bp (Figure 16). Agarose gel electrophoresis results were analyzed and quantified using Image Lab software, and a histogram was plotted using GraphPad Prism 9 software (Figure 17). The results showed that no exon skipping occurred in the PBS group. In the EXON23 PMO-treated group (G2), less than 10% exon 23 skipping was detected in the heart, diaphragm, quadriceps femoris, and gastrocnemius muscles on day 7, and in the quadriceps femoris muscle on day 14. In the MAOC-001-treated group (G3), 10-30% exon 23 skipping was detected in all six muscle tissues sampled. MAOC-001 exhibited significantly better exon skipping efficacy than EXON23 PMO.

[0262] Example 12: In vitro DMD double exon skipping experiment

[0263] 200,000 mouse myoblasts C2C12 were seeded / well in a 12-well plate and incubated overnight in an incubator at 37°C with 5% CO2 in DMEM + 10% FBS. The next day, the medium was replaced with differentiation medium DMEM + 2% Horse Serum (Procell, Cat#164215) + 1uM insulin (Procell, Cat#PB180432). The cell plates were placed in an incubator and incubated for 3-5 days, with the medium refreshed every 2 days. A 10-fold concentration of EXON22 and EXON23 PMO was prepared, 100 μl was added to 900 μl of fresh differentiation medium, and the cells were incubated. After 48 hours of drug treatment, the cells were harvested and total RNA was extracted. The methods for total RNA extraction and exon skipping analysis were the same as in Example 11. The primers used are listed in Table 6:

[0264] Table 6 Double exon skipping primers

[0265] PCR products were analyzed using 3% TAE agarose gel. The wild-type (WT) DMD product was 547 bp, the exon 22 skipping DMD product was 401 bp, the exon 23 skipping DMD product was 334 bp, and the double exon 22 and 23 skipping DMD product was 188 bp ( FIG18 ). Analysis of the experimental results showed that in samples treated with only 5μM EXON22 PMO, only the band of exon skipping was observed; in samples treated with only 5μM EXON23 PMO, both the band of exon skipping and the band of double exon skipping of exons 22 and 23 were observed, but the proportion of the exon skipping band was larger; in samples treated with 2.5μM EXON22 and EXON23 PMO at the same time, the band of exon skipping, the band of exon skipping and the band of double exon skipping of exons 22 and 23 were observed, but the proportion of the double exon skipping band was larger.

[0266] The four DNA fragments were gel-recovered and sequenced to verify whether the jump position was correct ( FIG. 19 ). The sequencing results showed that the four sequences were consistent with the DMD sequence before and after the jump.

[0267] Example 13: Analysis of the delivery efficiency of two oligonucleotides of MAOC-002 in the muscle tissue of hTfR1 humanized mice

[0268] For the in vivo double exon skipping experiment in mice, hTfR1 humanized C57BL / 6 mice weighing 19-25g and aged 6-8 weeks were used. Eighteen mice were randomly divided into three groups of six based on body weight and each group received a single injection of 600 nmol / kg of the test drug. A PBS group served as a control. Tissue samples were collected at various time points to analyze drug tissue distribution and double exon skipping in vivo. Detailed dosing information is shown in Table 7.

[0269] Table 7 - Grouping and Dosing Note: a: Single dose refers to administration once on the day of grouping;

[0270] All mice underwent clinical observation daily, including but not limited to their condition and diet. Body weight was measured twice weekly. Monitoring data showed that the body weight of all mice remained within the normal range during the experiment, indicating that the mice tolerated the drug well at the doses tested (Figure 20). At the end of the experiment or at a humane endpoint, animals were euthanized using an overdose of CO2.

[0271] On days 4, 7, 14, and 21 after administration, three mice were collected from each group and anesthetized and perfused intracardially with pre-chilled PBS. Tissue samples of the heart, diaphragm, quadriceps femoris, gastrocnemius, soleus, and tibialis anterior muscles were quickly collected. Each tissue sample was divided into two aliquots: the first aliquot was weighed and snap-frozen in liquid nitrogen and stored at -80°C for PMO content analysis; the second aliquot was weighed and immersed in 10 volumes of RNAsolid (Servicebio, Cat#G3019) solution, immersed overnight at 4°C, and then frozen at -80°C for total RNA extraction and exon skipping analysis.

[0272] After grinding the tissue samples, ELISA was used to measure the concentrations of EXON22 and EXON23 PMO in the tissues, using the same assay method as in Example 9. Histograms ( Figures 21 and 22 ) were plotted based on the experimental data. The results showed that the levels of EXON22 and EXON23 PMO in all tissue samples, including the heart, diaphragm, quadriceps femoris, gastrocnemius, and tibialis anterior, were significantly higher in the MAOC-002-treated group (G3) than in the combined EXON22 + EXON23 PMO group (G2).

[0273] Example 14: Pharmacodynamic Analysis of MAOC-002 in DMD Double Exon Skipping in hTfR1 Humanized Mice

[0274] After grinding each tissue, total RNA was extracted and exon skipping analysis was performed using the same primers as in Example 12. PCR products were analyzed on 3% TAE agarose gels. The wild-type (WT) DMD product was 547 bp, the exon 22 skipping DMD product was 401 bp, the exon 23 skipping DMD product was 334 bp, and the double exon 22 and 23 skipping DMD product was 188 bp ( Figure 23 ). Agarose gel electrophoresis results were analyzed and quantified using Image Lab software, and histograms were plotted using GraphPad Prism 9 software.

[0275] The experimental results are shown in Figure 24. No exon skipping occurred in the PBS group. In the EXON22 PMO and EXON23 PMO (1:1) co-administration group (EXON22+EXON23, abbreviated as G2), essentially no double exon skipping was detected in any of the six organs or tissues. Only a small amount (approximately 5%) of double exon skipping was detected in muscle tissue at certain time points after administration.

[0276] Unexpectedly, the MAOC-002-administered group (G3) of the present invention showed very significant double exon skipping (approximately 10-20%) in all six muscle tissues sampled. This indicates that MAOC-002 successfully co-loaded with both EXON22 PMO and EXON23 PMO, and that MAOC-002 co-loaded with both EXON22 PMO and EXON23 PMO was significantly more effective in double exon skipping than the combined administration of EXON22 PMO and EXON23 PMO.

[0277] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A polypeptide-oligonucleotide polymer complex, characterized in that, The complex is a polymer formed by the complexation of n monomers with complementary nucleic acid backbones, where n is a positive integer from 2 to 8; Among them, the monomers include: (a) Polypeptide monomers, the polypeptide monomers include: (a1) polypeptide elements and (a2) a first backbone nucleic acid single strand; and the polypeptide element is covalently linked to the first backbone nucleic acid single strand; (b) Oligonucleotide monomers, the oligonucleotide monomers include: (b1) oligonucleotide pharmacodynamic elements and (b2) a second backbone nucleic acid single strand; and the oligonucleotide pharmacodynamic element is covalently linked to the second backbone nucleic acid single strand; and (c) Optionally, pure backbone monomers, the pure backbone monomers include a third backbone nucleic acid single strand, and the third backbone nucleic acid single strand is not covalently linked to a targeting element (such as an antibody) nor to a pharmacodynamic element (such as an oligonucleotide drug); Moreover, in the polymer, the backbone nucleic acid single strand of each monomer forms a complementary double strand with the backbone nucleic acid single strands of other monomers, thereby forming a complementary nucleic acid backbone complex structure; And a complex carries at least one (a1) polypeptide element and at least one (b1) oligonucleotide pharmacodynamic element.

2. The polypeptide-oligonucleotide polymer complex according to claim 1, wherein The polypeptide element is selected from the group consisting of: polypeptide elements with targeting functions, polypeptide elements for extending the half-life, or combinations thereof.

3. The polypeptide-oligonucleotide polymer complex according to claim 1, wherein, The polypeptide monomer has the structure of formula I: Z1-W1 (I) In the formula, Z1 is a polypeptide moiety; W1 is a first backbone nucleic acid single strand; "-" is a linker or a bond.

4. The polypeptide-oligonucleotide polymer complex according to claim 1, wherein The oligonucleotide monomer has the structure of formula II: Y1-W2-Y2 (II) In the formula, Y1 is none or a first oligonucleotide pharmacodynamic element; Y2 is none or a second oligonucleotide pharmacodynamic element; W2 is a second backbone nucleic acid single strand; "-" is a linker or a bond; wherein, Y1 and Y2 are not both none, and the first oligonucleotide pharmacodynamic element and the second oligonucleotide pharmacodynamic element are the same or different.

5. The polypeptide-oligonucleotide polymer complex according to claim 1, wherein, The pure backbone monomer has the structure of formula III: V1-W3-V2 (III) In the formula, V1 is none or a first detectable label group; V2 is none or a second detectable label group; W3 is a third backbone nucleic acid single strand; "-" is a linker or a bond.

6. The polypeptide-oligonucleotide polymer complex according to claim 1, wherein The complex contains the structure shown in formula i: (A-Y-)m-PNSC-(-Z-R)n (i) Wherein, A contains a polypeptide element with a targeting function; R contains an oligonucleotide pharmacodynamic element; PNSC is a complementary nucleic acid backbone complex structure; Y is a bond or a first linker; Z is a bond or a second linker; and m and n are each independently a positive integer ≥ 1.

7. The polypeptide-oligonucleotide polymer complex according to claim 6, wherein The PNSC contains the structure shown in formula 1: C1-R1-C2-R2-C3 (1) Wherein, R1 is a base complementary pairing region 1; R2 is a base complementary pairing region 2; C1, C2 and C3 are each independently none or redundant nucleic acids; "-" is a bond.

8. A monomer library, characterized in that, The monomer library includes: (a) The first monomer, the first monomer is a polypeptide monomer, which includes: (a1) polypeptide elements and (a2) a first backbone nucleic acid single strand; and the polypeptide element is covalently linked to the first backbone nucleic acid single strand; (b) A second monomer, which is an oligonucleotide monomer and includes: (b1) an oligonucleotide pharmacodynamic element and (b2) a second backbone nucleic acid single strand; and the oligonucleotide pharmacodynamic element is covalently linked to the second backbone nucleic acid single strand; and (c) A third monomer, which is a pure backbone monomer and includes a third backbone nucleic acid single strand, and the third backbone nucleic acid single strand is not covalently linked to a targeting element (such as an antibody) nor to a pharmacodynamic element (such as an oligonucleotide drug).

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: (i) The polypeptide-oligonucleotide polymer complex according to claim 1 as an active ingredient; and (ii) A pharmaceutically acceptable carrier.

10. Use of the polypeptide-oligonucleotide polymer complex according to claim 1, characterized in that, For the preparation of a drug for treating diseases caused by abnormal gene expression.

11. A set of single-stranded nucleic acid sequences for forming a complementary nucleic acid backbone composite structure of a hexamer, characterized in that, The set of single-stranded nucleic acid sequences is selected from the set of backbone nucleic acid single-strand sequences shown by the numbers 6-1 to 6-20 in Table B.

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