Peptide docking vector for targeted delivery of nucleic acids

Abstract

The present invention provides a peptide docking carrier composition comprising a therapeutic compound (such as an siRNA molecule) and a targeting ligand, and methods for preparing and using the same. The composition and method allow the targeted delivery of the therapeutic compound to the cells / tissues of a subject, wherein the subject can be an animal or a human, by connecting the targeting ligand to the compound to provide an enhanced therapeutic benefit.

Description

[0001] summary

[0002] The present invention provides a peptide docking carrier composition comprising a therapeutic compound (such as an siRNA molecule) and a targeting ligand, and methods for preparing and using the same. The composition and method allow the targeted delivery of the therapeutic compound to the cells / tissues of a subject, wherein the subject can be an animal or a human, by connecting the targeting ligand to the compound to provide an enhanced therapeutic benefit. Field of the Invention

[0003] The present invention relates to a delivery system comprising a single or multiple nucleic acids covalently linked to an endosomal release peptide and further coupled to at least one small molecule ligand. The present invention provides a new material composition, and methods for preparing and using the same. By selecting ligands and linkers, the delivery system can deliver nucleic acid molecules to the liver and other tissues.

[0004] Background of the Invention

[0005] Double-stranded RNA has been shown to silence gene expression (Farr et al., Nature. 391:806-811 (1998); El-Bashir et al., Nature. 411:494-498 (2001)), and this is referred to as RNA interference or "RNAi", or interfering RNA molecules (RNAi). RNAi regulation induced by short interfering RNA (siRNA) shows great potential in treating a variety of human diseases and other traditionally untreatable diseases. RNA-based therapeutics have three major advantages over traditional small molecule and antibody therapeutics. First, once designed for delivery to a specific cell type or tissue (e.g., delivery of siRNA / antisense oligonucleotides (ASOs) to hepatocytes; delivery of ASOs to the central nervous system (CNS)), it is very likely that every disease-causing gene in that cell type or tissue will be targeted. Second, RNA therapeutics can selectively target a single gene and can be easily designed to avoid regulating non-target genes, while small molecule inhibitors often hit multiple targets and have unknown off-target side effects. Third, unlike static small molecules and inhibitors, RNA therapeutics can change their sequence as a disease, whether cancer or pandemic flu, evolves. These properties offer great potential for RNA-based therapeutics to treat diseases for which there are currently no drug treatments, once delivery issues are properly addressed.

[0006] In this area, advances in ASO, RNAi, and mRNA chemistry have resulted in improved stability and avoidance of innate immune responses. Therapeutic ASO and RNA compounds allow for large-scale production via automated synthesizers at relatively low cost compared to antibody or small molecule development. However, two areas remain in need of further development: (a) targeting these molecules to specific cell types or tissues and, most importantly, (b) designing nontoxic endosomal escape agents. Dowdy, Nature Biotechnology. 35: 222-229 (2017).

[0007] Currently, there are two effective delivery methods for ASO or RNAi drugs on the market. One is lipid-based nanoparticles, namely liposomes, which contain multiple components; the other is the GalNAc molecule coupling method targeting ASGPR (asialoglycoproteins receptor, asialoglycoprotein receptor).

[0008] Delivery into cells remains a major challenge for RNA-based therapeutics, and all delivery routes ultimately lead to endosomal escape. Delivery of ASOs and siRNAs to the liver has been effectively addressed by GalNAc-siRNA conjugates targeting ASGPR. For unknown reasons, ASGPR has properties that are particularly suitable for the delivery of macromolecular drugs to hepatocytes, which express millions of ASGPRs on their cell surfaces, which circulate at an astonishing rate, every 10-15 minutes [Cummings R, McIver R. Glycobiology Fundamentals. Second Edition Chapter 31 Cold Spring Harbor Laboratory Press, 2009]. Fortunately, even if the endosomal escape rate is assumed to be less than 0.01%, the GalNAc delivery method is still effective. However, in addition to the liver, there is currently no clinically proven effective method to deliver oligonucleotides or RNA to tissues other than liver cells. Unfortunately, no other ligand-receptor system can express receptors at high levels like ASGPR or circulate into endosomes so quickly. One of the main reasons is that most cell surface receptors are expressed at levels between 10,000 and 100,000 (or lower), and caveolin- and clathrin-mediated endocytosis usually cycles once every 90 minutes. Giuliano RL, Delivery of therapeutic oligonucleotides. Nucleic Acids Res. 44: 6518-6548 (2016).

[0009] In addition to the success of GalNAc-RNA / ASO conjugates, endosomal escape remains a key issue that needs to be addressed in drug development, and this issue is also a problem that all RNA-based therapeutic drug development must address. This requires the development of new chemicals and materials to enhance endosomal escape so that cells or tissues other than liver cells can be targeted. The classic approach is to use small molecule endosomal lytic agents to destroy or lyse endosomes, such as chloroquine and similar molecules. However, at effective concentrations, these endosomal lytic agents will lyse all types of endosomes in the cell, resulting in significant toxicity.

[0010] Another approach to endosomal escape is to directly bind endosomolytic peptides or molecules to RNA, which will severely limit their effects on endosomes containing RNA therapeutics. Several clinical studies have used bimolecular dynamic polymer (DPC) systems containing cholesterol or membrane lytic functions to escape endosomal entrapment, but these clinical studies have been terminated due to toxicity. Woodall CL et al., Mol Ther. 21:973-985 (2013); Hou et al., Biotechnol Adv. 33:931-940 (2015).

[0011] The present invention provides an endosomal release peptide coupled to an oligonucleotide and a targeting ligand, the construct enhancing the escape of the oligonucleotide into the cytoplasm in a non-toxic manner. The construct of the present invention is referred to herein as a "peptide docking vector" (PDoV). Summary of the Invention

[0012] The present invention provides a chemical construct comprising a peptide docking vector (PDoV) covalently linked to (a) a targeting residue and (b) a first therapeutic oligonucleotide, and may further comprise a second therapeutic oligonucleotide that is the same or different from the first therapeutic oligonucleotide. Wherein, the PDoV may contain multiple repeating units of histidine and lysine. In one embodiment, the targeting residue binds to an asialoglycoprotein receptor (ASGPR); the oligonucleotide may comprise siRNA, an antisense oligonucleotide, miRNA, saRNA, shRNA, an aptamer, an RNase, a DNase, a decoy oligonucleotide or a CpG motif.

[0013] In these constructs, the PDoV construct may contain an endosomal release motif, which may contain at least two identical or different targeting residues, and / or, at least two identical or different therapeutic oligonucleotides.

[0014] The above construct may contain one of the structures shown below, wherein site X is used to bind a targeting ligand, site Y is used to bind an oligonucleotide, site X and site Y may be the same or different; A is a peptide sequence of H, K, R, HH, HHH, HHH, HHK, HHHK, or any other short peptide for endosomal release containing up to 5 amino acids, B is a peptide sequence of H, K, R, HH, HHH, HHH, HHK and HHHK, or any other short peptide containing up to 5 amino acids, any other amino acids, or a combination of amino acids and a linker, D is an oligonucleotide, R L is the ligand, R S A linker that binds to an oligonucleotide.

[0015]

[0016] D can be siRNA, mRNA or aptamer.

[0017] In these constructs, the above-mentioned PDoV peptide construct may have an amino acid sequence structure selected from the group consisting of KHHHCKH, HKHHHCKH, HHKHHHCKH, HHHKHHHKCHHHKHH, HHHKHHCKHHH, HHHKHHCRHHH, HKHHCKH, HKHCH, HKHCKH, HKHC, HHHK(S)HHCKHHH and HHK(S)HHKCHH(S)HHH, wherein R L Attached to the side chain of a cysteine ​​residue, R S Attached to the side chain of a lysine residue. Where (S) is:

[0018]

[0019] In other embodiments, the PDoV peptide construct may contain a structure as shown below, in which three ligands are individually attached to the polypeptide.

[0020]

[0021] Target residue R L It may contain a linker-1, wherein the linker-1 may contain one of the following structures:

[0022] Where n is 1, 2 or 3 and is passed through with OCH 2 The 1,5-triazole ring of the unit is connected to the bridge; or

[0023] Where n is 1, 2 or 3 and is connected with CH 2 OCH 2 The 1,5-triazole ring of the unit is connected to the bridge; or

[0024] Where n is 1, 2 or 3 and is connected with CH 2 OCH 2 The 1,5-triazole ring of the unit is connected to the bridge.

[0025] The linker between the ligand and the PDoV peptide may comprise a polyethylene glycol chain -(CH 2 CH 2 O)n- or -(CH 2 CH 2 )n-chain, wherein n is an integer between 2 and 15. Ligand R L The PDoV peptide may be linked via a linker which may comprise a thioether, amide or triazole linkage.

[0026] In other embodiments, the oligonucleotide is connected by a S -Linker 2-oligonucleotide structure residues are connected, wherein Linker 2 is an aliphatic chain, a polyethylene glycol chain, a hydrophobic lipid chain or a hydrophilic chain. The terminal group 2 is the reaction site for chemical binding with the siRNA end.

[0027]

[0028] The oligonucleotide can be an RNAi molecule of a single-stranded oligonucleotide of 10-29 bases in length, or can be a double-stranded RNAi molecule comprising two complementary single-stranded oligonucleotides, each of which is 10-29 bases or 19-27 bases in length. The oligonucleotide can comprise deoxyribonucleotides and / or ribonucleotides, and the oligonucleotide can comprise an siRNA molecule having at least one or more chemically modified nucleotides at the 2' position, for example, wherein the chemically modified nucleotides include 2'-O-methyl, 2'-F, 2'-O-methoxyethyl, 2'-O-allyl or 2'-H modified nucleotides.

[0029] In other embodiments, the oligonucleotide may comprise an siRNA molecule comprising at least one chemically modified linker that is:

[0030]

[0031] In other embodiments, the siRNA or oligonucleotide is further chemically modified at the 5' end or the 3' end with a linker 3, wherein the linker 3 comprises a complementary binding site group 3, which can covalently react with the linker 2 to link the ligand-PDoV and the nucleotide together.

[0032] In a further embodiment, the above construct may have a structure as shown below:

[0033]

[0034] In these constructs, the targeting ligand may be selected from the group consisting of N-acetylgalactosamine (GalNAc), galactose, galactosamine, N-acetalgalactosamine, N-propionylgalactosamine, N-butyrylgalactosamine and aptamers. In another embodiment, the targeting ligand is a peptide selected from the group consisting of cyclic (c) RGD, APRPG, NGR, F3 peptide, CGKRK, LyP-1, iRGD (CRGDRCPDC), iNGR, T7 peptide (HAIYPRH), MMP2 cleavable octapeptide (GPLGIAGQ), CP15 (VHLGYAT), FSH (FSH-p, 33-53 amino acids, YTRDLVKDPARPKIQKTF), LHRH (QHTSYkcLRP), gastrin releasing peptides (GRPs) (CGGNHWAVGHLM), RVG (YTWMPENPRPGTPCDIFTNRSGKRASNG), FMDV20 peptide sequence (NAVPNLRGDLQVLAQKVART), GLP or folic acid.

[0035] In any of these constructs, the peptide may comprise the amino acid sequence -HHK, -KHH, -HHH, -HHHK, -KHHH, -HHHHK, -KHHHH, -K(HHHK)n(n=1-8), -K(HHHHK)n(HHHK)m(n=1-4, m=1-8), K(HHHHK)n(HHHK)m-(n=1-4, m=1-8), for example, the construct may comprise a peptide having the sequence -K(HHHHK)n(HHHK)m or K(HHHHK)n(HHHK)m-, wherein n is 1 and m is 3. The peptide may be an endosomal releasing peptide or a cell penetrating peptide.

[0036] The present invention also provides a peptide having the formula (HnKm)oXpYq, wherein H is histidine, K is lysine, X and Y are functional units selected from amino acids and linkers, n is 1-10, m is 1-10, o is 1-10, p is 1-5, and q is 1-4. The peptide may include at least two binding sites for binding to a targeting ligand. Preferably, the binding site is lysine or cysteine. The peptide may include at least two binding sites for binding to an oligonucleotide. The construct may include the formula (HnKm)oXpYq n K m ) o X p Y qA peptide and at least one targeting ligand coupled to the peptide through at least one coupling site, and at least one oligonucleotide can be coupled to the peptide through at least one coupling site. The targeting ligand can be selected from the group consisting of cyclic (c) RGD, APRPG, NGR, F3 peptide, CGKRK, LyP-1, iRGD (CRGDRCPDC), iNGR, T7 peptide (HAIYPRH), MMP2 cleavable octapeptide (GPLGIAGQ), CP15 (VHLGYAT), FSH (FSH-p, 33-53 amino acids, YTRDLVKDPARPKIQKTF), LHRH (QHTSYkcLRP), gastrin releasing peptide (GRPs) (CGGNHWAVGHLM), RVG (YTWMPENPRPGTPCDIFTNRSGKRASNG), FMDV20 peptide sequence (NAVPNLRGDLQVLAQKVART) and GLP. The oligonucleotide may be selected from the group consisting of siRNA, antisense oligonucleotide, miRNA, saRNA, shRNA, aptamer, RNase, DNase, decoy oligonucleotide and CpG motif.

[0037] The present invention also provides a pharmaceutical composition containing the above construct and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier may include histidine-lysine-rich polypeptide polymer HKP, HKP(H) or liposomes, and / or may include water and one or more of the following salts or buffers: anhydrous potassium dihydrogen phosphate NF, sodium chloride USP, disodium hydrogen phosphate heptahydrate USP, glucose and phosphate buffered saline (PBS).

[0038] The present invention also provides a method for delivering an oligonucleotide to a cell, which method comprises contacting the cell with the above construct or pharmaceutical composition.

[0039] The present invention also provides a method for delivering an oligonucleotide to a hepatocyte, the method comprising contacting the cell with the above construct.

[0040] In these methods, the oligonucleotide may be a siRNA molecule and the oligonucleotide may be delivered to a cell or liver cell in a mammal, which may be a human cell.

[0041] The present invention also provides a method for gene therapy in a mammal, which comprises administering an effective therapeutic dose of the above construct to the mammal, wherein the mammal may be a human.

[0042] The present invention also provides a method for treating mammalian cancer, such as human cancer, comprising administering to the mammal an effective therapeutic amount of the above construct. The cancer may be a solid tumor, and may include liver cancer, colon cancer, or pancreatic cancer. The liver cancer may be hepatocellular carcinoma, metastatic colon cancer, or metastatic pancreatic cancer; the above construct may be directly injected into a tumor containing cancer cells.

[0043] BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 , G-PDoV technology: Design base map of [GalNAc] peptide docking vector, trivalent GalNAc is covalently bound to one docking site A, and oligonucleotide or siRNA is bound to another or two docking sites B respectively.

[0045] Figure 2 , G-PDoV technology: [ligand] The design base map of the peptide docking vector, the monovalent GalNAc is covalently bound to 1 to 4 docking sites A, and the oligonucleotide or siRNA is respectively bound to another one or two docking sites B.

[0046] Figure 3 The peptide docking vector (PDoV) is designed to be composed of multiple repeating units of histidine (H), lysine (K) and functional unit X (amino acid or functional linker) (H n K m ) o XY peptide backbone structure, where n = 1-10, m = 1-10, o = 1-10, p = 1-5, q = 1-5. HK repeating units have been shown to have good cell penetration ability and can promote endosomal release. Lysine or various functional units X are docking sites for binding ligands, and Y is a docking site for binding oligonucleotides through different covalent bonds. For example, site ① can only react in the presence of ligands such as GalNAc or other targeting ligands, and site ③ can only bind to oligonucleotides and siRNA under specific conditions.

[0047] Figure 4 The peptide docking vector (PDoV) with three ligand binding sites and multiple oligonucleotide sites is designed to be composed of multiple repeating units of histidine (H), lysine (K), functional units X and Y (amino acids or functional linkers) (H n K m ) o X p Y qPeptide backbone, where n=1-10, m=1-10, o=1-10, p=1-5, q=1-5. HK repeating units have been shown to have good cell penetration ability and can promote endosomal release. Lysine or various functional units X are docking sites for binding ligands, and Y is a docking site for binding oligonucleotides through different covalent bonds. For example, site ① can only react in the presence of ligands such as GalNAc or other targeting ligands, and site ③ can only bind to oligonucleotides and siRNA under specific conditions.

[0048] Figure 5 , the structure of the PDoV construct, the PDoV construct is a cell penetrating / endosome releasing peptide into which multiple binding sites X and Y are inserted, site X is used to bind a targeting ligand, and site Y is used to bind multiple oligonucleotides or nucleic acids. The following are some examples of PDoV constructs, A represents the sequence of K, R, H, HH, HHH, HHHH, HHK, HHHK or other short peptides, B represents the sequence of K, R, H, HH, HHH, HHHH, HHK, HHHK or other short peptides, other amino acids or a combination of amino acids and linkers, D represents oligonucleotides, siRNA, mRNA, aptamers, RL represents a ligand, and RS represents a linker bound to an oligonucleotide.

[0049] Figure 6 , Structural examples of first-generation PDoVs, which contain one or two oligonucleotide sites and one ligand coupling site.

[0050] Figure 7 , structural example of a second-generation PDoV, which contains two oligonucleotide sites and one multivalent ligand coupling site.

[0051] Figure 8 , an alternative structural example of PDoV, which comprises one or two oligonucleotide sites and a multivalent ligand binding site. The ligands can be coupled to the PDoV backbone one by one.

[0052] Fig.9A , the connection mode selection of the coupling site. The chemical group Rs represents the "click"-like reaction part with the oligonucleotide coupled to the PDoV peptide carrier, which can be amino, hydrazine, N-hydroxysuccinimide, azide, alkyne, carboxylic acid, thiol, maleimide and other chemically reactive parts.

[0053] Fig. 9B , representative examples of connection methods.

[0054] Fig. 9CThe linker 2 in the Rs-linker 2-siRNA conjugate is a chemical spacer between the peptide and the conjugation site, which enables the binding site to be attached to the terminal site of the linker. Linker 2 can be an aliphatic chain, a polyethylene glycol chain, other hydrophobic lipids or a hydrophilic chain, and the terminal group 2 is the reaction site for chemical bonding with the siRNA terminal.

[0055] Fig.10 , an example of the choice of connection mode for the ligand coupling site. L The connection mode of the ligand can be selected according to a monovalent GalNAc molecule, a divalent GalNAc molecule, or a trivalent GalNAc molecule, and the coupling site can be maleimide / thiol, or a structure selected from group 2 shown in FIG. 9 .

[0056] Fig.11 , Representative examples of siRNA-PDoV-ligand compound 1 structures.

[0057] Fig.12 , Representative examples of siRNA-PDoV-ligand compound 2 structures.

[0058] Fig.13 , Representative examples of siRNA-PDoV-ligand compound 3 structures.

[0059] Fig.14 , list of selected siRNA sequences and structural modifications.

[0060] Fig.15A In vitro serial dilution data for the mTTR1-PDoV3-GalNAc3 conjugate are shown.

[0061] Fig. 15B In vitro serial dilution data for the mTTR1-PDoV2-GalNAc3 conjugate are shown.

[0062] Fig. 15C Serial dilution data for the mTTR2-GalNAc3 positive control conjugate are shown.

[0063] Fig.15D In vitro serial dilution data for the Ahal-GalNAc3 positive control conjugate are shown.

[0064] Description of the Invention

[0065] definition:

[0066] As disclosed herein, "oligonucleotide" refers to a chemically modified or unmodified nucleic acid molecule (RNA or DNA) less than 100 nucleotides in length (e.g., less than 50, less than 30, or less than 25 nucleotides), which can be an siRNA, microRNA, antimicroRNA, microRNA mimic, dsRNA, ssRNA, aptamer, triple-stranded oligonucleotide. In one embodiment, the oligonucleotide is an RNAi active molecule.

[0067] As disclosed herein, "siRNA molecules" or "RNAi molecules" are double-stranded oligonucleotides, which are short double-stranded polynucleotides that interfere with the expression of genes in cells after the molecules are introduced into cells. For example, siRNA molecules target single-stranded target RNA molecules and bind to the target RNA molecules through the complementary nucleotide sequences therein. According to the practice in the art, when siRNA molecules recognize a specific nucleotide sequence, the sequence refers to the sense strand in the double-stranded molecule. One or more ribonucleotides that make up the molecule can be chemically modified by existing techniques. In addition to being modified at the level of one or more nucleotides, the backbone of the oligonucleotide can also be modified. Other modifications include the use of small molecules (such as sugar molecules), amino acids, polypeptides, cholesterol or other macromolecules to bind to the siRNA.

[0068] "Peptide Docking Vehicle" (PDoV) refers to a synthetic peptide made of a specific sequence of amino acids, which has multiple binding sites for binding to one or more targeting ligands and one or more oligonucleotides. The above-mentioned peptide docking vehicle contains functional groups, such as hydrophobic chains or pH-sensitive residues, which facilitate the release of oligonucleotides in the cell endosomes after PDoV is delivered to the cell.

[0069] The present invention provides compositions and methods for using interfering RNA molecules with enhanced therapeutic benefits. These compositions and methods enable targeted delivery of therapeutic compounds (such as siRNA molecules) to cells or tissues of a subject, which can be an animal or a human, by linking a targeting ligand to the compound.

[0070] In some embodiments, the targeting ligand can be conjugated to the endosomal release peptide by a bioorthogonal conjugation method. The targeting ligand can particularly improve the delivery of RNAi molecules to selected targets (such as the liver). In other embodiments, the targeting ligand allows the targeted delivery of RNAi molecules to other tissues, such as the skin and brain.

[0071] The targeting ligand may comprise one or more targeting residues and one or more connectors. The connector is covalently linked to the siRNA and the targeting ligand by click chemistry, thiol / maleimide chemistry or other bioorthogonal chemistry. The connector is preferably hydrophilic, for example, it may be a water-soluble flexible polyethylene glycol, which is sufficiently stable and limits the potential interactions between one or more targeting groups. Polyethylene glycol has been verified to be safe and compatible with the therapeutic purposes of clinical studies. In some embodiments, the connector may be a poly (L-lactic acid) n (where n = 5-20) of a determined molecular weight, wherein the ester bond is enzymatically active or hydrolytically active. The targeting ligand may include one or more targeting residues, one or more connectors having reactive linking residues, which are covalently linked to the siRNA and the targeting ligand by click chemistry, thiol / maleimide chemistry or other bioorthogonal chemistry. The reactive linking residues of the connector include, but are not limited to, thiol-maleimide connectors, alkynyl-azidotriazole connectors, and amine-NHS amide connectors. All of these connectors are suitable for covalently linking targeting ligands and therapeutic compounds.

[0072] In some embodiments, the targeting ligand disclosed herein comprises one or more targeting residues, one or more linkers having reactive linking residues. The linkers include thiol residues, maleimide residues, carboxylic acids, amines, azide groups or alkyne groups, and the like.

[0073] In some embodiments, the compounds disclosed herein that target specific RNAs can be directly bound to endosomal release docking peptides via the 3' or 5' end of the RNA, and targeting ligands (such as N-acetylgalactosamine) can also be bound to the same docking peptide in a compatible manner.

[0074] In some embodiments, the compounds disclosed herein that target specific RNA can also be directly bound to a targeting ligand (such as N-acetylgalactosamine), for example, to the 3' or 5' end of the RNA. In some embodiments, the RNA can contain one or more modified nucleotides, such as 3'-O-methyl, 3'-F or 3'-MOE. In some embodiments, the RNA can be an RNAi active molecule, such as a double-stranded RNAi active molecule. In some embodiments, the targeting ligand disclosed herein is connected to the 5' or 3' end of the sense strand of the double-stranded RNAi active molecule or the 5' or 3' end of the antisense strand of the double-stranded RNAi active molecule. Alternatively, the targeting ligand can be connected to the 3' / 3", 3' / 5' or 5' / 5' ends of both the sense strand and the antisense strand of the double-stranded RNAi active molecule.

[0075] The targeting ligand can be covalently bonded to the RNAi active molecule via the phosphate, phosphorothioate or phosphate group at the 3' or 5' end of the sense strand of the double-stranded RNAi active molecule. In some embodiments, the targeting specific RNA compound disclosed herein is a specific compound for inhibiting the mRNA expression of TGFβ1 or ApoB100.

[0076] PDoV enhances the escape of macromolecules into the cytoplasm in a non-toxic manner, which enables the effective delivery of RNAi therapy. The present invention provides an endosomal escape peptide (PDoV) that can promote the escape of macromolecules, such as siRNA molecules, into the cytoplasm in a non-toxic manner. Figure 1-4 Various examples of the PDoV platform are presented, in which the endosomal escape peptide acts as a docking site linker for both RNA and targeting ligand. In this approach, the endosomal escape peptide acts as a docking site linker for both ASO / RNA and targeting ligand, and multiple RNA molecules can be bound to the same construct to achieve the coordinated delivery of siRNA molecules against different target mRNAs, providing a synergistic benefit for silencing multiple disease-related genes. Histidine- and lysine-rich polypeptides or histidine- and lysine-rich linear peptides have been demonstrated to be effective cell penetrating and endosomal release agents when delivering RNA. The peptide contains a histidine-rich domain in which the imidazole ring of the histidine residue is protonated at low pH (pH < ~6) and acts as a proton sponge in the endosome, resulting in the lysis of the endosomal lipid bilayer to release the RNA. The binding sites on the PDoV are described in more detail below.

[0077] In the following sections, each component will be discussed in detail, including RNAi active molecules, targeting ligands, linkers between RNAi active molecules and peptides, linkers between ligands and peptides, and endosomal release docking peptides.

[0078] RNAi active molecules

[0079] The RNAi active molecule is a double-stranded compound, for example, the double-stranded siRNA can be anti-TGFβ1 or anti-ApoB100, which can be unmodified or have a 2'-O-CH 3 , 2'-F or 2'-O-MOE chemical modification, -P(O) at the 5' position 2 =S (phosphorothioate diester) modification, or other chemical modifications known in the art, such as gelation or lipid functionalization to improve the overall stability and bioavailability of RNAi.

[0080] In a specific embodiment, the double-stranded siRNA can be Fig.14A derivative duplex consisting of 24, 23, 22, 21, 20, 19, 18, 17 or 16 consecutive base pairs of any one or more duplexes.

[0081] Targeting ligands

[0082] The targeting ligand can be, for example, N-acetylgalactosamine (GalNAc), galactose, galactosamine, N-acetal galactosamine, N-propionylgalactosamine, N-butyrylgalactosamine, cRGD, GLP peptide or other small molecules. The targeting ligand is covalently linked to the peptide via a covalent bond, and the number of ligands can be 1, 2 or 3. The targeting ligand disclosed herein has the following structure:

[0083]

[0084] Linkers between RNAi active molecules and peptides

[0085] Linker Rs for ligand binding: The chemical group Rs can be various "click"-like reactive parts used to bind the oligonucleotide to the PDoV peptide vector. It can be an amino group, hydrazine, N-hydroxysuccinimide, azide, alkyne, carboxylic acid, thiol, maleimide or other chemically reactive residues known in the art. Representative examples are shown in Figure 9.

[0086] Coupled body R S The linker 2 in -linker2-siRNA is a chemical spacer located between the peptide and the binding site, which enables the binding site to be attached to the terminal site of the linker. Linker 2 can be an aliphatic chain, a polyethylene glycol chain, other hydrophobic lipids or a hydrophilic chain, and the terminal group 2 is the reactive site for chemical bonding with the end of the siRNA.

[0087] Linkers between ligand and peptide

[0088] The targeting ligand and RNAi molecular entity disclosed herein include a linker-1, which is directly connected to the siRNA and the bridge containing the linker 3-ligand, and is directly connected to the RNAi molecule through the 3' or 5' end of the sense strand. The spacer of the linker-1 is a structure selected from linear polyethylene glycol, where n can be the number of ethylene glycol units of 1-50, or a structure of poly (L-lactic acid), where n can be the number of ethyl ester repeating units of 1-50, or a unit repeating number with an average molecular weight between 100-3500.

[0089] The binding site can be maleimide / thiol or Fig.10 Select from the Group 2 list.

[0090] Endosomal release of docking peptides

[0091] Preferably, the peptide docking vector (PDoV) has a ligand binding site and multiple oligonucleotide sites. PDoV has a peptide backbone with a general structure: (H n K m ) o X p Y q , consisting of histidine (H), lysine (K) and multiple repeating units of functional units X and Y, wherein X or Y is an amino acid or selected from linkers 1 and Fig.10 Amino acid derivatives of the functional groups shown, where n = 1-10, m = 1-10, o = 1-10, p = 1-5, q = 1-5. It should be noted that Y in PDoV is not a tyrosine amino acid - but a defined functional amino acid or linker. HK repeating units have been shown to have good cell penetration and can promote endosomal release. Lysine residues or functional units X can serve as docking sites for ligand binding, while Y provides docking sites for binding oligonucleotides through different covalent linkages. Figure 3 Schematic diagram showing how PDoV is conjugated. For example, site ① can only react in the presence of ligands such as GalNAc or other targeting ligands, and site ③ can only bind to oligonucleotides and siRNA under specific conditions, such as Fig.10 Example shown.

[0092] Alternatively, PDoV may have three ligand binding sites and multiple oligonucleotide sites (see Figure 4 ): It is composed of multiple repeating units of histidine (H), lysine (K), functional units X and Y (amino acids or functional linkers) (H n K m ) o X p Y q Peptide backbone, where n=1-10, m=1-10, o=1-10, p=1-5, q=1-5. HK repeating units have been shown to have good cell penetration ability and can promote endosomal release. Lysine or various functional units X are docking sites for binding ligands, and Y is a docking site for binding oligonucleotides through different covalent bonds. For example, site ① can only react in the presence of ligands such as GalNAc or other targeting ligands, and site ③ can only bind to oligonucleotides and siRNA under specific conditions. See Figure 4 shown.

[0093] In the structural design, the PDoV construct is an endosomal-release peptide with multiple binding sites X and Y inserted, where site X is used to bind targeting ligands and site Y is used to bind multiple oligonucleotides or nucleic acids. Figure 5Some examples of PDoV constructs are shown, wherein A represents a sequence of K, R, H, HH, HHH, HHHH, HHK, HHHK or other short peptides, B represents a sequence of K, R, H, HH, HHH, HHHH, HHK, HHHK or other short peptides, other amino acids or a combination of amino acids and a linker, D represents an oligonucleotide, siRNA, mRNA or aptamer, RL represents a ligand, and RS represents a linker bound to an oligonucleotide. In some embodiments, the length of the peptide is a 5-15 amino acid sequence.

[0094] In some embodiments, the PDoV has Figure 6 The structure shown. Specific implementation plan

[0095] Example 1. Representative schematic examples of siRNA-PDoV-ligand compound structures, in which one or two siRNAs can be conjugated to a PDoV peptide.

[0096] Step 1. Azide modification of peptides

[0097]

[0098] Step 2. Coupling of targeting ligand to peptide

[0099]

[0100] Step 3 and Step 4. Coupling of Azide-PDoV-GalNAc3 to Oligonucleotides

[0101]

[0102] Example 2. Representative schematic examples of siRNA-PDoV-ligand structures.

[0103] Step 1. Azide modification of peptides

[0104]

[0105] Step 2. Coupling of targeting ligand to peptide

[0106]

[0107] Step 3 and Step 4. Coupling of Azide-PDoV-GalNAc3 to Oligonucleotides

[0108] Embodiment 3,

[0109] Synthesis and Characterization of Azide-PDoV1(1)

[0110] The azido-PDoV peptide (sequence HHH{LYS(PEG4-N3)}HHCKHHH) was synthesized by an automated peptide synthesizer using standard amino acids in the sequence and a lysine-PEG4-N3 modifier through outsourcing. The peptide was purified using C-18 reverse phase HPLC and characterized by mass spectrometry and H NMR ( 1 H NMR) characterization data were consistent with the expected structure.

[0111] Embodiment 4,

[0112] Synthesis of PDoV2 and Azide-PDoV2

[0113] Synthesis of PDoV2, whose sequence is HHHKHHCRHHH. The peptide PDoV2 (HHHKHHCRHHH) was synthesized by an automated peptide synthesizer using standard amino acids in the sequence through outsourcing services. The peptide was purified using C-18 reverse phase HPLC and characterized by mass spectrometry, and the analytical data were consistent with the expected structure.

[0114] Synthesis of Azide-PDoV2(3):

[0115]

[0116] The azide linker was connected to the peptide docking support 2 (PDoV2) by forming an amide bond between the ester activated carboxylic acid of the azide linker and the primary amine (2) of the lysine side chain of PDoV2 to form compound 3. PDoV2 peptide HHHKHHCRHHH (42 mg, 0.0280 mmol) was suspended in 1.0 mL DMF, triethylamine (39 uL, 10 eq) was added, and the mixture was stirred at room temperature for 20 minutes. A solution of azide-Peg4-NHS ester (54 mg, 0.140 mmol, 5 eq) dissolved in 20 uL of DMF was added to the reaction mixture. After 30 minutes, the reaction mixture slowly turned into a transparent solution and continued to stir at room temperature for 16 hours. The TLC pattern of the reaction mixture was monitored by HPLC pattern of complete conversion of PDoV2 ( Figure 1 ).

[0117] The reaction mixture was quenched with water (200uL), concentrated by rotary evaporator, and the crude extract was purified by HPLC on a semi-preparative RP-C18 column using a gradient of 10-90% buffer B (0.1% trifluoroacetic acid in acetonitrile). Azide-PDoV2 (2) was isolated as the main product with a retention time of 10.5-11.5 minutes. The sample fraction was freeze-dried to obtain a transparent residual oil of compound 2 (44 mg, yield 88%). The proton and mass spectrometry analysis results are as follows: 1 H NMR (400 MHz, D 2 Oh, Figure 2)δ8.74 (brd d, 8H) and δ7.35 (brd d, 8H) are consistent with the aromatic hydrogens associated with the 8 histidine tetrazoles in the peptide segment above δ6.00 ppm. The methylene hydrogens on the α carbons of all 11 amino acids at δ4.75-4.30 (brd t, 11H), the ethylene hydrogens associated with the polyethylene group at δ3.9-3.75 (m, 100H); the ethylene hydrogens at δ3.6-2.75 (m, 53H) and δ1.8-1.3 (m, 12H) are associated with the side chain protons of lysine, arginine and cysteine. In total, 104 hydrogens were observed in the non-aromatic region, with 41 excess hydrogens, which correspond to the 2 azide peg4 groups. The NMR data are consistent with the ESI-MS data: the predicted m / z is 1775.9 Da and the observed m / z is 2323.5 Da ( Figure 3 ), the observed mass-to-charge ratio is 547.6 Da units more than the predicted value, and the extra mass units are consistent with the masses of the two azide-linkers.

[0118] Embodiment 5,

[0119] Synthesis and Characterization of Azide-PDoV3 Peptide (4)

[0120] The azido-PDoV3 peptide (sequence ({LYS(PEG4-N3)})HHHCHH) was synthesized by solid phase automated synthesis using standard amino acids in the sequence and a lysine-PEG4-N3 modifier. The peptide was purified by C-18 reverse phase HPLC and characterized by mass spectrometry and 1 The H NMR characterization data were consistent with the expected structure.

[0121] Embodiment 6,

[0122] Synthesis and characterization of PDoV1-GalNAc3(5)

[0123]

[0124] Scheme 1: Synthesis of PDoV1-GalNAc3(5)

[0125] PEG6-GalNAc3(9) (3.0 mg, 1.56 μmol) in dry DMF (400 uL) was added to N3-phosphate buffer (1 mL, pH = 7.4) containing PDoV1 2 (3.54 mg, 2.03 μmol), and the resulting mixture was stirred at 25 ° C overnight under nitrogen. After removing the solvent under reduced pressure, the sample was desalted and purified by a PD-10 column to obtain pure PDoV1-GalNAc35 (5.1 mg, white solid, yield 90%). The product was analyzed by HPLC using a reverse phase C18 column and a gradient elution with 0.1% trifluoroacetic acid in acetonitrile. The retention time Rt = 4.877 min and the purity was greater than 90%. Mass spectrometry analysis (ESI, positive ion mode): C 154 H 240 N 48 O 55 The calculated value of S is 3673.7 and the observed value is 3674.8.

[0126] Embodiment 7,

[0127] Synthesis and characterization of PDoV1-GalNAc3 6 and 7

[0128]

[0129] Scheme 2: Preparation of PDoV2-linker-GalNAc3

[0130] Preparation of PDoV2-Peg6-GalNAc3 (Compound 6): The nucleophile, compound 2 (49.8 mg, 0.0243 mmol) was dissolved in 1.0 mL of degassed PBS buffer at pH 7.4, and the trivalent GalNAc ligand (9) (30.8 mg, 0.0160 mmol) was dissolved in 400 uL of dry DMF. The reaction mixture was degassed again under dry argon and stirred overnight at room temperature. The reaction mixture was quenched with water (100 μL) and then desalted using a 1.0 μmol Sephadex Nap column according to the guidelines recommended by Glen Research. The eluate was lyophilized and the crude extract was eluted by HPLC using a semi-preparative RP-C18 column using a gradient of 10-90% buffer B (0.1% trifluoroacetic acid in acetonitrile) ( Figure 7 The product had a retention time of 4.0 min and was isolated as an oil (39 mg, 60% yield). Mass spectrometry analysis of the modified oligonucleotide confirmed that the synthesis of the PDoV2-peg6-GalNAc3 construct was successful.

[0131] Preparation of PDoV2-Peg12-GalNAc3 (Compound 7): Compound 7 Azide-PDoV2 (4.9 mg, 2.74 μmol) was dissolved in 1.0 mL of degassed PBS buffer at pH 7.4. The GalNAc ligand (3.0 mg, 1.37 μmol) was dissolved in 400 uL of dry DMF. The reaction mixture was degassed again under dry argon and stirred overnight at room temperature. The reaction mixture was quenched with water (100 μL) and then desalted with a 1.0 μmol Sephadex Nap column according to the guidelines recommended by Glen Research. The eluate was lyophilized and the crude extract was eluted by HPLC on a semi-preparative RP-C18 column using a gradient of 10-90% buffer B (0.1% trifluoroacetic acid in acetonitrile) ( Figure 4 The product was formed as the major product with a retention time of 4.2 minutes and was isolated as a white powder (4.4 mg, yield 81%). Proton NMR and MS analysis: H NMR (400 MHz, D 2 O) δ7.94 (s, 3H, triazole), δ8.57-δ7.19 (m, 18H, 8-histidine aromatic hydrocarbons), δ4.75-4.30 (m, 14H, amino acids), δ4.5 (d, 3H, galactose), δ2.05 (s, 9H). NMR data are consistent with ESI-MS data. ESI-MS (positive ion mode, m / z): C 168 H 268 N 50 O 61 The calculated value of S is 3968.2 and the observed value is 3968.2.

[0132] Embodiment 8,

[0133] Synthesis of PDoV3-GalNAc3(8)

[0134]

[0135] Scheme 3: Synthesis of PDoV3-GalNAc3(8)

[0136] Synthesis of PDoV3-GalNAc3 (Compound 8): At 25°C, under nitrogen conditions, DMF (1.5 mL) containing compound 4 azide-PDoV3 (47.0 mg, 38.9 μmol) was added to a mixture of phosphate buffer (4 mL, pH 7.4) containing trivalent GalNAc (9) (50.0 mg, 25.9 μmol). The resulting reaction mixture was stirred at 25°C for 12 hours. The reaction was monitored by HPLC until GalNAc9 was completely consumed. The solvent was removed by lyophilization, and the crude extract was purified by gel permeation column chromatography PD-10 column to obtain pure PDoV3-GalNAc3 compound 8 (70 mg, yield 86.4%). Gradient elution was performed by HPLC on a reverse phase C-18 column using a gradient of 10-90% buffer B (0.1% trifluoroacetic acid in acetonitrile), with a retention time Rt = 5.038 minutes. MS (ESl, positive ion mode), formula C 130 H 207 N 37 O 51 The molecular mass of S is 3134.45, and the observed value is 3136.35. The analytical data are consistent with the expected structure.

[0137] Embodiment 9,

[0138] Synthesis of control 3-GalNAc3 (11)

[0139]

[0140] Scheme 4: Synthesis of control 3-GalNAc3 (11)

[0141] Preparation of PDoV3-control 3-GalNAc3:

[0142] Compound Azide-Control 3 Peptide 11 (sequence {LYS(PEG4-N3)}SSSCSS)) (2.6 mg, 2.59 μmol) was dissolved in 1.0 mL of degassed PBS buffer at pH 7.4, and GalNAc-ligand (5.0 mg, 2.59 μmol) was dissolved in 500 uL of dry DMF. The reaction mixture was degassed again under dry argon and stirred overnight at room temperature. The reaction mixture was quenched with water (100 μL) and desalted by passing through a 1.0 μmol Sephadex Nap column. Multiple eluent fractions were collected and lyophilized to obtain the desired compound Control 3-GalNAc3 10. The compound was analyzed using an analytical HPLC C18 RP column with a gradient of 10-90% buffer B (0.1% trifluoroacetic acid in acetonitrile), and the product had a retention time of 3.80 minutes and was isolated as a transparent oil (4.9 mg, yield 67%). Mass spectrometry analysis of the modified oligonucleotides confirmed the structure of the PDoV3-control3-peg6-GalNAc construct.

[0143] Embodiment 10

[0144] Synthesis and Characterization of ApoB100-PDoV1-GalNAc3(12)

[0145]

[0146] Scheme 5. Synthesis of ApoB100-PDoV1-GalNAc3 (12)

[0147] DMF (95 μL) containing PDoVl-GalNAc3 5 (381 μg, 0.104 μmol) was added to an anhydrous RNAse (250 μL) solution containing ApoB100-sense chain-5'-DBCO (500 μg, 0.06.9 μmol), and the resulting mixture was stirred at 25°C for 2 hours. After removing the solvent under reduced pressure, the crude extract was purified by Glen Gel PaK column chromatography to provide pure ApoB100-PDoV3-GalNAc3 compound 12 (0.66 mg, yield 88%). HPLC was performed on a PA200 ion exchange column using a phosphate buffer at pH = 11, Rt = 13.015 minutes, purity > 85%. MS (ESI, positive ion mode), formula C 388 H 510 N 125 O 208 P 22 The calculated value of S is 10911.66, and the observed value is 10929.3 [M+H 2O]. After 1:1 annealing with the ApoB100 antisense strand (95°C for 5 minutes, cooled to room temperature at a rate of about 1°C / min, and then stored at -20°C), the final conjugated double-stranded ApoB100-PDoV1-GalNAc3 (12) was obtained. The dye Alexa647-labeled ApoB100-PDoV1-GalNAc3 was prepared by replacing the ApoB100 antisense strand with the dye-labeled antisense strand ApoB100-Alexa647 (5'-uuuGfTaaucgucgauAfcccugcucg-Alexia647-3'). The product was characterized by HPLC and MS, and the analytical data were consistent with the expected structure.

[0148] Embodiment 11,

[0149] Synthesis and characterization of ApoB100-PDoV2-GalNAc3

[0150]

[0151] ApoB100-PDoV2-GalNAc3 13 was prepared by reacting PDoV2-GalNAc (6, or 7, 0.0341 μmol, 1.5 equiv.) with APOB100-SS-5'C6NHS (DBCO) (0.0227 μmol), and the preparation method was similar to that of ApoB100-PDoV1-GalNAc3 12. First, azide-PDoV2-GalNAc was dissolved in DMSO at a ratio of 1 mg azide per 150 μL DMSO; secondly, the obtained azide solution was added to 100 μL of anhydrous RNAse containing 10 OD of 5'-DBCO labeled oligonucleotide, and the reaction solution mixture was incubated at room temperature for 3-4 hours; then, the reaction mixture was incubated in a Glen Gel-Pak Tm Desalting was performed on a desalting column to remove the organic solvent and any unbound peptide. For example, when PDoV2-PEG12-GalNAc 7 was the reactant, the final ApoB100-PDoV2-GalNAc3 14 was a white solid with a yield of 77%. HPLC was performed on a PA200 ion exchange column using a pH = 11 phosphate buffer, Rt = 12.077 minutes, and the purity was > 85%. MS (ESI, positive ion mode) calculated value was 11551.4 and observed value was 11553.5. After 1:1 annealing with the antisense strand (95°C for 5 minutes, cooled to room temperature at a rate of about 1°C / min, and then stored at -20°C), the final conjugated double-stranded ApoB100-PDoV1-GalNAc3 (14) was obtained.

[0152] ApoB100-PDoV1-GalNAc3 labeled with dye Alexa647 was prepared by replacing the antisense strand of ApoB100 with the antisense strand labeled with dye ApoB100-Alexa647 (5'-uuuGfTaaucgucgauAfcccugcucg-Alexia647-3'). The product was characterized by HPLC and MS, and the analytical data were consistent with the expected structure.

[0153] Embodiment 12

[0154] Synthesis and characterization of ApoB100-PDoV3-GalNAc3 15

[0155]

[0156] PDoV3-GalNAc3 8 (150 μg, 0.148 μmol) was added to anhydrous RNAse (200 μL) containing 5'-DBCO-C6HN-APOB100 positive chain (230.7 μg, 0.0319 μmol). The mixture was stirred at 25°C for 3 hours, and after lyophilization to remove the solvent, the crude extract was purified with a Glen Gel Pak column to obtain pure compound 15 positive chain ApoB100-PDoV3-GalNAc3 (298 μg, yield 90%). HPLC was performed on a PA200 ion exchange column using a phosphate buffer at pH = 11, Rt = 12.002 minutes, and the purity was > 89%. 1:1 annealing with the antisense (95°C for 5 minutes, cooling to room temperature at a rate of approximately 1°C / min, and then storing at -20°C) provided the final doublet ApoB100-PDoV3-GalNAc3. After 1:1 annealing with the antisense strand (95°C for 5 minutes, cooling to room temperature at a rate of approximately 1°C / min, and then storing at -20°C), the final conjugated double-stranded ApoB100-PDoV1-GalNAc3 was obtained. Dye Alexa647-labeled ApoB100-PDoV1-GalNAc3 was prepared by replacing the ApoB100 antisense strand with the dye-labeled antisense strand ApoB100-Alexa647 (5'-uuuGfTaaucgucgauAfcccugcucg-Alexia647-3'). The product was characterized by HPLC and MS, and the analytical data were consistent with the expected structure.

[0157] Embodiment 13

[0158] Synthesis and characterization of mTTR1-PDoV2-GalNAc3 16

[0159]

[0160] mTTR1-PDoV2-GalNAc3 was prepared by using PDoV2-peg6-GalNAc 6 (0.547 mg, 0.1374 μmol) and DMSO / water solvent containing DBCO-labeled mTTR1 positive chain (0.5 mg, 0.0687 μmol) according to the steps similar to the synthesis of mTTR1-PDoV3-GalNAc3. The crude extract was purified by gel permeation column chromatography G25 column to obtain the pure product with a yield of about 78%. After 1:1 annealing with the antisense chain (95°C for 5 minutes, cooling to room temperature at a rate of about 1°C / min, and then stored at -20°C), the final conjugated double-stranded mTTR1-PDoV2-GalNAc3 (16) was obtained.

[0161] Embodiment 14

[0162] Synthesis and characterization of mTTR1-PDoV3-GalNAc3 17

[0163]

[0164] A DMSO (99.6 μL) solution containing PDoV3-GalNAc3 8 (996 μg, 0.317 μmol) was added to anhydrous RNAse (100 μL) containing mTTR1-sense chain-5-DBCO (1.54 mg, 0.212 μmol). The resulting mixture was stirred at 25°C for 2 hours. After the solvent was removed under reduced pressure, the crude extract was purified by gel permeation column chromatography PD-10 column to obtain pure sense chain mTTR1-PDoV3-GalNAc3 compound 17 (1.9 mg, yield 85%). HPLC was performed on a PA200 ion exchange column using a phosphate buffer at pH = 11, Rt = 14.744 minutes, and purity > 85%. After 1:1 annealing with the antisense strand (95°C for 5 minutes, cooled to room temperature at a rate of about 1°C / min, and then stored at -20°C), the final conjugated double-stranded mTTR1-PDoV3-GalNAc3 was obtained.

[0165] Example 15

[0166] In vitro studies of ApoB100-PDOVn-GalNAc3 conjugates:

[0167] In vitro cell uptake experiments were performed using Alexa647-labeled siRNA-GalNAc conjugates (ApoB100-PDOV1-GalNAc3-Alexa647, ApoB100-PDOV2-GalNAc3-Alexa647, ApoB100-PDOV2-GalNAc3-Alexa647), where Alexa 647-labeled siRNA delivered by Lipofectamine was used as a control group. In transfection experiments, ASGPR-overexpressing HepG2 cells and Huh7 cells were compared with wild-type cells. 5000 cells were seeded in 96-well plates overnight, and cells were transfected with 50nM siRNA for 2, 24, and 48 hours. The nuclei were stained with Hoechst dye, and the cells were imaged on a Cytation imaging station in PBS, and the images were taken at ×20 magnification. The data obtained showed that ASGPR-overexpressing HepG2 cells took up much greater amounts of fluorescently labeled substances than wild-type HepG2 cells lacking ASGPR overexpression.

[0168] ASGPR-overexpressing Huh7 cells also showed greater uptake compared to Huh7 cells without ASGPR receptor overexpression, but the labeling was not as intense as that of wild-type HepG2 cells.

[0169] Both PDoV1 and PDoV2 demonstrated delivery of labeled siRNA to ASGPR overexpressing HepG2 cells, but showed little or no labeling of wild-type cells. In the absence of GalNac on the construct, we did not see any uptake in ASGPR overexpressing cells.

[0170] In subsequent experiments, we examined the delivery of PDoV1, PDoV2, and PDoV3 in ASGPR-overexpressing Huh7 cells. PDoV2 and PDoV3 showed some specific uptake into these cells compared to wild-type cells. PDoV1 also showed some delivery into these cells, but not as great as PDoV2 and PDoV3, with PDoV2 showing significant uptake after 24 hours of contact.

[0171] Example 16

[0172] In vitro studies of mTTR-PDoVx-GalNAc in primary mouse hepatocytes:

[0173] Primary mouse hepatocytes were used to perform a dose response screening of GalNAc-coupled siRNAs (mTTR1-PDOV3-GalNAc3, mTTR1-PDoV2-GalNAc3, mTTR2-GalNAc3 positive control, mTTR2-GalNAc3 positive control) against the target TTR. The cell density was 40,000 cells / 96 wells, the concentration was diluted 5 times from the highest 1μM to the lowest 3pM, and the cells were transfected by direct incubation for 72 hours. The data were read out by bDNA detection and analyzed by XLfit software. The TTR signal was normalized to gapdh, the average of four replicates, and the mock-treated cells were set to 1, and XLfit was used to calculate the IC50 value. The primary hepatocytes used were purchased from Primacyt, Germany (lot number MH181219), from CD1 mice, and were treated with siRNA immediately after thawing.

[0174] Both siRNAs are conjugated to a trivalent GalNAc cluster at the 3' end, and the mTTR2 siRNA sequence is a fully modified sequence with four additional PS linker modifications on the oligonucleotide compared to mTTR1. (Nair et al., J Am Chem Soc. 136:16958-16961 (2014)). Ahal-GaNAc3 positive control was used as a standard control, targeting the housekeeping ahsa1. Cell lysates were hybridized with probes specific to the screening target or ahsa1, which were used as negative and positive controls. Table e1 lists the siRNA sequences and the structures of the GalNAc clusters for the two controls.

[0175] Table el. Control siRNA sequences and targets.

[0176]

[0177]

[0178]

[0179] Table e2. Summary of dose response of in vitro screening of GalNAc-coupled siRNAs

[0180] Compound <![CDATA[IC 50 (μM)]]> <![CDATA[IC 80 (μM)]]> Max.KD(%) mTTR1-PDoV3-GalNAc3 0.0018 0.0048 97％ mTTR1-PDoV2-GalNAc3 0.0026 0.0071 97％ mTTR2-GalNAc3 positive control 0.0001 0.0003 96％ Aha1-GalNAc3 positive control 0.0015 0.0059 96％

[0181] In vitro serial dilution data of the mTTR1-PDoV3-GalNAc3 conjugate: Fig.15A :IC 50 = 1.82 nM IC of the same siRNA sequence as reported in the literature21 and chemical modifications on the oligonucleotide (Nair et al., supra) 50 =1.39nM equivalent; Fig. 15BIn vitro serial dilution data of mTTR1-PDoV2-GalNAc3 conjugate are shown; Fig. 15C The in vitro serial dilution data of the mTTR2-GalNAc3 positive control conjugate are as follows: the mTTR2 siRNA sequence is a fully modified sequence with 4 additional PS-linked modifications on the oligonucleotide (Nair et al., supra); Fig.15D Figure 2 shows the in vitro serial dilution data of the Ahal-GalNAc3 positive control conjugate.

[0182] All publications listed herein, including issued or pending patents, and all database entries accessible by link address, code or other means, are hereby incorporated by reference in their entirety.

[0183] Although the present invention has been described with particular specific embodiments and many details are listed for the purpose of clarity, it is apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain specific details herein may be described in a variety of different forms without departing from the basic principles of the invention.

Claims

1. A chemical construct, characterized in that The chemical construct consists of a peptide docking vector covalently linked to a targeting ligand and an oligonucleotide, wherein the peptide docking vector is azide-PDoV1: HHH{LYS(PEG4-N3)}HHCKHHH, azide-PDoV2: HHH{LYS(PEG4-N3)}HHCRHHH or azide-PDoV3: {LYS(PEG4-N3)}HHHCHH, the targeting ligand is linked to the side chain of the cysteine ​​residue of the peptide docking vector, the oligonucleotide is linked to N3 of LYS(PEG4-N3) of the peptide docking vector, and the targeting ligand is N-acetylgalactosamine.

2. The chemical construct according to claim 1, characterized in that The oligonucleotide comprises siRNA, antisense oligonucleotide, miRNA, saRNA, shRNA, aptamer or decoy oligonucleotide.

3. The chemical construct according to claim 2, characterized in that The oligonucleotide is a siRNA or miRNA molecule.

4. The chemical construct according to claim 1, characterized in that The chemical construct comprises at least two identical or different targeting ligands, and / or, at least two identical or different oligonucleotides.

5. The chemical construct according to claim 1, characterized in that The targeting ligand comprises a connector-1, wherein the connector-1 comprises one of the following structures: , wherein n is 1, 2 or 3 and is connected to the bridge via a 1,5-triazole ring bearing a CH2OCH2 unit; or , wherein n is 1, 2 or 3 and is connected to the bridge via a 1,5-triazole ring bearing a CH2OCH2 unit; or , where n is 1, 2 or 3 and is connected to the bridge via a 1,5-triazole ring with a CH2OCH2 unit.

6. The chemical construct according to claim 5, characterized in that The targeting ligand further comprises a polyethylene glycol chain -(CH2CH2O) n -or-(CH2CH2) n -chain, where n is an integer between 2 and 15.

7. The chemical construct according to claim 6, characterized in that The targeting ligand has the following structure: 。 8. The chemical construct according to claim 1, characterized in that The oligonucleotide is provided by a S -Linker 2-oligonucleotide structure residue connection, wherein Linker 2 is an aliphatic chain, a polyethylene glycol chain, a hydrophobic lipid chain or a hydrophilic chain, and the group 2 at the end of Linker 2 is a reaction site for chemical binding with the end of siRNA.

9. The chemical construct according to claim 8, characterized in that 。 10. The chemical construct according to claim 8, characterized in that Wherein linker 2 is: ， Group 2 is: 。 11. The chemical construct according to claim 1, characterized in that The oligonucleotide is a single-stranded oligonucleotide RNAi molecule with a length of 10-29 bases.

12. The chemical construct according to claim 11, characterized in that The oligonucleotide is a double-stranded RNAi molecule comprising two complementary single-stranded oligonucleotides, each of which has a length of 10-29 bases or 19-27 bases.

13. The chemical construct according to claim 1, characterized in that The oligonucleotide comprises deoxyribonucleotides.

14. The chemical construct according to claim 1, characterized in that The oligonucleotide comprises ribonucleotides.

15. The chemical construct according to claim 1, characterized in that The oligonucleotide comprises deoxyribonucleotides and ribonucleotides.

16. The chemical construct according to claim 1, characterized in that The oligonucleotide comprises a siRNA molecule having at least one or more chemically modified nucleotides at the 2' position.

17. The chemical construct according to claim 16, characterized in that The chemically modified nucleotides include 2'-O-methyl, 2'-F, 2'-O-methoxyethyl, 2'-O-allyl or 2'-H modified nucleotides.

18. The chemical construct according to claim 8, characterized in that The oligonucleotide comprises an siRNA molecule having at least one chemically modified linker, wherein the linker is: 。 19. The chemical construct according to claim 8, characterized in that The oligonucleotide is chemically modified with a connector 3 at the 5' or 3' end, and the structure is as follows: Wherein, the linker 3 comprises a complementary binding site group 3, and the group 3 can covalently react with the linker 2 to connect the ligand-PDoV and the nucleotide together.

20. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the chemical construct of any one of claims 1 to 19 and a pharmaceutically acceptable carrier.

21. The pharmaceutical composition according to claim 20, characterized in that The pharmaceutically acceptable carrier comprises histidine-lysine-rich polypeptide polymer HKP, HKP(H) or liposome.

22. The pharmaceutical composition according to claim 20, characterized in that The pharmaceutically acceptable carrier comprises water and one or more of the following salts or buffers: anhydrous potassium dihydrogen phosphate, sodium chloride, disodium hydrogen phosphate heptahydrate, dextrose and phosphate-buffered saline.

23. Use of a chemical construct as claimed in any one of claims 1 to 19 for the preparation of a medicament for delivering an oligonucleotide to a cell.

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