CN117881427A - Conjugates of saponins, oligonucleotides and GALNAC - Google Patents

Abstract

The present invention relates to a conjugate comprising a saponin covalently linked to a ligand of ASGPR and comprising an oligonucleotide covalently linked to the saponin and the ligand of ASGPR, wherein the ligand comprises at least one GalNAc moiety. Furthermore, the present invention relates to pharmaceutical compositions comprising the conjugates of the invention. Furthermore, the present invention relates to a pharmaceutical composition of the present invention for use as a medicament. The invention also relates to a pharmaceutical composition of the invention for use in the treatment or prevention of diseases or health problems wherein the expression product involves, for example, the following genes: apoB, HSP17, TTR, PCSK9, ALAS1, AT3, GO, CC5, HBV X gene, HBV S gene, AAT and LDH, and for use in the treatment or prevention of, for example, cancer, infectious diseases, viral infections, hypercholesterolemia, primary hyperoxalic acid urea, hemophilia a, hemophilia B, AAT-associated liver disease, acute hepatoporphyria, TTR amyloidosis, complement-mediated diseases, hepatitis b infection or autoimmune diseases. The invention also relates to an in vitro or ex vivo method of transferring the oligonucleotide conjugate of the invention from outside the cell to inside said cell.

Description

Conjugates of saponins, oligonucleotides and GALNAC

Technical Field

The present invention relates to an oligonucleotide conjugate comprising a saponin covalently linked to an ASGPR ligand (ASGPR ligand) and further comprising a covalently linked oligonucleotide, i.e. (saponin, oligonucleotide, ASGPR ligand) conjugate comprising a saponin covalently linked to an ASGPR ligand comprising at least one GalNAc, wherein the saponin-oligonucleotide-ASGPR ligand conjugate further comprises a covalently bound oligonucleotide, such as an siRNA or an antisense oligonucleotide, referred to as an oligonucleotide conjugate of the invention. Furthermore, the present invention relates to a pharmaceutical composition of the invention comprising said saponin-oligonucleotide-ASGPR ligand conjugate, for use as a medicament. The invention also relates to a pharmaceutical composition of the invention for use in the treatment or prevention of diseases or health problems wherein the expression product involves, for example, the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AT3, GO, CC5, X gene of HBV, S gene of HBV, AAT, miR-122, hepatitis b virus HbsAg, LDHA, CEBPA and LDH, for use in the treatment or prevention of: such as cancer, infectious disease, viral infection, hypercholesterolemia, cardiovascular disease, primary hyperoxaluria, hemophilia a, hemophilia B, AAT-associated liver disease, acute hepatic porphyria, TTR-mediated amyloidosis, hereditary TTR amyloidosis (hATTR), complement-mediated disease, hepatitis b infection, hepatitis c infection, alpha 1-antitrypsin deficiency, beta-thalassemia, or autoimmune disease. The invention also relates to a method for producing the oligonucleotide conjugates of the invention. Finally, the present invention relates to an in vitro or ex vivo method for transferring the saponin-oligonucleotide-ASGPR ligand conjugate of the invention (also referred to as the oligonucleotide conjugate of the invention) from outside the cell into said cell.

Background

The ability to inhibit protein function, both in humans and in pathogens, is an integral part of new drug discovery. Traditional small molecule drugs and antibodies are strongly limited to a subset of available targets. The small molecules bind primarily to cofactor sites or transmitter sites, which are effective sites designed to accommodate the small molecules. Antibodies can bind to a wider variety of proteins, but are typically limited to extracellular targets.

Oligonucleotide therapy is a relatively young and rapidly evolving field aimed at overcoming many of the problems encountered with small molecule drugs or antibody drugs by directly manipulating the gene transcription and translation pathways. The potency and versatility of oligonucleotides, particularly the prospect of inhibiting genes encoding proteins that are "non-drug-forming" as classical small molecule drugs, makes them attractive drug candidates. The first lot of oligonucleotide drugs are based on antisense technology whereby single stranded nucleic acid molecules bind their complementary mRNA targets in a sequence specific manner, triggering degradation of the duplex by the rnase H system.

In 1998, andrew Fire and Craig Mello published an original paper that identified double-stranded RNA (dsRNA) as the cause of post-transcriptional gene silencing (PTGS) in caenorhabditis elegans (Caenorhabditis elegans), which phenomenon was called RNA interference (RNAi). RNAi findings explain the confusing observations of gene silencing in plants and fungi and have led to a revolution in biology, ultimately suggesting that non-coding RNA is a central regulator of gene expression in multicellular organisms. Shortly thereafter, it was found that small dsrnas (typically 15-30 base pairs (bp)) could catalytically induce RNAi silencing in mammalian cells without eliciting a non-specific interferon response. Targeting the RNAi pathway by small dsRNA such as small interfering RNAs (sirnas) and short hairpin RNAs (shrnas) has several theoretical advantages over antisense, which is significantly more efficient (catalytic) and produces longer inhibition of gene expression. This may translate into lower doses and lower costs and less frequent dosing. Lower exposure may also mean fewer siRNA toxicity issues.

However, before siRNA reaches its in vivo target, it faces a number of significant obstacles that hinder its way to the RNA-induced silencing complex (RISC) mechanism. After siRNA enters the blood stream, it is easily degraded by endogenous nucleases and excreted by the kidneys due to its small size and highly anionic nature. In addition, the siRNA must be tightly endothelial linked through the blood vessels and diffuse through the extracellular matrix before reaching its target cells. Because siRNA has a large negative charge, it does not readily bind to or cross cell membranes, and once inside a cell, it must escape from the endosome to interact with its intracellular protein targets.

Thus, the success of oligonucleotide (e.g., siRNA) therapies is greatly dependent on the efficient delivery of drugs from outside the cell into the cytoplasm. Accordingly, much attention has been focused on developing delivery systems that improve the delivery of oligonucleotides (e.g., siRNA and antisense oligonucleotides). In addition to viral delivery, the primary method for enhancing delivery of siRNA (or other oligonucleotides) to cells uses liposomes, cell Penetrating Peptides (CPPs) and their mimics, or nanoparticles (good, matt et al, chemical biology & drug design [ Chemical biology and drug design ]80.6 (2012): 787-809). Safety issues such as insertional mutation and the possibility of immune development are believed to limit the future of viral methods.

Liposomes are the most commonly used delivery vehicles for oligonucleotides (e.g., siRNA), wherein the oligonucleotide is encapsulated within a lipid bilayer. Liposome oligonucleotide delivery suffers from several problems, such as oxygen radical mediated toxicity (typical for cationic liposomes), cytotoxicity, effects on gene regulation, and inflammatory responses. Furthermore, in vivo delivery using lipids appears to target primarily the liver and spleen.

Cell penetrating peptides (CCPs), also known as protein transduction domains, are short peptides (typically <30 amino acid residues in length) with specific properties that are able to cross the cell membrane. CPP can enhance cellular uptake of an oligonucleotide by covalent attachment to the oligonucleotide or by forming a non-covalent complex with the oligonucleotide. The field of CPP-mediated oligonucleotide delivery is extremely complex because it combines challenges presented by both oligonucleotide technology and peptide technology (both fields are still far from mature) in a single drug. In addition to the problems associated with oligonucleotides, typical challenges encountered by CPPs relate to the in vivo stability (lack) of peptide chains, often requiring the use of non-natural peptide derivatives that may be complex to synthesize and/or exhibit reduced cell penetrating activity.

Despite the enhanced cell delivery, overcoming endosomal entrapment is one of the major challenges in designing efficient CPPs and other transport systems (good et al).

Nanoparticles and nanocarriers are nanoscale oligonucleotide delivery systems, typically composed of a polymer, a biostabilizer, and a cell targeting ligand complexed with an oligonucleotide. An exemplary siRNA nanocarrier system is known under the name siRNA dynamic immunoconjugates. The system is based on an amphiphilic polymer linked to polyethylene glycol (PEG) as a biostabilizer and a hepatocyte-targeting ligand, wherein the siRNA is covalently bound to the polymer via disulfide bonds. The targeting moiety and the PEG moiety are attached to the polymer via a maleamide linkage, forming negatively charged nanoparticles that do not bind to serum proteins. After internalization by endocytosis, the maleamide bond readily hydrolyzes upon acidification of the endosome, exposing the cationic amine groups of the polymer and inducing endosome escape via proton sponge effect. Nanocarriers are also known to be in the form of cationic polymers such as Polyethylenimine (PEI), sometimes in combination with cyclodextrins, which can form electrostatic complexes with oligonucleotides such as siRNA, provide protection against degradation and aid internalization. However, the high concentration of toxicity resulting from membrane disruption and apoptosis induction may limit the use of cationic polymers as therapeutic delivery agents. In addition to the complex preparation of nanoparticles and nanocarriers, their long-term stability is a major obstacle to commercial availability.

Oligonucleotides can be used to modulate gene expression through a range of processes including RNAi, target degradation by rnase H mediated cleavage, splice modulation, non-coding RNA inhibition, gene activation, and procedural gene editing. Oligonucleotides are nucleic acid polymers that have the potential to treat or control a variety of diseases. While most oligonucleotide therapies focus on gene silencing, other strategies are under investigation, including splice regulation and gene activation, expanding the range of possible targets beyond what is typically achievable with conventional drug modalities.

Examples of drug oligonucleotides are single-stranded antisense oligonucleotides and double-stranded short interfering RNAs (sirnas), which have the same rationale: the oligonucleotide binds to the target RNA by watson-crick base pairing, and the resulting duplex directs degradation of the target messenger RNA (mRNA). In the cytoplasm (in the case of siRNA) or in the cell nucleus (in the case of antisense oligonucleotides), the oligonucleotides can modulate expression of homologous RNAs. Antisense oligonucleotides (ASOs) are small (-18-30 nucleotides), synthetic, single stranded nucleic acid polymers with a variety of chemical properties that can be used to regulate gene expression by a variety of mechanisms. ASOs can be divided into two main categories: rnase H-competent (rnase H command) and sterically hindered (steric block). The sterically hindered oligonucleotide is an ASO intended to bind to the target transcript with high affinity, but does not induce degradation of the target transcript due to the lack of rnase H ability. Sterically hindered oligonucleotides can mask specific sequences in the target transcript, thereby interfering with transcript RNA-RNA and/or RNA-protein interactions. siRNA molecules are effector molecules of RNAi and typically consist of a characteristic 19+2mer structure (i.e., a duplex of two 21-nucleotide RNA molecules with 19 complementary bases and a terminal 2-nucleotide 3' overhang). One strand (the guide or antisense strand) of the siRNA is complementary to the target transcript, while the other strand is referred to as the passenger or sense strand. The function of siRNA is to direct Argonaute2 protein (AGO 2), which is part of the RNA-induced silencing complex (RISC), to a complementary target transcript. Complete complementarity between the siRNA and the target transcript results in cleavage (i.e., slicing activity) of the guide strand at target relative positions 10-11, catalyzed by AGO2, resulting in gene silencing. The siRNA methods include Dicer substrate siRNA, small internal segmented siRNA, self-delivered siRNA (asymmetric and hydrophobic), single stranded siRNA, and bivalent siRNA. Sterically hindered ASOs competitively inhibit mirnas by directly binding to small RNA species in the RISC complex. Such ASOs are known as anti-miRNA oligonucleotides, anti-miR or antagomers. The first anti-miRNA drug entering the clinical trial was miravirsen (SpC 3649), an ASO, aimed at treating chronic Hepatitis C Virus (HCV) infection by targeting liver-specific mirnas mir-122. Other classes of oligonucleotide therapies modulate RNA function by binding to splice sites on pre-mRNA, which results in-for example-skipping of exons that contain mutations in diseases such as muscular dystrophies.

Other examples of oligonucleotides for clinical use are micrornas (mirnas) which are involved in endogenous RNAi triggers in the following, such as cancer, cell cycle progression, infectious diseases, immunity, diabetes, metabolism, myogenesis and muscular dystrophy. The miRNA hairpins embedded in long primary miRNA transcripts are processed sequentially by two rnase III family enzymes DICER1 (DICER) and DROSHA, which release the hairpins and then cleave the loop sequence, respectively. The resulting double stranded RNA (similar to siRNA) is loaded into an Argonaute protein (e.g. AGO 2) and one strand is discarded to yield a mature single stranded miRNA species. As with siRNA, mirnas direct RISC to target sequences where they initiate gene silencing. In contrast to siRNA, mirnas typically bind with partial complementarity and induce silencing by a slice-independent mechanism.

Therapeutic oligonucleotides are typically 15 to 30 nucleotides in length and are intended to complement or modulate a messenger RNA (mRNA) encoding a disease-associated protein. Following parenteral administration, the oligonucleotides enter the cells and bind to any complementary RNA. Once an oligonucleotide drug binds to its complementary mRNA or pre-mRNA, a series of events occur. The outcome depends in part on the nature of the sequence targeted and comprises the destruction of mRNA by: enzymatic cleavage (which is helpful when the mRNA is mutated and encodes a pathogenic protein), altering the pre-mRNA splice pattern (which is helpful when the "default" splice pattern produces a pathogenic product), or a change in the function of the regulatory RNA.

An example of a drug molecule comprising an oligonucleotide is an siRNA-GalNAc conjugate, wherein the siRNA moiety targets PCSK9 enzyme. This enzyme binds to and degrades low density lipoprotein receptor (when bound to low density lipoprotein) and is a target for the treatment of cardiovascular disease. Another example is GalNAc conjugate comprising an antisense oligonucleotide against apolipoprotein (a) expressed in the liver. Furthermore, the gene DMD is also a target for oligonucleotide-based drug molecules. Duchenne muscular dystrophy is a uniformly fatal disease caused by a mutation in the gene DMD encoding duchenne muscular dystrophy. Spinal muscular atrophy is an autosomal recessive genetic disease caused by mutations in SMN1 that result in loss of SMN1 protein function. The target of oligonucleotide-based therapies is the gene SMN2.

Oligonucleotide drugs that use sequence-driven cleavage mechanisms to reduce the levels of disease-associated mRNA and its protein products are, for example, mipramine (mipomersen) and incarviland (incarvian), which use cleavage mechanisms to alter cholesterol distribution. The two drugs each target a different gene product, each of which is important in hypercholesterolemia. This results in cleavage of the target mRNA at the hybridization site due to activation of endogenous enzymes as a common factor for each mechanism, as a result of hybridization of each oligonucleotide drug to the target. Milpomace is a single stranded oligonucleotide whose sequence is complementary to a portion of the RNA encoding apolipoprotein B (apoB), a component of Low Density Lipoprotein (LDL) cholesterol produced in the liver. When miphene hybridizes with the pre-mRNA of apolipoprotein B, the presence of the DNA-RNA heteroduplex attracts and activates rnase H, which cleaves the mRNA in the heteroduplex. Cleavage inactivates the apolipoprotein B mRNA, thereby reducing the amount of apolipoprotein B produced. As a result, very low density lipoprotein cholesterol output from the liver decreases, and eventually, the circulating level of LDL cholesterol decreases. Inkexin induces cleavage of mRNA encoding proprotein convertase subtilisin-kexin type 9 (PCSK 9), an enzyme that down-regulates LDL receptor (LDLR) levels. A human with naturally occurring genetic variation that reduces PCSK9 activity has increased LDLR levels, reduced LDL cholesterol levels, and reduced cardiovascular risk compared to a human without these variations. The infliximab cleaves and inactivates PCSK9 mRNA, which has the effect of lowering PCSK9 levels and thus increases LDLR levels and LDL cholesterol clearance, and lowers LDL cholesterol circulating levels. Infliximab is a double-stranded small interfering RNA (siRNA). One RNA strand of infliximab is complementary to a portion of PCSK9 mRNA. Once inflicted into the cell, the complementary strand (or guide strand) is loaded into the RNA-induced silencing complex (RISC), a protein complex that displays the strand to the intracellular environment. Once the nearly fully complementary sequence (within the mRNA molecule) hybridizes to a portion of the guide strand, the enzyme that is part of RISC cleaves the mRNA. mRNA cleavage products cannot be translated and PCSK9 protein levels are therefore reduced.

Oligonucleotide-based drugs that trigger rnase H-mediated and RISC-mediated cleavage (inotersen and patisiran, respectively) are being used to Treat Transthyretin (TTR) amyloidosis. In this form of amyloidosis, mutations in TTR can lead to misfolding of the protein product, leading to the formation of amyloid deposits in a variety of tissues including peripheral neurons and the heart.

FDA approved liver targeting oligonucleotide therapeutics are miphene (targeting gene apoB for the treatment of homozygous familial hypercholesterolemia), defibrinode (hepatic vein occlusion disease), patosiran TTR (hereditary thyroxine transporter amyloidosis, polyneuropathy), inotersen TTR (hereditary thyroxine transporter amyloidosis, polyneuropathy), and givosiran. Givosiran is GalNAc conjugate for use in the treatment of acute hepatic porphyria; the target gene is ALAS1. Oligonucleotide candidates targeting genes in the liver in clinical development are for example miravirsen targeting miR-122 (hepatitis c infection), RG-101 targeting miR-122 (hepatitis c infection), AB-729 targeting hepatitis b virus HBsAg (hepatitis b infection), ARO-AAT targeting AAT (alpha 1-antitrypsin deficiency), SLN124 targeting the gene TMPRSS6 (beta-thalassemia), DCR-PHXC targeting LDHA (primary hyperoxalic acid) and MTL-CEPBA targeting CEBPA (hepatocellular carcinoma).

Despite considerable advances, there are two major obstacles that have hampered the widespread use of oligonucleotide therapies: drug safety and delivery. The inability to deliver oligonucleotide drug candidates to organs and cells expressing disease-related RNAs has proven to be the greatest obstacle to successful development of oligonucleotide therapeutics. Oligonucleotides are much larger than traditional small molecule drug candidates. Their size, coupled with their highly anionic nature, makes them difficult to diffuse across the cell membrane to the cytoplasmic and nuclear compartments. Oligonucleotides are typically large hydrophilic polyanions (single stranded ASO is-4-10 kDa, double stranded siRNA is-14 kDa), meaning that they do not readily cross the plasma membrane. Furthermore, difficulties arise when considering the adequate release and delivery of the oligonucleotides in the cytoplasm and/or nucleus, when considering that the oligonucleotides escape from the lysosomal system before lysosomal degradation or are re-exported by exocytosis in order to be able to reach the correct intracellular site of action. Current oligonucleotide cell delivery strategies are inefficient, resulting in drug excretion from the kidneys or delivery of a large portion of the drug to cells and tissues that are not therapeutically significant. Although some of the above delivery systems may enable improved oligonucleotide (e.g., siRNA) delivery of cells, endosomal escape remains a major obstacle to oligonucleotide-based therapeutics (e.g., RNAi-based therapeutics). Only a very small portion of the endocytic oligonucleotide escapes into the cytoplasmic space where it can perform its intended function, while the vast majority of the endocytic oligonucleotides remain trapped in the endocytic compartment and are inactive. Recent literature suggests that the passive siRNA escape rate is <0.01% (Setten, ryan L. Et al, nature Reviews Drug Discovery [ Natural review drug discovery ]18.6 (2019): 421-446). Furthermore, the ability of cells to functionally internalize oligonucleotides to produce a target effect (e.g., knockdown) has been demonstrated to appear independent of the extent to which a large number of oligonucleotides are internalized by the cell. These observations led to the hypothesis of separate "productive" and "non-productive" free uptake pathways, although the molecular mechanisms that distinguish these pathways remain unclear (Setten, ryan l., john j. Rossi, and Si-ping han. Nature Reviews Drug Discovery [ natural review drug discovery ]18.6 (2019): 421-446).

In summary, there remains an urgent need for improved oligonucleotide delivery systems that produce increased potency (e.g., target knockdown), less toxicity, and/or less off-target effects, regardless of the underlying mechanism (improved endosomal escape, increased "productive" pathway uptake, or another mechanism).

An emerging strategy requires conjugation of antisense oligonucleotides (ASOs) to receptor ligands to increase the efficacy of the oligonucleotides and distribution to selected tissues. For example, conjugation of tri-antennary N-acetylgalactosamine (GalNAc) to oligonucleotide therapeutic agents results in a 10-30 fold increase in potency in isolated hepatocytes as well as in vivo liver. GalNAc was found on damaged glycoproteins, which lost terminal sialic acid residues from their dangling oligosaccharides. The liver is protected by a very high level on the surface of hepatocytes (10 ⁵ -10 ⁶ Order of magnitude/cell) express trimeric asialoglycoprotein receptor (ASGPR) to clear these proteins from the systemic circulation. ASGPR specifically binds GalNAc at neutral pH for endocytosis of circulating macromolecules from blood and releases GalNAc at acidic pH (-5-6) for load unloading in early endosomes. The free ASGPR is then recovered on the cell surface for reuse. About one third of the RNAi agents in current clinical trials are single molecule, chemically modified RNAi triggers conjugated to multivalent GalNAc ligands targeting ASGPR. The appropriate liver physiology, unique properties of ASGPR, non-toxic nature of GalNAc ligands, and simplicity of GalNAc-siRNA conjugates make them an attractive method for systemic RNAi delivery to hepatocytes.

By targeted delivery of oligonucleotide therapeutics (oligonucleotide bioconjugates) to hepatocytes via asialoglycoprotein receptor (ASGPR) mediated uptake, interactions between the conjugates and their corresponding cell surface receptor proteins are facilitated, leading to subsequent internalization by receptor-mediated endocytosis. The bioconjugate interacts with cell type related receptors to enable targeted delivery to specific tissues or cell types within the tissues. ASGPR binds and internalizes tri-antennary N-acetylgalactosamine (GalNAc), which is conjugated, for example, to therapeutic antisense oligonucleotides (these oligonucleotides are internalized and then released into the cell where they can hybridize RNA (mRNA) to their cognate precursor signals and induce cleavage of RNA-DNA heteroduplex), or to small interfering RNAs (siRNA) directed against disease-related genes, for example. After release of siRNA from endosomal or lysosomal compartments, now cytoplasmic siRNA can be loaded into RNA-induced silencing complex (RISC). The loaded RISC can then scan the sequence complementarity of all expressed RNAs. When complementary sequences are detected by hybridization, enzymes that are part of RISC cleave target mRNA, thereby reducing expression of disease-associated proteins. RNAi refers to RNA interference. This receptor-mediated uptake allows lower doses than are required for therapeutic delivery of unconjugated oligonucleotides. Single-and double-stranded oligonucleotides can be delivered to hepatocytes using GalNAc conjugates. For example, drugs for the treatment of diseases such as hemophilia a and hemophilia B were developed based on oligonucleotide-GalNAc conjugates, as well as siRNA-GalNAc conjugates targeting gene PCSK9, and siRNA-GalNAc conjugates givosiran targeting gene ALAS 1.

Despite advances in technology, achieving efficient oligonucleotide delivery remains a major translational limitation. For oligonucleotide-based drug platforms, methods aimed at improving oligonucleotide delivery are used, such as chemical modification of oligonucleotides, bioconjugation, and delivery of oligonucleotides using nanocarriers, but delivery challenges still need to be improved. There remains a need for improved delivery systems that further increase the efficacy, reduce toxicity, and/or reduce off-target effects of ASGPR-targeted oligonucleotide systems (e.g., galNAc-oligonucleotide conjugates).

Disclosure of Invention

One aspect of the invention relates to oligonucleotide conjugates comprising at least one saponin, the at least one soapCovalent attachment of the glycoside to a ligand of an asialoglycoprotein receptor (ASGPR), wherein the ligand of ASGPR comprises at least one N-acetylgalactosamine (GalNAc) moiety, preferably three or four GalNAc moieties, more preferably the ligand of ASGPR comprises (GalNAc) ₃ Tris or consists thereof, and is also covalently linked to an oligonucleotide, wherein the at least one saponin is selected from the group consisting of a monosaccharide-chain triterpenoid saponin and a disaccharide-chain triterpenoid saponin.

One embodiment is an oligonucleotide conjugate of the invention comprising at least one saponin covalently linked to a ligand of an asialoglycoprotein receptor (ASGPR), wherein the ligand of ASGPR comprises at least one N-acetylgalactosamine (GalNAc) moiety, preferably three or four GalNAc moieties, more preferably three GalNAc moieties, more preferably the ligand of ASGPR comprises (GalNAc) ₃ Tris or consists thereof, and is also covalently linked to an oligonucleotide, wherein the at least one saponin is a 12, 13-dehydrooleanane type monosaccharide or disaccharide chain pentacyclic triterpene saponin, preferably having an aldehyde function at the C-23 position of the aglycone core structure of the saponin, wherein the oligonucleotide conjugate comprises 1-16 saponin moieties, preferably 1-8 saponin moieties, more preferably 1, 4 or 8 saponin moieties.

An embodiment is an oligonucleotide conjugate of the invention, wherein at least one GalNAc moiety, preferably three GalNAc moieties, at least one saponin, preferably 1-16 saponin moieties, more preferably 1-8 saponin moieties (e.g. 1, 4 or 8 saponin moieties) and the oligonucleotide are covalently bound via a trifunctional linker, preferably wherein each of one or more GalNAc moieties, one or more saponin moieties and the oligonucleotide is covalently bound to a separate arm of the trifunctional linker.

An embodiment is an oligonucleotide conjugate of the invention comprising one saponin moiety, or 4 saponin moieties, preferably 4 saponin moieties, covalently bound to a dendron, preferably a G2 dendron, such as e.g. N, N' - ((9S, 19S) -14- (6-aminocaproyl) -1-mercapto-9- (3-mercaptopropanamido) -3,10,18-trioxo-4,11,14,17-tetraazaditridecane-19, 23-diyl) bis (3-mercaptopropanamide), or 8 saponin moieties, preferably 8 saponin moieties, covalently bound to a dendron, preferably a G3 dendron, such as e.g. (2S) -N- [ (1S) -1- { [2- (6-amino-N- {2- [ (2S) -2, 6-bis (3-sulfanylpropanamido) hexanamido ] ethyl } hexanamido) ethyl ] carbamoyl } -5- [ (2S) -2, 6-bis (3-sulfanylpropanamido) hexanamido ] 3-hexanamido ] propanamido.

An embodiment is an oligonucleotide conjugate of the invention, wherein at least one saponin moiety is linked via a hydrazone linkage or via a semicarbazone linkage. At least one saponin is preferably covalently bound in the oligonucleotide conjugate via a semicarbazone linkage. Such hydrazone bond or semicarbazone bond (with linker) is, for example, an aldehyde group at the C-23 atom formed as sapogenin core structure involving saponins or as silk-mangosteen sapogenin core structure of saponins.

Preferred oligonucleotide conjugates of the invention, wherein the conjugate comprises an oligonucleotide defined as a nucleic acid of no more than 150nt, preferably wherein the oligonucleotide has a size of 5-150nt, preferably 8-100nt, most preferably 10-50nt.

One aspect of the invention relates to a pharmaceutical composition comprising an oligonucleotide conjugate of the invention, and optionally a pharmaceutically acceptable excipient and/or optionally a pharmaceutically acceptable diluent.

One aspect of the invention relates to an oligonucleotide conjugate of the invention or a pharmaceutical composition comprising an oligonucleotide conjugate of the invention for use as a medicament.

One aspect of the invention relates to a pharmaceutical composition comprising an oligonucleotide conjugate of the invention for use in the treatment or prevention of a disease or health problem in which the expression product relates to any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AT3, GO, CC5, X gene of HBV, S gene of HBV, AAT, miR-122, hepatitis B virus HbsAg, LDHA, CEBPA and LDH; and/or for use in the treatment or prevention of diseases or health problems involving any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AT3, GO, CC5, X gene of HBV, S gene of HBV, AAT, miR-122, hepatitis B virus HbsAg, LDHA, CEBPA and LDH.

One aspect of the invention relates to a pharmaceutical composition comprising an oligonucleotide conjugate of the invention for use in the treatment or prevention of a disease or health problem in which the expression product relates to any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AAT, miR-122, hepatitis B virus HbsAg, LDHA and CEBPA; and/or for use in the treatment or prevention of diseases or health problems involving any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AAT, miR-122, hepatitis B virus HbsAg, LDHA and CEBPA.

One aspect of the invention relates to a pharmaceutical composition comprising an oligonucleotide conjugate of the invention for use in the treatment or prevention of a disease or health problem in which the expression product relates to any one or more of the following genes: expression products of HSP27 and apoB, preferably apoB; and/or for use in the treatment or prevention of diseases or health problems involving any one or more of the following genes: HSP27 and apoB, preferably apoB.

One aspect of the invention relates to a pharmaceutical composition comprising an oligonucleotide conjugate of the invention for use in the treatment or prevention of: cancer, infectious disease, viral infection, hypercholesterolemia, cardiovascular disease, primary hyperoxaluria, hemophilia a, hemophilia B, AAT-associated liver disease, acute hepatic porphyrin, TTR-mediated amyloidosis, hereditary TTR amyloidosis (hATTR), complement-mediated disease, hepatitis b infection, hepatitis c infection, alpha 1-antitrypsin deficiency, beta-thalassemia, or autoimmune disease.

One aspect of the invention relates to a pharmaceutical composition comprising an oligonucleotide conjugate of the invention for use in the treatment or prevention of: cancer (e.g., endometrial, breast, lung, or hepatocellular carcinoma); and/or cardiovascular diseases such as hypercholesterolemia, preferably hypercholesterolemia.

One aspect of the invention relates to an in vitro or ex vivo method for transferring an oligonucleotide conjugate of the invention from outside a cell into said cell, preferably followed by transferring an oligonucleotide comprised by the oligonucleotide conjugate of the invention into the cytoplasm and/or nucleus of said cell, comprising the steps of:

a) Providing a cell expressing ASGPR, preferably ASGPR1, on its surface, preferably selected from the group consisting of liver cells, virus-infected mammalian cells and mammalian tumor cells, wherein preferably the cell is a human cell;

b) Providing an oligonucleotide conjugate of the invention for transfer into the cell provided in step a);

c) Contacting the cells of step a) with the oligonucleotide conjugate of step b), preferably in a liquid medium, in vitro or ex vivo, thereby effecting transfer of the oligonucleotide conjugate from outside the cell into said cell, and optionally and preferably thereby subsequently effecting transfer of the oligonucleotide comprised by the oligonucleotide conjugate into the cytoplasm and/or nucleus of said cell.

One aspect of the invention relates to an in vitro or ex vivo method for transferring an oligonucleotide conjugate of the invention from outside a cell into said cell, preferably subsequently transferring an oligonucleotide comprised by said oligonucleotide conjugate into the cytoplasm of said cell, comprising the steps of:

a) Providing a cell expressing ASGPR on its surface, preferably selected from the group consisting of a liver cell, a virus-infected cell and a tumor cell, and providing an oligonucleotide conjugate of the invention for transfer into the provided cell;

b) Contacting the cell of step a) with the oligonucleotide conjugate of step a) in vitro or ex vivo, thereby effecting transfer of the oligonucleotide conjugate from outside the cell into said cell, and preferably subsequently effecting transfer of the oligonucleotide comprised by the oligonucleotide conjugate into the cytoplasm of said cell.

Preferably, the cell is a human cell. Preferably, the cell is a liver cell.

One aspect of the invention relates to a saponin conjugate comprising a ligand covalently linked to an asialoglycoprotein receptor (ASGPR)At least one saponin, wherein the ligand of ASGPR comprises at least one N-acetylgalactosamine (GalNAc) moiety, preferably three or four GalNAc moieties, more preferably the ligand of ASGPR comprises (GalNAc) ₃ Tris or consists thereof, wherein at least one saponin is selected from the group consisting of a monosaccharide-chain triterpenoid saponin and a disaccharide-chain triterpenoid saponin.

One aspect of the invention relates to a pharmaceutical combination comprising:

-a first pharmaceutical composition comprising a saponin conjugate of the invention and optionally comprising a pharmaceutically acceptable excipient and/or a pharmaceutically acceptable diluent; and

-a second pharmaceutical composition comprising a second conjugate of an effector molecule and an ASGPR ligand, or a third conjugate of an effector molecule and a binding molecule, and optionally comprising a pharmaceutically acceptable excipient and/or a pharmaceutically acceptable diluent, wherein the ASGPR ligand preferably comprises at least one GalNAc moiety, preferably three or four GalNAc moieties, more preferably the ligand of ASGPR comprises (GalNAc) ₃ Tris or consists of the same, the binding molecule comprising a binding site for a cell surface molecule.

One aspect of the present invention relates to a pharmaceutical composition comprising:

-the saponin conjugate of the invention;

-a second conjugate of an effector molecule and an ASGPR ligand, or a third conjugate of an effector molecule and a binding molecule, and optionally comprising a pharmaceutically acceptable excipient and/or a pharmaceutically acceptable diluent, wherein the ligand of ASGPR preferably comprises at least one GalNAc moiety, preferably three or four GalNAc moieties, more preferably the ligand of ASGPR comprises (GalNAc) ₃ Tris or consists of the same, the binding molecule comprising a binding site for a cell surface molecule.

One aspect of the present invention relates to the pharmaceutical combination of the invention comprising the saponin conjugate of the invention, or to the pharmaceutical composition of the invention comprising the saponin conjugate of the invention, for use as a medicament.

One aspect of the present invention relates to the use of the pharmaceutical combination of the present invention comprising the saponin conjugate of the present invention, or the pharmaceutical composition of the present invention comprising the saponin conjugate of the present invention, for the treatment or prevention of a disease or health problem wherein the expression product relates to any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AT3, GO, CC5, X gene of HBV, S gene of HBV, AAT, miR-122, hepatitis B virus HbsAg, LDHA, CEBPA and LDH.

One aspect of the invention relates to an in vitro or ex vivo method for transferring the second or third conjugate of the invention from outside the cell into said cell, preferably followed by transferring the effector molecule comprised by the second or third conjugate of the invention into the cytoplasm of said cell, comprising the steps of:

a) Providing a cell expressing ASGPR on its surface and expressing a cell surface molecule when transferring a third conjugate into the cell, wherein the third conjugate comprises a binding molecule for binding to said cell surface molecule, the cell preferably being selected from the group consisting of a liver cell, a virus-infected cell and a tumor cell;

b) Providing a second conjugate or a third conjugate according to any one of the invention for transfer into the cell provided in step a);

c) Providing a saponin conjugate of the present invention;

d) Contacting the cell of step a) with the second or third conjugate of step b) and the saponin conjugate of step c) in vitro or ex vivo, thereby effecting transfer of the second or third conjugate from outside the cell into the cell, and preferably thereby effecting subsequent transfer of the second or third conjugate into the cytoplasm of the cell, or preferably thereby effecting subsequent transfer of at least the effector molecule comprised by the second or third conjugate into the cytoplasm of the cell.

An aspect of the invention relates to a method of providing an oligonucleotide conjugate of the invention comprising the steps of:

a) Providing at least one saponin moiety comprising a covalently bound first linker, wherein the first linker comprises at least one first reactive group for covalent binding to a second reactive group on a second linker or a seventh reactive group on a seventh linker;

b) Providing an oligonucleotide comprising a covalently bound third linker, wherein the third linker comprises a third reactive group for covalent binding with a fourth reactive group on a fourth linker or an eighth reactive group on a seventh linker;

c) Providing at least one GalNAc moiety comprising a covalently bound fifth linker, wherein the fifth linker comprises a fifth reactive group for covalent binding to a sixth reactive group on the sixth linker or a ninth reactive group on the seventh linker; and

(d1) Ligating a first linker to a second linker by forming a covalent bond between the first reactive group and the second reactive group, ligating a third linker to a fourth linker by forming a covalent bond between the third reactive group and the fourth reactive group, ligating a fifth linker to a sixth linker by forming a covalent bond between the fifth reactive group and the sixth reactive group, and covalently ligating the second linker, the fourth linker, and the sixth linker together to provide an oligonucleotide,

or (d 2) linking the first linker to the seventh linker by forming a covalent bond between the first reactive group and the seventh reactive group, linking the third linker to the seventh linker by forming a covalent bond between the third reactive group and the eighth reactive group, and linking the fifth linker to the seventh linker by forming a covalent bond between the fifth reactive group and the ninth reactive group, thereby providing the oligonucleotide conjugate.

An embodiment is a method for providing an oligonucleotide conjugate of the invention, wherein the seventh linker is a trifunctional linker, such as the trifunctional linker of formula (XXI):

preferred are methods of providing oligonucleotide conjugates of the invention, wherein at least one of the saponin moieties is 1-16 saponin moieties, preferably 1-8 saponin moieties (e.g., 1, 4 or 8 saponin moieties).

Preferred are methods of providing the oligonucleotide conjugates of the invention wherein the saponin is SO1861, SO1832, QS-21 or any functional derivative thereof, preferably SO1861 or SO1832.

Preferred are methods of providing oligonucleotide conjugates of the invention, wherein the saponin moiety or moieties are covalently linked via a hydrazone bond or a semicarbazone bond.

Preferred are methods of providing the oligonucleotide conjugates of the invention, wherein at least one GalNAc moiety is 1-4 GalNAc moieties, preferably 1 or 3 GalNAc moieties.

One embodiment is an oligonucleotide conjugate of the invention, wherein the saponin comprised by the oligonucleotide conjugate is isolated from a plant. Preferably, the saponins are isolated from a part of the plant (e.g. root), or from a part of the tree (e.g. bark). Preferably, the saponins are isolated from roots from plants.

Definition of the definition

The term "GalNAc" has its conventional scientific meaning and refers herein to N-acetylgalactosamine and its IUPAC name: 2- (acetamido) -2-deoxy-D-galactose.

The term "(GalNAc) ₃ Tris "has its conventional scientific meaning in the field of siRNA-based therapies, for example, and herein refers to a moiety comprising three GalNAc units, each of which is covalently bound to a hydroxy group of Tris (hydroxymethyl) aminomethane (Tris) (IUPAC name: 2-amino-2- (hydroxymethyl) propane-1, 3-diol), preferably via at least one linker, respectively. (GalNAc) ₃ Tris may be present as a molecule comprising free amine, or may be further functionalized via the remaining amine binding sites, e.g., to form the inclusion (GalNAc) described herein ₃ Conjugates of Tris moieties.

The term "oligonucleotide" has its conventional scientific meaning and refers herein to a string of two or more nucleotides, i.e., an oligonucleotide is a short oligomer consisting of ribonucleotides or deoxyribonucleotides. Examples are RNA and DNA, and any warpModified RNA or DNA, e.g.a strand of nucleic acid comprising nucleotide analogues, e.g.bridging nucleic acid (BNA), also known as Locked Nucleic Acid (LNA) or 2'-O,4' -C-aminoethylene or 2'-O,4' -C-aminomethylene Bridging Nucleic Acid (BNA) ^NC ) Wherein the nucleotide is a ribonucleotide or a deoxyribonucleotide.

As used herein, the terms "nucleic acid", "oligonucleotide" and "polynucleotide" are synonymous with each other and should be interpreted to encompass any polymer molecule composed of units comprising nucleobases (or simply "bases", for example standard nucleobases such as adenine (a), cytosine (C), guanine (G), thymine (T), or uracil (U), or any known non-standard, modified or synthetic nucleobase such as 5-methylcytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 7-methylguanine, 5, 6-dihydrouracil, etc.) or functional equivalents thereof, which enable the polymer molecule to undergo hydrogen bond-based nucleobase pairing (such as watson-crick base pairing) with naturally occurring nucleic acids (such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)) under suitable hybridization conditions, wherein naturally occurring nucleic acids are understood to be polymer molecules composed of nucleotide units.

Thus, from a chemical perspective, the term nucleic acid under this definition can be interpreted to encompass polymer molecules that are chemically DNA or RNA, as well as polymer molecules of nucleic acid analogs, also known as heteronucleic acids (XNA) or artificial nucleic acids, which are polymer molecules in which one or more (or all) units are modified nucleotides or functional equivalents of nucleotides. Nucleic acid analogs are well known in the art and are widely used in research and medicine due to various properties, such as increased specificity and/or affinity, higher binding strength to their targets and/or increased in vivo stability. Typical examples of nucleic acid analogs include, but are not limited to, locked Nucleic Acids (LNAs) (also known as Bridging Nucleic Acids (BNA)), phosphodiamide morpholino oligomers (PMOs, also known as morpholinos), peptide Nucleic Acids (PNAs), glycol Nucleic Acids (GNAs), threose Nucleic Acids (TNA), hexitol Nucleic Acids (HNA), 2' -deoxy-2 ' -fluoroarabinonucleic acids (FANA or FNA), 2' -deoxy-2 ' -fluororibonucleic acids (2 ' -F RNA or FRNA); a Qu Tang Alcohol Nucleic Acid (ANA), cyclohexene nucleic acid (CeNA), and the like.

According to the specification, the length of a nucleic acid is expressed herein as the number of units that constitute a single strand of the nucleic acid. Because each unit corresponds exactly to one nucleobase capable of participating in a base pairing event, the length is often expressed in so-called "base pairs" or "bp", regardless of whether the nucleic acid in question is a single-stranded (ss) or double-stranded (ds) nucleic acid. In naturally occurring nucleic acids, 1bp corresponds to 1 nucleotide, abbreviated as 1nt. For example, a single-stranded nucleic acid consisting of 1000 nucleotides (or a double-stranded nucleic acid consisting of two complementary strands each consisting of 1000 nucleotides) is described as being 1000 base pairs or 1000bp in length, which can also be expressed as 1000nt or 1 kilobase abbreviated as 1 kb. The 2 kilobases or 2kb is equal to 2000 base pairs in length, which is equal to 2000 nucleotides of single stranded RNA or DNA. However, to avoid confusion, the length of a nucleic acid will be preferentially expressed herein in terms of "bp" or "kb" rather than "nt" as is also common in the art, considering the fact that a nucleic acid as defined herein may comprise or consist of units that are not only chemically nucleotides but also functionally equivalent thereto.

In an advantageous embodiment, the nucleic acids disclosed herein are not longer than 1kb, preferably not longer than 500bp, most preferably not longer than 250bp.

In a particularly advantageous embodiment, the nucleic acid is an oligonucleotide (or oligo) which is defined as a nucleic acid of no more than 100bp, i.e. any polymer molecule consisting of no more than 100 units according to the definition provided above, wherein each unit comprises a nucleobase or functional equivalent thereof which enables hydrogen bond based nucleobase pairing of said oligonucleotide with DNA or RNA under suitable hybridization conditions. Within the scope of the definition, it is immediately understood that the oligonucleotides disclosed herein may comprise or consist of units that are not only nucleotides but also synthetic equivalents thereof. In other words, from a chemical perspective, the term oligonucleotide as used herein will be interpreted as possibly comprising or consisting of RNA, DNA or nucleic acid analogues such as, but not limited to, LNA (BNA), PMO (morpholino), PNA, GNA, TNA, HNA, FANA, fRNA, ANA, ceNA and/or the like.

The term "RNAi-mediated gene targeting" has its conventional scientific meaning and refers herein to an in vivo, ex vivo, or in vitro method of affecting the function of a gene in a cell by transferring into said cell an oligonucleotide (e.g., a small double stranded RNA molecule) that targets an mRNA involved in gene transcription: for example, small double stranded RNA molecules can trigger RNAi silencing of a particular gene with high efficiency.

The term "bridging nucleic acid", or abbreviated "BNA", or "locking nucleic acid", or abbreviated "LNA", or 2'-O,4' -C-aminoethylene or 2'-O,4' -C-aminomethylene Bridging Nucleic Acid (BNA) ^NC ) Has its usual scientific meaning and is referred to herein as a modified RNA nucleotide. BNA is also called a 'restricted RNA molecule' or an 'inaccessible RNA molecule'. BNA monomers can comprise five-, six-, or even seven-membered bridging structures with "fixed" C3; -internal sugar folding. The bridge is incorporated synthetically at the 2',4' -position of the ribose to provide a 2',4' -BNA monomer. BNA monomers can be incorporated into oligonucleotide polymer structures using standard phosphoramidite chemistry known in the art. BNA is a structurally rigid oligonucleotide with increased binding affinity and stability. The term "BNA" also refers to BNA ^NC Or 2',4″ BNA ^NC (2 '-O,4' -aminoethylene bridged nucleic acids) and have their usual scientific meaning and are also referred to herein as oligonucleotides containing one or more nucleotide building blocks having a six-membered bridging structure with an N-O bond and bearing an (N-H) or (N-Me) residue.

The term antisense oligonucleotide has its conventional scientific meaning and may be abbreviated in the specification as "AON" or "ASO".

The term "BNA-based antisense oligonucleotide", or simply "BNA AON", has its conventional scientific meaning, herein referring to a string of antisense nucleotides, wherein at least one of said nucleotides is BNA.

The term "prion" has its conventional scientific meaning and refers herein to a protein-like molecule, meaning that the molecule has the physicochemical properties of a protein to some extent, has, is associated with, comprises, belongs to, consists of, is a protein, or is a protein. The term "prion" as used in, for example, "prion molecule" refers to a molecule in which at least a portion is similar to or is a protein, wherein "protein" is understood to comprise a chain of amino acid residues at least two residues in length, thus comprising a combination of peptides, polypeptides and proteins, as well as protein or protein domains. In prion molecules, at least two amino acid residues are linked, for example, via one or more amide bonds, such as one or more peptide bonds. In prion molecules, the amino acid residues are natural amino acid residues and/or artificial amino acid residues, e.g., modified natural amino acid residues. In a preferred embodiment, the prion molecule is a molecule comprising at least two amino acid residues, preferably from two to about 2.000 amino acid residues. In one embodiment, the prion molecule is a molecule comprising from 2 to 20 (typical for peptides) amino acids. In one embodiment, a prion molecule is a molecule comprising 21 to 1.000 amino acids (typical for polypeptides, proteins, protein domains (e.g., antibodies, fab, scFv, one or more Vh domains), ligands for receptors such as EGF). Preferably, the amino acid residues are (typically) linked by one or more peptide bonds. According to the invention, the amino acid residue is or comprises a (modified) (non) natural amino acid residue.

The term "effector molecule" or "effector moiety" when referring to an effector molecule as part of, for example, a covalent conjugate, has its conventional scientific meaning and refers herein to a molecule that can selectively bind to and modulate the biological activity of, for example, any one or more of the following target molecules: proteins, peptides, carbohydrates, sugars (e.g. glycans), (phospho) lipids, nucleic acids such as DNA, RNA, enzymes. Effector molecules are, for example, molecules selected from any one or more of the following: small molecules (e.g., drug molecules), toxins (e.g., protein toxins), oligonucleotides (e.g., BNA), heterologous nucleic acids or siRNA, enzymes, peptides, proteins, or any combination thereof. Thus, for example, an effector molecule or effector moiety is a molecule or moiety selected from any one or more of the following: a small molecule (e.g., a drug molecule), a toxin (e.g., a protein toxin), an oligonucleotide (e.g., BNA), a heterologous nucleic acid or siRNA, an enzyme, a peptide, a protein, or any combination thereof, which molecule or moiety can selectively bind to any one or more of the following target molecules: proteins, peptides, carbohydrates (e.g., glycans), (phospho) lipids, nucleic acids (e.g., DNA, RNA), enzymes, and upon binding to the target molecule, modulate the biological activity of such one or more target molecules. Typically, effector molecules may exert biological effects within cells, e.g., mammalian cells, e.g., human cells, such as in the cytoplasm of the cells. Thus, typical effector molecules are drug molecules, plasmid DNA, toxins (e.g. toxins composed of antibody-drug conjugates (ADCs)), oligonucleotides (e.g. nucleic acids composed of siRNA, BNA, antibody-oligonucleotide conjugates (AOCs)). For example, effector molecules are molecules that can act as ligands capable of increasing or decreasing (intracellular) enzymatic activity, gene expression, or cell signaling. The effector moiety is not the saponin or saponin derivative on which the conjugates of the invention are based. The effector moiety is not a conjugate of the invention.

The term "health problem" has its conventional scientific meaning and refers herein to any condition of the body of a subject (e.g., a human patient), or a portion thereof, organs, muscles, veins, arteries, skin, limbs, blood, cells, etc., when compared to the condition of the body or portion thereof of a healthy subject, thereby impeding the normal function and/or health of the subject, e.g., impairing the normal function of the human body.

The term "GalNAc modified oligonucleotide drug" has its conventional scientific meaning and refers herein to an oligonucleotide for interfering with gene transcription, wherein one or more GalNAc units are coupled to the oligonucleotide, e.g., via at least one linker.

The term "locked nucleic acid", abbreviated "LNA", is a bridging nucleic acid, as known in the art, having its conventional scientific meaning and refers herein to an oligonucleotide comprising one or more nucleotide building blocks, wherein the additional methylene bridge immobilizes the ribose moiety in either a C3 '-internal conformation (β -D-LNA) or a C2' -internal conformation (α -L-LNA).

The term "chemically modified, metabolically stable siRNA" has its conventional scientific meaning and refers herein to an siRNA molecule comprising chemical modifications that render the modified siRNA molecule more resistant, i.e. more stable, to metabolic degradation, digestion, enzymatic cleavage, etc., than an RNA oligonucleotide consisting of naturally occurring nucleotides.

The term "saponin" has its conventional scientific meaning and refers herein to a group of amphiphilic glycosides comprising one or more hydrophilic aglycone moieties bound to a lipophilic aglycone core, which is sapogenin. Saponins may be naturally occurring or synthetic (i.e., non-naturally occurring). The term "saponin" encompasses naturally occurring saponins, derivatives of naturally occurring saponins, saponins synthesized de novo by chemical and/or biotechnological synthetic routes.

The term "saponin derivative" (also referred to as "modified saponin") has its conventional scientific meaning and refers herein to a compound corresponding to a naturally occurring saponin that has been derivatized by one or more chemical modifications, such as oxidation of a functional group, reduction of a functional group, and/or formation of a covalent bond with another molecule. Preferred modifications comprise derivatization of the aldehyde groups of the aglycone core; derivatization of carboxyl groups of sugar chains or derivatization of acetoxy groups of sugar chains. Typically, the saponin derivative does not have a natural counterpart, i.e. the saponin derivative is not naturally produced by, for example, plants or trees. The term "saponin derivative" encompasses derivatives obtained by derivatization of naturally occurring saponins, as well as derivatives synthesized de novo by chemical and/or biotechnological synthetic routes, which synthesis yields compounds corresponding to naturally occurring saponins that have been derivatized by one or more chemical modifications.

The term "aglycone core structure" has its conventional scientific meaning and refers herein to the aglycone core of a saponin, which does not have one or two carbohydrate antennas or sugar chains (glycans) bound thereto. For example, saponaric acid is the aglycone core structure of SO1861, QS-7 and QS-21.

The term "sugar chain" has its conventional scientific meaning and refers herein to any one of the following: glycans, carbohydrate antennas, single sugar moieties (monosaccharides) or chains comprising multiple sugar moieties (oligosaccharides, polysaccharides). The sugar chain may consist of only sugar moieties or may further comprise additional moieties, for example any of the following: 4E-methoxy cinnamic acid, 4Z-methoxy cinnamic acid and 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid, such as are present in QS-21, for example.

The terms "SO1861" and "SO1862" refer to the same saporin (Saponaria officinalis) saponin, respectively, albeit in the deprotonated or api form. The molecular weight is 1862 daltons, as this mass is the form mass comprising protons at glucuronic acid. At neutral pH, the molecule is deprotonated. When mass spectrometry was used to measure mass in negative ion mode, the measured mass was 1861 daltons. Similarly, the terms "SO1831" and "SO1832" refer to the same saporin, respectively, albeit in the deprotonated or api form. The molecular weight is 1832 daltons because the mass is a formal mass containing protons at glucuronic acid. At neutral pH, the molecule is deprotonated. When mass spectrometry is used to measure mass in negative ion mode, the measured mass is 1831 daltons.

The term "Api/xl-" or "Apl-or xl-" in the context of the name of the sugar chain has its conventional scientific meaning and refers herein to a sugar chain comprising apiose (Api) moieties or comprising xylose (Xyl) moieties.

The term "antibody-drug conjugate" or "ADC" has its conventional scientific meaning and refers herein to any conjugate of an antibody (e.g., igG, fab, scFv, immunoglobulin fragment, one or more Vh domains, etc.), as well as any molecule (e.g., active pharmaceutical ingredient, toxin, oligonucleotide, enzyme, small molecule pharmaceutical compound, etc.) that can exert a therapeutic effect when contacted with cells of a subject (e.g., a human patient).

The term "antibody-oligonucleotide conjugate" or "AOC" has its conventional scientific meaning and refers herein to any conjugate of an antibody (e.g., igG, fab, scFv, immunoglobulin fragment, one or more Vh domains, etc.) with any oligonucleotide molecule that can exert a therapeutic effect when contacted with a cell of a subject (e.g., a human patient), such as an oligonucleotide selected from the group consisting of a natural or synthetic nucleic acid strand comprising DNA, modified DNA, RNA, modified RNA, synthetic nucleic acid, which is presented as a single-or double-stranded molecule (e.g., BNA, allele-specific oligonucleotides (ASOs), short or small interfering RNAs (siRNA; silencing RNAs), antisense DNA, antisense RNA, etc.).

The term "conjugate" has its conventional scientific meaning and refers herein to at least a first molecule covalently bound to at least a second molecule, thereby forming an assembly comprising or consisting of a covalent coupling of the first molecule and the second molecule. Typical conjugates are (GalNAc) ₃ Tris-siRNA, ADC, AOC and SO1861-EMCH (EMCH linked to the aldehyde group of the aglycone core structure of the saponins).

The term "moiety" has its conventional scientific meaning and refers herein to a molecule that binds, links, conjugates with, and thereby forms part of a larger molecule, conjugate, molecular assembly, etc., to another molecule, linker, molecular assembly, etc. Typically, a moiety is a first molecule covalently bound to a second molecule, involving one or more chemical groups initially present on the first molecule and present on the second molecule. For example, saporin is a typical effector molecule. Saporin is a typical effector moiety in ADCs as part of antibody-drug conjugates. As part of the antibody-oligonucleotide conjugate, BNA or siRNA is a typical effector moiety in AOC.

The term 'S' is used to denote a 'stable linker' that remains intact in endosomes and cytoplasm, as in an antibody-saponin conjugate or a construct comprising a linker.

The term ' L ' is used, for example, in an antibody-saponin conjugate or a construct comprising a linker, to denote a linker ' that is ' labile ' in the endosome under slightly acidic conditions.

The terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements, compositions, components in a composition, or separate method steps and not necessarily for describing a sequential or chronological order. Unless otherwise indicated, the terms are interchangeable under appropriate circumstances and the embodiments of the invention can operate in other sequences than described or illustrated herein.

The embodiments of the invention described herein may be combined and operated in concert unless otherwise indicated.

Furthermore, while various embodiments are referred to as being "preferred" or "e.g." for example "or" in particular "(in partial) etc., these embodiments should be construed as exemplary ways in which the invention may be practiced, without limiting the scope of the invention.

The term "comprising" as used in the claims should not be interpreted as being limited to, for example, elements or method steps or ingredients of the compositions listed thereafter; it does not exclude other elements or method steps or components of a composition. It should be interpreted as specifying the presence of the stated features, integers, (method) steps or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps or groups thereof. Thus, the scope of the expression "a method comprising steps a and B" should not be limited to a method consisting of only steps a and B, but rather, with respect to the present invention, only steps of the method are listed as a and B, and the claims should be further interpreted as comprising equivalents of those method steps. Thus, the scope of expression of "a composition comprising components a and B" should not be limited to a composition consisting of only components a and B, but rather, with respect to the present invention, only components a and B of the composition are enumerated, and the claims should be further interpreted to include equivalents of those components.

Furthermore, references to an element or component by the indefinite article "a/an" do not exclude the possibility that more than one element or component is present, unless the context clearly requires that there be one and only one of the elements or components. Thus, the indefinite article "a" or "an" generally means "at least one" (at least one).

The term "DAR" generally represents the drug-to-antibody ratio and refers to the average drug-to-antibody ratio for a given formulation of an Antibody Drug Conjugate (ADC), and herein refers to the ratio of the number of bound SO1861 moieties, or SPT001, or total saponin moieties, or bound loads (e.g., AON, such as ApoB BNA) relative to the conjugate molecule.

Brief Description of Drawings

FIGS. 1A-C: synthesis of trivalent-GalNAc.

Fig. 2A-B: synthesis of SO 1861-DBCO.

Fig. 3: synthesis of monovalent-GalNAc-SO 1861.

Fig. 4: synthesis of trivalent-GalNAc-SO 1861.

Fig. 5A-C: synthesis of monovalent-GalNAc-BNA.

Fig. 6A-B: synthesis of trivalent-GalNAc-BNA.

Fig. 7: synthesis of trivalent-GalNAc-BNA.

Fig. 8A-G: synthesis of trivalent GalNAc.

Fig. 9A and 9B: general toxicity (MTS) of GalNAc-SO1861, SO1861-EMCH and trivalent-GalNAc-SO 1861 to HepG2 (A) and Huh7 (B) cell lines. The legend shown alongside fig. 9B also applies to fig. 9A.

Fig. 10A and 10B: cell killing assay (MTS) HepG2 (a) and Huh7 (B) cell lines. The legend shown next to fig. 10B also applies to fig. 10A.

Fig. 11A and 11B: gene expression analysis of HepG2 (A) and Huh7 (B) cell lines.

Fig. 12A and 12B: gene expression analysis of HepG2 (A) and Huh7 (B) cell lines. The legend shown alongside fig. 12B also applies to fig. 12A.

Fig. 13A and 13B: cell viability assay for HepG2 (a) and Huh7 (B) cell lines. The legend shown alongside fig. 13B also applies to fig. 13A.

Fig. 14A and 14B: gene expression analysis of HepG2 (A) and Huh7 (B) cell lines. The legend shown next to fig. 14B also applies to fig. 14A.

Fig. 15A and 15B: cell viability assay for HepG2 (a) and Huh7 (B) cell lines. The legend shown alongside fig. 15B also applies to fig. 15A.

Fig. 16A and 16B: gene expression analysis of HepG2 (A) and Huh7 (B) cell lines. The legend shown next to fig. 16B also applies to fig. 16A.

Fig. 16C and 16D: cell viability assay for HepG2 (C) and Huh7 (D) cell lines. The legend shown next to fig. 16D also applies to fig. 16C.

Fig. 17A and 17B: gene expression analysis of HepG2 (A) and Huh7 (B) cell lines. The legend shown next to fig. 17B also applies to fig. 17A.

Fig. 18A: relative cell viability of cells versus the following primary human hepatocytes following contact (as compared to untreated cells set at 100% ('un r')): a series of concentrations of trivalent GalNAc ((GN) 3-SPT) conjugated to SO1861, a 10pM monoclonal antibody conjugated to saporin anti-CD 71 (CD 71-SPRN) and a series of concentrations of SO1861 (SPT-EMCH) bound to aglycone aldehyde groups via EMCH, a 10pM CD71-SPRN and a series of concentrations (GN) ₃ Combination of SPT, as well as 10pM CD71-SPRN and a range of concentrations conjugated to dendrites with four SO1861 moieties covalently bound thereto (GN) ₃ Is (dar=4, i.e. ratio (GN) ₃ Dendritic is 1:1 and proportional dendritic: SO1861 is 1:4) ((GN) ₃ -dSPT4)。

Fig. 18B: relative expression of apoB in cells with the following primary human hepatocytes following contact (compared to untreated cells set at 100% ('un'): a range of concentrations of and targeting apoB mRNA ((GalNAc) ₃ -antisense oligonucleotide conjugated trivalent GalNAc of ApoB), 300nM trivalent GalNAc conjugated with saponin SO1861 ((GalNAc) 3-SPT 001) and a range of concentrations (GalNAc) ₃ -a combination of ApoB, or a conjugate of trivalent GalNAc ((GalNAc) conjugated to a dendrimer having four SO1861 moieties (dar=4) covalently bound thereto and conjugated to an antisense oligonucleotide targeting ApoB ₃ -dSPT ₄ -ApoB). Throughout the description and claims, SO1861 is also referred to as 'SPT001' and 'dptt'.

Fig. 19A and 19B: cell viability of cells with the following primary human hepatocytes (a) and Huh7 hepatocytes line (B) after contact (both compared to untreated cells set at 100% ('un r'): a combination of a range of concentrations of trivalent GalNAc conjugated with SO1861 (trivalent GalNAc-SPT 001), 10pM monoclonal antibody anti-CD 71 conjugated with saporin (CD 71-saporin), and a range of concentrations of the covalent conjugate trivalent GalNAc-SPT 001.

Fig. 19C: sketch of covalent trivalent GalNAc-SPT001 conjugate (dar=1 of saponin SO 1861). 'S' is SPT001 SO1861 in the sketch.

Fig. 19D: sketch of an anti-CD 71 antibody-saporin conjugate (OKT-9), wherein 'T' is a toxin saporin covalently linked to the heavy chain of the antibody.

Fig. 19E: covalent trivalent (GalNAc) ₃ dSPT4 (or GN) ₃ -spt 4) sketch of conjugate (dar=4 for saponin SO 1861). The 'S' in the sketch is SPT001 SO1861; as described elsewhere for conjugation, dpt 4 describes a dendron with four SO1861 molecules.

Fig. 20A and B: relative apoB expression in primary human hepatocytes (a) and Huh7 hepatocyte line (B) following contacting the cells with: a range of concentrations of trivalent GalNAc (trivalent GalNAc-ApoBBNA) conjugated with antisense oligonucleotides for silencing ApoB mRNA and ApoB protein expression, i.e. targeting ApoB, 300nM trivalent GalNAc (trivalent GalNAc-SPT 001) conjugated with SO1861 (for combined SPT001 (saponin SO 1861), dar=1) in combination with a range of concentrations of trivalent GalNAc-ApoB BNA, or a range of concentrations of trivalent GalNAc (trivalent GalNAc-ApoBBNA-SPT 001) conjugated with antisense oligonucleotides for silencing ApoB mRNA and ApoB protein expression, i.e. targeting ApoB, with four SO1861 molecules conjugated with a single dendrimer moiety to GalNAc/oligonucleotide conjugates.

Fig. 20C: sketch of covalent trivalent GalNAc-ApoBBNA conjugate. The 'A' in the sketch is the antisense oligonucleotide ApoB (ApoBBNA).

Fig. 20D: sketch of trivalent GalNAc-ApoBBNA-SPT 001. The 'S' in the sketch is SPT001, also known as SO1861; "E" in the sketch is an effector moiety antisense oligonucleotide targeting apoB (ApoB BNA).

Fig. 20E: (GalNAc) ₃ Sketch of the conjugation of dSPT4-ApoB to dendrites, where four SO1861 moieties were covalently bound to dendrites (dar=4). The 'S' in the sketch is SPT001, also known as SO1861; "E" in the sketch is an effector moiety antisense oligonucleotide targeting apoB (ApoB BNA).

Fig. 21A and 21B: absolute apoB protein expression in primary human hepatocytes (a) and Huh7 hepatocyte line (B) under the influence of contacting cells with: a combination of a range of concentrations of trivalent GalNAc-ApoBBNA, 300nM of trivalent GalNAc-SPT001 and a range of concentrations of trivalent GalNAc-ApoBBNA, or a range of concentrations of trivalent GalNAc-ApoBBNA-SPT001; here four SO1861 molecules were conjugated to the GalNAc/oligonucleotide conjugate using a single dendritic moiety (hence the name (GalNAc) 3-dSPT 4-ApoB).

Fig. 22A-C: saponin-oligonucleotide-ASGPR ligand conjugate (i.e., trivalent GalNAc-ApoB BNA-SPT001, also known as (GalNAc) ₃ -SO 1861-ApoB).

Fig. 23A: synthesis of SO1861-OEG-NHS (SPT 001-L-NHS, molecule 12, where 'L' refers to an unstable cleavable linker).

Fig. 23B: dendrites (SPT 001) ₄ Synthesis of NH2 (molecule 15 from molecules 13 and 14).

Fig. 23C: dendrites (SPT 001) ₄ Synthesis of azide (molecule 19 from molecules 15 and 18).

Fig. 23D-E: dendrites (SPT 001) ₄ Synthesis of trivalent GalNAc (molecule 24 from molecules 19 and 23).

Molecule 21 shown in fig. 23D is intermediate 7 (as indicated by the symbol) shown in fig. 8G.

Fig. 24A-B: DBCO-TCO-trivalent GalNAc (molecule 29) is synthesized from molecule 27 and molecule 28 (trifunctional linker), wherein molecule 27 is GalNAc-thioacetate obtained from the reaction between molecule 22, formate of trivalent GalNAc-amine, and molecule 26 (4-nitrophenyl 3- (acetylthio) propionate).

Fig. 24C: DBCO-L-ApoB BNA oligomer-trivalent GalNAc (molecule 31) was synthesized by conjugating methyl tetrazine-L-ApoB BNA oligonucleotide (molecule 30) with DBCO-TCO-trivalent GalNAc (molecule 29).

Fig. 24D: by combining molecule 31 (DBCO-L-ApoB BNA oligonucleotide-trivalent GalNAc) with molecule 19 (dendrite (SPT 001) ₄ -azide) conjugation, synthesis of dendrons (-L-SO 1861) ₄ L-ApoB BNA oligonucleotide-trivalent GalNAc (molecule 34; 'trivalent GalNAc-ApoB BNA-SPT001 conjugate').

Fig. 25: analysis of ApoB expression in liver tissue of C57BL/6J mice.

Fig. 26: serum apoB protein analysis in C57BL/6J mice. 0.1mg/kg of ApoB#02 (196 hr), 0.01mg/kg (GalNAc) ₃ -ApoB+5mg/kg(GalNAc) ₃ -SO1861 (196 hr and 336 hr) and 0.1mg/kg (GalNAc) ₃ -ApoB+5mg/kg(GalNAc) ₃ The level of-SO 1861 (196 hours) was not reported.

Fig. 27: serum LDL-cholesterol (LDL-C) analysis in C57BL/6J mice.

Fig. 28: serum ALT analysis in C57BL/6J mice.

Fig. 29A-C: apoB expression analysis and cell viability assays for primary human hepatocytes, hepG2 and Huh7 cells for ApoB#02 conjugates.

Fig. 30: apoB expression analysis and cell viability assay for primary human hepatocytes for ApoB#02 conjugates.

Fig. 31: analysis of ApoB expression in liver tissue of C57BL/6J mice.

Fig. 32: serum ApoB protein analysis in C57BL/6J mice.

Fig. 33: serum LDL-cholesterol (LDL-C) analysis in C57BL/6J mice.

Fig. 34: serum ALT analysis in C57BL/6J mice.

Fig. 35A-D: trifunctional linkers- (L-hydrazone-SO 1861) - (L-BNA oligonucleotides) - (trivalent-GalNAc) were synthesized.

Fig. 36A-E: trifunctional linkers- (dendrite (-L-hydrazone-SO 1861) 4) - (L-BNA oligonucleotide) - (trivalent-GalNAc) synthesis. Note that the plot of fig. 36E contains two pages, labeled fig. 36E and fig. 36E (sequential), which together show conjugation of molecule 21 to molecule 19, thereby forming conjugate molecule 22.

Fig. 37A-C: trifunctional linker- (dendrite (-L-hydrazone-SO 1861) 8) - (BNA oligonucleotide) - (trivalent-GalNAc) synthesis. Note that the plot of fig. 37B contains two pages, labeled fig. 37B and fig. 37B (sequential), which together show conjugation of molecule 24 to molecule 18, thereby forming conjugate molecule 25 (intermediate 12). Note that the figure of fig. 37C contains two pages, labeled fig. 37C and fig. 37C (sequential), which together show conjugation of molecule 25 to molecule 21, thereby forming conjugate molecule 26.

Fig. 38A-C: trifunctional linkers- (L-semicarbazone-SO 1861) - (BNA oligonucleotides) - (trivalent-GalNAc) were synthesized.

Fig. 39A-F: trifunctional linkers- (dendrite (L-semicarbazone-SO 1861) 4) - (L-BNA oligonucleotide) - (trivalent-GalNAc) synthesis. Note that the plot of fig. 39E contains two pages, labeled fig. 39E and fig. 39E (sequential), which together show conjugation of molecule 40 to molecule 20, thereby forming conjugate intermediate 24.

Fig. 40A-D: trifunctional linkers- (dendrite (L-semicarbazone-SO 1861) 8) - (L-BNA oligonucleotide) - (trivalent-GalNAc) synthesis. Note that the plot of fig. 40A contains two pages, labeled fig. 40A and 40A (continuation), which together show conjugation of molecule 43 to molecule 37, thereby forming conjugate (molecule 44) intermediate 25. Note that the diagram of fig. 40B contains two pages, labeled fig. 40B and 40B (sequential), which together show conjugation of molecule 44 to molecule 18, thereby forming conjugate molecule 45 (intermediate 26). Note that the plot of fig. 40C contains two pages, labeled fig. 40C and fig. 40C (sequential), which together show conjugation of molecule 45 to molecule 20, thereby forming conjugate molecule 46 (intermediate 27).

Fig. 41: trivalent linker- (L-SPT 001) - (blocked TCO) - (trivalent GalNAc) synthesis.

Fig. 42A-B: trivalent linker- (blocked DBCO) - (L-BNA oligonucleotide) - (trivalent GalNAc) synthesis.

Fig. 43A-B: conjugate overview obtainable using TFL (trifunctional linker) method. General protocols for the family of oligonucleotide conjugates of the invention 'conjugate C1' were synthesized by covalently conjugating GalNAc, saponin (here SO 1861) and oligonucleotide (here BNA) to separate arms of a trifunctional linker (a). Conjugate family conjugate C1 synthesized according to the general scheme of (a), wherein option R1 is designated for conjugated saponin and option R2 is designated for conjugated BNA (B). Together, the diagrams of fig. 43A and 43B show conjugation of trifunctional linker-labeled molecule 4 with one of targeting ligand-labeled molecule 41, effector-labeled molecule 12, and enhancer-labeled molecules 3, 19, 25, 28, 40, and 45, or one of blocking agent-labeled molecules 5, 12a, and 50 for control, thereby forming a conjugate having the general conjugate structure 'conjugate C1' as shown in fig. 43B, comprising a group R1 and a group R2, wherein R1 is any of the groups R1 described below for conjugate C1, and R2 in conjugate C1 is any of the groups R2 described below for conjugate C1 in fig. 43B.

Detailed description of the preferred embodiments

The present invention will be described with respect to particular embodiments, but the invention is not limited thereto but only by the claims.

The first aspect of the present invention relates to a saponin conjugate comprising at least one saponin covalently linked to a ligand of an asialoglycoprotein receptor (ASGPR), wherein the ligand of ASGPR comprises at least one N-acetylgalactosamine (GalNAc) moiety, preferably three or four GalNAc moieties, more preferably the ligand of ASGPR comprises (GalNAc) ₃ Tris or consists thereof, wherein at least one saponin is selected from the group consisting of a monosaccharide-chain triterpenoid saponin and a disaccharide-chain triterpenoid saponin. Preferably, ASGPR is ASGPR1. One aspect of the invention relates to an oligonucleotide conjugate comprising at least one saponin covalently linked to a ligand of an asialoglycoprotein receptor (ASGPR), wherein the ligand of ASGPR comprises at least one N-acetylgalactosamine (GalNAc) moiety, preferably three or four GalNAc moieties, more preferably the ligand of ASGPR comprises (GalNAc) ₃ Tris or consists thereof, and is also covalently linked to an oligonucleotide, wherein the at least one saponin is selected from the group consisting of a monosaccharide-chain triterpenoid saponin and a disaccharide-chain triterpenoid saponin. Typically, ASGPR is ASGPR1. Triterpene saponins are typically and preferably 12, 13-dehydrooleanane-type triterpene saponins, such as triterpene saponins belonging to the 12, 13-dehydrooleanane-type having an aldehyde functional group at the C-23 position or disaccharide chain triterpene saponins. Preferred are the mono-or disaccharide pentacyclic triterpenoid saponins of the type 12, 13-dehydrooleanane (preferably having an aldehyde function at the C-23 position). Preferred are oligonucleotide conjugates of the invention, wherein the oligonucleotide comprised by the conjugate is defined as a nucleic acid of not more than 150nt, preferably wherein the oligonucleotide has a size of 5-150nt, preferably 8-100nt, most preferably 10-50nt.

Unexpectedly, when the therapeutic oligonucleotide is covalently conjugated to a saponin such as SO1861 (which is covalently conjugated to a ligand of the liver cell specific receptor ASGPR1 (such as trivalent-GalNAc (GN ₃ ) (i.e., conjugates comprising three GalNAc moieties)) in the presence of a therapeutic oligonucleotide, the inventors of the present application determined that the efficacy of the gene silencing oligonucleotide (ASO, AON, siRNA, BNA) is enhanced by at least a factor of 10, that is, at least a factor of 100 or even more than a factor of 1000, when considering mRNA expression and/or protein expression associated with a target gene targeted by the therapeutic oligonucleotide, when contacted with a mammalian cell (e.g., a human cell, such as a human liver cell) carrying ASGPR. Combining a therapeutic oligonucleotide, such as siRNA or ASO, which is involved in treating cancer (e.g. by silencing HSP27 expression) or in treating too high plasma (LDL-) cholesterol levels (by targeting apoB genes), with a saponin (e.g. provided as a GalNAc-containing conjugate separate from the oligonucleotide, or e.g. provided as a saponin moiety covalently linked to a conjugate of an ASGPR ligand and an oligonucleotide) widens the therapeutic window of the therapeutic oligonucleotide. When the target cells are contacted with an oligonucleotide coupled to a ligand of ASGPR1, effective gene silencing (any relevant degree of silencing) is achieved at lower doses of the oligonucleotide in the presence of the ASGPR1 targeting ligand comprising covalently linked saponin compared to gene silencing obtained in the absence of the (ASGPR 1 targeting) saponin. That is, when a cell bearing a receptor is contacted with an oligonucleotide conjugate of the invention in vitro or in vivo, the inventors of the present application established near complete (defined as at least 95% or more) inhibition of RNA expression. Furthermore, the inventors of the present application have also achieved (near) complete inhibition Protein expression, e.g., for apoB protein, then results in (near) complete clearance of LDL-cholesterol in serum. When the saponin does not comprise a payload (effector moiety, here an oligonucleotide) and an ASGPR ligand, here ASGPR1 (e.g. GN ₃ ) Such unexpected levels and efficacy of gene silencing are not achievable under the influence of the oligonucleotide conjugates of the invention when part of the conjugates. When evaluating mRNA expression and/or evaluating protein expression associated with a target gene, and indirectly evaluating LDL-cholesterol levels (because LDL particles contain one copy of apoB protein (more specifically apoB100 protein), the expression of which is silenced when a cell is contacted with an oligonucleotide conjugate of the invention), the efficacy of gene silencing after contacting ASGPR 1-expressing target (liver) cells with a saponin-containing conjugate of the invention is significantly improved. When the target cells are contacted with the same dose of an oligonucleotide coupled to a ligand of ASGPR1, improved gene silencing is obtained at the fixed oligonucleotide dose in the presence of an ASGPR1 targeting ligand comprising a covalently linked saponin, compared to the gene silencing efficacy obtained in the absence of the saponin. The inventors of the present application found that the therapeutic oligonucleotide was conjugated to an ASGPR1 ligand such as (GalNAc) compared to the efficacy obtained in the absence of the saponin conjugate ₃ (also known as GN ₃ Or (GN) ₃ ) Conjugates of (C) and saponins with ASGPR1 ligands such as (GalNAc) ₃ Which when contacted with ASGPR 1-bearing cells such as human hepatocytes results in unexpectedly high improvement of gene silencing efficacy. The inventors of the present application have also found that covalent coupling of saponins to ligands of therapeutic oligonucleotides and ASGPR1 (e.g. (GalNAc) compared to the efficacy obtained in the absence of saponins in GalNAc-oligonucleotide conjugates ₃ ) Above, this combination, when contacted with ASGPR 1-bearing cells (e.g., human liver cells), resulted in an unexpected increase in gene silencing efficacy.

Triterpenoid glycoside type saponins have the ability to stimulate the delivery of effector molecules from outside the target cell into the cell and subsequently into the cytoplasm of the cell. Saponins facilitate, for example, receptor-mediated uptake of certain effector molecules, such as protein toxins contained in ADCs. Endocytosis of toxins as part of ADCs delivers the toxins in the endosomes and/or lysosomes of target cells. Without wishing to be bound by any theory, the triterpene saponins used in the present invention stimulate or mediate the subsequent release of ADC or at least toxins from the endosome or the lysosome in vitro and into the cytoplasm of the cell. When a target cell is contacted with a toxin at too low a dose of the toxin to exert intracellular effects, co-administration of the toxin (e.g., part of the ADC) and free saponin allows the target cell to be contacted with both the ADC and the saponin, which can result in efficient toxin-mediated killing of the target cell at the same toxin dose that would be ineffective in contact with the cell in the absence of the saponin. Thus, the potency of the toxin is increased under the influence of free saponins. The toxin dose required for efficient and adequate target cell killing may still be accompanied by undesired off-target effects, e.g. also expressed on cells, e.g. in healthy cells, elsewhere in the patient's body, or present in the same organ in which the target cells are located, when the ADC-targeted tumor cell receptor is not a true (exclusive) tumor cell specific receptor. Furthermore, the required free saponin dose needs to reach a sufficient degree of toxin activation (delivery of toxin into the target cell cytoplasm; mediating the saponin threshold of target delivery) may be at a level that induces undesirable off-target effects. When considering target (tumor) cells to be treated with a toxin, since free saponins are administered systemically and non-targeted, a relatively high saponin dose will be administered to a patient in need of toxin treatment to achieve a local saponin dose at the target cell level. Furthermore, another problem and disadvantage encountered when free saponins are co-administered with e.g. ADCs is that it is difficult to optimise the synchronisation of adc+spt001 (free saponins) activity at the correct time and location in the target cells of the patient. The inventors of the present application now provide a 1-component conjugate solution that provides a solution to this problem.

The inventors of the present application have now surprisingly found that the amount of saponin required to enhance the effect of a toxin on tumor cells can be reduced by a factor of 10, or even by a factor of about 100 to 1000, when the saponin is conjugated to a target cell targeting moiety such as a receptor ligand (e.g. EGF or a cytokine), an antibody, or a binding fragment or domain thereof. For tumor cells, a range of cell surface receptors are currently known to be specifically expressed on the surface of such abnormal cells. The inventors of the present application successfully coupled saponins to antibodies and ligands to bind receptors such as trastuzumab, cetuximab, etc. that are typically enriched on tumor cells. Such targeted saponin conjugates do be capable of enhancing, for example, the cytotoxic effect of the toxin within the target cell, requiring at least 10-fold (e.g., 100-fold to 1000-fold) lower effective dosages of the conjugate than would be required for the same saponin in free form (i.e., without the tumor cell targeting moiety).

The inventors of the present application have now also invented that target cell specific delivery and subsequent endocytosis of (targeted) saponins is also possible for non-abnormal cells or abnormal cells not associated with e.g. cancer (tumour cells), autoimmune diseases, virus infected cells etc. That is, the inventors of the present application surprisingly found that ASGPR provides a suitable target receptor for cells having this receptor, such as liver cells, for the access of saponins provided with ligands of ASGPR, in particular ASGPR1 (see for example fig. 3: saponin SO1861 conjugated to a single GalNAc moiety (also referred to as 'SPT 001'). Known ligands such as monovalent GalNAc and trivalent GalNAc (GN) ₃ Successfully conjugated with one or more (2, 4, 8, etc.) saponin molecules, providing a saponin conjugate comprising an ASGPR ligand and comprising at least one saponin moiety, preferably a 12, 13-dehydrooleanane-type disaccharide chain pentacyclic triterpene saponin having an aldehyde functional group at position C-23.

Contacting cells (e.g., liver cells) expressing ASGPR (e.g., ASGPR 1) with a saponin conjugate comprising a ligand of ASGPR (see, e.g., fig. 4 and 19C) surprisingly results in uptake of the saponin, as is evident when such cells are co-targeted with: such as a conjugate comprising a gene silencing nucleic acid and a binding molecule for a target cell surface molecule (e.g. CD71, ASGPR), resulting in improved saponin dependent gene silencing in a target cell, or a conjugate comprising such a binding molecule for a target cell and a toxin, resulting in saponin dependent cytotoxicity. Contacting cells (e.g., liver cells) expressing ASGPR (e.g., ASGPR 1) with a saponin conjugate comprising an ASGPR ligand unexpectedly results in uptake of the saponinIt is apparent, however, that such cells are contacted with such saponin conjugates comprising a saponin, a ligand of ASGPR1 (e.g., galNAc or (GN) ₃ ) And also comprises an oligonucleotide for silencing mRNA expression and/or silencing protein expression in a cell (i.e., an oligonucleotide conjugate of the invention comprising at least one saponin, a ligand of ASGPR1, and an oligonucleotide, which are covalently linked together, such as by a trifunctional linker). Providing a ligand of ASGPR to a saponin results in an effect in a target cell exerted by co-administered effector molecules such as targeted oligonucleotides or toxins (e.g., anti-CD 71 antibodies or ASGPR ligands conjugated to nucleic acids or toxins) or by oligonucleotides that are also conjugated to ligands of saponin and ASGPR1 to oligonucleotide conjugates of the invention (see also fig. 22A-C and 24D). Such effects are clearly to a much lower extent in the absence of targeted saponins when the target cells are contacted with effector molecules (such as free oligonucleotides or oligonucleotides conjugated with, for example, trivalent GalNAc), if any. Thus, the saponin conjugates of the invention and the oligonucleotide conjugates of the invention improve the efficacy of effector molecules such as oligonucleotides when: target cells comprising ASGPR on their surface are contemplated, and such target cells are contacted with a saponin-ASGPR ligand of the invention and a (targeted) effector molecule, or with an oligonucleotide conjugate comprising a saponin, galNAc (as ligand for ASGPR 1), an oligonucleotide (as effector moiety), which saponin serves to enhance endosomal escape of the effector moiety from endosomes and/or lysosomes to the cytoplasm and/or eventually to the endosome of the nucleus of the target cell. Thereby, the therapeutic window of the effector molecule is improved: at the same dose, there is a higher therapeutic effect under the influence of co-administration of the saponin conjugates or under the influence of the oligonucleotide conjugates; similar therapeutic effects at lower doses under the influence of co-administration of saponin conjugates or under the influence of oligonucleotide conjugates, wherein the oligonucleotide is an effector moiety. The inventors of the present application have also found that, in order to achieve a certain level of therapeutic effect in target ASGPR expressing cells (e.g. liver cells) when compared to the amount of saponin required to achieve a similar effect when the saponin is not provided with a ligand of ASGPR, The dosage of saponin conjugate or oligonucleotide conjugate required is at least 10-fold (e.g., about 100-fold to 1000-fold) lower, and the therapeutic effect is induced by co-administered (targeted (ASGPR 1) effector molecules (e.g., ASO, siRNA, BNA, toxins such as protein toxins), or by oligonucleotides contained as effector moieties in the oligonucleotide conjugate. The inventors of the present application have also found that when at least one saponin moiety (e.g., SO1861 moiety or SO1832 moiety) is covalently linked to an oligonucleotide conjugate comprising at least one GalNAc (e.g., trivalent GalNAc), at least 10-fold (e.g., about 100-fold to 1000-fold) lower doses of the oligonucleotide are required in target ASGPR expressing cells (e.g., liver cells) to achieve a level of therapeutic effect induced by ((ASGPR 1) -targeted) oligonucleotides (e.g., ASO, siRNA, BNA) administered to the cells.

Thus, surprisingly, the inventors of the present application now provide improved therapeutic methods using ASGPR as target receptor for the saponin-mediated delivery of effector molecules (e.g. therapeutic oligonucleotides) in the cytoplasm of ASGPR expressing cells, resulting in the effector molecules inducing similar therapeutic effects at lower doses than possible in the absence of the saponin conjugates of the present invention or in the absence of covalently linked saponins in the oligonucleotide conjugates. Thus, when considering the delivery of effector molecules into the cytoplasm or nucleus of (e.g., hepatocytes), the present invention is achieved by providing a saponin-ASGPR ligand conjugate of the present invention and by providing an oligonucleotide conjugate of the present invention (e.g., saponins, oligonucleotides, (GalNAc) covalently linked together in a single conjugate) ₃ ) And provides improved therapeutic uses for saponin stimulation. The advantages achieved by the inventors of the present application are in particular: lower doses of effector molecules such as oligonucleotides (siRNA or ASO) are required by co-administration of ((ASGPR 1) -targeted) effector molecules with saponins or by administration of oligonucleotide conjugates comprising: galNAc moiety for targeting ASGPR1, comprising an oligonucleotide (e.g. siRNA or ASO, for silencing mRNA expression associated with a selected gene and/or for silencing protein expression by targeting a selected gene) and comprising at least one saponin (for once the oligonucleotide has been conjugated toThe ASGPR1 ligand in the compound is engulfed and delivered to the endosome/lysosome of a cell carrying ASGPR1, such as a hepatocyte, after binding to cell ASGPR1, stimulating endosome escape of the oligonucleotide); and the required dosage of saponin is lower by: saponins are provided as covalent conjugates of ASGPR ligands (e.g., trivalent GalNAc), and one or more saponin moieties (saponin conjugates of the invention), and effector moieties, such as therapeutic oligonucleotides (e.g., siRNA or ASO) (oligonucleotide conjugates of the invention). Surprisingly, the ligand of ASGPR can be the same in the saponin conjugate and in the targeted effector molecule conjugate, producing a highly potent effector molecule mediated effect in the target cells under the stimulatory influence of saponins. That is, a saponin conjugate and an effector molecule conjugate (both comprising a covalently linked ligand of ASGPR, such as trivalent GalNAc) can be co-administered to a target cell (e.g., a liver cell having ASGPR exposed at its surface) to efficiently obtain the desired therapeutic effect of the effector molecule, while both conjugates target the same receptor and enter the cell using the same endocytosis pathway. This surprising finding is of great benefit for hepatocyte-targeted therapies, such as therapies involving cytoplasmic delivery of nucleic acids (ASO, BNA, siRNA, just to name a few), because hepatocytes do express ASGPR, which is specific for hepatocytes (e.g., ASGPR 1), while there is virtually no additional hepatocyte receptor specific enough for hepatocyte-targeted therapies.

Thus, the present invention opens up a new way to improve hepatocyte-directed therapies, where effector molecules such as ASO and siRNA must be delivered and localized within the cell for their mode of action. The targeted saponins provided as the saponin conjugates of the invention and the oligonucleotide conjugates of the invention broaden the therapeutic window of such effector molecules (oligonucleotides such as siRNA, ASO) and at the same time, by providing the saponin with a hepatocyte-targeted binding molecule, the therapeutic window of the saponin is also improved according to the invention. Thus, the inventors of the present application achieved efficient oligonucleotide delivery, providing a solution for the major translational limitations evident in the therapeutic oligonucleotide field. For oligonucleotide-based drug platforms, the inventors of the present application provide solutions based on the bioconjugates of the present invention for improved oligonucleotide delivery resulting in improved oligonucleotide delivery. The improved delivery systems of the invention result in increased efficacy of the oligonucleotides, which may help reduce toxicity and/or may help reduce off-target effects of ASGPR targeting oligonucleotide systems (e.g., galNAc-oligonucleotide conjugates).

The inventors of the present application sought to synthesize a non-prion conjugate consisting of two or three bioactive small molecules, e.g. when compared to the molecular size (molecular weight) of the antibodies used in antibody-based (antibody-drug) conjugates, such as IgG, the conjugates of the present invention remain bioactive when considering the bioactivity of each of the two or three small molecules comprised by the conjugate: the saponin conjugates of the invention and the oligonucleotide conjugates of the invention. The conjugates of the invention are relatively small and, despite the fact that two or three small molecules are covalently conjugated together in a single molecule, are still capable of exerting biological activity associated with the nature of each small molecule. That is, saponins enhance endosomal and lysosomal escape of oligonucleotides taken up by cells. That is, ASGPR-carrying cells ingest GalNAc of the present invention comprising a saponin conjugate and an oligonucleotide conjugate. That is, the gene silencing effect is evident when ASGPR-bearing cells are contacted with the oligonucleotide conjugates of the invention. The conjugates of the invention comprising a saponin and GalNAc provide endosomal escape enhancing activity against an oligonucleotide when co-administered to the same cell with a conjugate comprising an oligonucleotide and GalNAc. Both saponins and oligonucleotides are taken up by these cells, which is mediated by GalNAc binding to ASGPR on the target cells. Oligonucleotides delivered to the cytoplasm to a higher extent exert their intracellular biological activity under the influence of saponins in endosomes/lysosomes, manifested by silencing of oligonucleotide-targeted genes (e.g., targeted genes HSP27 and apoB). The oligonucleotide conjugates of the invention provide endosomal escape enhancing activity against the oligonucleotides comprised by the oligonucleotide conjugates. Both saponins and oligonucleotides are taken up by those cells as part of the oligonucleotide conjugates of the invention by GalNAc and Binding of ASGPR on target cells is mediated. Oligonucleotides delivered to the cytoplasm to a higher extent exert their intracellular biological activity under the influence of saponins in endosomes/lysosomes, manifested by silencing of oligonucleotide-targeted genes (e.g. targeted genes HSP27 or apoB). Thus, while relatively small molecules are conjugated together to form a single conjugate, at least one saponin (e.g., one, four, eight saponin moieties), galNAc moiety, or cluster of GalNAc moieties (e.g., GN) ₃ ) And each of the oligonucleotides remains biologically active as compared to the biological activity measured when the cell is contacted with, for example, free saponin (e.g., in combination with a molecule that exerts its activity in the cytoplasm), galNAc-oligonucleotide, free oligonucleotide. Although saponins, galnacs and oligonucleotides are relatively small molecules, conjugating these molecules together does not interfere with their biological activity.

Saponin conjugate-introduction

The present invention provides conjugates of saponins and ligands of asialoglycoprotein receptor (ASGPR), referred to herein as "saponin conjugates". The ASGPR ligand comprises at least one N-acetylgalactosamine (GalNAc) moiety. According to a preferred embodiment of the present invention, each GalNAc moiety binds to the remainder of the ASGPR ligand via a covalent bond with oxygen at position "1" as indicated in formula (I), or to a saponin moiety in case the ASGPR ligand consists of a single GalNAc moiety:

Such as (II) _S The ASGPR ligand is represented by a linker L, preferably via a saponin moiety _S A single GalNAc moiety bound to saponin moiety S.

ASGPR ligands may comprise more than one GalNAc moiety, such as 2, 3, 4, 5 or 6 GalNAc moieties, preferably 3 or 4GalNAc moieties, more preferably 3 GalNAc moieties. In such cases, it is preferred that each GalNAc moiety is covalently bound to a central bridging moiety B, respectively via oxygen at position "1", which is effective to form a bridge between the GalNAc moiety and the saponin moiety, preferably via a saponin moiety linker L _S A bridge is effectively formed between the GalNAc moiety and the saponin moiety. Such as (III) _S As shown in (a), galNAc moiety may be directly bound to bridging moiety B:

wherein n is an integer greater than or equal to 2, L _S Is a saponin moiety linker, and S is a saponin moiety. More preferably, as formula (IV) _S In (a), galNAc moiety is via GalNAc linker L _GAL In combination with bridging moiety B:

wherein n is an integer greater than or equal to 2, L _S Is a saponin moiety linker, and S is a saponin moiety. Although each occurrence of L can be independently selected _GAL But one skilled in the art will understand that at each occurrence L _GAL In the case of representing the same moiety, the synthesis of the saponin conjugate is simplified.

Saponin conjugate-linker L _S

Saponin part linker L _S The expression is suitable for making saponin and the formula (II) _S GalNAc or and the like of the formula (III) _S And (IV) _S Any chemical moiety to which bridging moiety B is covalently bound. The nature and size of the linker are not particularly limited, and the saponin is allowed to react with the compound of formula (II) _S GalNAc or and the like of the formula (III) _S And (IV) _S Covalent binding of bridging moiety B in (c) may be through a 'conventional' chemical functional group (e.g. an ester linkage) and through a "click chemistry" linker typically having a long chain length (resulting in inclusion examples)E.g. a linker E of more than 10 or more than 20 carbon atoms _S ) To realize the method. Suitable linkers L for binding molecules to each other _S And related coupling reactions are described in the handbook Hermanson, greg T.bioconjugate technologies [ bioconjugation techniques ]]Academic press [ Academic Press ]]2013.

As will be appreciated by those skilled in the art, the saponin moiety linker L _S Will typically be at least a first precursor L _S1 With a second precursor L _S2 Results of coupling reactions between them (e.g. "click chemistry" type), the first precursor L _S1 Covalently binding to GalNAc or bridging moiety B, said second precursor L _S2 Covalently bound to a saponin moiety. The principle is represented by the formula (II) _S Is shown in the following reaction scheme for the compounds of (a):

wherein L is _S Is a saponin part linker, L _S1 Is a saponin moiety linker L covalently bound to GalNAc or to bridging moiety B _S And L is _S2 Is a saponin moiety linker L covalently bound to a saponin moiety _S And S is a saponin moiety.

Having (III) _S The corresponding reaction scheme for the compounds of (2) is as follows:

wherein L is _S Is a saponin part linker, L _S1 Is a saponin moiety linker L covalently bound to GalNAc _S And L is _S2 Is a saponin moiety linker L covalently bound to a saponin moiety _S N is an integer greater than or equal to 2, B is a bridging moiety, and S is a saponin moiety.

Having (IV) _S The corresponding reaction scheme for the compounds of (2) is as follows:

wherein L is _S Is a saponin part linker, L _S1 Is a saponin moiety linker L covalently bound to GalNAc _S And L is _S2 Is a saponin moiety linker L covalently bound to a saponin moiety _S N is an integer greater than or equal to 2, B is a bridging moiety, S is a saponin moiety and L _GAL Is a GalNAc linker.

Saponin part linker L _S May be at least a first precursor L _S1 With a second precursor L _S2 As a result of the coupling reaction between the first precursor L _S1 Covalently binding to GalNAc or bridging moiety B, said second precursor L _S2 Covalently bound to a saponin moiety, wherein the coupling reaction is, for example, an azide-alkyne cycloaddition, thiol maleimide coupling, staudinger reaction, nucleophilic ring opening of strained heterocyclic electrophiles such as aziridine, epoxide, cyclic sulfate, aziridine (aziridinium) ion, carboxylate ion, carbonyl reactions of non-aldol type such as urea, thiourea, hydrazone, oxime ether, amide or aromatic heterocyclic formation, or addition of carbon-carbon double bonds such as epoxidation, aziridine, dihydroxylation, sulfonyl halide addition, nitrosyl halide addition, or michael addition, preferably wherein the coupling reaction is an azide-alkyne cycloaddition, thiol maleimide coupling, staudinger reaction, nucleophilic ring opening of strained heterocyclic electrophiles, more preferably wherein the coupling reaction is an azide-alkyne cycloaddition or thiol maleimide coupling.

According to some embodiments of the invention, the saponin moiety linker L _S Comprising semicarbazones and/or hydrazones and/or 1,2, 3-triazoles, preferably hydrazones and 1,2, 3-triazoles or at least one semicarbazone. Whenever 1,2, 3-triazole is mentioned in the context of the linker of the present application, this preferably means 1H-1,2, 3-triazole.

Such hydrazones are the result of, for example, coupling between a terminal hydrazide and an aldehyde-containing compound (e.g., an aldehyde-containing saponin). This hydrazide/aldehyde coupling is a 'click' chemical tool known in the art of bioconjugation and is described, for example, in Hermanson, greg t.bioconjugate technologies [ bioconjugation techniques ]. Academic press [ Academic press ], 2013. Such semicarbazones are the result of, for example, coupling between a terminal semicarbazone and an aldehyde-containing compound (e.g., an aldehyde-containing saponin).

Such 1,2, 3-triazoles are the result of, for example, coupling between azides and alkyne-containing compounds. This azide/alkyne coupling is a commonly known 'click' chemical tool in the field of bioconjugation and is described, for example, in Hermanson, greg t.bioconjugate techniques, academic press, 2013.

Therefore, it is preferred that the saponin moiety linker L _S Is the first precursor L _S1 With a second precursor L _S2 As a result of the coupling reaction between the first precursor L _S1 Covalently bound to GalNAc or bridging moiety B, first precursor L _S1 Comprises an azide; the second precursor L _S2 Covalently bound to the saponin moiety, a second precursor L _S2 Comprising alkynes and preferably comprising hydrazones resulting from the aldehyde coupling of a hydrazide/aldehyde with a saponin moiety or preferably comprising semicarbazones resulting from the aldehyde coupling of a semicarbazide/aldehyde with a saponin moiety.

Having (V) _S Precursor L present in the compound of (2) _S1 An embodiment of the structure of (a) is the following azide:

wherein a represents an integer greater than or equal to 0, preferably a represents an integer selected from 1, 2 and 3, more preferably a represents 2.

An embodiment of the structure of the precursor LS1 present in the compound of formula (VII) S or (VIII) S is the following azide:

wherein c represents an integer greater than or equal to 0, preferably c represents an integer in the range of 5-15, more preferably c represents 9.

Having (VI) _S Precursor L present in the compound of (2) _S2 An embodiment of the structure of (a) is the following hydrazone:

wherein a represents an integer greater than or equal to 0, preferably a represents an integer in the range of 2-6, more preferably a represents 4, and wherein L _S2a Representing an alkyne-containing moiety. As will be appreciated by those of skill in the art in light of the present disclosure, the hydrazone described in formula (XIX) results from the reaction of a hydrazide with an aldehyde of the saponin moiety.

L _S2a Preferably containing less than 20 carbon atoms, more preferably L _S2a Represents a moiety according to formula (XX):

thus, in some embodiments, the saponin conjugates are of formula (II) as described herein _S 、(III) _S Or (IV) _S Wherein the saponin moiety linker L _S Is provided with (V) _S 、(VII) _S Or (VIII) _S The compounds of formula (VI) _S Results of a coupling reaction between compounds of (1) wherein the first precursor L _S1 Is an azide, for example an azide of formula (XVII) or (XVIII), and wherein the second precursor L _S2 Is composed of alkyne-containing part L _S2a (e.g., alkynes having the formula (XX)) and hydrazones produced from the reaction of a hydrazide with an aldehyde of a saponin moiety.

Saponin conjugate-saponins

One aspect of the invention relates to a saponin conjugate comprising at least one saponin covalently linked to a ligand of an asialoglycoprotein receptor (ASGPR), wherein the ligand of ASGPR comprises at least one N-acetylgalactosamine (GalN)Ac) moiety, preferably three or four GalNAc moieties, more preferably three GalNAc moieties (also referred to as tri-GalNAc and (GN) ₃ ) More preferably the ligand of ASGPR comprises (GalNAc) ₃ Tris or consists thereof, wherein the at least one saponin is selected from the group consisting of a monosaccharide chain triterpenoid saponin and a disaccharide chain triterpenoid saponin.

One aspect of the invention relates to oligonucleotide conjugates comprising at least one saponin (preferably 1-32, more preferably 1-16, most preferably 1-8 saponin moieties, such as 1, 4 or 8 saponin moieties) covalently linked to a ligand of an asialoglycoprotein receptor (ASGPR), wherein the ligand of ASGPR comprises at least one N-acetylgalactosamine (GalNAc) moiety, preferably three or four GalNAc moieties, more preferably three GalNAc moieties, more preferably the ligand of ASGPR comprises (GalNAc) ₃ Tris or consists thereof, and is also covalently linked to an oligonucleotide, wherein the at least one saponin is selected from the group consisting of a monosaccharide-chain triterpenoid saponin and a disaccharide-chain triterpenoid saponin. When evaluating the intracellular effects of molecules such as protein toxins (e.g., saporin, carnation) or gene silencing oligonucleotides such as HSP27 BNA when contacted with target cells in the presence or absence of saponins, saponins are selected because of their endosomal escape enhancing activity. For the saponins used in the conjugates of the invention, typically, when the toxin or oligonucleotide is initially co-localized with the saponin in the endosome, the presence of the saponin in the endosome of the cell increases the biological activity (e.g., gene silencing, toxicity) in the cytoplasm of the cell by at least a factor of 10.

An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the saponin is a pentacyclic triterpenoid saponin of the 12, 13-dehydrooleanane type, preferably a 12, 13-dehydrooleanane type pentacyclic triterpenoid saponin having an aldehyde functional group at the C-23 position of the aglycone core structure of the saponin.

An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the saponin is a 12, 13-dehydrooleanane type monosaccharide chain or disaccharide chain pentacyclic triterpene saponin, preferably a 12, 13-dehydrooleanane type monosaccharide chain or disaccharide chain pentacyclic triterpene saponin having an aldehyde functional group at position C-23 of the aglycone core structure of the saponin.

An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the saponin comprises an aglycone core structure selected from the group consisting of:

2 alpha-hydroxy oleanolic acid;

16 alpha-hydroxy oleanolic acid;

hederagenin (23-hydroxy oleanolic acid);

16 alpha, 23-dihydroxyoleanolic acid;

silk carnation sapogenin;

soap skin acid;

escin-21 (2-methylbut-2-enoate) -22-acetate;

23-oxo-staurogenin C-21, 22-bis (2-methylbut-2-enoate);

23-oxo-staurogenin C-21 (2-methylbut-2-enoate) -16, 22-diacetate;

digitonin;

3,16,28-trihydroxy oleanane-12-ene;

carnation acid;

and

The derivatives of these and the derivatives thereof,

preferably, the sapogenins comprise a aglycone core structure selected from the group consisting of saponaric acid and serrulaspin or derivatives thereof, more preferably the sapogenins core structure is saponaric acid or derivatives thereof. Such preferred aglycones (or their derivatives) comprise an aldehyde group at the C-23 atom or their derivatives as described elsewhere herein. Without wishing to be bound by any theory, such aldehyde groups contribute to the endosomal escape enhancing activity of triterpenoid glycosides comprising aglycones selected from group C (especially saponaric acid and sericin).

One embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein at least one saponin comprises a first sugar chain, said first sugar chain being associated with the C of the aglycone core structure of said at least one saponin ₃ Atoms or C ₂₈ Atomic bond, preferably with C ₃ Atomic binding, and/or wherein the at least one saponin comprises a second sugar chain, the second sugar chain being associated with C of the aglycone core structure of the at least one saponin ₂₈ The atom-binding, preferably, the saponin comprises a first sugar chain and a second sugar chain. Thus, when the saponin conjugate of the present invention or the oligonucleotide conjugate of the present invention comprises a saponin having two glycans (sugar chains), the first sugar chain is bound at position C of the aglycone core structure ₃ At a position C where the second sugar chain is bound to the aglycone core structure ₂₈ Where it is located.

One embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein

The saponin comprises a sugar chain bound to the aglycone core structure, said sugar chain being selected from group a:

GlcA-、

Glc-、

Gal-、

Rha-(1→2)-Ara-、

Gal-(1→2)-[Xyl-(1→3)]-GlcA-、

Glc-(1→2)-[Glc-(1→4)]-GlcA-、

Glc-(1→2)-Ara-(1→3)-[Gal-(1→2)]-GlcA-、

Xyl-(1→2)-Ara-(1→3)-[Gal-(1→2)]-GlcA-、

Glc-(1→3)-Gal-(1→2)-[Xyl-(1→3)]-Glc-(1→4)-Gal-、

Rha-(1→2)-Gal-(1→3)-[Glc-(1→2)]-GlcA-、

Ara-(1→4)-Rha-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-、

Ara-(1→4)-Fuc-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-、

Ara-(1→4)-Rha-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-、

Ara-(1→4)-Fuc-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-、

Ara-(1→4)-Rha-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-、

Ara-(1→4)-Fuc-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-、

Ara-(1→4)-Rha-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-、

Ara-(1→4)-Fuc-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-、

Xyl-(1→4)-Rha-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-、

Xyl-(1→4)-Fuc-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-、

Xyl-(1→4)-Rha-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-、

Xyl-(1→4)-Fuc-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-、

Xyl-(1→4)-Rha-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-、

Xyl-(1→4)-Fuc-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-、

Xyl-(1→4)-Rha-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-、

xyl- (1- > 4) -Fuc- (1- > 2) -Gal- (1- > 2) -Fuc- (1- > 2) -GlcA-

The derivatives of these and the derivatives thereof,

or (b)

The saponin comprises a sugar chain bound to the aglycone core structure, said sugar chain being selected from group B:

Glc-，

Gal-，

Rha-(1→2)-[Xyl-(1→4)]-Rha-，

Rha-(1→2)-[Ara-(1→3)-Xyl-(1→4)]-Rha-，

Ara-，

Xyl-，

Xyl- (1.fwdarw.4) -Rha- (1.fwdarw.2) - [ R1- (. Fwdarw.4) ] -Fuc-, wherein R1 is 4E-methoxy cinnamic acid,

xyl- (1.fwdarw.4) -Rha- (1.fwdarw.2) - [ R2- (. Fwdarw.4) ] -Fuc-, wherein R2 is 4Z-methoxycinnamic acid,

Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-4-OAc-Fuc-，

xyl- (1.fwdarw.4) - [ Glc- (1.fwdarw.3) ] -Rha- (1.fwdarw.2) -3, 4-di-OAc-Fuc-,

xyl- (1.fwdarw.4) - [ Glc- (1.fwdarw.3) ] -Rha- (1.fwdarw.2) - [ R3- (. Fwdarw.4) ] -3-OAc-Fuc-, wherein R3 is 4E-methoxycinnamic acid,

Glc-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-4-OAc-Fuc-，

Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-4-OAc-Fuc-，

(Ara-or Xyl-) (1.fwdarw.3) - (Ara-or Xyl-) (1.fwdarw.4) - (Rha-or Fuc-) (1.fwdarw.2) - [4-OAc- (Rha-or Fuc-) (1.fwdarw.4) ] - (Rha-or Fuc-),

Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Qui-(1→4)]-Fuc-，

Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-Fuc-，

Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-Fuc-，

Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-Fuc-，

Ara/Xyl-(1→4)-Rha/Fuc-(1→4)-[Glc/Gal-(1→2)]-Fuc-，

api- (1.fwdarw.3) -Xyl- (1.fwdarw.4) - [ Glc- (1.fwdarw.3) ] -Rha- (1.fwdarw.2) - [ R4- (. Fwdarw.4) ] -Fuc-, wherein R4 is 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid,

api- (1- & gt 3) -Xyl- (1- & gt 4) -Rha- (1- & gt 2) - [ R5- (& gt 4) ] -Fuc-, wherein R5 is 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid), api- (1- & gt 3) -Xyl- (1- & gt 4) -Rha- (1- & gt 2) - [ Rha- (1- & gt 3) ] -4-OAc-Fuc-,

Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4-OAc-Fuc-，

6-OAc-Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3-OAc-Rha-(1→3)]-Fuc-，

Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3-OAc--Rha-(1→3)]-Fuc-，

Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Qui-(1→4)]-Fuc-，

Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-[Qui-(1→4)]-Fuc-，

Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc-，

xyl- (1.fwdarw.3) -Xyl- (1.fwdarw.4) -Rha- (1.fwdarw.2) - [3, 4-di-OAc-Qui- (1.fwdarw.4) ] -Fuc-,

Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-Fuc-，

6-OAc-Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-Fuc-，

Glc-(1→3)-[Xyl-(1→3)-Xyl-(1→4)]-Rha-(1→2)-Fuc-，

Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc-，

Api/Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4OAc-Fuc-，

Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4OAc-Fuc-，

Api/Xyl- (1.fwdarw.3) -Xyl- (1.fwdarw.4) - [ Glc- (1.fwdarw.3) ] -Rha- (1.fwdarw.2) - [ R6- (. Fwdarw.4) ] -Fuc-, wherein R6 is 5-O- [5-O-Rha- (1.fwdarw.2) -Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid,

Api/Xyl- (1.fwdarw.3) -Xyl- (1.fwdarw.4) - [ Glc- (1.fwdarw.3) ] -Rha- (1.fwdarw.2) - [ R7- (. Fwdarw.4) ] -Fuc-, wherein R7 is 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid,

Api/Xyl- (1.fwdarw.3) -Xyl- (1.fwdarw.4) - [ Glc- (1.fwdarw.3) ] -Rha- (1.fwdarw.2) - [ R8- (. Fwdarw.4) ] -Fuc-, wherein R8 is 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid,

api- (1.fwdarw.3) -Xyl- (1.fwdarw.4) -Rha- (1.fwdarw.2) - [ R9- (. Fwdarw.4) ] -Fuc-, wherein R9 is 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid,

xyl- (1- & gt 3) -Xyl- (1- & gt 4) -Rha- (1- & gt 2) - [ R10- (& gt 4) ] -Fuc-, wherein R10 is 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid,

api- (1.fwdarw.3) -Xyl- (1.fwdarw.4) -Rha- (1.fwdarw.2) - [ R11- (. Fwdarw.3) ] -Fuc-, wherein R11 is 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid,

xyl- (1- > 3) -Xyl- (1- > 4) -Rha- (1- > 2) - [ R12- (. Fwdarw.3) ] -Fuc-, wherein R12 is 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid,

glc- (1.fwdarw.3) - [ Glc- (1.fwdarw.6) ] -Gal-, and a process for preparing the same

The derivatives of these and the derivatives thereof,

or (b)

The saponin is a disaccharide chain triterpene glycoside (disaccharide chain triterpene saponin, preferably pentacyclic saponin, more preferably 12, 13-dehydrooleanane type saponin, preferably 12, 13-dehydrooleanane type saponin having an aldehyde functional group at position C-23 of the sapogenin core structure) comprising a first sugar chain selected from group a bound to the aglycon core structure and comprising a second sugar chain selected from group B bound to the aglycon core structure.

Thus, when the saponin conjugate of the present invention or the oligonucleotide conjugate of the present invention comprises a saponin having two glycans (sugar chains), the first sugar chain is bound to C of the aglycone core structure ₃ At position C of the aglycone core structure, and a second sugar chain is bound at position C of the aglycone core structure ₂₈ Where it is located.

An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the aglycone core structure of the saponin is selected from any one or more of the following: saponaric acid, sericin and derivatives thereof, preferably wherein the aglycone core structure of the saponin is saponaric acid or sericin, more preferably saponaric acid.

One embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the saponin is at least one disaccharide saponin comprising a first sugar chain comprising a terminal glucuronic acid residue and a second sugar chain comprising at least four sugar residues in a branched configuration; preferably, wherein the first sugar chain is Gal- (1→2) - [ xy- (1→3) ] -GlcA and/or wherein the branched second sugar chain of at least four sugar residues comprises terminal fucose residues and/or terminal rhamnose residues.

An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the saponin comprises a first sugar chain at the C-3 position of the aglycone core structure of the saponin and/or a second sugar chain at the C-28 position of the aglycone core structure of the saponin; preferably, wherein the first sugar chain is a carbohydrate substituent on a C-3 β -OH group of the sapogenin core structure and/or wherein the second sugar chain is a carbohydrate substituent on a C-28-OH group of the sapogenin core structure.

Without wishing to be bound by any theory, the presence of aldehyde groups or their derivatives in the aglycone core structure (also referred to herein as 'aglycone') is beneficial to the ability of such saponins to stimulate and/or enhance endosomal escape of (effector) molecules (e.g. oligonucleotides) when such saponins are co-located with the (effector) molecules in the endosomes of the cells. Thus, the saponin conjugates of the present invention or the oligonucleotide conjugates of the present invention comprising saponins having aglycones with aldehyde groups are preferred. In saponaric acid and sericin, the aldehyde group is located at the C23 atom of the aglycone.

An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the saponin is selected from the group consisting of: quillaja saponaria saponins, dipsacus asperoides saponin B, saikosaponin A, saikosaponin D, macranthosaponin A, phytolaccatin, escin salt, AS6.2, NP-005236, AMA-1, AMR, alpha-hederagenin, NP-012672, NP-017777, NP-017778, NP-017774, NP-018110, NP-017772, NP-018109, NP-017888, NP-017889, NP-018108, SA1641, AE X55, NP-017674, NP-017810, AG1, NP-003881, NP-017676, NP-017677 NP-017706, NP-017705, NP-017773, NP-017775, SA1657, AG2, SO1861, GE1741, SO1542, SO1584, SO1658, SO1674, SO1832, SO1862, SO1904, QS-7, QS1861, QS-7api, QS1862, QS-17, QS-18, QS-21A-apio, QS-21A-xylo, QS-21B-apio, QS-21B-xylo, beta-escin, escin Ia, tea seed saponin I, tea seed saponin J, assam saponin F, digitonin, primrose acid 1 and AS64R, their stereoisomers, their derivatives, and combinations thereof; preferably the saponins are selected from: QS-21, QS-21 derivatives, SO1861 derivatives, SO1832 derivatives, SA1641 derivatives, GE1741 derivatives, and combinations thereof; more preferably the saponins are selected from: QS-21 derivatives, SO1861 derivatives, and combinations thereof, most preferably the saponin is a SO1861 derivative. More preferably, the saponins are selected from: QS-21, QS-21 derivatives, SO1861 derivatives, SA1641 derivatives, GE1741 derivatives, and combinations thereof; more preferably the saponins are selected from: QS-21 derivatives, SO1861 and combinations thereof; most preferably the saponin is a SO1861 derivative or SO1861, or a SO1832 derivative or SO1832. Such derivatives of saponins are endosomal escape enhancing derivatives to the same or similar extent as their non-derivatized natural counterparts.

A preferred embodiment is an oligonucleotide conjugate of the invention and/or a saponin conjugate of the invention, wherein the conjugate comprises a saponin that is any one or more of the following:

a) A saponin selected from any one or more of list a:

-a mixture of sapogenins of the quillaja saponaria, or saponins isolated from the quillaja saponaria, such as Quil-A, QS-17-api, QS-17-xyl, QS-21A, QS-21B, QS-7-xyl;

-a mixture of carnation saponins, or saponins isolated from carnation;

-a mixture of saponinium album saponins, or saponins isolated from saponinium album;

-a mixture of soapstock saponins, or saponins isolated from soapstock; and

-a saponaria saponins mixture, or saponins isolated from saponaria barks, such as Quil-A, QS-17-api, QS-17-xyl, QS-21A, QS-21B, QS-7-xyl; or (b)

b) A saponin comprising a silk diabolo sapogenin core structure selected from list B:

SA1641, carnation saponin A, NP-017772, NP-017774, NP-017777, NP-017778, NP-018109, NP-017888, NP-017889, NP-018108, SO1658 and phytolaccagenin; or (b)

c) A saponin comprising a sapogenin core structure selected from list C:

AG1856, AG1, AG2, agrostemmoside E, GE1741, silk bamboo saponin 1 (Gyp 1), NP-017674, NP-017810, NP-003881, NP-017676, NP-017677, NP-017705, NP-017706, NP-017773, NP-017775, SA1657, saponaria oside B, SO1542, SO1584, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862, SO 4, QS-7api, QS-17, QS-18, QS-21A-apio, QS-21A-xylo, QS-21B-apio and QS-21B-xylo; or (b)

d) A sapogenin core structure comprising a 12, 13-dehydrooleanane type selected from list D, having no aldehyde group at the C-23 position of the aglycone:

escin Ia, escin salt, alpha-hederagenin, AMA-1, AMR, AS6.2, AS64R, assam saponin F, dipsacus asperosaponin B, esculentoside A, lonicera macranthoides saponin A, NP-005236, NP-012672, primula acid 1, saikosaponin A, saikosaponin D, tea seed saponin I and tea seed saponin J;

preferably, the saponin is any one or more of the saponins selected from the list A, B or C, more preferably selected from the list B or C,

even more preferably, the saponins are selected from list C.

Preferred are oligonucleotide conjugates of the invention and/or saponin conjugates of the invention, wherein the saponin is any one or more of the following: AG1856, GE1741, saponins isolated from Quil-A, QS-17, QS-21, QS-7, SA1641, saponins isolated from soapbark, saponaria oside B, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862 and SO1904; preferably, wherein the saponin is any one or more of the following: QS-21, SO1832, SO1861, SA1641 and GE1741; more preferably wherein the saponin is QS-21, SO1832 or SO1861; most preferably SO1861.

A more specific embodiment is an oligonucleotide conjugate of the invention and/or a saponin conjugate of the invention, wherein the saponin is a saponin isolated from saponaria, preferably wherein the saponin is any one or more of the following: saponarioside B, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862 and SO1904; more preferably, wherein the saponin is any one or more of the following: SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862 and SO1904; even more preferably, wherein the saponin is any one or more of: SO1832, SO1861 and SO1862; even more preferably, wherein the saponins are SO1832 and SO1861; most preferably SO1861.

Such triterpenoid glycoside-type saponins are capable of enhancing endosomal escape of (effector) molecules present in the endosome (or lysosome) of a cell when such saponins are co-located within the cell with such (effector) molecules (e.g., oligonucleotides comprised by the oligonucleotide conjugates of the invention). The inventors of the present application established that when the saponin conjugates of the present invention comprise a saponin or when the oligonucleotide conjugates of the present invention comprise a saponin, the endosomal escape enhancing activity of these saponins is at least about 5-fold more potent, that is, at least about 10-fold more potent (e.g., 10-1000-fold more potent or about 100-1000-fold more potent), when the saponin is contacted with a cell. When such cells are contacted with (effector) molecules and saponins at a concentration of saponins 100-1000 times higher than the concentration of the same saponins comprised by the saponin conjugates of the invention or comprised by the oligonucleotide conjugates of the invention that is required to achieve the same degree of delivery of the (effector) molecules, such as oligonucleotides selected from ASO or siRNA, from outside the cell into the endosome and eventually into the cytoplasm and/or nucleus of said cells, the free saponins are capable of stimulating the delivery of the (effector) molecules in the cytoplasm of the cells. Saponins exhibiting such endosomal escape enhancing activity, as well as saponins having a high structural similarity to those having established the cytoplasmic delivery capacity of the enhancing (effector) molecules, are listed in table A1. The effectiveness (potency) of targeted delivery of a saponin into the endosome of a target cell (preferably a liver cell) is thus at least 5-10 fold (e.g. about 100 to 1000 fold) higher after binding of a ligand of ASGPR (e.g. ASGPR 1) such as a single GalNAc moiety or a cluster of three covalently coupled GalNAc moieties to a cell surface binding site on said cell and after endocytosis, when the saponin is part of a saponin conjugate of the invention or of an oligonucleotide conjugate of the invention, compared to contacting the same cell with a free, non-targeted saponin (which does not provide a ligand for ASGPR binding to the ASGPR of the target cell).

Typically, for the saponin conjugates of the invention and the oligonucleotide conjugates of the invention, once the conjugate is taken up by ASGPR-carrying cells and transported into endosomes, the covalently bound saponin of the conjugate is cleaved (released) from the conjugate. In the endosome, chemical conditions and/or pH are such that covalent bonds between one or more saponins and the remainder of the conjugate are broken and the saponins are released into the endosome in free form. Preferably, the cleaved saponin has its natural chemical structure. For example, when the aldehyde group of the saponin is implicit in the covalent coupling of the saponin to the GalNAc moiety (typically via a linker) and/or the oligonucleotide (typically via a linker), preferably the saponin released from the conjugate in the endosome again comprises this aldehyde group formed under the influence of the uncoupling chemistry. Without wishing to be bound by any theory, it is believed that the saponin in free form (preferably with freely accessible aldehyde groups, preferably with an aldehyde function at position C-23 of the aglycone core structure of the saponin) exerts optimal endosomal escape enhancing activity in the endosome. For example, under the influence of an acidic pH in the endosome of a cell (e.g. a mammalian cell, such as a human cell, e.g. a tumour cell, a liver cell), the saponins are cleaved from the conjugate, for example by hydrazone bonds or semicarbazone bonds. Thus, preferably, in the conjugates of the invention, the saponins are covalently bound by cleavable bonds, preferably by cleavable covalent bonds that cleave inside the endosome, e.g. bonds that are cleaved due to acidic conditions in the endosome.

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Preferably, the saponins of the saponin conjugates and/or the saponins of the oligonucleotide conjugates of the present invention are plant-derived saponins. The saponin comprised by the saponin conjugates of the invention and/or the saponin comprised by the oligonucleotide conjugates of the invention is preferably a saponin isolated from a plant. The term "plant" is understood to include plants and trees. For example, saponins are isolated from the roots of plants or the bark of trees. For example, saponins are isolated from roots of plants. Examples of such plants from which saponins are extracted (isolated) are quillaja saponaria, carnation (Gypsophila paniculate l.), saponaria officinalis (Saponaria officinalis) (e.g., saponaria officinalis l.), and carnation (Gypsophila elegans) (e.g., carnation m.bieb). Preferably, the saponin conjugates and/or oligonucleotide conjugates comprise a single type of saponin, preferably a single type of saponin derived from plant material, such as plant roots, such as SO1861, SO1862 or SO1832 from soapstock (e.g. soapstock l., preferably soapstock l.) (roots), or such as QS-21, QS-7 or QS-17 from quillaja (roots).

Suitable sources for isolating saponins according to the invention, i.e. those which show enhanced endosomal escape activity, are quillaja saponaria, carnation, soapstock and carnation, and quillaja bark. Thus, saponins suitable for the saponin derivative of the invention and for the saponin conjugate of the invention are for example:

Quil-A, QS-17-api, QS-17-xyl, QS-21A, QS-21B, QS-7-xyl,

saponin album, from which saponins are isolated.

Soapstock saponins, saponins isolated from soapstock (preferred),

Quil-A, QS-17-api, QS-17-xyl, QS-21A, QS-21B, QS-7-xyl.

Furthermore, in addition to QS-21, the individual saponins present in QS-21 are also suitable saponins for use in the saponin conjugates of the present invention, i.e., the saponins depicted as the saponins of scheme Q:

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thus, for all embodiments of the invention, the saponins are preferably of a single type, preferably saponins isolated from plant material (e.g. plant derived roots).

One embodiment is an oligonucleotide conjugate of the invention, wherein the saponin comprised by the oligonucleotide conjugate is isolated from a plant. Preferably, the saponins are isolated from a part of a plant (such as a root), or from a part of a tree (such as bark). Preferably, the saponins are isolated from roots derived from plants.

In embodiments, the saponin conjugate or oligonucleotide conjugate comprises at least one saponin, wherein the saponin is a derivative, wherein

i. The aglycone core structure of the saponin comprises a derivatized aldehyde group;

the sugar chain of the saponin (preferably selected from group a sugar chains) comprises a derivatized carboxyl group;

the sugar chain of the saponin, preferably selected from group B, comprises a derivatized acetoxy (Me (CO) O-) group; or (b)

Any combination of derivatizations (i), (ii) and/or (iii) is present.

Exemplary derivatization of aldehyde groups of the aglycone core structure (i) comprises reduction to an alcohol, such as a primary alcohol; conversion to hydrazones (functional groups) or semicarbazones (functional groups); conversion to the hemiacetal; is converted into acetal; oxidizing to carboxyl; conversion to an imine; conversion to oxime; conversion to beta-hydroxyketone; or to an ketene, preferably reduced to an alcohol, such as a primary alcohol; or to hydrazones (functional groups) or semicarbazones (functional groups). Once present in the endosome of the cell that ingests the saponin or oligonucleotide conjugates of the present invention, the derivatized saponins of the conjugates of the present invention still exert their endosomal escape enhancing activity.

In embodiments, the saponin is a derivative wherein the aglycone core structure comprises an aldehyde group that has been derivatized by:

reduction to alcohols, such as primary alcohols; or (b)

-conversion to a compound of formula R ^b HC＝NNHR ^a Hydrazones of (2); preferably into a compound of formula R ^b HC＝NNH(CO)R ^a Hydrazones of (2)

Wherein R is ^a Is a group containing less than 20 carbon atoms, preferably less than 10 carbon atoms, and R ^b Is the remainder of the saponins. PreferablyGround, R ^a Is an N-alkylated maleimide. In certain embodiments, the aldehyde group has been converted to a hydrazone bond by reaction with: n-epsilon-maleimidocaaproic acid hydrazide (EMCH), N- [ beta-maleimidopropionic acid]Hydrazide (BMPH), or N- [ kappa-maleimido undecanoic acid]Hydrazide (KMUH).

Exemplary derivatization of carboxyl groups of sugar chains (ii) comprises: reduction to alcohols, such as primary alcohols; conversion to an amide; conversion to esters; conversion to amines, such as primary or secondary amines; or decarboxylated, preferably reduced to an alcohol, such as a primary alcohol; conversion to an amide; or to an ester; more preferably to an amide.

Preferably the derivatised carboxyl group is part of a glucuronic acid moiety, and preferably the sugar chain is selected from group a.

In an embodiment, the saponin is a derivative wherein the sugar chain (preferably a sugar chain selected from group a) comprises a carboxyl group (preferably of the glucuronic acid moiety) that has been derivatized by:

reduction to alcohols, such as primary alcohols;

-conversion to a compound of formula R ^a NH(CO)R ^b Is an amide of (a); or (b)

-conversion to a compound of formula R ^a O(CO)R ^b Esters of (2)

Wherein R is ^a Is a group containing less than 20 carbon atoms, preferably less than 10 carbon atoms, and R ^b Is the remainder of the saponins. Preferably, R ^a Is an N-alkylated maleimide or diol. In particular embodiments, the carboxyl group has been derivatized by conversion to an amide bond by reaction with 2-amino-2-methyl-1, 3-propanediol (AMPD) or N- (2-aminoethyl) maleimide (AEM).

Exemplary derivatization of the acetoxy groups of the sugar chains (iii) comprises: conversion to an alcohol, such as a secondary alcohol, by deacetylation, optionally followed by conversion of the resulting alcohol to an ether; esters or ketones; preferably to alcohols, such as secondary alcohols, by deacetylation.

Preferably, the derivatized acetoxy group is part of a sugar chain selected from group B.

In an embodiment, the saponin is a derivative wherein the sugar chain (preferably a sugar chain selected from group B) comprises an acetoxy group that has been derivatized by:

conversion to alcohols, such as secondary alcohols, by deacetylation; or (b)

Conversion by deacetylation to an alcohol, such as a secondary alcohol, followed by conversion of the resulting alcohol to a compound of formula R ^a OR ^b Ethers of the formula R ^a (CO)OR ^b Or an ester of formula R ^a (CO)R ^b Ketone of (2)

Wherein R is ^a Is a group containing less than 20 carbon atoms, preferably less than 10 carbon atoms, and R ^b Is the remainder of the saponins. Preferably, R ^a Is an N-alkylated maleimide or diol.

Thus, in embodiments, the saponin is a derivative, wherein:

the aglycone core structure comprises aldehyde groups which have been derivatised by:

-reduction to an alcohol;

-conversion to hydrazone bonds by reaction with N-epsilon-maleimidocaaproic acid hydrazide (EMCH);

-conversion to hydrazone bonds by reaction with N- [ β -maleimidopropionic acid ] hydrazide (BMPH); or (b)

-conversion to hydrazone bonds by reaction with N- [ kappa-maleimido undecanoic acid ] hydrazide (KMUH);

selecting a group a sugar chain comprising a carboxyl group, preferably a carboxyl group of a glucuronic acid moiety, which has been derivatised by reaction with 2-amino-2-methyl-1, 3-propanediol (AMPD) or N- (2-aminoethyl) maleimide (AEM) to an amide bond;

sugar chains selected from group B comprise an acetoxy group (Me (CO) O-), which acetoxy group has been derivatized by conversion to a hydroxyl group (HO-); or (b)

Any combination of derivatizations (i), (ii) and/or (iii) is present.

One embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the saponin is a saponin derivative, wherein

i. The saponin derivative comprises an aglycone core structure comprising a derivatized aldehyde group;

The saponin derivative comprises a sugar chain, preferably selected from group a, comprising a derivatized carboxyl group;

the saponin derivative comprises a sugar chain, preferably selected from group B, comprising a derivatized acetoxy (Me (CO) O-) group; or (b)

The saponin derivative comprises any combination of derivatizations i, ii, and iii, preferably any combination of derivatizations of two of i, ii, and iii.

An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the saponin is any one or more of the following: SO1861, SA1657, GE1741, SA1641, QS-21A, QS-21A-api, QS-21A-xyl, QS-21B, QS-21B-api, QS-21B-xyl, QS-7-api, QS-17-xyl, QS1861, QS1862, quillaja saponin, saponin album, QS-18, quil-A, gyp1, marshall saponin A, AG, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, SO1904, stereoisomers thereof, derivatives thereof, and combinations thereof; preferably the saponins are selected from: QS-21, QS-21 derivatives, SO1861 derivatives, SO1832, SA1641 derivatives, GE1741 derivatives, and combinations thereof; more preferably the saponins are selected from: QS-21 derivatives, SO1861 derivatives, and combinations thereof; most preferably the saponin is a SO1861 derivative. Most preferably the saponins are selected from: QS-21 derivatives, SO1861 and combinations thereof; even more preferably the saponin is a SO1861 derivative or SO1861, or a SO1832 derivative or SO1832.

An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the saponin is a saponin derivative of a saponaric acid saponin or a silk diabolo sapogenin saponin according to the invention and is represented by molecule 1:

wherein the method comprises the steps of

A ₁ Represents hydrogenA monosaccharide or a linear or branched oligosaccharide, preferably A ₁ Represents a sugar chain selected from group A, more preferably A ₁ Represents a sugar chain selected from group A and A ₁ Comprising or consisting of glucuronic acid moieties;

A ₂ represents hydrogen, monosaccharides or linear or branched oligosaccharides, preferably A ₂ Represents a sugar chain selected from group B, more preferably A ₂ Represents a sugar chain selected from group B and A ₂ Comprising at least one acetoxy (Me (CO) O-) group (e.g., one, two, three or four acetoxy groups),

wherein A is ₁ And A ₂ At least one of which is not hydrogen, preferably A ₁ And A ₂ Are oligosaccharide chains;

and R is hydrogen in sericin or hydroxyl in saponaric acid;

wherein the saponin derivative corresponds to the saponin represented by molecule 1, wherein at least one, preferably one or two, more preferably one of the following derivatizations is present:

i. c of saponaric acid or serrulate sapogenin ₂₃ The aldehyde group at the position has been derivatized;

when A ₁ Represents a sugar chain selected from group A and A ₁ When comprising or consisting of glucuronic acid moieties, A ₁ Has been derivatized with carboxyl groups of glucuronic acid moieties; and

When A2 represents a sugar chain selected from group B and A2 comprises at least one acetoxy group, one sugar moiety of A2 or one or more (preferably all) of the acetoxy groups of two or more sugar moieties have been derivatized.

Embodiments are saponin conjugates of the invention or oligonucleotide conjugates of the invention, wherein A ₁ Represents a sugar chain selected from group A and comprises or consists of a glucuronic acid moiety, and wherein A ₁ Has been derivatized with carboxyl groups of glucuronic acid moieties, and/or wherein A ₂ Represents a sugar chain selected from group B and A ₂ Comprises at least one acetoxy group, and wherein A ₂ At least one acetoxy group of (2)The radical of the radical has been derivatized.

An embodiment is the saponin conjugate of the present invention or the oligonucleotide conjugate of the present invention, wherein the saponin represented by molecule 1 is a disaccharide chain triterpenoid saponin.

An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the saponin derivative corresponds to a saponin represented by molecule 1, wherein at least one, preferably one or two, more preferably one of the following derivatizations is present:

i. C of saponaric acid or serrulate sapogenin ₂₃ The aldehyde group at the position has been derivatized by:

-reduction to an alcohol;

-converting to hydrazone linkage by reaction with N-epsilon-maleimidocaaproic acid hydrazide (EMCH) to provide saponin-Ald-EMCH, such as SO1861-Ald-EMCH or QS-21-Ald-EMCH, wherein the maleimide group of EMCH is optionally derivatized by forming a thioether bond with mercaptoethanol;

-conversion to hydrazone linkage by reaction with N- [ β -maleimidopropionic acid ] hydrazide (BMPH), wherein the maleimide group of BMPH is optionally derivatized by forming a thioether bond with mercaptoethanol; or (b)

-conversion to hydrazone bond by reaction with N- [ kappa-maleimido undecanoic acid ] hydrazide (KMUH), wherein the maleimide group of KMUH is optionally derivatized by forming a thioether bond with mercaptoethanol;

when A ₁ Represents a sugar chain selected from group A and A ₁ When comprising or consisting of glucuronic acid moieties, A ₁ The carboxyl group of the glucuronic acid moiety of (a) has been derivatized by reaction with 2-amino-2-methyl-1, 3-propanediol (AMPD) or N- (2-aminoethyl) maleimide (AEM) to an amide bond, to provide a saponin-Glu-AMPD (e.g., QS-21-Glu-AMPD or SO 1861-Glu-AMPD) or a saponin-Glu-AEM (e.g., QS-21-Glu-AEM or SO 1861-Glu-AEM); and

When A ₂ Represents a sugar chain selected from group B and A ₂ When at least one acetoxy group is included, A ₂ Is a sugar moiety or acetoxy group of two or more sugar moietiesOne or more, preferably all, of which have been derivatized by conversion to hydroxyl groups (HO-) by deacetylation.

One embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein A ₁ Is Gal- (1- > 2) - [ Xyl- (1- > 3)]-GlcA and/or A ₂ Is Glc- (1.fwdarw.3) -Xyl- (1.fwdarw.4) -Rha- (1.fwdarw.2) - [ Xyl- (1.fwdarw.3) -4-OAc-Qui- (1.fwdarw.4)]-Fuc; preferably the saponin represented by molecule 1 is 3-O-beta-D-galactopyranosyl- (1.fwdarw.2) - [ beta-D-xylopyranosyl- (1.fwdarw.3)]-beta-D-glucuronopyranosyl soap pinoic acid 28-O-beta-D-glucopyranosyl- (1.fwdarw.3) -beta-D-xylopyranosyl- (1.fwdarw.4) -alpha-L-rhamnopyranosyl- (1.fwdarw.2) - [ beta-D-xylopyranosyl- (1.fwdarw.3) -4 OAc-beta-D-quiniopyranosyl- (1.fwdarw.4)]-beta-D-fucopyranoside; more preferably the saponin is any one or more of the following: SO1861, SO1832, GE1741, SA1641 and QS-21 or derivatives thereof; most preferred is SO1861 or a derivative thereof, or SO1832 derivative or SO1832.

One embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the saponin is a saponin derivative, wherein

i. The saponin derivative comprises a aglycone core structure comprising aldehyde groups which have been derivatized by:

-reduction to an alcohol;

-converting to a hydrazone bond by reaction with N-epsilon-maleimidocaproyl hydrazide (EMCH) to provide a saponin-Ald-EMCH, such as SO1861-Ald-EMCH or QS-21-Ald-EMCH, wherein the maleimide group of the EMCH is optionally derivatized by forming a thioether bond with mercaptoethanol;

-conversion to hydrazone linkage by reaction with N- [ β -maleimidopropionic acid ] hydrazide (BMPH), wherein the maleimide group of BMPH is optionally derivatized by forming a thioether bond with mercaptoethanol; or (b)

-conversion to hydrazone bond by reaction with N- [ kappa-maleimido undecanoic acid ] hydrazide (KMUH), wherein the maleimide group of KMUH is optionally derivatized by forming a thioether bond with mercaptoethanol;

the saponin derivative comprises a sugar chain, preferably selected from group a, comprising a carboxyl group, preferably a carboxyl group of a glucuronic acid moiety, which has been derivatised by conversion to an amide bond by reaction with 2-amino-2-methyl-1, 3-propanediol (AMPD) or N- (2-aminoethyl) maleimide (AEM), thereby providing a saponin-Glu-AMPD (such as QS-21-Glu-AMPD or SO 1861-Glu-AMPD) or a saponin-Glu-AEM (such as QS-21-Glu-AEM or SO 1861-Glu-AEM);

The saponin derivative comprises a sugar chain, preferably selected from group B, comprising an acetoxy (Me (CO) O-) group that has been derivatized by deacetylation to a hydroxyl group (HO-); or (b)

The saponin derivative comprises any combination of derivatizations i, ii, and iii, preferably any combination of derivatizations of two of i, ii, and iii;

preferably, the sapogenin derivative comprises an aglycone core structure, wherein the aglycone core structure comprises an aldehyde group that has been derivatised by reaction with EMCH to convert to a hydrazone bond, wherein the maleimide group of EMCH is optionally derivatised by formation of a thioether bond with mercaptoethanol.

One embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the saponin is a saponin derivative, wherein

i. The saponin derivative comprises a aglycone core structure comprising an aldehyde group that has been derivatized by reaction with N-epsilon-maleimidocaaproic acid hydrazide (EMCH) to a hydrazone bond, thereby providing a saponin-aldemch (e.g., SO 1861-aldemch or QS-21-aldemch);

the saponin derivative comprises a sugar chain, preferably selected from group a, comprising a carboxyl group, preferably a carboxyl group of a glucuronic acid moiety, which has been derivatised by conversion to an amide bond by reaction with N- (2-aminoethyl) maleimide (AEM), thereby providing a saponin-Glu-AEM (e.g. QS-21-Glu-AEM or SO 1861-Glu-AEM); or (b)

The saponin derivative comprises a combination of derivatizations i.and ii..

An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the saponin derivative comprises a aglycone core structure, wherein the aglycone core structure comprises an aldehyde group, and wherein the saponin derivative comprises a sugar chain, preferably selected from group a, comprising a carboxyl group, preferably a carboxyl group of a glucuronic acid moiety, which glucuronic acid moiety has been derivatized by conversion to an amide bond by reaction with N- (2-aminoethyl) maleimide (AEM).

An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the saponin derivative is represented by molecule 2:

or wherein the saponin derivative is represented by molecule 3:

one embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein at least one of the saponins and the ligand of ASGPR are covalently linked directly or via at least one linker. An embodiment is an oligonucleotide conjugate of the invention, wherein the at least one saponin and the ligand of ASGPR are covalently linked directly or via at least one linker, and/or wherein the at least one saponin and the oligonucleotide are covalently linked directly or via at least one linker, and/or wherein the ligand of ASGPR and the oligonucleotide are covalently linked directly or via at least one linker, preferably the at least one saponin, the ligand of ASGPR and the oligonucleotide are linked via at least one linker.

One embodiment is a saponin conjugate of the invention, wherein the GalNAc moiety is preferably via a saponin linker L _s Combined with saponin S, as shown in formula (II) _S Is represented by:

one embodiment is a saponin conjugate of the invention wherein each GalNAc moiety is covalently bound to a central bridging moiety B via oxygen at position "1" of the GalNAc moiety, respectively, said central bridging moiety B being effective to form a bridge between the GalNAc moiety and the saponin moiety, preferably via a saponin moiety linker L _S Effectively forming a bridge between the GalNAc moiety and the saponin moiety, as in formula (III) _S As shown in:

wherein n is an integer greater than or equal to 2, preferably n is 3, L _S Is a saponin moiety linker, and S is the saponin moiety.

One embodiment is a saponin conjugate of the invention, wherein the GalNAc moiety is via a GalNAc linker L _GAL Combined with bridging moiety B, e.g. of formula (IV) _S As shown in:

wherein n is an integer greater than or equal to 2, preferably n is 3, L _S Is a saponin moiety linker, and S is the saponin moiety.

An embodiment is a saponin conjugate of the invention, wherein the saponin moiety linker Ls represents a moiety suitable for use in reacting a saponin with a compound of formula (II) _S GalNAc or and the like of the formula (III) _S And (IV) _S Any chemical moiety to which bridging moiety B is covalently bound.

One embodiment is a saponin conjugate of the invention, wherein the saponin moiety linker L _S Is at least a first precursor L _S1 With a second precursor L _S2 As a result of the coupling reaction between the first precursor L _S1 Covalently binding to GalNAc or bridging moiety B, said second precursor L _S2 Covalently bound to a saponin moiety, wherein L _S1 Is a saponin moiety linker L covalently bound to GalNAc or to bridging moiety B _S And L is _S2 Is a saponin moiety linker L covalently bound to a saponin moiety _S Is a precursor of (a).

One embodiment is a saponin conjugate of the invention, wherein the saponin moiety linker L _S Is at least a first precursor L _S1 With a second precursor L _S2 As a result of the coupling reaction between the first precursor L _S1 Covalently binding to GalNAc or bridging moiety B, said second precursor L _S2 Covalently bound to a saponin moiety, wherein the coupling reaction is selected from the group consisting of: azide-alkyne cycloaddition, thiol maleimide coupling, staudinger reaction, nucleophilic ring opening of strained heterocyclic electrophiles, carbonyl reactions of non-aldol type, addition of carbon-carbon double bonds, preferably wherein the coupling reaction is azide-alkyne cycloaddition, thiol maleimide coupling, staudinger reaction, nucleophilic ring opening of strained heterocyclic electrophiles, more preferably wherein the coupling reaction is azide-alkyne cycloaddition or thiol maleimide coupling.

One embodiment is a saponin conjugate of the invention, wherein the saponin moiety linker L _S Is the first precursor L _S1 With a second precursor L _S2 As a result of the coupling reaction between the first precursor L _S1 Covalently binding to GalNAc or bridging moiety B, said first precursor L _S1 Comprises an azide; the second precursor L _S2 Covalently bound to the saponin moiety, the second precursor L _S2 Comprising alkynes and preferably hydrazones resulting from the coupling of a hydrazide/aldehyde with the aldehyde of the saponin moiety.

An embodiment is a saponin conjugate of the present invention, wherein the structure of precursor LS1 is the following azide having formula (XVII):

wherein a represents an integer greater than or equal to 0, preferably a represents an integer selected from 1, 2 and 3, more preferably a represents 2, or

Wherein precursor L _S1 Comprises an azide of formula (XVIII):

wherein c represents an integer greater than or equal to 0, preferably c represents an integer in the range of 5-15, more preferably c represents 9.

One embodiment is a saponin conjugate of the present invention, wherein precursor L _S2 Comprises hydrazones of the following formula (XIX):

wherein a represents an integer greater than or equal to 0, preferably a represents an integer in the range of 2-6, more preferably a represents 4, and wherein L _S2a Representing an alkyne-containing moiety.

One embodiment is a saponin conjugate of the invention, wherein L _S2a Containing less than 20 carbon atoms, preferably L _S2a Represents a moiety according to formula (XX):

an embodiment is a saponin conjugate of the present invention, wherein the bridging moiety is a compound having the formula (XV):

wherein the oxygen atom of the compound having the formula (XV) is bonded to GalNAc or a GalNAc linker L _GAL Is bound to, and has a nitrogen atom of the compound of formula (XV) bonded to a saponin moiety linker L _S And (5) combining.

One embodiment is a saponin conjugate of the invention, wherein L _GAL Represents any chemical moiety suitable for covalently binding GalNAc to bridging moiety B.

One embodiment is a saponin conjugate of the invention, wherein L _GAL Containing 2 to 25 carbon atoms, preferably 7 to 15 carbon atoms, more preferably 11 carbon atoms, and wherein L _GAL Comprising at least one, preferably two amide moieties.

One embodiment is a saponin conjugate of the invention, wherein L _GAL Is a compound according to formula (XVI):

an embodiment is an oligonucleotide conjugate of the invention, wherein at least one GalNAc moiety, at least one saponin and the oligonucleotide are covalently bound via a trifunctional linker, preferably each of the GalNAc moiety, the saponin and the oligonucleotide are covalently bound to separate arms of the trifunctional linker. Preferred oligonucleotide conjugates of the invention are oligonucleotide conjugates in which at least one GalNAc moiety, preferably three GalNAc moieties, at least one saponin, preferably 1-16 saponin moieties, more preferably 1-8 saponin moieties (e.g. 1, 4 or 8 saponin moieties) and the oligonucleotide are covalently bound by a trifunctional linker, preferably each of one or more GalNAc moieties, saponin/saponin moieties and the oligonucleotide is covalently bound to a separate arm of the trifunctional linker. Examples of trifunctional linkers suitable for incorporation into the oligonucleotide conjugates of the invention are trifunctional linkers represented by formula (XXI):

An embodiment is an oligonucleotide conjugate of the invention, wherein a trifunctional linker, e.g. represented by formula (XXI), is covalently bound to one or more saponin moieties, preferably 1-16 saponin moieties, more preferably 1-8 saponin moieties (e.g. 1, 4 or 8 saponin moieties), by a first arm of the linker, is covalently bound to at least one GalNAc moiety, preferably 1-4 GalNAc moieties, more preferably 3 GalNAc moieties, by a second arm of the linker, via a third arm of the trifunctional linker, to an oligonucleotide, preferably an AON, e.g. BNA or siRNA.

One embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the ligand of ASGPR is represented by molecule (DD 3) or molecule (DD 4) (GalNAc) ₃ Tris：

Or wherein the ligand of ASGPR is single GalNAc represented by molecule II':

an embodiment is a saponin conjugate of the present invention, wherein at least one saponin is covalently bound to a molecule (DD 3) or a molecule (DD 4) or a molecule II 'of the present invention via a linker represented by molecule III':

wherein the hydrazide moiety of molecule III 'forms a covalent hydrazone bond with the aldehyde group in the saponin, and wherein the dibenzocyclooctyne group forms a covalent bond with the azide group of molecule (DD 3) or molecule (DD 4) or molecule II'.

An embodiment is an oligonucleotide conjugate of the invention, wherein at least one saponin is covalently bound to a ligand of ASGPR via at least one cleavable linker, and/or wherein at least one saponin is covalently bound to the oligonucleotide via at least one cleavable linker.

One embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein at least one saponin is covalently bound to a ligand of ASGPR via at least one cleavable linker.

An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the cleavable linker is subjected to cleavage under acidic, reducing, enzymatic and/or photoinduced conditions, and preferably the cleavable linker comprises a cleavable bond selected from the group consisting of: hydrazone bonds and hydrazide bonds that undergo cleavage under acidic conditions, and/or bonds that are readily proteolytically (e.g., proteolytically by cathepsin B), and/or bonds that are readily cleavable under reducing conditions (e.g., disulfide bonds).

One embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the cleavable linker is subjected to in vivo cleavage under acidic conditions, e.g. as present in endosomes and/or lysosomes of mammalian cells, preferably human cells, preferably the cleavable linker is subjected to in vivo cleavage at a pH of 4.0-6.5, and more preferably at a pH of +.5.5.

An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the conjugate comprises 1, 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, 128 or 1-100 saponin moieties, or any number of saponin moieties in between, such as 7, 9, 12 saponin moieties, and preferably 1, 4 or 8 saponin moieties. Preferably the saponin SO1861 or SO1832 or a functional derivative thereof, for example QS-21, is also a suitable saponin or a functional derivative thereof.

One embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the conjugate comprises 1 saponin moiety. One embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the conjugate comprises 4 or 8 saponin moieties.

One of the many benefits of the conjugates of the invention, as well as the use of the saponin conjugates and oligonucleotide conjugates of the invention, is that the number of saponin moieties in the conjugates suitable for achieving improved intracellular oligonucleotide activity (e.g., gene silencing activity) can be selected and screened when compared to the activity of the oligonucleotide when taken up by cells that are not co-contacted with the oligonucleotide. The inventors of the present application demonstrated (see example section) that the incorporation of a single saponin moiety in the conjugates of the present invention was sufficient to achieve improved intracellular oligonucleotide activity as measured by gene silencing. In further examples, it has been demonstrated that gene silencing is further improved by, for example, including 4 or 8 saponin moieties in the conjugates of the invention. The saponin conjugates and oligonucleotide conjugates provide the freedom to couple 1-64 or even more (e.g. 128) saponin moieties to one or more GalNAc moieties (preferably 3 GalNAc moieties for the saponin conjugates) or to one or more GalNAc moieties and oligonucleotides (for the oligonucleotide conjugates). As described above, the inventors of the present application have unexpectedly found that the combination of saponins and galnacs, or the combination of saponins, galnacs and oligonucleotides, does not interfere with the biological activity of any component in the conjugate. Furthermore, increasing the number of saponin moieties in the conjugate may increase the oligonucleotide activity. Without wishing to be bound by any theory, an increase in the number of saponin moieties increases endosomal escape enhancing activity of the saponin once taken up by the cell and transferred into the endosome. The administered oligonucleotides present in the endosome together with the saponin (either as part of the oligonucleotide conjugate or separate as GalNAc-oligonucleotide conjugate) are more efficiently transferred from the endosome into the cytoplasm due to the increased activity of the saponin-mediated endosome escape, resulting in increased gene silencing activity. For example, the possibility of incorporating one or more saponin moieties in the conjugate allows tailoring of the conjugate design when optimal gene silencing with selected oligonucleotides is considered. Oligonucleotides that are relatively less efficient in transferring from endosomes to the cytoplasm, e.g., 1% -2% efficient, benefit from the presence of a single saponin moiety in the conjugate (the ratio in the oligonucleotide conjugate is 1:1), whereas oligonucleotides that are released to a lesser extent from endosomes into the cytoplasm can be increased in transferring to the cytoplasm by the use of more than one single saponin moiety in the conjugate, e.g., 2-100 moieties, 2-64, 2-34, 2-16, 4-12, 4-8 moieties. Suitable methods for incorporating more than one saponin moiety are, for example, incorporating dendrons, such as G2, G3 or G4 dendrons, into the conjugates of the invention, for example for incorporating 4 or 8 or 16 saponin moieties together in the conjugate. In the examples section, examples of improved gene silencing (degree of silencing at selected time points, duration of target gene silencing) are provided for oligonucleotide conjugates comprising a single saponin moiety, 4 saponin moieties and 8 saponin moieties. The inventors of the present application determined that by contacting a cell with an oligonucleotide conjugate of the present invention, the extent of gene silencing at a point in time is improved and the duration of gene silencing is prolonged compared to gene silencing in a cell contacted with the oligonucleotide in the absence of the saponin.

For the oligonucleotide conjugates of the invention, it is suitable (and preferred) that the conjugate comprises 1 or 3 GalNAc moieties, preferably 3 GalNAc moieties.

According to the invention, the saponins are typically one or more SO1861 or SO1832 (also referred to as SO 1831) moieties, e.g. 1-32 SO1861 or SO1832 moieties, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 moieties, more preferably 1, 2, 4, 8, 12 or 16 moieties, most preferably 1, 4 or 8 moieties, e.g. 1, 4 or 8 SO1861 or 1, 4 or 8 SO1832. The inventors of the present application determined that SO1832 is approximately the same as SO1861 in terms of endosomal escape enhancing activity against oligonucleotides (or protein toxins) in the endosome. As described above, the number of saponin moieties may be optimized for the conjugates of the invention when considering the extent and duration of gene silencing when the cells are contacted with the oligonucleotides. For the oligonucleotide conjugates of the invention, this also applies to the number of GalNAc moieties in the conjugates of the invention. Typically, when a conjugate of the invention comprises 1 or 3 GalNAc moieties, optimal gene silencing (degree and/or duration) is established.

The saponin SO1832 consists of a compound with a carbohydrate substituent Gal- (1.fwdarw.2) - [ Xyl- (1.fwdarw.3) at the C-3. Beta. -OH group]-GlcA-and the carbohydrate substituent Xyl- (1.fwdarw.3) -Xyl- (1.fwdarw.4) -Rha- (1.fwdarw.2) - [ Xyl- (1.fwdarw.3) -4-OAc-Qui- (1.fwdarw.4) at the C-28-OH group]-Fuc-sapogenin core (see also table A1). The chemical formula is C ₈₂ H ₁₂₈ O ₄₅ The exact mass was 1832,77 daltons. The saponin structure of SO1832 is according to the molecule (SO 1832):

those skilled in the art will appreciate that if the present inventionThe saponins contained in the saponin conjugates are of formula (II) as described herein _S 、(III) _S Or (IV) _S Wherein the saponin moiety linker L _S Is the first precursor L _S1 With a second precursor L _S2 As a result of the coupling reaction between the first precursor L _S1 Covalently binding to GalNAc or a bridging moiety, said second precursor L _S2 Covalently bound to a saponin moiety, said second precursor L _S2 Comprising hydrazones resulting from aldehyde coupling of a hydrazide/aldehyde to a saponin moiety or comprising semicarbazone functionalities resulting from aldehyde coupling of a semicarbazide/aldehyde to a saponin moiety, as described herein, means that the aldehyde groups have been occupied by linkers such that suitable saponin derivatives are those in which the saponin is derivatized by (ii) a carboxyl group of a sugar chain as described herein and/or (iii) an acetoxy group of a sugar chain as described herein.

Effector molecule conjugates-introduction

Certain pharmaceutical combinations and certain pharmaceutical compositions of the invention comprise a second conjugate of an effector molecule and a ligand for an asialoglycoprotein receptor (ASGPR), referred to herein as an "effector molecule conjugate". When an effector molecule binds to the remainder of the effector molecule conjugate, it is referred to herein as an "effector moiety". ASGPR ligands comprise at least one N-acetylgalactosamine (GalNAc) moiety and preferably three GalNAc moieties (GN) ₃ . According to a preferred embodiment of the present invention, each GalNAc moiety binds to the remainder of the ASGPR ligand via a covalent bond with oxygen at position "1" as indicated in formula (I), or to an effector moiety in case the ASGPR ligand consists of a single GalNAc moiety:

as shown in formula (II) E, ASGPR ligand consists of a single GalNAc moiety bound to effector moiety E, preferably via effector moiety linker L _E 。

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ASGPR ligands may comprise more than one GalNAc moiety, such as 2, 3, 4, 5 or 6 GalNAc moieties, preferably 3 or 4 GalNAc moieties, most preferably three moieties. In such a case, it is preferred that each GalNAc moiety is covalently bound via oxygen at position "1", respectively, to a central bridging moiety B, which is preferably effective to form a bridge between the GalNAc moiety and the effector moiety via an effector moiety linker LE. GalNAc moieties can be bound directly to bridging moiety B, e.g. of formula (III) _E As shown in:

wherein n is an integer greater than or equal to 2, L _E Is an effector moiety linker, and E is an effector moiety. More preferably, the GalNAc moiety is via a GalNAc linker L _GAL Combined with bridging moiety B, e.g. of formula (IV) _E As shown in:

wherein n is an integer greater than or equal to 2, L _E Is an effector moiety linker, and E is an effector moiety. Although each occurrence of L can be independently selected _GAL But one skilled in the art will understand that at each occurrence L _GAL In the case of representing the same moiety, the synthesis of the conjugate is simplified.

Effector molecule conjugate-linker L _E

Effector moiety linker L _E Representing a suitable compound for bringing effector moieties into association with a compound of formula (II) _E GalNAc or and the like of the formula (III) _E And (IV) _E Any chemical moiety to which bridging moiety B is covalently bound. The nature and size of the linker is not particularly limited and the effector molecule is allowed to react with the molecule of formula (II) _E GalNAc or and the like of the formula (III) _E And (IV) _E Covalent bonding of bridging moiety B in (a) may be via a 'conventional' chemical functional group (e.g. an ether linkage) or via a "click chemistry" linker typically having a long chain length (yielding a linker E comprising e.g. more than 10 or more than 20 carbon atoms) _L ) To realize the method. Suitable effector moiety linker L _E And related coupling reactions are described in the handbook Hermanson, greg T.bioconjugate technologies [ bioconjugation techniques ]]Academic press [ Academic Press ]]2013.

As will be appreciated by those skilled in the art, effector moiety linker L _E Will typically be at least a first precursor L _E1 With a second precursor L _E2 Results of coupling reactions between them (e.g., of the "click chemistry" type), the first precursor L _E1 Covalently binding to GalNAc or bridging moiety B, said second precursor L _E2 Covalently bound to an effector moiety. This principle is demonstrated in the following reaction scheme for compounds having formula (II):

wherein L is _E Is an effector moiety linker, L _E1 Effector moiety linker L which is covalently bound to GalNAc _E And L is _E2 An effector moiety linker L covalently bound to an effector moiety _E And E is an effector moiety.

The corresponding reaction scheme for the compounds of formula (III) is as follows:

wherein L is _E Is an effector moiety linker, L _E1 Effector moiety linker L which is covalently bound to GalNAc _E And L is _E2 An effector moiety linker L covalently bound to an effector moiety _E N is an integer greater than or equal to 2, and E is an effector Part(s).

The corresponding reaction scheme for compounds having formula (IV) E is as follows:

wherein L is _E Is an effector moiety linker, L _E1 Effector moiety linker L which is covalently bound to GalNAc _E And L is _E2 An effector moiety linker L covalently bound to an effector moiety _E N is an integer greater than or equal to 2, E is an effector moiety, and L _GAL Is a GalNAc linker and B is a bridging moiety.

Effector moiety linker L _E May be at least a first precursor L _E1 With a second precursor L _E2 As a result of the coupling reaction between the first precursor L _E1 Covalently binding to GalNAc or a bridging moiety, said second precursor L _E2 Covalently bound to an effector moiety, wherein the coupling reaction is, for example, an azide-alkyne cycloaddition, thiol maleimide coupling, a staudinger reaction, nucleophilic ring opening of strained heterocyclic electrophiles (e.g., aziridine, epoxide, cyclic sulfate, aziridine ion, carboxylate ion), carbonyl reactions of non-aldol type (e.g., urea, thiourea, hydrazone, oxime ether, amide or aromatic heterocyclic formation), or addition of carbon-carbon double bonds (e.g., epoxidation, aziridine, dihydroxylation, sulfonyl halide addition, nitrosyl halide addition, or michael addition), preferably wherein the coupling reaction is an azide-alkyne cycloaddition, thiol maleimide coupling, staudinger reaction, nucleophilic ring opening of strained heterocyclic electrophiles, more preferably wherein the coupling reaction is an azide-alkyne cycloaddition or thiol maleimide coupling.

According to some embodiments of the invention, effector moiety linker L _E Comprising a succinimide thioether moiety. Such succinimide thioethers are the result of, for example, thiol maleimide coupling between an N-substituted maleimide and a thiol-or thiol-containing compound. The thiol maleimide coupling is a biological prefix"click" chemistry tools are generally known in the art and are described, for example, in Hermanson, greg T.bioconjugate technologies [ bioconjugation techniques ]]Academic press [ Academic Press ]]2013 page 289. Therefore, effector moiety linker L is preferred _E Is the first precursor L _E1 With a second precursor L _E2 As a result of the coupling reaction between the first precursor L _E1 Covalently binding to GalNAc or a bridging moiety, said first precursor L _E1 Comprising an N-substituted maleimide; the second precursor L _E2 Covalently bound to an effector moiety, said second precursor L _E2 Comprising a thiol or precursor thereof. Suitable thiol precursors are disulfides, which can be cleaved (e.g., in situ) via reduction to the corresponding thiol.

Exist in a compound (V) _E 、(VII) _E Or (VIII) _E Precursor L in the compound of (2) _E1 The preferred structure of (a) is the following terminal N-substituted maleimide:

wherein X represents any linker suitable for covalently binding the terminal N-substituted maleimide to GalNAc or bridging moiety B. As will be appreciated by those skilled in the art, X may be the result of a coupling reaction between a first moiety covalently bound to GalNAc or bridging moiety and a second moiety covalently bound to maleimide. X may comprise hydrazone and/or 1,2, 3-triazole. Whenever 1,2, 3-triazole is mentioned in the context of the linker of the present application, this preferably means 1H-1,2, 3-triazole.

Exist in a compound (V) _E 、(VII) _E Or (VIII) _E Precursor L in the compound of (2) _E1 An embodiment of the structure of (a) is the following terminal N-substituted maleimide:

wherein a and b each independently represent an integer greater than or equal to 0, preferably a and b each independently represent an integer selected from 0, 1,2 and 3, more preferably a and b represent 2, and wherein L _E1a Represents hydrazone and/or 1,2, 3-triazole, preferably 1,2, 3-triazole, and wherein L _E1b Represents hydrazone and/or 1,2, 3-triazole, preferably hydrazone;

wherein b and c each independently represent an integer greater than or equal to 0, preferably b represents an integer selected from 0, 1,2 and 3 and c represents an integer in the range of 5-15, more preferably b represents 2 and c represents 9, wherein L _E1a Represents hydrazone and/or 1,2, 3-triazole, preferably 1,2, 3-triazole, and wherein L _E1b Represents hydrazone and/or 1,2, 3-triazole, preferably hydrazone;

and

Wherein c represents an integer greater than or equal to 0, preferably c represents an integer in the range of 5-15, more preferably c represents 9, wherein L _E1c Represents hydrazone and/or 1,2, 3-triazole, preferably 1,2, 3-triazole.

L _E1a 、L _E1b And L _E1c Each preferably contains less than 20 carbon atoms, more preferably L _E1a 、L _E1b And L _E1c Each independently represents a moiety according to formula (XIII), (XIV) or (XV), preferably L _E1a Is a 1,2, 3-triazole of the formula (XIII), L _E1b Is a hydrazone of formula (XIV), and L _E1c Is a 1,2, 3-triazole having the formula (XV):

thus, in some embodiments, the effector molecule conjugate is as described herein having the formulaII) _E 、(III) _E Or (IV) _E Wherein the effector moiety linker L _E Is provided with (V) _E 、(VII) _E Or (VIII) _E The compounds of formula (VI) _E Results of a coupling reaction between compounds of (1) wherein the first precursor L _E1 Is a terminal N-substituted maleimide of formula (X), (XI) or (XII), wherein L _E1a Is, for example, a 1,2, 3-triazole of the formula (XIII), L _E1b Is, for example, a hydrazone of the formula (XIV), and L _E1c Is, for example, a 1,2, 3-triazole of the formula (XV).

Effector molecule conjugate-effector moiety

One aspect of the invention relates to a pharmaceutical combination comprising:

-a first pharmaceutical composition comprising a saponin conjugate of the invention and optionally comprising a pharmaceutically acceptable excipient and/or a pharmaceutically acceptable diluent; and

-a second pharmaceutical composition comprising a second conjugate of an effector molecule and a ligand of ASGPR, or a third conjugate of an effector molecule and a binding molecule, and optionally comprising a pharmaceutically acceptable excipient and/or a pharmaceutically acceptable diluent, wherein the ligand of ASGPR preferably comprises at least one GalNAc moiety, preferably three or four GalNAc moieties, more preferably the ligand of ASGPR comprises (GalNAc) ₃ Tris or consists of the same, the binding molecule comprising a binding site for a cell surface molecule.

One aspect of the present invention relates to a pharmaceutical composition comprising:

-the saponin conjugate of the invention;

-a second conjugate of an effector molecule and a ligand of ASGPR, or a third conjugate of an effector molecule and a binding molecule, and optionally comprising a pharmaceutically acceptable excipient and/or a pharmaceutically acceptable diluent, wherein the ligand of ASGPR preferably comprises at least one GalNAc moiety, preferably three or four GalNAc moieties, more preferably the ligand of ASGPR comprises (GalNAc) ₃ Tris or consists of the same, the binding molecule comprising a binding site for a cell surface molecule.

An embodiment is the pharmaceutical combination of the invention comprising the saponin conjugate of the invention or the pharmaceutical composition of the invention comprising the saponin conjugate of the invention, wherein the effector molecule comprises or consists of at least one of the following: a small molecule (e.g., a drug molecule), a toxin (e.g., a protein toxin), an oligonucleotide (e.g., an AON, such as BNA), a heterologous nucleic acid or siRNA, an enzyme, a peptide, a protein, or any combination thereof, preferably, the effector molecule is a toxin, an enzyme, or an oligonucleotide.

An embodiment is the pharmaceutical combination of the invention comprising the saponin conjugate of the invention or the pharmaceutical composition of the invention comprising the saponin conjugate of the invention, wherein the ligand of ASGPR comprises (GalNAc) ₃ Tris or (GalNAc) ₃ Tris, and/or wherein the ligand of ASGPR and the effector molecule are conjugated via a covalent bond, preferably via at least one linker.

An embodiment is the pharmaceutical combination of the invention comprising the saponin conjugate of the invention or the pharmaceutical composition of the invention comprising the saponin conjugate of the invention, wherein the effector molecule is an oligonucleotide selected from any one or more of the following: short interfering RNA (siRNA), short hairpin RNA (shRNA), anti-hairpin microRNA (miRNA), single-stranded RNA, aptamer RNA, double-stranded RNA (dsRNA), anti-microRNA (anti-miRNA, anti-miR), antisense oligonucleotide (ASO), DNA, antisense DNA, locked Nucleic Acid (LNA), bridged Nucleic Acid (BNA), 2'-O,4' -aminoethylene Bridged Nucleic Acid (BNA) ^NC ) BNA-based siRNA, and BNA-based antisense oligonucleotide (BNA-aON).

An embodiment is the pharmaceutical combination of the invention comprising the saponin conjugate of the invention or the pharmaceutical composition of the invention comprising the saponin conjugate of the invention, wherein the effector molecule is an oligonucleotide selected from any one or more of the following: anti-miRNA, BNA-AON or siRNA, such as BNa-based siRNA, selected from chemically modified siRNA, metabolically stable siRNA, and chemically modified metabolically stable siRNA.

An embodiment is the pharmaceutical combination of the invention comprising the saponin conjugate of the invention or the pharmaceutical composition of the invention comprising the saponin conjugate of the invention, wherein the effector molecule is an oligonucleotide capable of silencing any of the following genes, e.g. when present in a mammalian cell: apolipoprotein B (apoB), HSP27, thyroxine Transporter (TTR), proprotein convertase subtilisin/kexin type 9 (PCSK 9), delta-aminolevulinate synthase 1 (ALAS 1), antithrombin 3 (AT 3), glycolate Oxidase (GO), complement component C5 (CC 5), the X gene of Hepatitis B Virus (HBV), the S gene of HBV, alpha-1 antitrypsin (AAT) and Lactate Dehydrogenase (LDH), and/or oligonucleotides capable of targeting aberrant mirnas, for example, when present in mammalian cells. An embodiment is the pharmaceutical combination of the invention comprising the saponin conjugate of the invention or the pharmaceutical composition of the invention comprising the saponin conjugate of the invention, wherein the effector molecule is an oligonucleotide capable of silencing any of the following genes, e.g. when present in a mammalian cell: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AT3, GO, CC5, X gene of HBV, S gene of HBV, AAT, miR-122, hepatitis b virus HbsAg, LDHA, CEBPA and LDH, and/or for use in the treatment or prevention of diseases or health problems involving any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AT3, GO, CC5, X gene of HBV, S gene of HBV, AAT, miR-122, hepatitis B virus HbsAg, LDHA, CEBPA and LDH; and/or an oligonucleotide capable of targeting an aberrant miRNA when present in a mammalian cell, for example. Preferred target genes are HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AAT, miR-122, hepatitis B virus HbsAg, LDHA and CEBPA, more preferably the genes HPS27 and apoB.

An embodiment is the pharmaceutical combination of the invention comprising the saponin conjugate of the invention or the pharmaceutical composition of the invention comprising the saponin conjugate of the invention, wherein the effector molecule is an oligonucleotide, e.g. an mRNA capable of targeting expression of any one of the following proteins when present in a mammalian cell: apoB, HSP27, TTR, PCSK9, ALAS1, AT3, GO, CC5, expression products of the X gene of HBV, expression products of the S gene of HBV, AAT and LDH, or are capable of antagonizing or restoring miRNA function, such as inhibiting oncogenic miRNA (onco-miR) or repressing expression of onco-miR, for example, when present in mammalian cells. An embodiment is the pharmaceutical combination of the invention comprising the saponin conjugate of the invention or the pharmaceutical composition of the invention comprising the saponin conjugate of the invention, wherein the effector molecule is an oligonucleotide, e.g. an mRNA capable of targeting expression of any one of the following proteins when present in a mammalian cell: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AT3, GO, CC5, X gene of HBV, S gene of HBV, AAT, miR-122, hepatitis B virus HbsAg, LDHA, CEBPA and LDH, preferably HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AAT, miR-122, hepatitis B virus HbsAg, LDHA and CEBPA, or is capable of antagonizing or restoring miRNA function, such as inhibiting oncogenic miRNA (onco-miR) or suppressing expression of onco-miR, for example, when present in a mammalian cell.

An embodiment is the pharmaceutical combination of the invention comprising the saponin conjugate of the invention or the pharmaceutical composition of the invention comprising the saponin conjugate of the invention, wherein the effector molecule is or comprises a toxin comprising or consisting of at least one molecule selected from any one or more of the following: peptides, proteins, enzymes (such as urease and Cre recombinase), prions, ribosome inactivating proteins, and/or bacterial toxins, plant toxins; more preferably selected from any one or more of the following: viral toxins, such as apoptotic proteins; bacterial toxins, such as shiga toxin, shiga-like toxin, pseudomonas aeruginosa exotoxin (PE) or exotoxin a of PE, full length or truncated Diphtheria Toxin (DT), cholera toxin; mycotoxins, such as α -sarcin; a plant toxin comprising a ribosome inactivating protein and a chain of type 2 ribosome inactivating protein, such as carnation toxin protein, e.g. carnation toxin protein-30 or carnation toxin protein-32, saporin, e.g. saporin-S3 or saporin-S6, deluge ribosome inactivating protein or deimmunized derivative of the same, deluge ribosome inactivating protein, shiga-like toxin a, pokeweed antiviral protein, ricin a chain, boea samara a chain, abrin a chain, mistletoe lectin A chain A; or animal or human toxins, such as frog ribonucleases, or granzyme B or angiogenic proteins from human, or any fragment or derivative thereof; preferably, the protein toxin is carnation toxin and/or saporin, and/or comprises or consists of at least one of the following: ribosome-targeting toxins, extension factor-targeting toxins, tubulin-targeting toxins, DNA-targeting toxins, and RNA-targeting toxins; more preferably any one or more of the following: enmei, pa Shu Tuo, maytansinoid derivative DM1, maytansinoid derivative DM4, monomethyl auristatin E (MMAE, vitamin D), monomethyl auristatin F (MMAF, mo Futing), calicheamicin, N-acetyl-gamma-calicheamicin, pyrrolobenzodiazepine(PBD) dimer, benzodiazepine +.>CC-1065 analog, docamicin, doxorubicin, paclitaxel, docetaxel, cisplatin, cyclophosphamide, etoposide, docetaxel, 5-fluorouracil (5-FU), mitoxantrone, tubulysin, indolinone benzodiazepine>AZ13599185, candidiasis, rhizobia, methotrexate, anthracyclines, camptothecin analogues, SN-38, DX-8951f, escitalopram mesylate, truncated forms of pseudomonas aeruginosa exotoxin (PE 38), docamicin derivatives, amanitine, alpha-amanitine, splice inhibitors, telavancin, ozunamicin, teslin, ambastatin 269 and grommet, or derivatives thereof.

Conjugate-bridging moiety B

Exists in the formula (III) described herein _S Or (IV) _S Is of formula (III) _E Or (IV) _E Bridging moiety B in the effector molecule conjugate of (2) represents a moiety suitable for covalent binding of 2 or more, preferablyOptionally 3 or 4, more preferably 3 GalNAc moieties and a saponin moiety linker L _S Or effector moiety linker L _E Any portion of (2).

According to a preferred embodiment, bridging moiety B is a compound having formula (XV):

wherein the oxygen atom of the compound of formula (XV) and GalNAc (corresponding to the compound of formula (III) _S Or (III) _E Compounds of (2) or with GalNAc linker L _GAL (corresponding to the formula (IV) _S Or (IV) _E A compound of formula (XV) and having a nitrogen atom of the compound of formula (XV) bonded to a saponin moiety linker L _S Or effector moiety linker L _E And (5) combining.

Those skilled in the art will appreciate that having the formula (III) _S 、(III) _E 、(IV) _S Or (IV) _E Wherein the bridging moiety B is a compound of formula (XV) corresponds to a compound wherein the ASGPR ligand consists of (GalNAc) ₃ Tris-composed effector molecule conjugates.

Conjugate-linker L _GAL

GalNAc linker L _GAL Represents a compound suitable for use in the reaction of GalNAc with the formula (IV) _S Or (IV) _E Any chemical moiety to which the bridging moiety of (a) is covalently bound. The nature and size of the linker is not particularly limited.

According to an embodiment, galNAc linker L _GAL Each containing from 2 to 25 carbon atoms, preferably from 7 to 15 carbon atoms, more preferably 11 carbon atoms. Preferably GalNAc linker L _GAL Comprising at least one, preferably two amide moieties. Particularly preferred GalNAc linker L _GAL Is a compound according to formula (XVI):

an embodiment is a pharmaceutical combination of the invention comprising a saponin conjugate of the invention or a pharmaceutical composition of the invention comprising a saponin conjugate of the invention, wherein the binding molecule comprising a binding site for a cell surface molecule comprised by the third conjugate is a ligand of a cell surface molecule or an antibody comprising a binding site for a cell surface molecule or at least one domain or fragment thereof comprising the binding site.

An embodiment is the pharmaceutical combination of the invention comprising the saponin conjugate of the invention or the pharmaceutical composition of the invention comprising the saponin conjugate of the invention, wherein the third conjugate is any one or more of the following: antibody-toxin conjugates, receptor-ligand-toxin conjugates, antibody-drug conjugates, receptor-ligand-drug conjugates, antibody-nucleic acid conjugates, or receptor-ligand-nucleic acid conjugates.

An embodiment is a pharmaceutical combination of the invention comprising a saponin conjugate of the invention or a pharmaceutical composition of the invention comprising a saponin conjugate of the invention, wherein the binding molecule comprising a binding site for a cell surface molecule comprised by the third conjugate is capable of binding to any of the following cell surface molecules: CD71, CD63, CA125, epCAM (17-1A), CD52, CEA, CD44V6, FAP, EGF-IR, integrin, aggrecan-1, angiopoietin alpha-V beta-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, canag, integrin-alpha V, CA6, CD33, mesothelin, cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25, ephrin A4, MUC1, trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, FGF 3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, asialoglycoprotein receptor (ASP); preferably any one of the following: HER2, CD71, ASGPR and EGFR, more preferably CD71.

An embodiment is the pharmaceutical combination of the invention comprising the saponin conjugate of the invention or the pharmaceutical composition of the invention comprising the saponin conjugate of the invention, wherein the binding molecule comprising a binding site for a cell surface molecule comprised by the third conjugate is or comprises any one of the following: antibodies, preferably monoclonal antibodies such as human monoclonal antibodies, igG, molecules comprising or consisting of: single domain antibodies, at least one VHH domain, preferably camelid VH, variable heavy chain neoantigen receptor (VNAR) domain, fab, scFv, fv, dAb, F (ab) 2 and Fcab fragments.

One aspect of the invention relates to the pharmaceutical combination of the invention comprising the saponin conjugate of the invention or to the pharmaceutical composition of the invention comprising the saponin conjugate of the invention, for use as a medicament. One aspect of the invention relates to an oligonucleotide conjugate of the invention for use as a medicament.

One aspect of the present invention relates to the pharmaceutical combination of the present invention comprising the saponin conjugate of the present invention or the pharmaceutical composition of the present invention comprising the saponin conjugate of the present invention for use in the treatment or prevention of a disease or health problem wherein the expression product relates to any one or more of the following genes: apoB, TTR, PCSK9, ALAS1, AT3, GO, CC5, X gene of HBV, S gene of HBV, AAT and LDH. One aspect of the present invention relates to the pharmaceutical combination of the present invention comprising the saponin conjugate of the present invention or the pharmaceutical composition of the present invention comprising the saponin conjugate of the present invention for use in the treatment or prevention of a disease or health problem wherein the expression product relates to any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AT3, GO, CC5, X gene of HBV, S gene of HBV, AAT, miR-122, hepatitis B virus HbsAg, LDHA, CEBPA and LDH; preferably HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AAT, miR-122, hepatitis B virus HbsAg, LDHA and CEBPA; more preferably HSP27, apoB.

An embodiment is the use of the pharmaceutical combination of the invention comprising the saponin conjugate of the invention or the pharmaceutical composition comprising the saponin conjugate of the invention or the saponin conjugate of the following for the invention, for the treatment or prevention of: cancer, infectious disease, viral infection, hypercholesterolemia, cardiovascular disease, primary hyperoxaluria, hemophilia a, hemophilia B, AAT-associated liver disease, acute hepatic porphyrin disease, TTR-mediated amyloidosis, hereditary TTR amyloidosis (hATTR), complement-mediated disease, hepatitis b infection, hepatitis c infection, alpha 1-antitrypsin deficiency, beta-thalassemia or autoimmune disease.

An embodiment is a pharmaceutical combination comprising a saponin conjugate of the invention for use in the invention or a pharmaceutical composition comprising an oligonucleotide conjugate of the invention, wherein the saponin is a saponin derivative, preferably a QS-21 derivative or a SO1861 derivative or a SO1832 derivative according to the invention.

One aspect of the invention relates to an in vitro or ex vivo method for transferring a second or third conjugate of the invention from the outside of a cell into said cell, preferably followed by transferring an effector molecule comprised by the second or third conjugate according to the invention into the cytoplasm of said cell, comprising the steps of:

a) Providing a cell that expresses ASGPR on its surface and that expresses a cell surface molecule when transferring a third conjugate into the cell, wherein the third conjugate comprises a binding molecule for binding to the cell surface molecule, the cell preferably being selected from the group consisting of a hepatocyte, a virus-infected cell and a tumor cell;

b) Providing a second conjugate or a third conjugate according to the invention for transfer into the cell provided in step a);

c) Providing a saponin conjugate of the present invention;

d) Contacting the cell of step a) with the second or third conjugate of step b) and the saponin conjugate of step c) in vitro or ex vivo, thereby effecting transfer of the second or third conjugate from outside the cell into the cell, and preferably thereby effecting subsequent transfer of the second or third conjugate into the cytoplasm of the cell, or preferably thereby effecting subsequent transfer of at least the effector molecule comprised by the second or third conjugate into the cytoplasm of the cell.

An embodiment is an oligonucleotide conjugate of the invention, wherein the oligonucleotide is any one of BNA, a heterologous nucleic acid, siRNA, an antisense oligonucleotide.

An embodiment is an oligonucleotide conjugate of the invention, wherein the oligonucleotide is selected from any one or more of the following: short interfering RNA (siRNA), short hairpin RNA (shRNA), anti-hairpin microRNA (miRNA), single-stranded RNA, aptamer RNA, double-stranded RNA (dsRNA), anti-microRNA (anti-miRNA, anti-miR), antisense oligonucleotide (ASO), DNA, antisense DNA, locked Nucleic Acid (LNA), bridged Nucleic Acid (BNA), 2'-O,4' -aminoethylene Bridged Nucleic Acid (BNA) ^NC ) BNA-based siRNA, and BNA-based antisense oligonucleotide (BNA-aON).

An embodiment is an oligonucleotide conjugate of the invention, wherein the oligonucleotide is selected from any one or more of the following: anti-miRNA, BNA-AON or siRNA, such as BNa-based siRNA, selected from chemically modified siRNA, metabolically stable siRNA, and chemically modified metabolically stable siRNA.

An embodiment is an oligonucleotide conjugate of the invention, wherein the oligonucleotide is an oligonucleotide capable of silencing any of the following genes, e.g., when present in a mammalian cell and preferably when present in a human cell: apolipoprotein B (apoB), HSP27, thyroxine Transporter (TTR), proprotein convertase subtilisin/kexin type 9 (PCSK 9), TMPRSS6, deltA-Aminolevulinate synthase 1 (ALAS 1), antithrombin 3 (AT 3), glycolate Oxidase (GO), complement component C5 (CC 5), the X gene of Hepatitis B Virus (HBV), the S gene of HBV, alpha-1 antitrypsin (AAT), miR-122, hepatitis B virus HbsAg, LDHA, CEBPA and Lactate Dehydrogenase (LDH), and/or oligonucleotides capable of targeting aberrant mirnas, for example, when present in mammalian cells. Preferably, the gene is any one of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AAT, miR-122, hepatitis B virus HbsAg, LDHA and CEBPA for expressing the proteins HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AAT, miR-122, hepatitis B virus HbsAg, LDHA and CEBPA.

An embodiment is an oligonucleotide conjugate of the invention, wherein the oligonucleotide is an oligonucleotide capable of silencing any of the following genes, e.g., when present in a mammalian cell and preferably when present in a human cell: apolipoprotein B (apoB) and HSP27.

An embodiment is an oligonucleotide conjugate of the invention, wherein the oligonucleotide is an oligonucleotide capable of targeting mRNA involved in the expression of any of the following proteins, e.g. when present in a mammalian cell and preferably when present in a human cell: apoB, HSP27, TTR, PCSK9, TMPRSS6, ALAS1, AT3, GO, CC5, expression products of X genes of HBV, expression products of S genes of HBV, AAT, miR-122, hepatitis B virus HbsAg, LDHA, CEBPA and LDH; or are capable of antagonizing or restoring miRNA function, such as inhibiting oncogenic miRNA (onco-miR) or repressing expression of onco-miR, for example, when present in mammalian cells and preferably when present in human cells. Preferably, the protein is a liver protein selected from the group consisting of: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AAT, miR-122, hepatitis B virus HbsAg, LDHA and CEBPA.

An embodiment is an oligonucleotide conjugate of the invention, wherein the oligonucleotide is an oligonucleotide capable of targeting mRNA involved in the expression of any of the following proteins, e.g. when present in a mammalian cell and preferably when present in a human cell: apoB and HSP27.

An aspect of the invention relates to a method of providing an oligonucleotide conjugate of the invention comprising the steps of:

(a) Providing at least one saponin moiety comprising a covalently bound first linker, wherein the first linker comprises at least one first reactive group for covalent binding to a second reactive group on a second linker or a seventh reactive group on a seventh linker;

(b) Providing an oligonucleotide comprising a covalently bound third linker, wherein the third linker comprises a third reactive group for covalent binding with a fourth reactive group on a fourth linker or an eighth reactive group on a seventh linker;

(c) Providing at least one GalNAc moiety comprising a covalently bound fifth linker, wherein the fifth linker comprises a fifth reactive group for covalent binding to a sixth reactive group on the sixth linker or a ninth reactive group on the seventh linker; and

(d1) Ligating a first linker to a second linker by forming a covalent bond between the first reactive group and the second reactive group, ligating a third linker to a fourth linker by forming a covalent bond between the third reactive group and the fourth reactive group, ligating a fifth linker to a sixth linker by forming a covalent bond between the fifth reactive group and the sixth reactive group, and covalently ligating the second linker, the fourth linker and the sixth linker together to provide an oligonucleotide,

Or (b)

(d2) The first linker is linked to the seventh linker by forming a covalent bond between the first reactive group and the seventh reactive group, the third linker is linked to the seventh linker by forming a covalent bond between the third reactive group and the eighth reactive group, and the fifth linker is linked to the seventh linker by forming a covalent bond between the fifth reactive group and the ninth reactive group, thereby providing the oligonucleotide conjugate.

An embodiment is a method of providing an oligonucleotide conjugate of the invention, wherein the seventh linker is a trifunctional linker, e.g., a trifunctional linker of formula (XXI):

preferred are methods of providing oligonucleotide conjugates of the invention, wherein at least one of the saponin moieties is 1-16 saponin moieties, preferably 1-8 saponin moieties (e.g., 1, 4 or 8 saponin moieties).

Preferred are methods of providing the oligonucleotide conjugates of the invention wherein the saponin is SO1861, SO1832, QS-21 or any functional derivative thereof, preferably SO1861 or SO1832.

Preferred are methods of providing oligonucleotide conjugates of the invention, wherein one or more saponin moieties are covalently linked by hydrazone or semicarbazone linkages.

Preferred are methods for providing the oligonucleotide conjugates of the invention, wherein at least one GalNAc moiety is 1-4 GalNAc moieties, preferably 1 or 3 GalNAc moieties.

One aspect of the invention relates to a pharmaceutical composition comprising an oligonucleotide conjugate of the invention, optionally together with a pharmaceutically acceptable excipient and/or optionally together with a pharmaceutically acceptable diluent.

One aspect of the invention relates to a pharmaceutical composition comprising an oligonucleotide conjugate of the invention, optionally together with a pharmaceutically acceptable excipient and/or optionally together with a pharmaceutically acceptable diluent for use as a medicament. One aspect of the invention relates to an oligonucleotide conjugate of the invention for use as a medicament.

One aspect of the invention relates to a pharmaceutical composition comprising an oligonucleotide conjugate of the invention and optionally a pharmaceutically acceptable excipient and/or optionally a pharmaceutically acceptable diluent or to the use of an oligonucleotide conjugate of the invention for the treatment or prevention of a disease or health problem in which the expression product relates to any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AT3, GO, CC5, X gene of HBV, S gene of HBV, AAT, miR-122, hepatitis B virus HbsAg, LDHA, CEBPA and LDH; and/or for use in the treatment or prevention of diseases or health problems involving any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AT3, GO, CC5, X gene of HBV, S gene of HBV, AAT, miR-122, hepatitis B virus HbsAg, LDHA, CEBPA and LDH. For example, the disease is a cancer associated with (over) expression of HSP27 in tumor cells. Administration of an oligonucleotide conjugate of the invention comprising an oligonucleotide for silencing an HSP27 gene to a patient suffering from a tumor whose growth is associated with (over) expression of HSP27 results in silencing of the HSP27 gene and thereby stopping tumor growth. For example, a health problem associated with apoB expression is a health problem associated with elevated LDL-cholesterol levels in the blood of a human subject, such as a patient at risk of atherosclerosis and/or hypercholesterolemia, and/or a patient suffering from such atherosclerosis and/or hypercholesterolemia. Administration of an oligonucleotide of the invention comprising an oligonucleotide for silencing apoB gene to the human subject or patient results in inhibition or reduction of apoB expression and thus reduction of LDL (-cholesterol) because apoB is the protein component of LDL particles. The reduction in circulating LDL levels is associated with a reduction in circulating LDL-cholesterol and thereby reduces the health risk and disease symptom risk associated with LDL-cholesterol, which is evident in patients suffering from atherosclerosis and/or hypercholesterolemia or in subjects at risk of developing atherosclerosis and/or hypercholesterolemia under the influence of LDL-cholesterol at higher than normal blood levels.

One aspect of the invention relates to a pharmaceutical composition comprising the oligonucleotide conjugate of the invention or to the use of the oligonucleotide conjugate of the invention for the treatment or prevention of a disease or health problem in which the expression product relates to any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AAT, miR-122, hepatitis B virus HbsAg, LDHA and CEBPA; and/or for use in the treatment or prevention of diseases or health problems involving any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AAT, miR-122, hepatitis B virus HbsAg, LDHA and CEBPA.

An embodiment is a pharmaceutical composition for use according to the invention comprising an oligonucleotide conjugate according to the invention, or an oligonucleotide conjugate according to the invention for use according to the invention, wherein the use is the treatment or prevention of a disease or health problem in which the expression product is involved in any one or more of the following genes: HSP27 and apoB, preferably apoB; and/or for use in the treatment or prevention of diseases or health problems involving any one or more of the following genes: HSP27 and apoB, preferably apoB.

An embodiment is a pharmaceutical composition for use according to the invention comprising an oligonucleotide conjugate according to the invention, or an oligonucleotide conjugate according to the invention for use according to the invention, wherein the use is the treatment or prevention of: cancer, infectious disease, viral infection, hypercholesterolemia, primary hyperoxaluria, hemophilia a, hemophilia B, AAT-associated liver disease, acute hepatic porphyria, TTR-mediated amyloidosis, hereditary TTR amyloidosis (hATTR), complement-mediated disease, hepatitis b infection, or autoimmune disease.

An embodiment is a pharmaceutical composition for use according to the invention comprising or for use according to the invention, wherein the use is the treatment or prevention of cancer (e.g. endometrial cancer, breast cancer, lung cancer) and hypercholesterolemia, preferably hypercholesterolemia.

One aspect of the invention relates to a pharmaceutical composition comprising an oligonucleotide conjugate of the invention, or to an oligonucleotide conjugate of the invention for use in the treatment or prevention of: cancer, infectious disease, viral infection, hypercholesterolemia, cardiovascular disease, primary hyperoxaluria, hemophilia a, hemophilia B, AAT-associated liver disease, acute hepatic porphyrin disease, TTR-mediated amyloidosis, hereditary TTR amyloidosis (hATTR), complement-mediated disease, hepatitis b infection, hepatitis c infection, alpha 1-antitrypsin deficiency, beta-thalassemia or autoimmune disease.

One aspect of the invention relates to a pharmaceutical composition comprising an oligonucleotide conjugate of the invention, or to an oligonucleotide conjugate of the invention for use in the treatment or prevention of: cancer (e.g., endometrial, breast, lung, or hepatocellular carcinoma) and/or cardiovascular disease (e.g., atherosclerosis and/or hypercholesterolemia, preferably atherosclerosis and/or hypercholesterolemia).

For example, the disease is a cancer associated with (over) expression of HSP27 in tumor cells. Administration of an oligonucleotide conjugate of the invention comprising an oligonucleotide for silencing an HSP27 gene to a patient suffering from a tumor whose growth is associated with (over) expression of HSP27 results in silencing of the HSP27 gene and thereby stopping tumor growth. For example, a health problem associated with apoB expression is a health problem associated with elevated LDL-cholesterol levels in the blood of a human subject, such as a patient at risk of atherosclerosis and/or hypercholesterolemia, and/or a patient suffering from such atherosclerosis and/or hypercholesterolemia. Administration of an oligonucleotide conjugate of the invention comprising an oligonucleotide for silencing apoB gene to the human subject or patient results in inhibition or reduction of apoB expression and thus reduction of LDL (-cholesterol) because apoB is the protein component of LDL particles. The reduction in circulating LDL levels is associated with a reduction in circulating LDL-cholesterol and thereby reduces the health risk and disease symptom risk associated with LDL-cholesterol, which is evident in patients suffering from atherosclerosis and/or hypercholesterolemia or in subjects at risk of developing atherosclerosis and/or hypercholesterolemia under the influence of LDL-cholesterol at higher than normal blood levels.

One aspect of the invention relates to a pharmaceutical composition comprising the oligonucleotide conjugate of the invention or to the use of the oligonucleotide conjugate of the invention for reducing LDL-cholesterol in a subject. One aspect of the invention relates to a pharmaceutical composition comprising the oligonucleotide conjugate of the invention or to the oligonucleotide conjugate of the invention for use in a method of reducing LDL-cholesterol concentration in the blood of a human subject. One aspect of the invention relates to a pharmaceutical composition comprising the oligonucleotide conjugate of the invention or to the oligonucleotide conjugate of the invention for use in a method of treating or preventing a cardiovascular disease. Typically, treatment or prophylaxis is performed in a human patient suffering from a cardiovascular disease or in a human subject at risk for suffering from a cardiovascular disease. Typically, cardiovascular disease is associated with elevated LDL-cholesterol blood levels, as compared to the upper end of the normal LDL-cholesterol level range.

One aspect of the invention relates to an in vitro or ex vivo method for transferring an oligonucleotide conjugate of the invention from the outside of a cell into said cell, preferably followed by transferring an oligonucleotide comprised by the oligonucleotide conjugate of the invention into the cytoplasm and/or nucleus of said cell, comprising the steps of:

a) Providing a cell expressing ASGPR, preferably ASGPR1, on its surface, preferably selected from the group consisting of hepatocytes, virus-infected mammalian cells and mammalian tumor cells, wherein preferably the cell is a human cell;

b) Providing an oligonucleotide conjugate of the invention for transfer into the cell provided in step a);

c) Contacting the cells of step a) in vitro or ex vivo with the oligonucleotide conjugate of step b), preferably in a liquid medium, thereby effecting transfer of the oligonucleotide conjugate from outside the cells into said cells, and optionally and preferably thereby effecting subsequent transfer of the oligonucleotides comprised by the oligonucleotide conjugate into the cytoplasm and/or nucleus of said cells.

Surprisingly, the inventors of the present application found that a saponin derivative based on a saponin comprising a triterpene aglycone core structure and a first sugar chain' R ¹ 'and second sugar chain' R ² At least one of (as defined herein, attached to the aglycone core structure, preferably saponaric acid), wherein the saponin derivative comprises an aglycone core structure comprising an aldehyde group, wherein the aldehyde group is converted to a semicarbazone functional group according to formula (I1):

wherein R is ¹ And R is ² Independently selected from the group consisting of hydrogen, monosaccharides, linear oligosaccharides and branched oligosaccharides,

x= O, P or S

Y=nr3r4, wherein R ³ And R is ⁴ Independently represents H, an unsubstituted C1-C10 linear, branched or cyclic alkyl, an unsubstituted C2-C10 linear, branched or cyclic alkenyl or an unsubstituted C2-C10 linear or branched alkynyl, or a covalently bonded linker, preferably R ³ And R is ⁴ One of them is H; or (b)

Wherein n and m are each integers independently selected from 1, 2 or 3,

Z＝CH ₂ o, S, P or NR ⁵ And (2) and

wherein R is ⁵ Represents H, an unsubstituted C1-C10 straight, branched or cyclic alkyl, an unsubstituted C2-C10 straight, branched or cyclic alkenyl, an unsubstituted C2-C10 straight or branched alkynyl, or a covalently bonded linker, or a maleimide moiety according to formula (II 1) a):

or an azide moiety according to formula (II 1) b

Wherein o is an integer selected from 0-10, preferably 2-7, more preferably 4-6, having reduced toxicity when considering the cell viability of the cells contacted with the saponin derivative; having activity when considering, for example, toxin cytotoxicity or enhancement of BNA-mediated gene silencing (without wishing to be bound by any theory: similar or improved endosomal escape enhancing activity involving modified saponins (i.e. saponin derivatives), if the aldehyde functionality is converted to a semicarbazone functionality according to formula (I1); and/or has reduced hemolytic activity compared to the toxicity, activity and hemolytic activity of the unmodified saponin. That is, the saponin derivative has at least one, preferably to three, more preferably all three of the following:

(i) Reduced toxicity when considering the cell viability of cells contacted with the saponin derivative;

(ii) Having enhanced activity when considering, for example, BNA-mediated enhancement of gene silencing (without wishing to be bound by any theory: similar or improved endosomal escape enhancing activity involving modified saponins), if the aldehyde functionality is converted to a semicarbazone functionality according to formula (I1); and/or

(iii) Reduced hemolytic activity when compared to the toxicity, activity and hemolytic activity of the unmodified saponin on which the saponin derivative is based. Thus, the inventors of the present application provide saponin derivatives with an improved therapeutic window in that for a saponin derivative the cytotoxicity is lower than the cytotoxicity determined for its naturally occurring counterpart, the hemolytic activity is lower than the hemolytic activity determined for its naturally occurring counterpart, the ratio between the IC50 value of the cytotoxicity and the IC50 value of e.g. toxin-enhanced IC50 value or gene silencing is similar or increased, and/or in that the ratio between the IC50 value of the saponin hemolytic activity and the IC50 value of e.g. toxin-enhanced IC50 value or gene silencing is similar or increased. The term "endosomal escape enhancing activity" may be abbreviated as 'activity'.

Furthermore, the inventors of the present application surprisingly established (tumor) cell killing by contacting such cells with ADCs based on saponin conjugates comprising a saponin derivative of a semicarbazone functional group and antibody-protein toxin conjugates or the like, despite the mid-to-low expression of cell surface receptors targeted by the cell surface molecule binding molecules comprised by the saponin conjugates (e.g. antibodies) and/or despite the mid-to-low expression of cell surface receptors targeted by the ADCs. The saponin derivative and the saponin conjugate comprise semicarbazone functional groups. For example, in the comparative example, when a saponin conjugate comprising the same cell surface molecule binding molecule (e.g., antibody) but comprising a hydrazone functionality (=n-N (H) -C (O) -) instead of a semicarbazone functionality is contacted with a cell, such cell killing cannot be established or is established only to a lesser extent. Thus, the inventors of the present application provide more effective saponin derivatives and saponin conjugates when considering the activation or enhancement of effector moieties such as those comprised by ADCs or AOCs.

Furthermore, the inventors of the present application have found that saponin derivatives comprising semicarbazone functionality hydrolyze faster and in higher amounts under acidic conditions (which are conditions present in the endosome and/or lysosome of a mammalian cell) than corresponding natural saponins comprising "free" aldehyde functionality, as known in the art for saponin derivatives comprising hydrazone functionality (=n-N (H) -C (O) -). This has the following benefits: lower amounts of the saponin derivatives of the present invention should be administered to patients in need of enhancement of e.g. ADC or AOC or gene silencing oligonucleotides (e.g. BNA coupled to one or more GalNAc moieties) compared to the required amount of saponin derivative comprising e.g. hydrazone functional groups (=n-N (H) -C (O) -) to obtain the same amount of native saponin comprising "free" aldehyde, thereby acting as endosomal escape enhancer for the targeted toxin or targeted oligonucleotide. Without wishing to be bound by any theory, the faster and more efficient release of a saponin comprising an aldehyde function from a saponin derivative comprising an semicarbazone function is the basis for the improved activity of a saponin conjugate comprising a saponin derivative comprising an semicarbazone function (e.g. a conjugate comprising a saponin, an oligonucleotide and a cell surface receptor ligand, such as one to three GalNAc moieties, for targeting ASGPR expressing (liver) cells) as determined by the inventors of the present application, when compared to the release of a saponin comprising an aldehyde function from a saponin derivative comprising an hydrazone function, wherein the activity is an enhancement of the activity of an effector moiety, such as a gene silencing oligonucleotide, within the cytoplasm or nucleus of the target cell by endosomal escape enhancement.

Unexpectedly, the conversion (derivatization) of the aldehyde group at sapogenin C-23 to a semicarbazone functional group according to formula (I1) results in reduced cytotoxicity when such saponin derivatives are contacted with cells (i.e., various types of cells). The following is therefore advantageous: these series of saponin derivatives are provided having reduced cytotoxicity relative to cytotoxicity measured with an unmodified naturally occurring saponin counterpart. The saponin derivative may be formed from such naturally occurring saponins, for example SO1861, equally active SO1832 and QS-21 (isoforms), preferably from SO1861 or SO 1832. When reduction of cytotoxicity is considered, a saponin derivative comprising a semicarbazone functional group is also suitable when a saponin having reduced cytotoxicity is to be provided.

Thus, the inventors of the present application provide saponin derivatives with improved therapeutic window when considering cytotoxicity and/or hemolytic activity, and when considering enhancement of oligonucleotides compared to corresponding underacting saponins (e.g. gene silencing). Such a saponin derivative comprising a semicarbazone functional group is for example particularly suitable for use in a therapeutic regimen involving a GalNAc-oligonucleotide conjugate in combination with a saponin derivative or the conjugate is an oligonucleotide conjugate of the invention comprising a covalently linked saponin as well as a GalNAc moiety and an oligonucleotide for use in the prevention or treatment of e.g. cancer, hypercholesterolemia, a cardiovascular disease or an elevation of LDL-cholesterol levels above normal in a healthy subject in need thereof (human) subject. The safety of such saponin derivatives is improved when considering cytotoxicity and/or haemolytic activity, in particular when such saponin derivatives are administered to a patient in need of treatment, e.g. with ADC or with AOC or with a conjugate comprising at least one GalNAc moiety and comprising an oligonucleotide (e.g. BNA for silencing genes such as HSP27 and apoB), or when a saponin derivative comprising a semicarbazone functionality is part of an oligonucleotide conjugate of the invention.

One embodiment is an oligonucleotide conjugate of the invention comprising one saponin moiety.

An embodiment is an oligonucleotide conjugate of the invention, wherein at least one GalNAc moiety, at least one saponin and an oligonucleotide are covalently bound by a trifunctional linker, preferably with each of the GalNAc moiety, the saponin and the oligonucleotide being covalently bound to a separate arm of the trifunctional linker. Preferred oligonucleotide conjugates of the invention are oligonucleotide conjugates in which at least one GalNAc moiety, preferably three GalNAc moieties, at least one saponin, preferably 1-16 saponin moieties, more preferably 1-8 saponin moieties (e.g. 1, 4 or 8 saponin moieties) are covalently bound to the oligonucleotide via a trifunctional linker, preferably each of the GalNAc moiety/moieties, saponin/saponin moiety and oligonucleotide is/are covalently bound to a separate arm of the trifunctional linker. Examples of trifunctional linkers suitable for incorporation into the oligonucleotide conjugates of the invention are trifunctional linkers represented by formula (XXI):

an embodiment is an oligonucleotide conjugate of the invention, wherein a trifunctional linker (e.g., a trifunctional linker represented by formula (XXI)) is covalently bound to a saponin moiety or moieties, preferably 1-16 saponin moieties, more preferably 1-8 saponin moieties (e.g., 1, 4 or 8 saponin moieties), via a first arm of the linker, to at least one GalNAc moiety, preferably 1-4 GalNAc moieties, more preferably 3 GalNAc moieties, via a second arm of the linker, and to an oligonucleotide (preferably an AON, such as BNA or siRNA) via a third arm of the trifunctional linker. Preferred are oligonucleotide conjugates of the invention, wherein at least one GalNAc moiety, preferably three GalNAc moieties, the at least one saponin, preferably 1-16 saponin moieties, more preferably 1-8 saponin moieties, e.g. 1, 4 or 8 saponin moieties, preferably 1, 4 or 8 saponin moieties, and the oligonucleotides are covalently bound by a trifunctional linker, preferably one GalNAc moiety or more GalNAc moieties, one saponin or more saponin moieties and each of the oligonucleotides are covalently bound to a separate arm of a trifunctional linker.

One embodiment is an oligonucleotide conjugate of the invention comprising at least one saponin covalently linked to a ligand of an asialoglycoprotein receptor (ASGPR), wherein the ligand of ASGPR comprises at least one N-acetylgalactosamine (GalNAc) moiety, preferably three or four GalNAc moieties, more preferably three GalNAc moieties, more preferably the ligand of ASGPR comprises (GalNAc) ₃ Tris or (GalNAc) ₃ Tris, and further covalently linked to an oligonucleotide, wherein at least one of the saponins is a 12, 13-dehydrooleanane type monosaccharide or disaccharide chain pentacyclic triterpene saponin, preferably having an aldehyde function at the C-23 position of the aglycone core structure of the saponin, wherein the oligonucleotide conjugate comprises 1-16 saponin moieties, preferably 1-8 saponin moieties, more preferably 1 saponin moiety, 4 saponin moieties or 8 saponin moieties.

An embodiment is an oligonucleotide conjugate of the invention, wherein at least one GalNAc moiety, preferably three GalNAc moieties, at least one saponin, preferably 1-16 saponin moieties, more preferably 1-8 saponin moieties, e.g. 1, 4 or 8 saponin moieties, and the oligonucleotide are covalently bound by a trifunctional linker, preferably to a separate arm of the trifunctional linker, to each of the GalNAc moiety or moieties, the saponin or the saponin moiety and the oligonucleotide.

One embodiment is an oligonucleotide conjugate of the invention comprising one saponin moiety, 4 saponin moieties or 8 saponin moieties.

An embodiment is an oligonucleotide conjugate of the invention, wherein at least one saponin moiety is linked by a hydrazone linkage or by a semicarbazone linkage. The at least one saponin is preferably covalently bound in the oligonucleotide conjugate via a semicarbazone linkage. Such hydrazone bond or semicarbazone bond (with linker) is formed, for example, as an aldehyde group at the C-23 atom of sapogenin core structure related to saponins or of silk-mangosteen sapogenin core structure of saponins (for these types of saponins, see table A1).

One aspect of the invention relates to an oligonucleotide conjugate according to molecule (EE):

wherein molecule (EE) is via a trifunctional linker according to formula (XXI):

/>

covalent conjugation products obtained with covalent conjugation of the following (1), (2) and (3): (1) a saponin derivative according to molecule (AA):

wherein

Represents a saponin moiety according to formula (SM):

wherein R is ¹ And R is ² Independently selected from the group consisting of hydrogen, monosaccharides, linear oligosaccharides and branched oligosaccharides, and wherein the saponin moiety according to formula (SM) is based on a saponin comprising an aldehyde group in the C-23 position,

And

(2) GalNAc conjugate according to molecule (FF):

wherein

Represents a tri-GalNAc conjugate according to molecule (DD 1) or according to molecule (DD 2):

wherein y1, y2 and y3 are each independently integers selected from 0-20, preferably 1-15, more preferably 2-12, even more preferably 2-10, even more preferably 2-8, most preferably 2 and 3, and preferably y1, y2 and y3 are the same, and y4 is an integer selected from 1-100, preferably 2-80, more preferably 3-70, even more preferably 4-60, even more preferably 4-50, even more preferably 4-40, even more preferably 4-30, even more preferably 4-20, even more preferably 4-6, most preferably 4-5, e.g. 4;

/>

wherein x1, x2 and x3 are each independently integers selected from 0 to 20, preferably 1 to 15, more preferably 2 to 12, even more preferably 2 to 10, even more preferably 2 to 8, most preferably 2 and 3, and preferably x1, x2 and x3 are the same, and x4 is an integer selected from 1 to 50, preferably 2 to 40, more preferably 3 to 30, even more preferably 4 to 20, even more preferably 5 to 15, most preferably 8 to 12, for example 9,

and preferably represents a tri-GalNAc conjugate according to molecule (DD 3) or according to molecule (DD 4):

and

(3) An oligonucleotide having a linker according to molecule (GG):

wherein molecule (GG) represents the conjugation product of a conjugation reaction between linker (E) -1- (4- ((2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoyl) hydrazino) methyl) benzamide) -N- (4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) benzyl) -3,6,9, 12-tetraoxapentadecane-15-amide and an oligonucleotide-linker molecule according to molecule (HH):

Such oligonucleotide conjugates of molecules (EE) according to the invention show unexpectedly improved potency when considering the endosomal escape enhancing activity of the saponin moiety. It is believed that the improvement in efficacy is associated with an improvement in the uncoupling (release) of the saponin from the conjugate by cleavage of the semicarbazone functional group under the influence of the weakly acidic pH in the endosome of the cell that has taken up the conjugate through the binding of the conjugate to ASGPR. Thus, endosomal escape of the oligonucleotide is enhanced and (gene silencing) activity of the oligonucleotide in the cytoplasm and/or nucleus is enhanced compared to the use of a similar conjugate (although without covalently attached saponin moiety).

An example of an oligonucleotide conjugate of the present invention comprising a single saponin moiety is the oligonucleotide shown as molecule 13 in fig. 35D. The synthesis of such an oligonucleotide of the invention comprising a single saponin moiety is illustrated by the detailed description of the synthesis of molecule 13 (fig. 35D) in the example section below and shown in fig. 35. The saponin moiety is linked to the trifunctional linker through a linker, wherein the saponin moiety is linked to the linker through a hydrazone bond. Another example of such an oligonucleotide conjugate of the present invention comprising a single saponin moiety is the oligonucleotide shown as molecule 31 in FIG. 38C. The synthesis of such oligonucleotide conjugates of the invention comprising a single saponin moiety is illustrated by the detailed description of the synthesis of molecule 31 (fig. 38C) in the example section below and shown in fig. 38. The saponin moiety is linked to the trifunctional linker through a linker to which the saponin moiety is linked through a semicarbazone linkage (see, e.g., molecule 28 in fig. 38A). See also the above saponin derivatives according to molecule (AA).

One embodiment is an oligonucleotide conjugate of the invention comprising four saponin moieties. One embodiment is an oligonucleotide conjugate of the invention comprising four saponin moieties covalently bound to a dendron, preferably a G2 dendron. An example of such a (G2) dendron is N, N' - ((9S, 19S) -14- (6-aminocaproyl) -1-mercapto-9- (3-mercaptopropionamido) -3,10,18-trioxo-4,11,14,17-tetraazatriacontane-19, 23-diyl) bis (3-mercaptopropionamide). Synthesis of dendrites linked to four saponin moieties is shown, for example, in FIG. 39B and in the example portion for the synthesis of intermediate 20 (molecule 39), the formation of molecule 37 (the maleimide-linked saponin involves a semicarbazone bond between the saponin (here SO 1861) and the linker with a maleimide group), and molecule 38, which is the dendrite N, N' - ((9S, 19S) -14- (6-aminocaproyl) -1-mercapto-9- (3-mercaptopropionamido) -3,10,18-trioxo-4,11,14,17-tetraazaditridec-19, 23-diyl) bis (3-mercaptopropionamide) formate. One aspect of the invention relates to oligonucleotide conjugates according to the molecule (PP):

wherein the molecule (PP) is a saponin-GalNAc conjugate according to the molecule (LL):

a covalent conjugation product obtained by covalent conjugation with:

An oligonucleotide having a linker according to molecule (GG):

wherein molecule (GG) represents the conjugation product of the conjugation reaction between linker (E) -1- (4- ((2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoyl) hydrazino) methyl) benzamide) -N- (4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) benzyl) -3,6,9, 12-tetraoxapentadecane-15-amide and an oligonucleotide-linker molecule according to molecule (HH):

wherein molecule (LL) is prepared by reacting a saponin derivative according to molecule (JJ):

a covalent conjugation product obtained by covalent conjugation with:

conjugate of trifunctional linker according to molecule (MM) and GalNAc:

wherein the molecule (JJ) is the conjugation product of N, N' - ((9 s,19 s) -14- (6-aminocaproyl) -1-mercapto-9- (3-mercaptopropionamido) -3,10,18-trioxo-4,11,14,17-tetraazaditridecane-19, 23-diyl) bis (3-mercaptopropionamide) conjugated to: first with a saponin derivative according to formula (KK):

wherein

Represents a saponin moiety according to formula (SM):

wherein R is ¹ And R is ² Independently selected from the group consisting of hydrogen, monosaccharides, linear oligosaccharides and branched oligosaccharides according to the invention, and wherein the saponin moiety according to formula (SM) is based on a saponin comprising an aldehyde group according to any of the invention at position C-23, and is for example listed in table A1, such as QS-21, SO1861, SO1832,

And then with 2, 5-dioxopyrrolidin-1-yl-1-azido-3, 6,9, 12-tetraoxapentadecane-15-oic acid ester,

and wherein the molecule (MM) is a conjugate of: a trifunctional linker according to formula (XXI):

and GalNAc conjugates according to molecule (NN):

wherein

Represents a tri-GalNAc conjugate according to the molecule (DD 1) or the molecule (DD 2) as detailed above, preferably the molecule (DD 3) or the molecule (DD 4) as detailed above, more preferably the molecule (DD 3) as detailed above.

An example of such an oligonucleotide conjugate of the present invention comprising four saponin moieties is the oligonucleotide conjugate shown as molecule 22 in fig. 36E. The synthesis of such oligonucleotide conjugates of the invention comprising four saponin moieties is illustrated by the detailed description of the synthesis of molecule 22 (fig. 36E) in the example section below and shown in fig. 36. The saponin moieties are linked to the dendrons by linkers, wherein each saponin moiety is linked to a linker by a hydrazone bond. Another example of such an oligonucleotide conjugate of the present invention comprising four saponin moieties is the oligonucleotide conjugate shown as molecule 42 in fig. 39F. The synthesis of such oligonucleotide conjugates of the invention comprising four saponin moieties is illustrated by the detailed description of the synthesis of molecule 42 (fig. 39F) in the example section below and shown in fig. 39. The saponin moieties are linked to the dendrons by linkers, wherein each saponin moiety is linked to a linker by a semicarbazone linkage. See also the above saponin derivatives according to the molecule (KK).

One embodiment is an oligonucleotide conjugate of the invention comprising eight saponin moieties. As mentioned, preferred are oligonucleotide conjugates of the invention, wherein at least one GalNAc moiety, preferably three GalNAc moieties, at least one saponin, preferably 1-16 saponin moieties, more preferably 1-8 saponin moieties, such as 8 saponin moieties, and the oligonucleotide is covalently bound via a trifunctional linker, preferably with one GalNAc moiety or with each of the GalNAc moieties, a single saponin or with more than one saponin moiety and the oligonucleotide is covalently bound to a separate arm of the trifunctional linker.

One embodiment is an oligonucleotide conjugate of the invention comprising eight saponin moieties covalently bound to a dendron, preferably a G3 dendron. An example of such a (G3) dendron is (2S) -N- [ (1S) -1- { [2- (6-amino-N- {2- [ (2S) -2, 6-bis (3-sulfanylpropionylamino) hexanoylamino ] ethyl } hexanoylamino) ethyl ] carbamoyl } -5- [ (2S) -2, 6-bis (3-sulfanylpropionylamino) hexanoylamino ] pentyl ] -2, 6-bis (3-sulfanylpropionylamino) hexanamide. The synthesis of a dendron linked to eight saponin moieties is shown for example in FIG. 40a and in the example moiety for the synthesis of intermediate 25 (molecule 44) forms molecule 37 (the maleimide linked saponin involves a semicarbazone bond between the saponin (here SO 1861) and the linker bearing a maleimide group), and molecule 43 which is a dendron (2S) -N- [ (1S) -1- { [2- (6-amino-N- {2- [ (2S) -2, 6-bis (3-sulfanylpropionamido) amido ] hexanamido ] ethyl } hexanamido) ethyl ] carbamoyl } -5- [ (2S) -2, 6-bis (3-sulfanylpropionamido) hexanamido ] pentyl ] -2, 6-bis (3-sulfanylpropionamido) hexanamide formate.

One aspect of the invention relates to oligonucleotide conjugates according to molecules (SS):

wherein the molecule (SS) is prepared by reacting a saponin-GalNAc conjugate with the molecule (RR):

a covalent conjugation product obtained by covalent conjugation with:

an oligonucleotide having a linker according to molecule (GG):

wherein molecule (GG) represents the conjugation product of a conjugation reaction between linker (E) -1- (4- ((2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoyl) hydrazino) methyl) benzamide) -N- (4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) benzyl) -3,6,9, 12-tetraoxapentadecane-15-amide and an oligonucleotide-linker molecule according to molecule (HH):

wherein the molecule (RR) is obtained by reacting a saponin derivative according to the molecule (QQ):

a covalent conjugation product obtained by covalent conjugation with:

conjugate of trifunctional linker according to molecule (MM) and GalNAc:

wherein the molecule (QQ) is the conjugation product of (2S) -N- [ (1S) -1- { [2- (6-amino-N- {2- [ (2S) -2, 6-bis (3-sulfanylpropionamido) hexanamido ] ethyl } hexanamido) ethyl ] carbamoyl } -5- [ (2S) -2, 6-bis (3-sulfanylpropionamido) hexanamido ] pentyl ] -2, 6-bis (3-sulfanylpropionamido) hexanamide formate conjugated to: first

(a) And a saponin derivative according to molecule (KK):

wherein

Represents a saponin moiety according to formula (SM):

wherein R is ¹ And R is ² Independently selected from the group consisting of hydrogen, monosaccharides, linear oligosaccharides and branched oligosaccharides according to the invention, and wherein the saponin moiety according to formula (SM) is based on a saponin comprising an aldehyde group at the C-23 position according to any of the saponins according to the invention, and is for example listed in table A1, such as QS-21, SO1861, SO1832,

and then

(b) With 2, 5-dioxopyrrolidin-1-yl-1-azido-3, 6,9, 12-tetraoxapentadecane-15-oic acid ester,

and wherein the molecule (MM) is a conjugate of: a trifunctional linker according to formula (XXI):

and GalNAc conjugates according to molecule (NN) (see above), wherein

Represents a tri-GalNAc conjugate as described above with respect to molecule (DD 1) or molecule (DD 2), preferably molecule (DD 3) or molecule (DD 4) as described above, more preferably molecule (DD 3) as described above.

An example of such an oligonucleotide conjugate of the invention comprising eight saponin moieties is the oligonucleotide conjugate shown as molecule 26 in fig. 37C. The synthesis of such oligonucleotide conjugates of the invention comprising eight saponin moieties is illustrated by the detailed description of the synthesis of molecule 26 (fig. 37C) in the examples section below and shown in fig. 37. The saponin moieties are linked to the dendrons by linkers, wherein each saponin moiety is linked to a linker by a hydrazone bond. Another example of such an oligonucleotide conjugate of the present invention comprising eight saponin moieties is the oligonucleotide conjugate shown as molecule 47 in fig. 40D. The synthesis of such oligonucleotide conjugates of the invention comprising eight saponin moieties is illustrated by the detailed description of the synthesis of molecule 47 (fig. 40D) in the example section below and shown in fig. 40. The saponin moieties are linked to the dendrons by linkers, wherein each saponin moiety is linked to a separate linker by a semicarbazone linkage. See also the above saponin derivatives according to the molecule (KK).

Preferred are oligonucleotide conjugates of the invention comprising one saponin moiety, or 4 saponin moieties, preferably 4 saponin moieties, covalently bound to a dendron, preferably a G2 dendron such as N, N' - ((9S, 19S) -14- (6-aminocaproylamino) -1-mercapto-9- (3-mercaptopropionylamino) -3,10,18-trioxo-4,11,14,17-tetraazaditridecane-19, 23-diyl) bis (3-mercaptopropionamide) or 8 saponin moieties, preferably 8 saponin moieties, covalently bound to a dendron, preferably a G3 dendron such as (2S) -N- [ (1S) -1- { [2- (6-amino-N- {2- [ (2S) -2, 6-bis (3-sulfanylpropionylamino) caproamido ] ethyl } caproamido {5- [ (2S) -2, 6-bis (3-sulfanylpropionylamino) caproamido ] ethyl } caproamide.

An embodiment is an oligonucleotide conjugate according to the invention and for example according to any of the molecules (EE), (PP) and (SS), wherein the oligonucleotide conjugate is based on a saponin according to any of the saponins listed above and in table A1.

Preferred are oligonucleotide conjugates according to the invention and for example according to any of the molecules (EE), (PP) and (SS), wherein the oligonucleotide conjugates are based on saponins selected from the group consisting of SO1861, SO1832 and QS-21, preferably SO1861 and SO1832, more preferably SO1861 or SO1832. When the saponin is SO1861, the saponin derivative according to molecule (VII) a:

Conjugated to an oligonucleotide and at least one GalNAc moiety to provide an oligonucleotide conjugate of the invention.

Also preferred are oligonucleotide conjugates according to the invention and e.g. according to any of the molecules (EE), (PP) and (SS), wherein the semicarbazone functionality undergoes hydrolysis in vivo under acidic conditions, which are present in the endosome and/or lysosome of a mammalian cell, preferably a human cell, preferably at a pH of 4.0-6.5, more preferably at a pH of +.5.5.

An embodiment is according to the invention, wherein the conjugate comprises a saponin that is any one or more of the following:

a) A saponin selected from any one or more of list a:

-a mixture of sapogenins of the quillaja saponaria, or saponins isolated from the quillaja saponaria, such as Quil-A, QS-17-api, QS-17-xyl, QS-21A, QS-21B, QS-7-xyl;

-a mixture of carnation saponins, or saponins isolated from carnation;

-a mixture of saponinium album saponins, or saponins isolated from saponinium album;

-a mixture of soapstock saponins, or saponins isolated from soapstock; and

-a saponaria saponins mixture, or saponins isolated from saponaria barks, such as Quil-A, QS-17-api, QS-17-xyl, QS-21A, QS-21B, QS-7-xyl; or (b)

b) A saponin comprising a silk diabolo sapogenin core structure selected from list B:

SA1641, carnation saponin A, NP-017772, NP-017774, NP-017777, NP-017778, NP-018109, NP-017888, NP-017889, NP-018108, SO1658 and phytolaccagenin; or (b)

c) A saponin comprising a sapogenin core structure selected from list C:

AG1856, AG1, AG2, agrostemmoside E, GE1741, caryophylloside 1 (Gyp 1), NP-017674, NP-017810, NP-003881, NP-017676, NP-017677, NP-017705, NP-017706, NP-017773, NP-017775, SA1657, saponaria oside B, SO1542, SO1584, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862, SO 4, QS-7, QS-1907 API, QS-17, QS-18, QS-21A-apio, QS-21A-xylo, QS-21B-apio and QS-21B-xylo; or (b)

d) A sapogenin core structure comprising a 12, 13-dehydrooleanane type selected from list D, having no aldehyde group at the C-23 position of the aglycone:

escin Ia, escin salt, alpha-hederagenin, AMA-1, AMR, AS6.2, AS64R, assam saponin F, dipsacus asperosaponin B, esculentoside A, lonicera macranthoides saponin A, NP-005236, NP-012672, primula acid 1, saikosaponin A, saikosaponin D, tea seed saponin I and tea seed saponin J;

Preferably, the saponin is any one or more of the saponins selected from the list A, B or C, more preferably, the saponins selected from the list B or C,

even more preferably, the saponins are selected from list C.

Preferred are oligonucleotide conjugates of the invention, wherein the saponin is any one or more of the following: AG1856, GE1741, saponins isolated from Quil-A, QS-17, QS-21, QS-7, SA1641, saponins isolated from soapbark, saponaria oside B, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862 and SO1904; preferably, wherein the saponins are any one or more of the following: is QS-21, SO1832, SO1861, SA1641 and GE1741; more preferably wherein the saponin is QS-21, SO1832 or SO1861; most preferred is SO1861.

Also preferred are oligonucleotide conjugates of the invention, wherein the saponin is a saponin isolated from soapberry, preferably wherein the saponin is any one or more of the following: saponarioside B, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862 and SO1904; more preferably, wherein the saponin is any one or more of the following: SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862 and SO1904; even more preferably, wherein the saponin is any one or more of the following: SO1832, SO1861 and SO1862; even more preferably, wherein the saponins are SO1832 and SO1861; most preferred is SO1861.

An embodiment is an oligonucleotide conjugate of the invention, wherein the saponin is SO1861, and wherein the oligonucleotide conjugate is provided by conjugation of a SO1861 saponin derivative according to molecule (VII) a:

conjugation to an oligonucleotide and at least one GalNAc moiety to provide an oligonucleotide conjugate.

An embodiment is an oligonucleotide conjugate of the invention, wherein the conjugate comprises an oligonucleotide defined as a nucleic acid of no more than 150nt, preferably wherein the oligonucleotide has a size of 5-150nt, preferably 8-100nt, most preferably 10-50nt.

An embodiment is an oligonucleotide conjugate according to the invention, wherein the semicarbazone functionality undergoes hydrolysis in vivo under acidic conditions, such as in endosomes and/or lysosomes of mammalian cells, preferably human cells, preferably at a pH of 4.0-6.5, and more preferably at a pH of < 5.5. Upon such hydrolysis, an aldehyde group of the saponin or saponin moiety comprised by the oligonucleotide conjugate is formed.

An embodiment is an oligonucleotide conjugate of the invention, wherein the hydrazone functionality undergoes hydrolysis in vivo under acidic conditions, such as in endosomes and/or lysosomes of mammalian cells, preferably human cells, preferably at a pH of 4.0-6.5, more preferably at a pH of 5.5 or less. Upon such hydrolysis, an aldehyde group of the saponin or saponin moiety comprised by the oligonucleotide conjugate is formed.

An embodiment is an oligonucleotide conjugate according to the invention and e.g. according to any of the molecules (EE), (PP) and (SS), comprising an oligonucleotide, wherein the oligonucleotide is an AON, e.g. any of BNA, a heterologous nucleic acid, siRNA.

An embodiment is an oligonucleotide conjugate according to the invention and for example according to any of the molecules (EE), (PP) and (SS), comprising an oligonucleotide, wherein the oligonucleotide is selected from any one or more of the following: short interfering RNA (siRNA), short hairpin RNA (shRNA), anti-hairpin microRNA (miRNA), single-stranded RNA, aptamer RNA, double-stranded RNA (dsRNA), anti-microRNA (anti-miRNA, anti-miR), antisense oligonucleotide (ASO), DNA, antisense DNA, locked Nucleic Acid (LNA), bridged Nucleic Acid (BNA), 2'-O,4' -aminoethylene bridgeNucleic Acid (BNA) ^NC ) BNA-based siRNA, and BNA-based antisense oligonucleotide (BNA-aON).

An embodiment is an oligonucleotide conjugate according to the invention and for example according to any of the molecules (EE), (PP) and (SS), comprising an oligonucleotide, wherein the oligonucleotide is selected from any one or more of the following: anti-miRNA, BNA-AON or siRNA, such as BNa-based siRNA, selected from chemically modified siRNA, metabolically stable siRNA, and chemically modified metabolically stable siRNA.

An embodiment is an oligonucleotide conjugate according to the invention and e.g. according to any of the molecules (EE), (PP) and (SS), comprising an oligonucleotide, wherein the oligonucleotide is an oligonucleotide capable of silencing e.g. when present in a mammalian cell and preferably when present in a human cell any of the following genes: apolipoprotein B (apoB), HSP27, thyroxine Transporter (TTR), proprotein convertase subtilisin/kexin type 9 (PCSK 9), TMPRSS6, delta-aminolevulinate synthase 1 (ALAS 1), antithrombin 3 (AT 3), glycolate Oxidase (GO), complement component C5 (CC 5), the X gene of Hepatitis B Virus (HBV), the S gene of HBV, alpha-1 antitrypsin (AAT), miR-122, hepatitis B virus HbsAg, LDHA, CEBPA and Lactate Dehydrogenase (LDH), and/or oligonucleotides capable of targeting aberrant mirnas, for example, when present in mammalian cells.

An embodiment is an oligonucleotide conjugate according to the invention and e.g. according to any of the molecules (EE), (PP) and (SS), comprising an oligonucleotide, wherein the oligonucleotide is an oligonucleotide capable of silencing e.g. when present in a mammalian cell and preferably when present in a human cell any of the following genes: apolipoprotein B (apoB) and HSP27.

An embodiment is an oligonucleotide conjugate according to the invention and e.g. according to any of the molecules (EE), (PP) and (SS), comprising an oligonucleotide, wherein the oligonucleotide is an oligonucleotide capable of targeting mRNA involved in the expression of any of the following proteins, e.g. when present in a mammalian cell and preferably when present in a human cell: apoB, HSP27, TTR, PCSK9, TMPRSS6, ALAS1, AT3, GO, CC5, expression products of X genes of HBV, expression products of S genes of HBV, AAT, miR-122, hepatitis B virus HbsAg, LDHA, CEBPA and LDH; or are capable of antagonizing or restoring miRNA function, such as inhibiting oncogenic miRNA (onco-miR) or repressing expression of onco-miR, for example, when present in mammalian cells and preferably when present in human cells.

An embodiment is an oligonucleotide conjugate according to the invention and e.g. according to any of the molecules (EE), (PP) and (SS), comprising an oligonucleotide, wherein the oligonucleotide is an oligonucleotide capable of targeting mRNA involved in the expression of any of the following proteins, e.g. when present in a mammalian cell and preferably when present in a human cell: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AAT, miR-122, hepatitis B virus HbsAg, LDHA and CEBPA.

An embodiment is an oligonucleotide conjugate according to the invention and e.g. according to any of the molecules (EE), (PP) and (SS), comprising an oligonucleotide, wherein the oligonucleotide is an oligonucleotide capable of targeting mRNA involved in the expression of any of the following proteins, e.g. when present in a mammalian cell and preferably when present in a human cell: apoB and HSP27.

An aspect of the invention relates to a second pharmaceutical composition comprising an oligonucleotide conjugate according to the invention and e.g. according to any of the molecules (EE), (PP) and (SS), optionally together with a pharmaceutically acceptable excipient and/or optionally with a pharmaceutically acceptable diluent.

One aspect of the invention relates to the second pharmaceutical composition of the invention or the oligonucleotide conjugate of the invention and for example according to any of the molecules (EE), (PP) and (SS), for use as a medicament.

An aspect of the invention relates to the use of the second pharmaceutical composition of the invention or the oligonucleotide conjugate of the invention and e.g. according to any of the molecules (EE), (PP) and (SS) for the treatment or prevention of diseases or health problems wherein the expression product is involved in any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AT3, GO, CC5, X gene of HBV, S gene of HBV, AAT, miR-122, hepatitis B virus HbsAg, LDHA, CEBPA and LDH; and/or for use in the treatment or prevention of diseases or health problems involving any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AT3, GO, CC5, X gene of HBV, S gene of HBV, AAT, miR-122, hepatitis B virus HbsAg, LDHA, CEBPA and LDH.

An aspect of the invention relates to the use of the second pharmaceutical composition of the invention or the oligonucleotide conjugate of the invention and e.g. according to any of the molecules (EE), (PP) and (SS) for the treatment or prevention of diseases or health problems wherein the expression product is involved in any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AAT, miR-122, hepatitis B virus HbsAg, LDHA and CEBPA; and/or for use in the treatment or prevention of diseases or health problems involving any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AAT, miR-122, hepatitis B virus HbsAg, LDHA and CEBPA.

An embodiment is the second pharmaceutical composition of the invention or the oligonucleotide conjugate of the invention and e.g. according to any of the molecules (EE), (PP) and (SS), for use as described above, wherein the use is for the treatment or prevention of a disease or health problem wherein the expression product is involved in any one or more of the following genes: HSP27 and apoB, preferably apoB, and/or for use in the treatment or prevention of diseases or health problems involving any one or more of the following genes: HSP27 and apoB, preferably apoB.

An embodiment is the second pharmaceutical composition of the invention or the oligonucleotide conjugate of the invention and e.g. according to any of the molecules (EE), (PP) and (SS), for use as described above for the treatment or prevention of use in: cancer, infectious disease, viral infection, hypercholesterolemia, cardiovascular disease, primary hyperoxaluria, hemophilia a, hemophilia B, AAT-associated liver disease, acute hepatic porphyrin disease, TTR-mediated amyloidosis, hereditary TTR amyloidosis (hATTR), complement-mediated disease, hepatitis b infection, hepatitis c infection, alpha 1-antitrypsin deficiency, beta-thalassemia or autoimmune disease.

An embodiment is the second pharmaceutical composition of the invention or the oligonucleotide conjugate of the invention and e.g. according to any of the molecules (EE), (PP) and (SS), for use as described above, wherein the use is in the treatment or prevention of: cancer (e.g., endometrial, breast, lung, or hepatocellular carcinoma) and/or cardiovascular disease (hypercholesterolemia, preferably hypercholesterolemia).

One aspect of the invention relates to the use of the second pharmaceutical composition of the invention or the oligonucleotide conjugate of the invention and e.g. according to any of the molecules (EE), (PP) and (SS) for reducing LDL-cholesterol in a subject. Preferably, the subject is a human subject.

Preferably, the saponins of the oligonucleotide conjugates of the invention are plant-derived saponins, e.g. according to any of the molecules (EE), (PP) and (SS). The saponin comprised by the oligonucleotide conjugate of the invention is preferably a saponin isolated from a plant. For example, saponins are isolated from roots of plants. Examples of such plants from which saponins are extracted (isolated) are quillaja saponaria, phyllostachys pratensis, saponaria officinalis (Saponaria officinalis) (e.g., saponaria officinalis l.) and phyllostachys pratensis (Gypsophila elegans) (e.g., phyllostachys pratensis m. Bieb). Preferably, the saponin conjugates and/or oligonucleotide conjugates comprise a single type of saponin, preferably a single type of saponin derived from plant material, such as plant roots, such as SO1861, SO1862 or SO1832 from soapstock (e.g. soapstock l., preferably soapstock l.) (roots), or such as QS-21, QS-7 or QS-17 from quillaja (roots). Suitable sources of isolated saponins according to the present invention, i.e. those that exhibit enhanced endosomal escape activity, are quillaja saponaria, carnation, soapberry and carnation, and quillaja bark.

An aspect of the invention relates to an in vitro or ex vivo method for transferring an oligonucleotide conjugate of any of the above outlined embodiments of the invention from outside of a cell into said cell, preferably subsequently transferring an oligonucleotide comprised by said oligonucleotide conjugate into the cytosol of said cell, comprising the steps of:

a) Providing a cell expressing ASGPR on its surface, preferably selected from the group consisting of a hepatocyte, a virus-infected cell and a tumor cell, and providing an oligonucleotide conjugate of the invention for transfer into the provided cell;

b) Contacting the cell of step a) in vitro or ex vivo with the oligonucleotide conjugate of step a), thereby effecting transfer of the oligonucleotide conjugate from outside the cell into said cell, and preferably subsequently effecting transfer of the oligonucleotide contained in the oligonucleotide conjugate into the cytosol of said cell.

While the invention has been described in terms of several embodiments, it is contemplated that alternatives, modifications, permutations and equivalents thereof will become apparent to those of ordinary skill in the art upon reading of the specification and study of the drawings. The invention is not in any way limited to the embodiments shown. Changes may be made without departing from the scope as defined in the following claims.

Embodiments of the invention

1. A saponin conjugate comprising at least one saponin covalently linked to a ligand of an asialoglycoprotein receptor (ASGPR), wherein the ligand of ASGPR comprises at least one N-acetylgalactosamine (GalNAc) moiety, preferably three or four GalNAc moieties, more preferably the ligand of ASGPR comprises (GalNAc) ₃ Tris or consists thereof, wherein at least one saponin is selected from the group consisting of a monosaccharide-chain triterpenoid saponin and a disaccharide-chain triterpenoid saponin.

2. The saponin conjugate of embodiment 1, wherein the saponin comprises a aglycone core structure selected from the group consisting of:

2 alpha-hydroxy oleanolic acid,

16 alpha-hydroxy oleanolic acid,

Hederagenin (23-hydroxy oleanolic acid),

16 alpha, 23-dihydroxyoleanolic acid,

The silk-stone bamboo sapogenin,

Soap scum acid,

Escin-21 (2-methylbut-2-enoate) -22-acetate,

23-oxo-yucygenin C-21, 22-bis (2-methylbut-2-enoate),

23-oxo-yucygenin C-21 (2-methylbut-2-enoate) -16, 22-diacetate,

Digitalis sapogenin,

3,16,28-Trihydroxyolean-12-ene,

Carnation acid,

And

The derivatives of these and the derivatives thereof,

preferably, the sapogenins comprise a aglycone core structure selected from the group consisting of saponaric acid and serrulaspin or derivatives thereof, more preferably the sapogenins core structure is saponaric acid or derivatives thereof.

3. The saponin conjugate of embodiment 1 or 2, wherein

Saponins comprise a sugar chain bound to the aglycone core structure, said sugar chain being selected from group a:

GlcA-、

Glc-；

Gal-；

Rha-(1→2)-Ara-、

Gal-(1→2)-[Xyl-(1→3)]-GlcA-、

Glc-(1→2)-[Glc-(1→4)]-GlcA-、

Glc-(1→2)-Ara-(1→3)-[Gal-(1→2)]-GlcA-、

Xyl-(1→2)-Ara-(1→3)-[Gal-(1→2)]-GlcA-、

Glc-(1→3)-Gal-(1→2)-[Xyl-(1→3)]-Glc-(1→4)-Gal-、

Rha-(1→2)-Gal-(1→3)-[Glc-(1→2)]-GlcA-、

Ara-(1→4)-Rha-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-、

Ara-(1→4)-Fuc-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-、

Ara-(1→4)-Rha-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-、

Ara-(1→4)-Fuc-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-、

Ara-(1→4)-Rha-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-、

Ara-(1→4)-Fuc-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-、

Ara-(1→4)-Rha-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-、

Ara-(1→4)-Fuc-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-、

Xyl-(1→4)-Rha-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-、

Xyl-(1→4)-Fuc-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-、

Xyl-(1→4)-Rha-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-、

Xyl-(1→4)-Fuc-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-、

Xyl-(1→4)-Rha-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-、

Xyl-(1→4)-Fuc-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-、

Xyl-(1→4)-Rha-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-、

xyl- (1.fwdarw.4) -Fuc- (1.fwdarw.2) -Gal- (1.fwdarw.2) -Fuc- (1.fwdarw.2) -GlcA-, and

The derivatives of these and the derivatives thereof,

or (b)

Saponins comprise a sugar chain bound to the aglycone core structure, said sugar chain being selected from group B:

Glc-，

Gal-，

Rha-(1→2)-[Xyl-(1→4)]-Rha-，

Rha-(1→2)-[Ara-(1→3)-Xyl-(1→4)]-Rha-，

Ara-，

Xyl-，

xyl- (1.fwdarw.4) -Rha- (1.fwdarw.2) - [ R1- (. Fwdarw.4) ] -Fuc-, wherein R1 is 4E-methoxy cinnamic acid,

xyl- (1.fwdarw.4) -Rha- (1.fwdarw.2) - [ R2- (. Fwdarw.4) ] -Fuc-, wherein R2 is 4Z-methoxycinnamic acid,

Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-4-OAc-Fuc-，

xyl- (1.fwdarw.4) - [ Glc- (1.fwdarw.3) ] -Rha- (1.fwdarw.2) -3, 4-di-OAc-Fuc-,

xyl- (1.fwdarw.4) - [ Glc- (1.fwdarw.3) ] -Rha- (1.fwdarw.2) - [ R3- (. Fwdarw.4) ] -3-OAc-Fuc-, wherein R3 is 4E-methoxycinnamic acid,

Glc-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-4-OAc-Fuc-，

Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-4-OAc-Fuc-，

(Ara-or Xyl-) (1.fwdarw.3) - (Ara-or Xyl-) (1.fwdarw.4) - (Rha-or Fuc-) (1.fwdarw.2) - [4-OAc- (Rha-or Fuc-) (1.fwdarw.4) ] - (Rha-or Fuc-),

Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Qui-(1→4)]-Fuc-，

Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-Fuc-，

Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-Fuc-，

Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-Fuc-，

Ara/Xyl-(1→4)-Rha/Fuc-(1→4)-[Glc/Gal-(1→2)]-Fuc-，

api- (1.fwdarw.3) -Xyl- (1.fwdarw.4) - [ Glc- (1.fwdarw.3) ] -Rha- (1.fwdarw.2) - [ R4- (. Fwdarw.4) ] -Fuc-, wherein R4 is 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid,

api- (1.fwdarw.3) -Xyl- (1.fwdarw.4) -Rha- (1.fwdarw.2) - [ R5- (. Fwdarw.4) ] -Fuc-, wherein R5 is 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid,

Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Rha-(1→3)]-4-OAc-Fuc-，

Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4-OAc-Fuc-，

6-OAc-Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3-OAc-Rha-(1→3)]-Fuc-，

Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3-OAc--Rha-(1→3)]-Fuc-，

Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Qui-(1→4)]-Fuc-，

Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-[Qui-(1→4)]-Fuc-，

Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc-，

xyl- (1.fwdarw.3) -Xyl- (1.fwdarw.4) -Rha- (1.fwdarw.2) - [3, 4-di-OAc-Qui- (1.fwdarw.4) ] -Fuc-,

Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-Fuc-，

6-OAc-Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-Fuc-，

Glc-(1→3)-[Xyl-(1→3)-Xyl-(1→4)]-Rha-(1→2)-Fuc-，

Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc-，

Api/Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4OAc-Fuc-，

Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4OAc-Fuc-，

Api/Xyl- (1.fwdarw.3) -Xyl- (1.fwdarw.4) - [ Glc- (1.fwdarw.3) ] -Rha- (1.fwdarw.2) - [ R6- (. Fwdarw.4) ] -Fuc-, wherein R6 is 5-O- [5-O-Rha- (1.fwdarw.2) -Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid,

Api/Xyl- (1.fwdarw.3) -Xyl- (1.fwdarw.4) - [ Glc- (1.fwdarw.3) ] -Rha- (1.fwdarw.2) - [ R7- (. Fwdarw.4) ] -Fuc-, wherein R7 is 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid,

Api/Xyl- (1.fwdarw.3) -Xyl- (1.fwdarw.4) - [ Glc- (1.fwdarw.3) ] -Rha- (1.fwdarw.2) - [ R8- (. Fwdarw.4) ] -Fuc-, wherein R8 is 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid,

api- (1.fwdarw.3) -Xyl- (1.fwdarw.4) -Rha- (1.fwdarw.2) - [ R9- (. Fwdarw.4) ] -Fuc-, wherein R9 is 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid,

xyl- (1- & gt 3) -Xyl- (1- & gt 4) -Rha- (1- & gt 2) - [ R10- (& gt 4) ] -Fuc-, wherein R10 is 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid,

api- (1.fwdarw.3) -Xyl- (1.fwdarw.4) -Rha- (1.fwdarw.2) - [ R11- (. Fwdarw.3) ] -Fuc-, wherein R11 is 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid,

xyl- (1- & gt 3) -Xyl- (1- & gt 4) -Rha- (1- & gt 2) - [ R12- (& gt 3) ] -Fuc-, wherein R12 is 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid), glc- (1- & gt 3) - [ Glc- (1- & gt 6) ] -Gal-, and

the derivatives of these and the derivatives thereof,

or (b)

Saponins are disaccharide chain triterpene glycosides comprising a first sugar chain selected from group a bound to the aglycone core structure and comprising a second sugar chain selected from group B bound to the aglycone core structure.

4. The saponin conjugate of any one of embodiments 1-3, wherein the saponin is selected from the group consisting of: quillaja saponaria saponins, dipsacus asperoides saponin B, saikosaponin A, saikosaponin D, macranthosaponin A, phytolaccatin, escin salt, AS6.2, NP-005236, AMA-1, AMR, alpha-hederagenin, NP-012672, NP-017777, NP-017778, NP-017774, NP-018110, NP-017772, NP-018109, NP-017888, NP-017889, NP-018108, SA1641, AE X55, NP-017674, NP-017810, AG1, NP-003881, NP-017676, NP-017677, NP-017706, NP-017705, NP-017773 NP-017775, SA1657, AG2, SO1861, GE1741, SO1542, SO1584, SO1658, SO1674, SO1832, SO1862, SO1904, QS-7, QS1861, QS-7api, QS1862, QS-17, QS-18, QS-21A-apio, QS-21A-xylo, QS-21B-apio, QS-21B-xylo, β -escin, escin Ia, tea seed saponin I, tea seed saponin J, assam saponin F, digitonin, primordic acid 1 and AS64R, stereoisomers thereof, derivatives thereof, and combinations thereof; preferably the saponins are selected from: QS-21, QS-21 derivatives, SO1861 derivatives, SA1641 derivatives, GE1741 derivatives, and combinations thereof; more preferably the saponins are selected from: QS-21 derivatives, SO1861 derivatives, and combinations thereof; most preferably the saponin is a SO1861 derivative.

5. The saponin conjugate of embodiment 3 or 4, wherein the saponin is a saponin derivative, wherein

i. The saponin derivative comprises an aglycone core structure comprising a derivatized aldehyde group;

the saponin derivative comprises a sugar chain, preferably selected from group a as defined in embodiment 3, said sugar chain comprising a derivatized carboxyl group;

the saponin derivative comprises a sugar chain, preferably selected from group B as defined in embodiment 3, said sugar chain comprising a derivatized acetoxy (Me (CO) O-) group; or (b)

The saponin derivative comprises any combination of derivatizations i, ii, and iii, preferably any combination of derivatizations of two of i, ii, and iii.

6. The saponin conjugate of any one of embodiments 1-5, wherein the saponin is any one or more of: SO1861, SA1657, GE1741, SA1641, QS-21A, QS-21A-api, QS-21A-xyl, QS-21B, QS-21B-api, QS-21B-xyl, QS-7-api, QS-17-xyl, QS1861, QS1862, quillaja saponin, saponin album, QS-18, quil-A, gyp1, marshall saponin A, AG, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, SO1904, stereoisomers thereof, derivatives thereof, and combinations thereof; preferably the saponins are selected from: QS-21, QS-21 derivatives, SO1861 derivatives, SA1641 derivatives, GE1741 derivatives, and combinations thereof; more preferably the saponins are selected from: QS-21 derivatives, SO1861 derivatives, and combinations thereof; most preferably the saponin is a SO1861 derivative.

7. The saponin conjugate of any one of embodiments 1-6, wherein the saponin is a saponin derivative of a saponaric acid saponin or a silk diabolo sapogenin saponin as described in embodiment 2, and is represented by molecule 1:

wherein the method comprises the steps of

A ₁ Represents hydrogen, monosaccharides or linear or branched oligosaccharides, preferably A ₁ Represents a sugar chain selected from group A as defined in embodiment 3, more preferably A ₁ Represents a sugar chain selected from group A as defined in embodiment 3 and A ₁ Comprising or consisting of glucuronic acid moieties;

A ₂ represents hydrogen, monosaccharides or linear or branched oligosaccharides, preferably A ₂ Represents a sugar chain selected from group B as defined in embodiment 3, more preferably A ₂ Is shown as being selected from the following embodimentsSugar chain of group B defined in case 3 and A ₂ Comprising at least one acetoxy (Me (CO) O-) group, such as one, two, three or four acetoxy groups,

wherein A is ₁ And A ₂ At least one of which is not hydrogen, preferably A ₁ And A ₂ Are oligosaccharide chains;

and R is hydrogen in sericin or hydroxyl in saponaric acid; wherein the saponin derivative corresponds to the saponin represented by molecule 1, wherein at least one, preferably one or two, more preferably one of the following derivatizations is present:

i. Position C of saponaric acid or serrulate sapogenin ₂₃ The aldehyde groups at which have been derivatized;

when A ₁ Represents a sugar chain selected from group A as defined in embodiment 3 and A ₁ When comprising or consisting of glucuronic acid moieties, A ₁ Has been derivatized with carboxyl groups of glucuronic acid moieties; and

When A ₂ Represents a sugar chain selected from group B as defined in embodiment 3 and A ₂ When at least one acetoxy group is included, A ₂ One or more, preferably all, of the acetoxy groups of one sugar moiety or two or more sugar moieties have been derivatized.

8. The saponin conjugate of embodiment 7, wherein A ₁ Represents a sugar chain selected from group A as defined in embodiment 3 and comprises or consists of a glucuronic acid moiety, and wherein A ₁ Has been derivatized with carboxyl groups of glucuronic acid moieties, and/or wherein A ₂ Represents a sugar chain selected from group B as defined in embodiment 3 and A ₂ Comprises at least one acetoxy group, and wherein A ₂ Has been derivatized with at least one acetoxy group.

9. The saponin conjugate of embodiment 7 or 8, wherein the saponin represented by molecule 1 is a disaccharide chain triterpenoid saponin.

10. The saponin conjugate of any one of embodiments 7-9, wherein the saponin derivative corresponds to a saponin represented by molecule 1, wherein at least one, preferably one or two, more preferably one of the following derivatizations is present:

i. c of saponaric acid or serrulate sapogenin ₂₃ The aldehyde group at the position has been derivatized by:

-reduction to an alcohol;

conversion to hydrazone linkage by reaction with N-epsilon-maleimidocaaproic acid hydrazide (EMCH) to provide saponin-aldemch (e.g., SO 1861-aldemch or QS-21-aldemch), wherein the maleimide group of EMCH is optionally derivatized by formation of a thioether bond with mercaptoethanol;

-conversion to hydrazone linkage by reaction with N- [ β -maleimidopropionic acid ] hydrazide (BMPH), wherein the maleimide group of BMPH is optionally derivatized by formation of thioether bond with mercaptoethanol; or (b)

-conversion to hydrazone bond by reaction with N- [ kappa-maleimido undecanoic acid ] hydrazide (KMUH), wherein the maleimide group of KMUH is optionally derivatized by forming a thioether bond with mercaptoethanol;

when A ₁ Represents a sugar chain selected from group A as defined in embodiment 3 and A ₁ When comprising or consisting of glucuronic acid moieties, A ₁ The carboxyl group of the glucuronic acid moiety of (a) has been derivatized by reaction with 2-amino-2-methyl-1, 3-propanediol (AMPD) or N- (2-aminoethyl) maleimide (AEM) to an amide bond, to provide a saponin-Glu-AMPD (e.g., QS-21-Glu-AMPD or SO 1861-Glu-AMPD), or a saponin-Glu-AEM (e.g., QS-21-Glu-AEM or SO 1861-Glu-AEM); and

When A ₂ Represents a sugar chain selected from group B as defined in embodiment 3 and A ₂ When at least one acetoxy group is included, A ₂ One or more, preferably all, of the acetoxy groups of one sugar moiety or two or more sugar moieties have been derivatized by deacetylation to hydroxyl groups (HO-).

11. The saponin conjugate of any one of embodiments 7-10, wherein a ₁ Is Gal- (1- > 2) - [ Xyl- (1- > 3)]-GlcA and/or A ₂ Is Glc- (1- & gt 3) -Xyl- (1- & gt 4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]Fuc, preferably the saponin represented by molecule 1 is 3-O-beta-D-galactopyranosyl- (1.fwdarw.2) - [ beta-D-xylopyranosyl- (1.fwdarw.3)]-beta-D-glucuronopyranosyl soap pinoic acid 28-O-beta-D-glucopyranosyl- (1.fwdarw.3) -beta-D-xylopyranosyl- (1.fwdarw.4) -alpha-L-rhamnopyranosyl- (1.fwdarw.2) - [ beta-D-xylopyranosyl- (1.fwdarw.3) -4 OAc-beta-D-quiniopyranosyl- (1.fwdarw.4) ]-beta-D-fucopyranoside, more preferably the saponin is any one or more of the following: SO1861, GE1741, SA1641 and QS-21, or derivatives thereof, most preferably SO1861 or derivatives thereof.

12. The saponin conjugate of any one of embodiments 5-11, wherein the saponin is a saponin derivative, wherein

i. The saponin derivative comprises a aglycone core structure comprising aldehyde groups which have been derivatised by:

-reduction to an alcohol;

-converting to hydrazone linkage by reaction with N-epsilon-maleimidocaaproic acid hydrazide (EMCH) to provide saponin-Ald-EMCH, such as SO1861-Ald-EMCH or QS-21-Ald-EMCH, wherein the maleimide group of EMCH is optionally derivatized by forming a thioether bond with mercaptoethanol;

-conversion to hydrazone linkage by reaction with N- [ β -maleimidopropionic acid ] hydrazide (BMPH), wherein the maleimide group of BMPH is optionally derivatized by formation of thioether bond with mercaptoethanol; or (b)

-conversion to hydrazone bond by reaction with N- [ kappa-maleimido undecanoic acid ] hydrazide (KMUH), wherein the maleimide group of KMUH is optionally derivatized by forming a thioether bond with mercaptoethanol;

the saponin derivative comprises a sugar chain, preferably selected from group a as defined in embodiment 3, comprising a carboxyl group, preferably a carboxyl group of a glucuronic acid moiety, which has been derivatised by conversion to an amide bond by reaction with 2-amino-2-methyl-1, 3-propanediol (AMPD) or N- (2-aminoethyl) maleimide (AEM), thereby providing a saponin-Glu-AMPD (such as QS-21-Glu-AMPD or SO 1861-Glu-AMPD) or a saponin-Glu-AEM (such as QS-21-Glu-AEM or SO 1861-Glu-AEM);

The saponin derivative comprises a sugar chain, preferably selected from group B as defined in embodiment 3, comprising an acetoxy (Me (CO) O-) group that has been derivatized by deacetylation to a hydroxyl group (HO-); or (b)

The saponin derivative comprises any combination of derivatizations i, ii, and iii, preferably any combination of derivatizations of two of i, ii, and iii;

preferably, the sapogenin derivative comprises an aglycone core structure, wherein the aglycone core structure comprises an aldehyde group that has been derivatised by reaction with EMCH to convert to a hydrazone bond, wherein the maleimide group of EMCH is optionally derivatised by formation of a thioether bond with mercaptoethanol.

13. The saponin conjugate of embodiment 12, wherein the saponin is a saponin derivative, wherein

i. The saponin derivative comprises an aglycone core structure comprising an aldehyde group that has been derivatized by reaction with N-epsilon-maleimidocaaproic acid hydrazide (EMCH) to a hydrazone bond, thereby providing a saponin-aldemch (e.g., SO 1861-aldemch or QS-21-aldemch);

the saponin derivative comprises a sugar chain, preferably selected from group a as defined in embodiment 3, comprising a carboxyl group, preferably a carboxyl group of a glucuronic acid moiety, which has been derivatized by conversion to an amide bond by reaction with N- (2-aminoethyl) maleimide (AEM), thereby providing a saponin-Glu-AEM (such as QS-21-Glu-AEM or SO 1861-Glu-AEM); or (b)

The saponin derivative comprises a combination of derivatizations i.and ii..

14. The saponin conjugate of embodiment 12, wherein the saponin derivative comprises a aglycone core structure, wherein the aglycone core structure comprises an aldehyde group, and wherein the saponin derivative comprises a sugar chain, preferably a sugar chain selected from group a as defined in embodiment 3, said sugar chain comprising a carboxyl group, preferably a carboxyl group of a glucuronic acid moiety, said glucuronic acid moiety having been derivatized by conversion to an amide bond by reaction with N- (2-aminoethyl) maleimide (AEM).

15. The saponin conjugate of any one of embodiments 1-12, wherein the saponin is a saponin derivative represented by molecule 2:

or wherein the saponin is a saponin derivative represented by molecule 3:

16. the saponin conjugate of any one of embodiments 1-15, wherein at least one ligand of saponin and ASGPR is covalently linked directly or via at least one linker.

17. The saponin conjugate of any one of embodiments 1-16, wherein a GalNAc moiety is bound to the saponin S, preferably via a saponin linker Ls, as in formula (II) _S As shown in:

18. the saponin conjugate of any one of embodiments 1-16, wherein GalNAc moieties are each covalently bound via oxygen at position "1" of the GalNAc moiety to a central bridging moiety B, respectively, effective to form a bridge between the GalNAc moiety and the saponin moiety, preferably via a saponin moiety linker L _S Effectively forming a bridge between the GalNAc moiety and the saponin moiety, as in formula (III) _S As shown in:

where n is an integer greater than or equal to 2, preferably n is 3, LS is a saponin moiety linker, and S is the saponin moiety.

19. The saponin conjugate of embodiment 18Wherein the GalNAc moiety is bound to bridging moiety B via a GalNAc linker LGAL, e.g. of formula (IV) _S As shown in:

wherein n is an integer greater than or equal to 2, preferably n is 3, L _S Is a saponin moiety linker, and S is the saponin moiety.

20. The saponin conjugate according to any one of embodiments 17-19, wherein the saponin moiety linker Ls represents GalNAc or GalNAc as in formula (II) S, suitable for use in contacting a saponin with a polypeptide of formula (III) _S And (IV) _S Any chemical moiety to which bridging moiety B is covalently bound.

21. The saponin conjugate of any one of embodiments 17-20, wherein the saponin moiety linker L _S Is at least a first precursor L _S1 With a second precursor L _S2 As a result of the coupling reaction between the first precursor L _S1 Covalently bound to GalNAc or to the bridging moiety B, a second precursor L _S2 Covalently bound to a saponin moiety, wherein L _S1 Is a saponin moiety linker L covalently bound to GalNAc or to bridging moiety B _S And L is _S2 Is a saponin moiety linker L covalently bound to a saponin moiety _S Is a precursor of (a).

22. The saponin conjugate of any one of embodiments 17-21, wherein the saponin moiety linker L _S Is at least a first precursor L _S1 With a second precursor L _S2 As a result of the coupling reaction between the first precursor L _S1 Covalently bound to GalNAc or bridging moiety B, a second precursor L _S2 Covalently bound to a saponin moiety, wherein the coupling reaction is selected from the group consisting of: azide-alkyne cycloaddition, thiol maleimide coupling, staudinger reaction, nucleophilic ring opening of strained heterocyclic electrophiles, carbonyl reactions of non-aldol type, addition of carbon-carbon double bonds, preferably wherein the coupling reaction is azide-alkyne cycloaddition, thiol maleimide coupling, staudinger reaction, nucleophilic ring opening of strained heterocyclic electrophiles, more preferably wherein coupling isThe coupling reaction is azide-alkyne cycloaddition or thiol maleimide coupling.

23. The saponin conjugate of any one of embodiments 17-22, wherein the saponin moiety linker L _S Is the first precursor L _S1 With a second precursor L _S2 As a result of the coupling reaction between the first precursor L _S1 Covalently bound to GalNAc or bridging moiety B, first precursor L _S1 Comprises an azide; second precursor L _S2 Covalently bound to the saponin moiety, a second precursor L _S2 Comprising alkynes and preferably hydrazones resulting from the coupling of a hydrazide/aldehyde with the aldehyde of the saponin moiety.

24. The saponin conjugate of any one of embodiments 21-23, wherein precursor L _S1 Is an azide having the following formula (XVII):

wherein a represents an integer greater than or equal to 0, preferably a represents an integer selected from 1, 2 and 3, more preferably a represents 2, or

Wherein precursor L _S1 Comprises an azide of formula (XVIII):

wherein c represents an integer greater than or equal to 0, preferably c represents an integer in the range of 5-15, more preferably c represents 9.

25. The saponin conjugate of any one of embodiments 21-24, wherein precursor L _S2 Comprises hydrazones of the following formula (XIX):

wherein a represents an integer greater than or equal to 0, preferably a represents an integer in the range of 2-6, more preferably a represents 4, and wherein L _S2a Representing an alkyne-containing moiety.

26. The saponin conjugate of embodiment 25, wherein L _S2a Containing less than 20 carbon atoms, preferably L _S2a Represents a moiety according to formula (XX):

27. the saponin conjugate of any one of embodiments 18-26, wherein the bridging moiety is a compound of formula (XV):

Wherein the oxygen atom of the compound having the formula (XV) is bonded to GalNAc or a GalNAc linker L _GAL Is bound to, and has a nitrogen atom of the compound of formula (XV) bonded to a saponin moiety linker L _S And (5) combining.

28. The saponin conjugate of any one of embodiments 19-27, wherein L _GAL Represents any chemical moiety suitable for covalently binding GalNAc to bridging moiety B.

29. The saponin conjugate of any one of embodiments 19-28, wherein L _GAL Containing 2 to 25 carbon atoms, preferably 7 to 15 carbon atoms, more preferably 11 carbon atoms, and wherein L _GAL Comprising at least one, preferably two amide moieties.

30. The saponin conjugate of any one of embodiments 19-29, wherein L _GAL Is a compound according to formula (XVI):

31. as in embodiments 17-30The saponin conjugate of any one of claims, wherein the ligand of ASGPR is represented by molecule (DD 3) or molecule (DD 4) (GalNAc) ₃ Tris：

/>

Or wherein the ligand of ASGPR is single GalNAc represented by molecule II':

32. the saponin conjugate of any one of embodiments 1-31, wherein at least one saponin is covalently bound to the molecule (DD 3) or the molecule (DD 4) or the molecule II 'of embodiment 31 via a linker represented by molecule III':

wherein the hydrazide moiety of molecule III 'forms a covalent hydrazone bond with the aldehyde group in the saponin, and wherein the dibenzocyclooctyne group forms a covalent bond with the azide group of molecule (DD 3) or molecule (DD 4) or molecule II'.

33. The saponin conjugate of any one of embodiments 1-32, wherein at least one saponin is covalently bound to a ligand of ASGPR via at least one cleavable linker.

34. The saponin conjugate of embodiment 33, wherein the cleavable linker is subject to cleavage under acidic, reducing, enzymatic, and/or photoinduced conditions, and preferably the cleavable linker comprises a cleavable bond selected from the group consisting of: hydrazone bonds and hydrazide bonds that undergo cleavage under acidic conditions, and/or bonds that are susceptible to proteolysis, such as bonds that are proteolytically cleaved by cathepsin B, and/or bonds that are susceptible to cleavage under reducing conditions (e.g., disulfide bonds).

35. The saponin conjugate of embodiments 33 or 34, wherein the cleavable linker is subjected to in vivo cleavage under acidic conditions, e.g., such as those present in the endosome and/or lysosome of a mammalian cell, preferably a human cell, preferably the cleavable linker is subjected to in vivo cleavage at a pH of 4.0-6.5, and more preferably at a pH of +.5.5.

36. The saponin conjugate of any one of embodiments 1-35, wherein the conjugate comprises 1, 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, 128, or 1-100 saponin moieties, or any number of saponin moieties therebetween (e.g., 7, 9, 12 saponin moieties).

37. A pharmaceutical combination comprising:

-a first pharmaceutical composition comprising the saponin conjugate of any one of embodiments 1-36 and optionally comprising a pharmaceutically acceptable excipient and/or a pharmaceutically acceptable diluent; and

-a second pharmaceutical composition comprising a second conjugate of an effector molecule and a ligand of ASGPR, or a third conjugate of an effector molecule and a binding molecule, and optionally comprising a pharmaceutically acceptable excipient and/or a pharmaceutically acceptable diluent, wherein the ligand of ASGPR preferably comprises at least one GalNAc moiety, preferably three or four GalNAc moieties, more preferably the ligand of ASGPR comprises (GalNAc) ₃ Tris or consists of the same, the binding molecule comprising a binding site for a cell surface molecule.

38. A pharmaceutical composition comprising:

-a saponin conjugate according to any one of embodiments 1 to 36;

-a second conjugate of an effector molecule and a ligand of ASGPR, or a third conjugate of an effector molecule and a binding molecule, and optionally comprising a pharmaceutically acceptable excipient and/or a pharmaceutically acceptable diluent, wherein the ligand of ASGPR preferably comprises or consists of at least one GalNAc moiety, preferably three or four GalNAc moieties, more preferably the ligand of ASGPR comprises (GalNAc) 3Tris, and the binding molecule comprises a binding site for a cell surface molecule.

39. The pharmaceutical combination of embodiment 37 or the pharmaceutical composition of embodiment 38, wherein the effector molecule comprises or consists of at least one of: a small molecule (e.g., a drug molecule), a toxin (e.g., a protein toxin), an oligonucleotide (e.g., an AON, such as BNA), a heterologous nucleic acid or siRNA, an enzyme, a peptide, a protein, or any combination thereof, preferably, the effector molecule is a toxin, an enzyme, or an oligonucleotide.

40. The pharmaceutical combination according to embodiment 37 or 39, or the pharmaceutical composition according to embodiment 38 or 39, wherein the ligand of ASGPR comprises (GalNAc) ₃ Tris or (GalNAc) ₃ Tris, and/or wherein the ligand of ASGPR and the effector molecule are conjugated via a covalent bond, preferably via at least one linker.

41. The pharmaceutical combination of any one of embodiments 37 or 39-40 or the pharmaceutical composition of any one of embodiments 38-40, wherein the effector molecule is an oligonucleotide selected from any one or more of: short interfering RNA (siRNA), short hairpin RNA (shRNA), anti-hairpin microRNA (miRNA), single-stranded RNA, aptamer RNA, double-stranded RNA (dsRNA), anti-microRNA (anti-miRNA, anti-miR), antisense oligonucleotide (ASO), DNA, antisense DNA, locked Nucleic Acid (LNA), bridged Nucleic Acid (BNA), 2'-O,4' -aminoethylene Bridged Nucleic Acid (BNA) ^NC ) BNA-based siRNA, and BNA-based antisense oligonucleotide (BNA-aON).

42. The pharmaceutical combination of any one of embodiments 37 or 39-41 or the pharmaceutical composition of any one of embodiments 38-41, wherein the effector molecule is an oligonucleotide selected from any one or more of: anti-miRNA, BNA-AON or siRNA, such as BNa-based siRNA, selected from chemically modified siRNA, metabolically stable siRNA, and chemically modified metabolically stable siRNA.

43. The pharmaceutical combination of any one of embodiments 37 or 39-42 or the pharmaceutical composition of any one of embodiments 38-42, wherein the effector molecule is an oligonucleotide capable of silencing any one of the following genes, e.g., when present in a mammalian cell: apolipoprotein B (apoB), HSP27, thyroxine Transporter (TTR), proprotein convertase subtilisin/kexin type 9 (PCSK 9), deltA-Aminolevulinate synthase 1 (ALAS 1), antithrombin 3 (AT 3), glycolate Oxidase (GO), complement component C5 (CC 5), X gene of Hepatitis B Virus (HBV), S gene of HBV, alpha-1 antitrypsin (AAT) and Lactate Dehydrogenase (LDH); and/or an oligonucleotide capable of targeting an aberrant miRNA, e.g., when present in a mammalian cell.

44. The pharmaceutical combination of any one of embodiments 37 or 39-43 or the pharmaceutical composition of any one of embodiments 38-43, wherein the effector molecule is an oligonucleotide, e.g., an mRNA capable of targeting expression of any one of the following proteins when present in a mammalian cell: apoB, HSP27, TTR, PCSK9, ALAS1, AT3, GO, CC5, expression product of X gene of HBV, expression product of S gene of HBV, AAT and LDH; or are capable of antagonizing or restoring miRNA function, such as inhibiting oncogenic miRNA (onco-miR) or repressing expression of onco-miR, for example, when present in mammalian cells.

45. The pharmaceutical combination of any one of embodiments 37 or 39-44 or the pharmaceutical composition of any one of embodiments 38-44, wherein the effector molecule is or comprises a toxin comprising or consisting of at least one molecule selected from any one or more of the following: peptides, proteins, enzymes (such as urease and Cre recombinase), prions, ribosome inactivating proteins, and/or bacterial toxins, plant toxins; more preferably selected from any one or more of the following: viral toxins, such as apoptotic proteins; bacterial toxins, such as shiga toxin, shiga-like toxin, pseudomonas aeruginosa exotoxin (PE) or exotoxin a of PE, full length or truncated Diphtheria Toxin (DT), cholera toxin; mycotoxins, such as α -sarcin; a plant toxin comprising a ribosome inactivating protein and a chain of type 2 ribosome inactivating protein, such as carnation toxin protein e.g. carnation toxin protein-30 or carnation toxin protein-32, saporin e.g. saporin-S3 or saporin-S6, bougainvillea, or deimmunized derivatives of bougainvillea, or a shizania ribosome inactivating protein, shizania A Hega sample toxin A, a pokeweed antiviral protein, ricin, a ricin A chain, a capsule root toxin A chain, abrin A chain, a capsule root toxin A chain, a mistletoe lectin A chain; or animal or human toxins, such as frog ribonucleases, or granzyme B or angiogenic proteins from human, or any fragment or derivative thereof; preferably, the protein toxin is carnation toxin and/or saporin, and/or comprises or consists of at least one of the following: ribosome-targeting toxins, extension factor-targeting toxins, tubulin-targeting toxins, DNA-targeting toxins, and RNA-targeting toxins; more preferably any one or more of the following: enmei, pa Shu Tuo, maytansinoid derivative DM1, maytansinoid derivative DM4, monomethyl auristatin E (MMAE, vitamin D), monomethyl auristatin F (MMAF, mo Futing), calicheamicin, N-acetyl-gamma-calicheamicin, pyrrolobenzodiazepine(PBD) dimer, benzodiazepine +.>CC-1065 analog, docamicin, doxorubicin, paclitaxel, docetaxel, cisplatin, cyclophosphamide, etoposide, docetaxel, 5-fluorouracil (5-FU), mitoxantrone, tubulysin, indolinone benzodiazepine >AZ13599185, candidiasis, rhizobia, methotrexate, anthracyclines, camptothecin analogues, SN-38, DX-8951f, escitalopram mesylate, truncated forms of pseudomonas aeruginosa exotoxin (PE 38), docamicin derivatives, amanitine, alpha-amanitine, splice inhibitors, telavancin, ozunamicin, teslin, ambastatin 269 and grommet, or derivatives thereof.

46. The pharmaceutical combination of any one of embodiments 37 or 39-45 or the pharmaceutical composition of any one of embodiments 38-45, wherein the binding molecule comprising a binding site for a cell surface molecule comprised by the third conjugate is a ligand for a cell surface molecule or an antibody comprising a binding site for a cell surface molecule or at least one domain or fragment thereof comprising a binding site.

47. The pharmaceutical combination of any one of embodiments 37 or 39-46 or the pharmaceutical composition of any one of embodiments 38-46, wherein the third conjugate is any one or more of the following: antibody-toxin conjugates, receptor-ligand-toxin conjugates, antibody-drug conjugates, receptor-ligand-drug conjugates, antibody-nucleic acid conjugates, or receptor-ligand-nucleic acid conjugates.

48. The pharmaceutical combination of any one of embodiments 37 or 39-47 or the pharmaceutical composition of any one of embodiments 38-47, wherein the binding molecule comprising a binding site for a cell surface molecule comprised by the third conjugate is capable of binding to any one of the following cell surface molecules: CD71, CD63, CA125, epCAM (17-1A), CD52, CEA, CD44V6, FAP, EGF-IR, integrin, aggrecan-1, angiopoietin alpha-V beta-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, canag, integrin-alpha V, CA6, CD33, mesothelin, cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25, ephrin A4, MUC1, trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, FGF 3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, asialoglycoprotein receptor (ASP); preferably any one of the following: HER2, CD71, ASGPR and EGFR, more preferably CD71.

49. The pharmaceutical combination of any one of embodiments 37 or 39-48 or the pharmaceutical composition of any one of embodiments 38-48, wherein the binding molecule comprising a binding site for a cell surface molecule comprised by the third conjugate is or comprises any one of: antibodies, preferably monoclonal antibodies such as human monoclonal antibodies, igG, molecules comprising or consisting of: single sheet Domain antibody, at least one V _HH Domain, preferably camel VH, variable heavy chain neoantigen receptor (V _NAR ) Domains, fab, scFv, fv, dAb, F (ab) 2 and Fcab fragments.

50. The pharmaceutical combination according to any one of embodiments 37, 39-49 or the pharmaceutical composition according to any one of embodiments 38-49 for use as a medicament.

51. The pharmaceutical combination according to any one of embodiments 37, 39-49 or the pharmaceutical composition according to any one of embodiments 38-49 for use in the treatment or prevention of a disease or health problem in which the expression product relates to any one or more of the following genes: apoB, TTR, PCSK9, ALAS1, AT3, GO, CC5, X gene of HBV, S gene of HBV, AAT and LDH.

52. The pharmaceutical combination according to any one of embodiments 37, 39-49 or the pharmaceutical composition according to any one of embodiments 38-49, or the pharmaceutical combination or pharmaceutical composition for use according to embodiment 51, for use in the treatment or prevention of: cancer, infectious disease, viral infection, hypercholesterolemia, primary hyperoxaluria, hemophilia a, hemophilia B, AAT-associated liver disease, acute hepatic porphyria, TTR-mediated amyloidosis, hereditary TTR amyloidosis (hATTR), complement-mediated disease, hepatitis b infection, or autoimmune disease.

53. A pharmaceutical combination for use as described in embodiment 51 or 52 or a pharmaceutical composition for use as described in embodiment 51 or 52 wherein the saponin is a saponin derivative, preferably a QS-21 derivative or a SO1861 derivative as described in any one of embodiments 4-15.

54. An in vitro or ex vivo method for transferring a second or third conjugate according to any one of embodiments 37-48 from outside of a cell into said cell, preferably followed by transferring an effector molecule comprised by the second or third conjugate according to any one of embodiments 37-48 into the cytoplasm of said cell, comprising the steps of:

a) Providing a cell expressing ASGPR on its surface and expressing a cell surface molecule when transferring a third conjugate into the cell, wherein the third conjugate comprises a binding molecule for binding to said cell surface molecule, the cell preferably being selected from the group consisting of a hepatocyte, a virus-infected cell and a tumor cell;

b) Providing a second conjugate or a third conjugate according to any of embodiments 37-48 for transfer into the cell provided in step a);

c) Providing a saponin conjugate of any one of embodiments 1-36;

d) Contacting the cell of step a) with the second or third conjugate of step b) and the saponin conjugate of step c) in vitro or ex vivo, thereby effecting transfer of the second or third conjugate from outside the cell into the cell, and preferably thereby effecting subsequent transfer of the second or third conjugate into the cytoplasm of the cell, or preferably thereby effecting subsequent transfer of at least the effector molecule comprised by the second or third conjugate into the cytoplasm of the cell.

One embodiment is an oligonucleotide conjugate of the invention and/or a saponin conjugate of the invention, wherein the oligonucleotide conjugate or the saponin conjugate comprises a saponin that is isolated from a plant. Preferably, the saponins are isolated from a part of a plant (such as a root), or from a part of a tree (such as bark). Preferably, the saponins are isolated from roots from plants.

Embodiments of the invention

I. An oligonucleotide conjugate comprising at least one saponin covalently linked to a ligand of an asialoglycoprotein receptor (ASGPR), wherein the ligand of ASGPR comprises at least one N-acetylgalactosamine (GalNAc) moiety, preferably three or four GalNAc moieties, more preferably the ligand of ASGPR comprises (GalNAc) ₃ Tris or consists thereof, and is also covalently linked to an oligonucleotide, wherein at least one saponin is selected from the group consisting of a monosaccharide chain triterpenoid saponin and a disaccharide chain triterpenoid saponin.

The oligonucleotide conjugate according to embodiment I, wherein the saponin is a pentacyclic triterpenoid saponin of the 12, 13-dehydrooleanane type, preferably a pentacyclic triterpenoid saponin of the 12, 13-dehydrooleanane type having an aldehyde functional group at position C-23 of the aglycone core structure of the saponin.

The oligonucleotide conjugate according to embodiment I or II, wherein the saponin is a 12, 13-dehydrooleanane type monosaccharide chain or disaccharide chain pentacyclic triterpene saponin, preferably a 12, 13-dehydrooleanane type monosaccharide chain or disaccharide chain pentacyclic triterpene saponin having an aldehyde function at position C-23 of the aglycone core structure of the saponin.

An oligonucleotide conjugate according to any of embodiments I-III, wherein the saponin comprises a aglycone core structure selected from:

2 alpha-hydroxy oleanolic acid,

16 alpha-hydroxy oleanolic acid,

Hederagenin (23-hydroxy oleanolic acid),

16 alpha, 23-dihydroxyoleanolic acid,

The silk-stone bamboo sapogenin,

Soap scum acid,

Escin-21 (2-methylbut-2-enoate) -22-acetate,

23-oxo-yucygenin C-21, 22-bis (2-methylbut-2-enoate),

23-oxo-yucygenin C-21 (2-methylbut-2-enoate) -16, 22-diacetate,

Digitalis sapogenin,

3,16,28-Trihydroxyolean-12-ene,

Carnation acid,

And their derivatives;

preferably, the saponins comprise an aglycone core structure selected from saponaric acid and sericin or derivatives thereof; more preferably, the sapogenin core structure is saponaric acid or a derivative thereof.

V. an oligonucleotide conjugate according to any of embodiments I-IV, wherein

The saponin comprises a sugar chain bound to the aglycone core structure, said sugar chain being selected from group a:

GlcA-、

Glc-、

Gal-、

Rha-(1→2)-Ara-、

Gal-(1→2)-[Xyl-(1→3)]-GlcA-、

Glc-(1→2)-[Glc-(1→4)]-GlcA-、

Glc-(1→2)-Ara-(1→3)-[Gal-(1→2)]-GlcA-、

Xyl-(1→2)-Ara-(1→3)-[Gal-(1→2)]-GlcA-、

Glc-(1→3)-Gal-(1→2)-[Xyl-(1→3)]-Glc-(1→4)-Gal-、

Rha-(1→2)-Gal-(1→3)-[Glc-(1→2)]-GlcA-、

Ara-(1→4)-Rha-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-、

Ara-(1→4)-Fuc-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-、

Ara-(1→4)-Rha-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-、

Ara-(1→4)-Fuc-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-、

Ara-(1→4)-Rha-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-、

Ara-(1→4)-Fuc-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-、

Ara-(1→4)-Rha-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-、

Ara-(1→4)-Fuc-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-、

Xyl-(1→4)-Rha-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-、

Xyl-(1→4)-Fuc-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-、

Xyl-(1→4)-Rha-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-、

Xyl-(1→4)-Fuc-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-、

Xyl-(1→4)-Rha-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-、

Xyl-(1→4)-Fuc-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-、

Xyl-(1→4)-Rha-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-、

xyl- (1.fwdarw.4) -Fuc- (1.fwdarw.2) -Gal- (1.fwdarw.2) -Fuc- (1.fwdarw.2) -GlcA-, and

the derivatives of these and the derivatives thereof,

or (b)

The saponin comprises a sugar chain bound to the aglycone core structure, said sugar chain being selected from group B:

Glc-，

Gal-，

Rha-(1→2)-[Xyl-(1→4)]-Rha-，

Rha-(1→2)-[Ara-(1→3)-Xyl-(1→4)]-Rha-，

Ara-，

Xyl-，

xyl- (1.fwdarw.4) -Rha- (1.fwdarw.2) - [ R1- (. Fwdarw.4) ] -Fuc-, wherein R1 is 4E-methoxy cinnamic acid,

xyl- (1.fwdarw.4) -Rha- (1.fwdarw.2) - [ R2- (. Fwdarw.4) ] -Fuc-, wherein R2 is 4Z-methoxycinnamic acid,

Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-4-OAc-Fuc-，

xyl- (1.fwdarw.4) - [ Glc- (1.fwdarw.3) ] -Rha- (1.fwdarw.2) -3, 4-di-OAc-Fuc-,

xyl- (1.fwdarw.4) - [ Glc- (1.fwdarw.3) ] -Rha- (1.fwdarw.2) - [ R3- (. Fwdarw.4) ] -3-OAc-Fuc-, wherein R3 is 4E-methoxycinnamic acid,

Glc-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-4-OAc-Fuc-，

Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-4-OAc-Fuc-，

(Ara-or Xyl-) (1.fwdarw.3) - (Ara-or Xyl-) (1.fwdarw.4) - (Rha-or Fuc-) (1.fwdarw.2) - [4-OAc- (Rha-or Fuc-) (1.fwdarw.4) ] - (Rha-or Fuc-),

Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Qui-(1→4)]-Fuc-，

Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-Fuc-，

Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-Fuc-，

Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-Fuc-，

Ara/Xyl-(1→4)-Rha/Fuc-(1→4)-[Glc/Gal-(1→2)]-Fuc-，

api- (1.fwdarw.3) -Xyl- (1.fwdarw.4) - [ Glc- (1.fwdarw.3) ] -Rha- (1.fwdarw.2) - [ R4- (. Fwdarw.4) ] -Fuc-, wherein R4 is 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid,

api- (1.fwdarw.3) -Xyl- (1.fwdarw.4) -Rha- (1.fwdarw.2) - [ R5- (. Fwdarw.4) ] -Fuc-, wherein R5 is 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid,

Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Rha-(1→3)]-4-OAc-Fuc-，

Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4-OAc-Fuc-，

6-OAc-Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3-OAc-Rha-(1→3)]-Fuc-，

Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3-OAc--Rha-(1→3)]-Fuc-，

Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Qui-(1→4)]-Fuc-，

Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-[Qui-(1→4)]-Fuc-，

Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc-，

xyl- (1.fwdarw.3) -Xyl- (1.fwdarw.4) -Rha- (1.fwdarw.2) - [3, 4-di-OAc-Qui- (1.fwdarw.4) ] -Fuc-,

Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-Fuc-，

6-OAc-Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-Fuc-，

Glc-(1→3)-[Xyl-(1→3)-Xyl-(1→4)]-Rha-(1→2)-Fuc-，

Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc-，

Api/Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4OAc-Fuc-，

Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4OAc-Fuc-，

Api/Xyl- (1.fwdarw.3) -Xyl- (1.fwdarw.4) - [ Glc- (1.fwdarw.3) ] -Rha- (1.fwdarw.2) - [ R6- (. Fwdarw.4) ] -Fuc-, wherein R6 is 5-O- [5-O-Rha- (1.fwdarw.2) -Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid,

Api/Xyl- (1.fwdarw.3) -Xyl- (1.fwdarw.4) - [ Glc- (1.fwdarw.3) ] -Rha- (1.fwdarw.2) - [ R7- (. Fwdarw.4) ] -Fuc-, wherein R7 is 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid,

Api/Xyl- (1.fwdarw.3) -Xyl- (1.fwdarw.4) - [ Glc- (1.fwdarw.3) ] -Rha- (1.fwdarw.2) - [ R8- (. Fwdarw.4) ] -Fuc-, wherein R8 is 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid,

api- (1.fwdarw.3) -Xyl- (1.fwdarw.4) -Rha- (1.fwdarw.2) - [ R9- (. Fwdarw.4) ] -Fuc-, wherein R9 is 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid,

Xyl- (1- & gt 3) -Xyl- (1- & gt 4) -Rha- (1- & gt 2) - [ R10- (& gt 4) ] -Fuc-, wherein R10 is 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid,

api- (1.fwdarw.3) -Xyl- (1.fwdarw.4) -Rha- (1.fwdarw.2) - [ R11- (. Fwdarw.3) ] -Fuc-, wherein R11 is 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid,

xyl- (1- & gt 3) -Xyl- (1- & gt 4) -Rha- (1- & gt 2) - [ R12- (& gt 3) ] -Fuc-, wherein R12 is 5-O- [5-O-Ara/Api-3, 5-dihydroxy-6-methyl-octanoyl ] -3, 5-dihydroxy-6-methyl-octanoic acid), glc- (1- & gt 3) - [ Glc- (1- & gt 6) ] -Gal-, and

the derivatives of these and the derivatives thereof,

or (b)

Saponins are disaccharide chain triterpene glycosides comprising a first sugar chain selected from group a bound to the aglycone core structure and comprising a second sugar chain selected from group B bound to the aglycone core structure.

An oligonucleotide conjugate according to any of embodiments I-V, wherein the saponin is selected from the group consisting of: quillaja saponaria saponins, dipsacus asperoides saponin B, saikosaponin A, saikosaponin D, macranthosaponin A, phytolaccatin, escin salt, AS6.2, NP-005236, AMA-1, AMR, alpha-hederagenin, NP-012672, NP-017777, NP-017778, NP-017774, NP-018110, NP-017772, NP-018109, NP-017888, NP-017889, NP-018108, SA1641, AE X55, NP-017674, NP-017810, AG1, NP-003881, NP-017676, NP-017677, NP-017706, NP-017705, NP-017773 NP-017775, SA1657, AG2, SO1861, GE1741, SO1542, SO1584, SO1658, SO1674, SO1832, SO1862, SO1904, QS-7, QS1861, QS-7api, QS1862, QS-17, QS-18, QS-21A-apio, QS-21A-xylo, QS-21B-apio, QS-21B-xylo, β -escin, escin Ia, tea seed saponin I, tea seed saponin J, assam saponin F, digitonin, primordic acid 1 and AS64R, stereoisomers thereof, derivatives thereof, and combinations thereof; preferably the saponins are selected from: QS-21, QS-21 derivatives, SO1861 derivatives, SA1641 derivatives, GE1741 derivatives, and combinations thereof; more preferably the saponins are selected from: QS-21 derivatives, SO1861 and combinations thereof; most preferably the saponin is a SO1861 derivative or SO1861.

The oligonucleotide conjugate according to embodiment V or VI, wherein the saponin is a saponin derivative, wherein

i. The saponin derivative comprises an aglycone core structure comprising a derivatized aldehyde group;

the saponin derivative comprises a sugar chain, preferably selected from group a as defined in embodiment V, said sugar chain comprising a derivatized carboxyl group;

the saponin derivative comprises a sugar chain, preferably selected from group B as defined in embodiment V, comprising a derivatized acetoxy (Me (CO) O-) group; or (b)

The saponin derivative comprises any combination of derivatizations i, ii, and iii, preferably any combination of derivatizations of two of i, ii, and iii.

The oligonucleotide conjugate according to any one of embodiments I-VII, wherein the saponin is any one or more of: SO1861, SA1657, GE1741, SA1641, QS-21A, QS-21A-api, QS-21A-xyl, QS-21B, QS-21B-api, QS-21B-xyl, QS-7-api, QS-17-xyl, QS1861, QS1862, quillaja saponin, saponin album, QS-18, quil-A, gyp1, marshall saponin A, AG, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, SO1904, stereoisomers thereof, derivatives thereof, and combinations thereof; preferably the saponins are selected from: QS-21, QS-21 derivatives, SO1861 derivatives, SA1641 derivatives, GE1741 derivatives, and combinations thereof; more preferably the saponins are selected from: QS-21 derivatives, SO1861 and combinations thereof; most preferably the saponin is a SO1861 derivative or SO1861.

IX. the oligonucleotide conjugate according to any one of embodiments I-VIII, wherein the saponin is a saponin derivative of a saponaric acid saponin or a silk diabolo sapogenin saponin as described in embodiment IV and represented by molecule 1:

wherein the method comprises the steps of

A ₁ Represents hydrogen, monosaccharides or linear or branched oligosaccharides, preferably A ₁ Represents a sugar chain selected from group A as defined in embodiment V, more preferably A ₁ Represents a sugar chain selected from group A as defined in embodiment V and A ₁ Comprising or consisting of glucuronic acid moieties;

A ₂ represents hydrogen, monosaccharides or linear or branched oligosaccharides, preferably A ₂ Represents a sugar chain selected from group B as defined in embodiment V, more preferably A ₂ Represents a sugar chain selected from group B as defined in embodiment V and A ₂ Comprising at least one acetoxy (Me (CO) O-) group, such as one, two, three or four acetoxy groups,

wherein A is ₁ And A ₂ At least one of which is not hydrogen, preferably A ₁ And A ₂ Are oligosaccharide chains;

and R is hydrogen in sericin or hydroxyl in saponaric acid;

wherein the saponin derivative corresponds to the saponin represented by molecule 1, wherein at least one, preferably one or two, more preferably one of the following derivatizations is present:

i. C of saponaric acid or serrulate sapogenin ₂₃ The aldehyde group at the position has been derivatized;

when A ₁ Represents a sugar chain selected from group A as defined in embodiment V and A ₁ When comprising or consisting of glucuronic acid moieties, A ₁ Has been derivatized with carboxyl groups of glucuronic acid moieties; and

When A ₂ Represents a sugar chain selected from group B as defined in embodiment V and A ₂ When at least one acetoxy group is included, A ₂ One or more, preferably all, of the acetoxy groups of one sugar moiety or two or more sugar moieties have been derivatized.

X. oligonucleotide conjugate according to embodiment IX, wherein A ₁ Represents a sugar chain selected from group A as defined in embodiment V and comprises or consists of glucuronic acid moieties, and wherein A ₁ Has been derivatized with carboxyl groups of glucuronic acid moieties, and/or wherein A ₂ Represents a sugar chain selected from group B as defined in embodiment V and A ₂ Comprises at least one acetoxy group, and wherein A ₂ Has been derivatized with at least one acetoxy group.

XI the oligonucleotide conjugate as described in embodiment IX or X, wherein the saponin represented by molecule 1 is a disaccharide chain triterpenoid saponin.

The oligonucleotide conjugate according to any one of embodiments IX-XI, wherein the saponin derivative corresponds to the saponin represented by molecule 1, wherein at least one, preferably one or two, more preferably one of the following derivatizations is present:

i. c of saponaric acid or serrulate sapogenin ₂₃ The aldehyde group at the position has been derivatized by:

-reduction to an alcohol;

conversion to hydrazone linkage by reaction with N-epsilon-maleimidocaaproic acid hydrazide (EMCH) to provide saponin-aldemch (e.g., SO 1861-aldemch or QS-21-aldemch), wherein the maleimide group of EMCH is optionally derivatized by formation of a thioether bond with mercaptoethanol;

-conversion to hydrazone linkage by reaction with N- [ β -maleimidopropionic acid ] hydrazide (BMPH), wherein the maleimide group of BMPH is optionally derivatized by formation of thioether bond with mercaptoethanol; or (b)

-conversion to hydrazone bond by reaction with N- [ kappa-maleimido undecanoic acid ] hydrazide (KMUH), wherein the maleimide group of KMUH is optionally derivatized by forming a thioether bond with mercaptoethanol;

when A ₁ Represents a sugar chain selected from group A as defined in embodiment V and A ₁ When comprising or consisting of glucuronic acid moieties, A ₁ The carboxyl group of the glucuronic acid moiety of (a) has been derivatized by reaction with 2-amino-2-methyl-1, 3-propanediol (AMPD) or N- (2-aminoethyl) maleimide (AEM) to an amide bond, to provide a saponin-Glu-AMPD (e.g., QS-21-Glu-AMPD or SO 1861-Glu-AMPD), or a saponin-Glu-AEM (e.g., QS-21-Glu-AEM or SO 1861-Glu-AEM); and

When A ₂ Represents a sugar chain selected from group B as defined in embodiment V and A ₂ When at least one acetoxy group is included, A ₂ One or more, preferably all, of the acetoxy groups of one sugar moiety or two or more sugar moieties have been derivatized by deacetylation to hydroxyl groups (HO-).

XIII an oligonucleotide conjugate according to any of embodiments IX-XII, wherein A ₁ Is Gal- (1- > 2) - [ Xyl- (1- > 3)]-GlcA and/or A ₂ Is Glc- (1.fwdarw.3) -Xyl- (1.fwdarw.4) -Rha- (1.fwdarw.2) - [ Xyl- (1.fwdarw.3) -4-OAc-Qui- (1.fwdarw.4)]-Fuc; preferably the saponin represented by molecule 1 is 3-O-beta-D-galactopyranosyl- (1.fwdarw.2) - [ beta-D-xylopyranosyl- (1.fwdarw.3)]-beta-D-glucuronopyranosyl soap pinoic acid 28-O-beta-D-glucopyranosyl- (1.fwdarw.3) -beta-D-xylopyranosyl- (1.fwdarw.4) -alpha-L-rhamnopyranosyl- (1.fwdarw.2) - [ beta-D-xylopyranosyl- (1.fwdarw.3) -4 OAc-beta-D-quiniopyranosyl- (1.fwdarw.4) ]-beta-D-fucopyranoside; more preferably the saponin is any one or more of the following: SO1861, GE1741, SA1641 and QS-21, or derivatives thereof, most preferably SO1861 or derivatives thereof.

XIV. the oligonucleotide conjugate according to any of embodiments VII-XIII, wherein the saponin is a saponin derivative, wherein

i. The saponin derivative comprises a aglycone core structure comprising aldehyde groups which have been derivatized by:

-reduction to an alcohol;

conversion to hydrazone linkage by reaction with N-epsilon-maleimidocaaproic acid hydrazide (EMCH) to provide saponin-aldemch (e.g., SO 1861-aldemch or QS-21-aldemch), wherein the maleimide group of EMCH is optionally derivatized by formation of a thioether bond with mercaptoethanol;

-conversion to hydrazone linkage by reaction with N- [ β -maleimidopropionic acid ] hydrazide (BMPH), wherein the maleimide group of BMPH is optionally derivatized by formation of thioether bond with mercaptoethanol; or (b)

-conversion to hydrazone bond by reaction with N- [ kappa-maleimido undecanoic acid ] hydrazide (KMUH), wherein the maleimide group of KMUH is optionally derivatized by forming a thioether bond with mercaptoethanol;

the saponin derivative comprises a sugar chain, preferably selected from group a as defined in embodiment V, comprising a carboxyl group, preferably a carboxyl group of a glucuronic acid moiety, which has been derivatised by conversion to an amide bond by reaction with 2-amino-2-methyl-1, 3-propanediol (AMPD) or N- (2-aminoethyl) maleimide (AEM), thereby providing a saponin-Glu-AMPD (such as QS-21-Glu-AMPD or SO 1861-Glu-AMPD) or a saponin-Glu-AEM (such as QS-21-Glu-AEM or SO 1861-Glu-AEM);

The saponin derivative comprises a sugar chain, preferably selected from group B as defined in embodiment V, comprising an acetoxy (Me (CO) O-) group that has been derivatized by deacetylation to a hydroxyl group (HO-); or (b)

The saponin derivative comprises any combination of derivatizations i, ii, and iii, preferably any combination of derivatizations of two of i, ii, and iii;

preferably, the sapogenin derivative comprises an aglycone core structure, wherein the aglycone core structure comprises an aldehyde group that has been derivatised by reaction with EMCH to convert to a hydrazone bond, wherein the maleimide group of EMCH is optionally derivatised by formation of a thioether bond with mercaptoethanol.

XV. the oligonucleotide conjugate according to embodiment XIV, wherein the saponin is a saponin derivative, wherein

a) The saponin derivative comprises a aglycone core structure comprising an aldehyde group that has been derivatized by reaction with N-epsilon-maleimidocaaproic acid hydrazide (EMCH) to a hydrazone bond, thereby providing a saponin-aldemch (e.g., SO 1861-aldemch or QS-21-aldemch);

b) The saponin derivative comprises a sugar chain, preferably selected from group a as defined in embodiment V, comprising a carboxyl group, preferably a carboxyl group of a glucuronic acid moiety, which has been derivatized by conversion to an amide bond by reaction with N- (2-aminoethyl) maleimide (AEM), thereby providing a saponin-Glu-AEM (such as QS-21-Glu-AEM or SO 1861-Glu-AEM); or (b)

c) The saponin derivative comprises a combination of derivatizations i.and ii..

The oligonucleotide conjugate according to embodiment XIV, wherein the sapogenin derivative comprises a aglycone core structure, wherein the aglycone core structure comprises an aldehyde group, and wherein the sapogenin derivative comprises a sugar chain, preferably a sugar chain selected from group a as defined in embodiment V, said sugar chain comprising a carboxyl group, preferably a carboxyl group of a glucuronic acid moiety, said glucuronic acid moiety having been derivatised by reaction with N- (2-aminoethyl) maleimide (AEM) to an amide bond.

XVII the oligonucleotide conjugate according to any of embodiments I-XIV, wherein the saponin is a saponin derivative represented by molecule 2:

or wherein the saponin is a saponin derivative represented by molecule 3:

the oligonucleotide conjugate according to any one of embodiments I-XVII, wherein at least one saponin and a ligand of ASGPR are covalently linked directly or via at least one linker, and/or wherein at least one saponin and an oligonucleotide are covalently linked directly or via at least one linker, and/or wherein a ligand of ASGPR and an oligonucleotide are covalently linked directly or via at least one linker, preferably at least one saponin, a ligand of ASGPR and an oligonucleotide are linked by at least one linker.

The oligonucleotide conjugate according to any one of embodiments I-XVIII, wherein at least one GalNAc moiety, at least one saponin and the oligonucleotide are covalently bound by a trifunctional linker, preferably each of the GalNAc moiety, the saponin and the oligonucleotide is covalently bound to a separate arm of the trifunctional linker.

XX. the oligonucleotide conjugate according to any one of embodiments I-XIX, wherein the ligand of ASGPR is (GalNAc) 3Tris represented by molecule (DD 3) or molecule (DD 4):

or wherein the ligand of ASGPR is single GalNAc represented by molecule II':

the oligonucleotide conjugate according to any one of embodiments I-XX, wherein at least one saponin is covalently bound to a ligand of ASGPR via at least one cleavable linker, and/or wherein at least one saponin is covalently bound to the oligonucleotide via at least one cleavable linker.

Xxii the oligonucleotide conjugate according to embodiment XIX, wherein at least one saponin is covalently bound to the arm of the trifunctional linker via at least one cleavable linker.

The oligonucleotide conjugate according to embodiment XXI or XXII, wherein the cleavable linker is subjected to cleavage under acidic, reducing, enzymatic and/or light-induced conditions, and preferably the cleavable linker comprises a cleavable bond selected from the group consisting of: hydrazone bonds and hydrazide bonds that undergo cleavage under acidic conditions, and/or bonds that are susceptible to proteolysis, such as bonds that are proteolytically cleaved by cathepsin B, and/or bonds that are susceptible to cleavage under reducing conditions (e.g., disulfide bonds).

The oligonucleotide conjugate according to any of embodiments XXI-XXIII, wherein the cleavable linker is subjected to in vivo cleavage under acidic conditions, e.g. such as those present in the endosome and/or lysosome of a mammalian cell, preferably a human cell, preferably the cleavable linker is subjected to in vivo cleavage at a pH of 4.0-6.5, and more preferably at a pH of +.5.5.

The oligonucleotide conjugate of any of embodiments I-XXIV, wherein the conjugate comprises 1, 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, 128, or 1-100 saponin moieties, or any number of saponin moieties therebetween (e.g., 7, 9, 12 saponin moieties).

Xxvi the oligonucleotide conjugate of any one of embodiments I-XXIV, wherein the oligonucleotide conjugate comprises 1 saponin moiety.

The oligonucleotide conjugate according to any one of embodiments I-XXVI, wherein the oligonucleotide is any one of BNA, a heterologous nucleic acid, siRNA, an antisense oligonucleotide.

Xxviii the oligonucleotide conjugate according to any one of embodiments I-XXVI, wherein the oligonucleotide is selected from any one or more of: short interfering RNA (siRNA), short hairpin RNA (shRNA), anti-hairpin microRNA (miRNA), single-stranded RNA, aptamer RNA, double-stranded RNA (dsRNA), anti-microRNA (anti-miRNA, anti-miR), antisense oligonucleotide (ASO), DNA, antisense DNA, locked Nucleic Acid (LNA), bridged Nucleic Acid (BNA), 2'-O,4' -aminoethylene Bridged Nucleic Acid (BNA) ^NC ) BNA-based siRNA, and BNA-based antisense oligonucleotide (BNA-aON).

The oligonucleotide conjugate according to any one of embodiments I-XXVI, wherein the oligonucleotide is selected from any one or more of the following: anti-miRNA, BNA-AON or siRNA, such as BNa-based siRNA, selected from chemically modified siRNA, metabolically stable siRNA, and chemically modified metabolically stable siRNA.

The oligonucleotide conjugate according to any of embodiments I-XXIX, wherein the oligonucleotide is an oligonucleotide capable of silencing any of the following genes, e.g. when present in a mammalian cell and preferably when present in a human cell: apolipoprotein B (apoB), HSP27, thyroxine Transporter (TTR), proprotein convertase subtilisin/kexin type 9 (PCSK 9), deltA-Aminolevulinate synthase 1 (ALAS 1), antithrombin 3 (AT 3), glycolate Oxidase (GO), complement component C5 (CC 5), the X gene of Hepatitis B Virus (HBV), the S gene of HBV, alpha-1 antitrypsin (AAT) and Lactate Dehydrogenase (LDH), and/or oligonucleotides capable of targeting aberrant mirnas, for example, when present in mammalian cells.

The oligonucleotide conjugate according to any of embodiments I-XXIX, wherein the oligonucleotide is an oligonucleotide capable of silencing any of the following genes, e.g. when present in a mammalian cell and preferably when present in a human cell: apolipoprotein B (apoB) and HSP27.

Xxxii. the oligonucleotide conjugate according to any of embodiments I-XXXI, wherein the oligonucleotide is an oligonucleotide capable of targeting mRNA involved in expression of any of the following proteins, e.g. when present in a mammalian cell and preferably when present in a human cell: apoB, HSP27, TTR, PCSK9, ALAS1, AT3, GO, CC5, expression products of the X gene of HBV, expression products of the S gene of HBV, AAT and LDH, or are capable of antagonizing or restoring miRNA function, such as inhibiting oncogenic miRNA (onco-miR) or repressing expression of onco-miR, for example, when present in mammalian cells and preferably when present in human cells.

Xxxiii an oligonucleotide conjugate according to any of claims I-XXXI, wherein the oligonucleotide is an oligonucleotide capable of targeting mRNA involved in expression of any of the following proteins, e.g. when present in a mammalian cell and preferably when present in a human cell: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AAT, miR-122, hepatitis B virus HbsAg, LDHA and CEBPA.

The oligonucleotide conjugate according to any of embodiments I-xxxiv, wherein the oligonucleotide is an oligonucleotide capable of targeting mRNA involved in expression of any of the following proteins, e.g. when present in a mammalian cell and preferably when present in a human cell: apoB and HSP27.

Xxxv. a pharmaceutical composition comprising an oligonucleotide conjugate as described in any of embodiments I-XXXIV, optionally together with a pharmaceutically acceptable excipient and/or optionally with a pharmaceutically acceptable diluent.

Xxxvi pharmaceutical composition as described in embodiment XXXV for use as a medicament.

Xxxvii a pharmaceutical composition as claimed in XXXV for use in the treatment or prophylaxis of a disease or health condition in which the expression product involves any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AT3, GO, CC5, X gene of HBV, S gene of HBV, AAT, miR-122, hepatitis B virus HbsAg, LDHA, CEBPA and LDH; and/or for use in the treatment or prevention of diseases or health problems involving any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AT3, GO, CC5, X gene of HBV, S gene of HBV, AAT, miR-122, hepatitis B virus HbsAg, LDHA, CEBPA and LDH.

Xxxviii the pharmaceutical composition of claim XXXV for use in the treatment or prevention of a disease or health problem in which the expression product involves any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AAT, miR-122, hepatitis B virus HbsAg, LDHA and CEBPA; and/or for use in the treatment or prevention of diseases or health problems involving any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AAT, miR-122, hepatitis B virus HbsAg, LDHA and CEBPA.

Xxxix. a pharmaceutical composition for use as described in claim xxxix or XXXVII for use in the treatment or prevention of a disease or health problem in which the expression product involves any one or more of the following genes: HSP27 and apoB, preferably apoB; and/or for use in the treatment or prevention of diseases or health problems involving any one or more of the following genes: HSP27 and apoB, preferably apoB.

XL. a pharmaceutical composition for use as described in any one of claims XXXVI-XXXIX for use in the treatment or prophylaxis of: cancer, infectious disease, viral infection, hypercholesterolemia, cardiovascular disease, primary hyperoxaluria, hemophilia a, hemophilia B, AAT-associated liver disease, acute hepatic porphyrin, TTR-mediated amyloidosis, hereditary TTR amyloidosis (hATTR), complement-mediated disease, hepatitis b infection, hepatitis c infection, alpha 1-antitrypsin deficiency, beta thalassemia, or autoimmune disease.

Xli. a pharmaceutical composition for use according to any one of claims XXXVI-XXXIX for use in the treatment or prevention of: cancers (e.g., endometrial, breast, lung, or hepatocellular carcinoma) and cardiovascular diseases (hypercholesterolemia, preferably hypercholesterolemia).

Xlii. an in vitro or ex vivo method for transferring an oligonucleotide conjugate according to any one of embodiments I-XXXIV from outside of a cell into said cell, preferably followed by transferring an oligonucleotide comprised by an oligonucleotide conjugate according to any one of embodiments I-XXXIV into the cytoplasm and/or nucleus of said cell, comprising the steps of:

a) Providing a cell expressing ASGPR, preferably ASGPR1, on its surface, preferably selected from the group consisting of hepatocytes, virus-infected mammalian cells and mammalian tumor cells, wherein preferably the cell is a human cell;

b) Providing an oligonucleotide conjugate as described in any one of embodiments I-XXXIV for transfer into a cell provided in step a);

c) Contacting the cells of step a) with the oligonucleotide conjugate of step b), preferably in a liquid medium, in vitro or ex vivo, thereby effecting transfer of the oligonucleotide conjugate from outside the cell into said cell, and optionally and preferably thereby effecting subsequent transfer of the oligonucleotide contained by the oligonucleotide conjugate into the cytoplasm and/or nucleus of said cell.

An aspect of the invention relates to a method of providing an oligonucleotide conjugate of the invention comprising the steps of:

(a) Providing at least one saponin moiety comprising a covalently bound first linker, wherein the first linker comprises at least one first reactive group for covalent binding to a second reactive group on a second linker or a seventh reactive group on a seventh linker;

(b) Providing an oligonucleotide comprising a covalently bound third linker, wherein the third linker comprises a third reactive group for covalent binding with a fourth reactive group on a fourth linker or an eighth reactive group on a seventh linker;

(c) Providing at least one GalNAc moiety comprising a covalently bound fifth linker, wherein the fifth linker comprises a fifth reactive group for covalent binding to a sixth reactive group on the sixth linker or a ninth reactive group on the seventh linker; and

(d1) Ligating a first linker to a second linker by forming a covalent bond between the first reactive group and the second reactive group, ligating a third linker to a fourth linker by forming a covalent bond between the third reactive group and the fourth reactive group, ligating a fifth linker to a sixth linker by forming a covalent bond between the fifth reactive group and the sixth reactive group, and covalently ligating the second linker, the fourth linker and the sixth linker together to provide an oligonucleotide,

Or (b)

(d2) The first linker is linked to the seventh linker by forming a covalent bond between the first reactive group and the seventh reactive group, the third linker is linked to the seventh linker by forming a covalent bond between the third reactive group and the eighth reactive group, and the fifth linker is linked to the seventh linker by forming a covalent bond between the fifth reactive group and the ninth reactive group, thereby providing the oligonucleotide conjugate.

An embodiment is a method of providing an oligonucleotide conjugate of the invention, wherein the seventh linker is a trifunctional linker, e.g., a trifunctional linker of formula (XXI):

preferred are methods of providing oligonucleotide conjugates of the invention, wherein at least one of the saponin moieties is 1-16 saponin moieties, preferably 1-8 saponin moieties (e.g., 1, 4 or 8 saponin moieties).

Preferred are methods of providing the oligonucleotide conjugates of the invention wherein the saponin is SO1861, SO1832, QS-21 or any functional derivative thereof, preferably SO1861 or SO1832.

Preferred are methods of providing oligonucleotide conjugates of the invention, wherein one or more saponin moieties are covalently linked by hydrazone or semicarbazone linkages.

Preferred are methods for providing the oligonucleotide conjugates of the invention, wherein at least one GalNAc moiety is 1-4 GalNAc moieties, preferably 1 or 3 GalNAc moieties.

One embodiment is an oligonucleotide conjugate of the invention, wherein the saponin comprised by the oligonucleotide conjugate is isolated from a plant. Preferably, the saponins are isolated from a part of a plant (such as a root), or from a part of a tree (such as bark). Preferably, the saponins are isolated from roots from plants.

The following is an overview of preferred embodiments of oligonucleotide conjugates according to the invention:

oligonucleotide conjugate comprising at least one saponin covalently linked to a ligand of an asialoglycoprotein receptor (ASGPR), wherein the ligand of ASGPR comprises at least one N-acetylgalactosamine (GalNAc) moiety, preferably three or four GalNAc moieties, more preferably three GalNAc moieties, more preferably the ligand of ASGPR comprises (GalNAc) ₃ Tris or consisting thereof, and also covalently linked to an oligonucleotide, wherein at least one saponin is 12, 13-dehydrooleaAn alkane-type monosaccharide or disaccharide-chain pentacyclic triterpene saponin preferably having an aldehyde functional group at the C-23 position of the aglycone core structure of the saponin,

wherein the oligonucleotide conjugate comprises 1-16 saponin moieties, preferably 1-8 saponin moieties, more preferably 1 saponin moiety, 4 saponin moieties or 8 saponin moieties.

The oligonucleotide conjugate according to embodiment 1A, wherein at least one GalNAc moiety, preferably three GalNAc moieties, at least one saponin, preferably 1-16 saponin moieties, more preferably 1-8 saponin moieties (e.g. 1, 4 or 8 saponin moieties) are covalently bound to the oligonucleotide via a trifunctional linker, preferably one GalNAc moiety or moieties, one saponin or each of the plurality of saponin moieties and the oligonucleotide are covalently bound to a separate arm of the trifunctional linker.

Oligonucleotide conjugates as described in embodiment 1A or 2A comprising one saponin moiety, or 4 saponin moieties, preferably 4 saponin moieties, covalently bound to a dendron, preferably a G2 dendron, such as N, N' - ((9S, 19S) -14- (6-aminocaproylamino) -1-mercapto-9- (3-mercaptopropionylamino) -3,10,18-trioxo-4,11,14,17-tetraazaditridecane-19, 23-diyl) bis (3-mercaptopropionamide) or 8 saponin moieties, preferably 8 saponin moieties, covalently bound to a dendron, preferably a G3 dendron, such as (2S) -N- [ (1S) -1- { [2- (6-amino-N- {2- [ (2S) -2, 6-bis (3-sulfanylpropionylamino) hexanylamino ] ethyl } hexanylamino) carbamoyl {5- [ (2S) -2, 6-bis (3-sulfanylpropionylamino ] ethyl } hexanylamino } -2-sulfanylpropionamide.

4A. The oligonucleotide conjugate according to any one of embodiments 1A-3A, wherein the oligonucleotide conjugate comprises a saponin moiety, and wherein the oligonucleotide conjugate is according to molecule (EE):

wherein molecule (EE) is via a trifunctional linker according to formula (XXI):

covalent conjugation products obtained with covalent conjugation of the following (1), (2) and (3):

(1) A saponin derivative according to molecule (AA):

wherein, wherein.

Represents a saponin moiety according to formula (SM):

/>

wherein R is ¹ And R is ² Independently selected from the group consisting of hydrogen, monosaccharides, linear oligosaccharides and branched oligosaccharides, and wherein the saponin moiety according to formula (SM) is based on a saponin comprising an aldehyde group at the C-23 position,

and

(2) GalNAc conjugate according to molecule (FF):

wherein, the method comprises the steps of, wherein,

represents a tri-GalNAc conjugate according to the molecule (DD 1) or the molecule (DD 2) as detailed above, preferably the molecule (DD 3) or the molecule (DD 4) as detailed above, more preferably the molecule (DD 3) as detailed above,

and

(3) An oligonucleotide having a linker according to molecule (GG):

wherein molecule (GG) represents the conjugation product of a conjugation reaction between linker (E) -1- (4- ((2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoyl) hydrazono) methyl) benzoylamino) -N- (4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) benzyl) -3,6,9, 12-tetraoxapentadecane-15-amide and an oligonucleotide-linker molecule according to molecule (HH):

The oligonucleotide conjugate according to any one of embodiments 1A-3A, comprising four saponin moieties, and wherein the oligonucleotide conjugate is according to molecule (PP):

wherein the molecule (PP) is a saponin-GalNAc conjugate according to the molecule (LL):

a covalent conjugation product obtained by covalent conjugation with:

the oligonucleotide having a linker according to molecule (GG):

wherein molecule (GG) represents the conjugation product of a conjugation reaction between linker (E) -1- (4- ((2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoyl) hydrazono) methyl) benzoylamino) -N- (4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) benzyl) -3,6,9, 12-tetraoxapentadecane-15-amide and an oligonucleotide-linker molecule according to molecule (HH):

wherein molecule (LL) is prepared by reacting a saponin derivative according to molecule (JJ):

a covalent conjugation product obtained by covalent conjugation with:

conjugate of trifunctional linker according to molecule (MM) and GalNAc:

wherein the molecule (JJ) is the conjugation product of N, N' - ((9 s,19 s) -14- (6-aminocaproyl) -1-mercapto-9- (3-mercaptopropionamido) -3,10,18-trioxo-4,11,14,17-tetraazaditridecane-19, 23-diyl) bis (3-mercaptopropionamide) conjugated to: first with a saponin derivative according to molecule (KK):

Wherein

Represents a saponin moiety according to formula (SM):

wherein R is ¹ And R is ² Independently selected from the group consisting of hydrogen, monosaccharides, linear oligosaccharides and branched oligosaccharides according to the invention, and wherein the saponin moiety according to formula (SM) is based on a saponin comprising an aldehyde group at the C-23 position,

and then with 2, 5-dioxopyrrolidin-1-yl-1-azido-3, 6,9, 12-tetraoxapentadecane-15-oic acid ester,

and wherein the molecule (MM) is a conjugate of: a trifunctional linker according to formula (XXI):

and GalNAc conjugates according to molecule (NN):

wherein

Represents a tri-GalNAc conjugate according to the molecule (DD 1) or the molecule (DD 2) as detailed above, preferably the molecule (DD 3) or the molecule (DD 4) as detailed above, more preferably the molecule (DD 3) as detailed above.

The oligonucleotide conjugate according to any one of embodiments 1A-3A, comprising eight saponin moieties, and wherein the oligonucleotide conjugate is according to molecule (SS):

wherein the molecule (SS) is prepared by reacting a saponin-GalNAc conjugate with the molecule (RR):

a covalent conjugation product obtained by covalent conjugation with: an oligonucleotide having a linker according to molecule (GG):

wherein molecule (GG) represents the conjugation product of the conjugation reaction of linker (E) -1- (4- ((2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoyl) hydrazono) methyl) benzoylamino) -N- (4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) benzyl) -3,6,9, 12-tetraoxapentadecane-15-amide and an oligonucleotide-linker molecule according to molecule (HH):

Wherein the molecule (RR) is obtained by reacting a saponin derivative according to the molecule (QQ):

a covalent conjugation product obtained by covalent conjugation with:

conjugate of trifunctional linker according to molecule (MM) and GalNAc:

wherein the molecule (QQ) is the conjugation product of (2S) -N- [ (1S) -1- { [2- (6-amino-N- {2- [ (2S) -2, 6-bis (3-sulfanylpropionamido) hexanamido ] ethyl } hexanamido) ethyl ] carbamoyl } -5- [ (2S) -2, 6-bis (3-sulfanylpropionamido) hexanamido ] pentyl ] -2, 6-bis (3-sulfanylpropionamido) hexanamide formate conjugated to: first

(a) And a saponin derivative according to molecule (KK):

wherein

Represents a saponin moiety according to formula (SM):

wherein R is ¹ And R is ² Independently selected from the group consisting of hydrogen, monosaccharides, linear oligosaccharides and branched oligosaccharides according to the invention, and wherein the saponin moiety according to formula (SM) is based on a saponin comprising an aldehyde group at the C-23 position,

and then

(b) With 2, 5-dioxopyrrolidin-1-yl-1-azido-3, 6,9, 12-tetraoxapentadecane-15-oic acid ester,

and wherein the molecule (MM) is a conjugate of: a trifunctional linker according to formula (XXI):

and GalNAc conjugates according to the molecule (NN) (see above, the molecule (NN) of embodiment 5A), wherein

Represents a tri-GalNAc conjugate according to the molecule (DD 1) or the molecule (DD 2) as detailed above, preferably the molecule (DD 3) or the molecule (DD 4) as detailed above, more preferably the molecule (DD 3) as detailed above.

The oligonucleotide conjugate of any one of embodiments 1A-6A, wherein the conjugate comprises a saponin that is any one or more of:

a) A saponin selected from any one or more of list a:

-a mixture of sapogenins of the quillaja saponaria, or saponins isolated from the quillaja saponaria, such as Quil-A, QS-17-api, QS-17-xyl, QS-21A, QS-21B, QS-7-xyl;

-a mixture of carnation saponins, or saponins isolated from carnation;

-a mixture of saponinium album saponins, or saponins isolated from saponinium album;

-a mixture of soapstock saponins, or saponins isolated from soapstock; and

-a saponaria saponins mixture, or saponins isolated from saponaria barks, such as Quil-A, QS-17-api, QS-17-xyl, QS-21A, QS-21B, QS-7-xyl; or (b)

b) A saponin comprising a silk diabolo sapogenin core structure selected from list B:

SA1641, carnation saponin A, NP-017772, NP-017774, NP-017777, NP-017778, NP-018109, NP-017888, NP-017889, NP-018108, SO1658 and phytolaccagenin; or (b)

c) A saponin comprising a sapogenin core structure selected from list C:

AG1856, AG1, AG2, agrostemmoside E, GE1741, silk bamboo saponin 1 (Gyp 1), NP-017674, NP-017810, NP-003881, NP-017676, NP-017677, NP-017705, NP-017706, NP-017773, NP-017775, SA1657, saponaria oside B, SO1542, SO1584, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862, SO 4, QS-7api, QS-17, QS-18, QS-21A-apio, QS-21A-xylo, QS-21B-apio and QS-21B-xylo; or (b)

d) A sapogenin core structure comprising a 12, 13-dehydrooleanane type selected from list D, having no aldehyde group at the C-23 position of the aglycone:

escin Ia, escin salt, alpha-hederagenin, AMA-1, AMR, AS6.2, AS64R, assam saponin F, dipsacus asperosaponin B, esculentoside A, lonicera macranthoides saponin A, NP-005236, NP-012672, primula acid 1, saikosaponin A, saikosaponin D, tea seed saponin I and tea seed saponin J;

preferably, the saponin is any one or more of the saponins selected from the list A, B or C, more preferably, the saponins selected from the list B or C,

even more preferably, the saponins are selected from list C.

The oligonucleotide conjugate according to any one of embodiments 1A-7A, wherein the saponin is any one or more of: AG1856, GE1741, saponins isolated from Quil-A, QS-17, QS-21, QS-7, SA1641, saponins isolated from soapbark, saponaria side B, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862 and SO1904; preferably, wherein the saponin is any one or more of the following: QS-21, SO1832, SO1861, SA1641 and GE1741; more preferably wherein the saponin is QS-21, so1832 or SO1861; most preferred is SO1861.

The oligonucleotide conjugate according to any one of embodiments 1A-8A, wherein the saponin is isolated from saponaria; preferably wherein the saponin is any one or more of the following: saponarioside B, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862 and SO1904; more preferably, wherein the saponin is any one or more of the following: SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862 and SO1904; even more preferably, wherein the saponin is any one or more of the following: SO1832, SO1861 and SO1862; even more preferably, wherein the saponins are SO1832 and SO1861; most preferred is SO1861.

The oligonucleotide conjugate of any one of embodiments 1A-9A, wherein the saponin is SO1861, and wherein the oligonucleotide conjugate is provided by conjugation to provide the oligonucleotide conjugate by: SO1861 saponin derivative according to molecule (VII) a:

conjugated to an oligonucleotide and at least one GalNAc moiety.

The oligonucleotide conjugate according to any one of embodiments 1A-10A, wherein the oligonucleotide comprised by the conjugate is defined as a nucleic acid of no more than 150nt, preferably wherein the oligonucleotide has a size of 5-150nt, preferably 8-100nt, most preferably 10-50nt.

A second pharmaceutical composition comprising the oligonucleotide conjugate of any one of embodiments 1A-11A, and optionally a pharmaceutically acceptable excipient and/or optionally a pharmaceutically acceptable diluent.

13a. a second pharmaceutical composition as described in embodiment 12A or an oligonucleotide conjugate as described in any one of embodiments 1A-11A for use as a medicament.

14a. a second pharmaceutical composition as described in embodiment 12A or an oligonucleotide conjugate as described in any one of embodiments 1A-11A for use in the treatment or prevention of a disease or health problem in which the expression product involves any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AT3, GO, CC5, X gene of HBV, S gene of HBV, AAT, miR-122, hepatitis B virus HbsAg, LDHA, CEBPA and LDH; and/or for use in the treatment or prevention of diseases or health problems involving any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AT3, GO, CC5, X gene of HBV, S gene of HBV, AAT, miR-122, hepatitis B virus HbsAg, LDHA, CEBPA and LDH.

15a. a second pharmaceutical composition as described in embodiment 12A or an oligonucleotide conjugate as described in any one of embodiments 1A-11A for use in the treatment or prevention of a disease or health problem in which the expression product involves any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AAT, miR-122, hepatitis B virus HbsAg, LDHA and CEBPA; and/or for use in the treatment or prevention of diseases or health problems involving any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AAT, miR-122, hepatitis B virus HbsAg, LDHA and CEBPA.

A second pharmaceutical composition as described in embodiment 12A or an oligonucleotide conjugate as described in any one of embodiments 1A-11A for use according to embodiment 14A or 15A, wherein the use is for the treatment or prevention of a disease or health problem wherein the expression product is involved in any one or more of the following genes: HSP27 and apoB, preferably apoB; and/or for use in the treatment or prevention of diseases or health problems involving any one or more of the following genes: HSP27 and apoB, preferably apoB.

The second pharmaceutical composition according to embodiment 12A or the oligonucleotide conjugate according to any one of embodiments 1A-11A for use in the treatment or prevention of cancer, infectious disease, viral infection, hypercholesterolemia, cardiovascular disease, primary hyperoxaluria, hemophilia a, hemophilia B, AAT-associated liver disease, acute hepatic porphyria, TTR-mediated amyloidosis, hereditary TTR amyloidosis (hATTR), complement-mediated disease, hepatitis b infection, hepatitis c infection, alpha 1-antitrypsin deficiency, beta-thalassemia or autoimmune disease according to any one of embodiments 14A-16A.

The second pharmaceutical composition according to embodiment 12A or the oligonucleotide conjugate according to any one of embodiments 1A-11A for use according to any one of embodiments 14A-17A, wherein the use is for the treatment or prophylaxis of cancer (such as endometrial cancer, breast cancer, lung cancer or hepatocellular carcinoma) and/or cardiovascular diseases (such as hypercholesterolemia, preferably hypercholesterolemia).

19a. a second pharmaceutical composition as described in embodiment 12A or an oligonucleotide conjugate as described in any one of embodiments 1A-11A for use in reducing LDL-cholesterol in a subject.

An in vitro or ex vivo method for transferring an oligonucleotide conjugate according to any of embodiments 1A-11A from outside a cell to inside said cell, preferably followed by transferring an oligonucleotide comprised by said oligonucleotide conjugate into the cytosol of said cell, comprising the steps of:

a) Providing a cell expressing ASGPR on its surface, preferably selected from the group consisting of a hepatocyte, a virus-infected cell and a tumor cell, and providing the oligonucleotide conjugate of any one of embodiments 1A-11A for transfer into the provided cell;

b) Contacting the cell of step a) in vitro or ex vivo with the oligonucleotide conjugate of step a), thereby effecting transfer of the oligonucleotide conjugate from outside the cell into said cell, and preferably subsequently effecting transfer of the oligonucleotide contained in the oligonucleotide conjugate into the cytosol of said cell.

Preferably, the subject is a human subject.

Preferably, the saponin of the oligonucleotide conjugate is a plant-derived saponin. The saponin comprised by the oligonucleotide conjugate of the invention is preferably a saponin isolated from a plant. For example, saponins are isolated from roots of plants. Examples of such plants from which saponins are extracted (isolated) are quillaja saponaria, phyllostachys pratensis, saponaria officinalis (Saponaria officinalis) (e.g., saponaria officinalis l.) and phyllostachys pratensis (Gypsophila elegans) (e.g., phyllostachys pratensis m. Bieb). Preferably, the saponin conjugates and/or oligonucleotide conjugates comprise a single type of saponin, preferably a single type of saponin derived from plant material, such as plant roots, such as SO1861, SO1862 or SO1832 from soapstock (e.g. soapstock l., preferably soapstock l.) (roots), or such as QS-21, QS-7 or QS-17 from quillaja (roots). Suitable sources of isolated saponins according to the present invention, i.e. those that exhibit enhanced endosomal escape activity, are quillaja saponaria, carnation, soapberry and carnation, and quillaja bark.

One embodiment is an oligonucleotide conjugate of the invention, wherein the saponin comprised by the oligonucleotide conjugate is isolated from a plant. Preferably, the saponins are isolated from a part of a plant (such as a root), or from a part of a tree (such as bark). Preferably, the saponins are isolated from roots from plants.

The invention has been described above with reference to a number of exemplary embodiments. Modifications are possible and are included within the scope of protection as defined in the appended claims. The invention is further illustrated by the following examples, which should not be construed as limiting the invention in any way.

Examples and exemplary embodiments

EXAMPLE 1 CD 71-saporin +4000nM trivalent GalNAc-L-SO1861

Trivalent GalNAc is a targeting ligand that recognizes and binds to ASGPR1 receptor on hepatocytes. Trivalent GalNAc (FIGS. 1A-C and 8A-G) was produced and SO1861-EMCH was conjugated to trivalent GalNAc (labile in a similar manner as described for underivatized SO1861 in FIGS. 2A-B and 4, where SPT001 is synonymous with SO 1861), DAR was 1, (trivalent GalNAc-SO 1861). SO1861-EMCH refers to SO1861 in which the aldehyde group is derivatized by reaction with N- ε -maleimidocaaproic acid hydrazide (EMCH) to a hydrazone bond, thereby providing SO1861-Ald-EMCH. The 'trivalent GalNAc' as described in FIGS. 1A-C and 8A-G is typical (GalNAc) suitable for coupling with effector molecules or with saponins ₃ Tris conjugate. As a control, SO1861-EMCH was also conjugated (labile) to monovalent GalNAc (FIG. 3), DAR was 1, (GalNAc-SO 1861). HepG2 (ASGPR 1) ⁺ ；CD71 ⁺ Table A3) and Huh7 (ASGPR 1) ^+/- ；CD71 ⁺ Table A3) cells were treated with a range of concentrations of trivalent GalNAc-L-SO1861 and GalNAc-L-SO1861 in the presence and absence of 10pM of CD 71-saporin (CD 71-targeting monoclonal antibody clone OKT-9 conjugated with the protein toxin saporin). Cell treatment in the absence of CD 71-saporin showed that trivalent-GalNAc-L-SO 1861 (or trivalent GalNAc) as a single compound was not toxic up to 15000nM (FIG. 9).

HepG2 and Huh7 cells were also treated with a combination of CD 71-saporin in the concentration range with fixed concentrations of trivalent GalNAc-SO1861 (DAR 1) of 1000nM or 4000 nM. Targeted protein toxin-mediated cell killing of ASGPR1/CD71 expressing cells (HepG 2 and Huh 7) was determined. This suggests that both cell lines have strong cell killing at low concentrations of CD 71-saporin (ic50=1 pM), whereas equivalent concentrations of CD 71-saporin, CD 71-saporin+1000 nM trivalent-GalNAc or CD 71-saporin+4000 nM trivalent-GalNAc can induce cell killing at high concentrations of CD 71-saporin (ic50=100 pM) in both cell lines only (fig. 10).

All of these demonstrate that the combination of trivalent GalNAc-SO1861 with low concentration of CD 71-saporin is effective in inducing cell killing in ASGPR1/CD71 expressing cells. Thus, trivalent GalNAc-SO1861 effectively induces endosomal escape of protein toxins in ASGPR 1-expressing cells.

EXAMPLE 1A trivalent GalNAc-L-SO1861, trivalent GalNAc- (L-SO 1861) 4, SO1861-EMCH at a concentration of the CD71-saporin+ series

Trivalent GalNAc (FIGS. 1A-C and 8A-G) was produced and SO1861-EMCH was conjugated to trivalent-GalNAc (labile in a similar manner as described for underivatized SO1861 in FIGS. 2A-B and 4, where SPT and SPT001 are synonymous with saponin SO 1861) (DAR 1) (trivalent-GalNAc-SO 1861 (GN) 3-SPT)), or DAR 4 was prepared by first coupling SO1861-EMCH to dendrites (trivalent-GalNAc-dendrites (SO 1861) ₄ The method comprises the steps of carrying out a first treatment on the surface of the (GN) 3-dSPT4; fig. 23D-E). Again, SO1861-EMCH refers to SO1861 in which the aldehyde group is derivatized by conversion to a hydrazone bond by reaction with N- ε -maleimidocaaproic acid hydrazide (EMCH), thereby providing SO1861-Ald-EMCH. The 'trivalent GalNAc' as described in FIGS. 1A-C and 8A-G is typical (GalNAc) suitable for coupling with effector molecules or with saponins ₃ Tris conjugate. As a control, SO1861-EMCH was also conjugated (labile) to monovalent GalNAc (FIG. 3), either as DAR 1 or as DAR 4 (using dendrites) (GalNAc-SO 1861 and GalNAc- (SO 1861) ₄ ). In the presence of 10pM of CD 71-saporin (CD 71-targeting monoclonal antibody OKT-9 conjugated with protein toxin saporin), a concentration range of trivalent GalNAc-L-SO1861, SO1861-EMCH or (GN) ₃ Treatment of primary hepatocytes highly expressing ASGPR1 with dpt 4 (ASGPR 1) ^{High height} ). Cell treatment in the absence of CD 71-saporin indicated that trivalent GalNAc-L-SO1861 as a single compound was not toxic up to at least 1.000nM (FIG. 18A; relative cell viability). Cell viability of cells treated with the combination of anti-CD 71 MoAb-saporin ('MoAb' =monoclonal antibody) and trivalent GalNAc conjugated to SO1861 (DAR 1 or DAR 4) was about 100-fold lower compared to that of cells treated with the combination of anti-CD 71 MoAb-saporin (CD 71-SPRN) and SO1861-EMCH (SPT-EMCH) (fig. 18A).

All of these demonstrate that the combination of trivalent GalNAc-SO1861 and trivalent GalNAc- (SO 1861) 4 with low concentrations of CD 71-saporin effectively induced cell killing in ASGPR1/CD71 expressing cells, whereas CD 71-saporin was ineffective in the absence of SO 1861. Thus, trivalent GalNAc-SO1861 effectively induces endosomal escape of protein toxins in ASGPR 1-expressing cells. Thus, both SPT001 and GalNAc-targeted SPT001 (DAR 1) increase the efficacy of industry standard GalNAc-targeted ASOs; when considering the cell viability of hepatocytes, SO1861-EMCH is about 10-fold, and both (GN) 3-SPT and (GN) 3-dSPT4 are about 100-fold.

EXAMPLE 1B trivalent GalNAc+trivalent GalNAc-L-SO1861 conjugated to ApoB antisense oligonucleotide, or a series of concentrations of conjugated trivalent GalNAc- (L-SO 1861) -ApoB antisense oligonucleotide (saponin-oligonucleotide-ASGPR ligand conjugate; FIGS. 22A-C)

Trivalent GalNAc (FIGS. 1A-C and 8A-G) was produced and SO1861-EMCH was conjugated to trivalent GalNAc (labile in a manner similar to that described for underivatized SO1861 in FIGS. 2A-B and 4, where SPT and SPT001 are synonymous with saponin SO 1861), DAR was 1, (trivalent GalNAc-SO1861; (GalNAc) ₃ -SPT 001); fig. 19C). Again, SO1861-EMCH refers to SO1861 in which the aldehyde group is derivatized by conversion to a hydrazone bond by reaction with N- ε -maleimidocaaproic acid hydrazide (EMCH), thereby providing SO1861-Ald-EMCH. In addition, covalent conjugates of trivalent GalNAc coupled to both the ApoB antisense BNA oligonucleotide ApoBBNA (SEQ ID NO: 2) and the dendrite comprising four covalently coupled SO1861-EMCH were prepared (FIG. 20E). For an overview of the preparation of conjugates, see also the following ' Synthesis of trivalent GalNAc-SO1861 and GalNAc-SO1861 (FIGS. 2A-B, 4) ' portions and ' Synthesis of trivalent GalNAc-BNA oligonucleotides (FIGS. 5A-B; FIGS. 6A-B, FIG. 7) ' portions and ' Synthesis of trivalent GalNAc-S-ApoB (or HSP 27) BNA oligonucleotides ' and ' Synthesis of trivalent GalNAc-L-ApoB (or HSP 27) BNA oligonucleotides ' portions and ' dendrites (-L-SO 1861) ₄ Synthesis of 'part and' dendrons (-L-SO 1861) of trivalent GalNAc and dendrons (-L-SO 1861) 8-trivalent GalNAc ₄ Synthesis of trivalent GalNAc 'and' dendrite (-L-SO 1861) ₄ -L-BNA oligonucleotide-trivalent GalNAc and dendrite (-L-SO 1861) ₈ Synthesis of L-BNA oligonucleotide-trivalent GalNAc 'and' dendrite (-L-SO 1861) ₄ -L-ApoB BNA oligonucleotide-a synthetic' moiety of trivalent GalNAc.

In the presence or absence of 300nM trivalent GalNAc-SO1861 ((GalNAc) ₃ -SPT 001) with a range of concentrations of trivalent GalNAc-ApoBBNA ((GalNAc) ₃ -ApoB treatment of primary hepatocytes (ASGPR 1) highly expressing ASGPR1 ^{High height} ) (FIG. 18B). Comparing absence and presence (GalNAc) ₃ Cell treatment at SPT001 showed that the cell was used alone (GalNAc) ₃ ApoB gene expression in ApoB treated cells trivalent-GalNAc-L-SO 1861 was very effective in increasing the potency of ASO to reduce ApoB gene expression in primary hepatocytes (fig. 18B).

All of these demonstrate that the combination of trivalent GalNAc-SO1861 with (GalNAc) 3-ApoB is effective in improving silencing of apoB gene expression in cells expressing ASGPR 1. Thus, trivalent GalNAc-SO1861 effectively induces endosomal escape of BNA in ASGPR 1-expressing cells.

In addition, primary hepatocytes were contacted with a range of concentrations of the saponin-oligonucleotide-ASGPR ligand conjugate, i.e., trivalent GalNAc ((GalNAc) conjugated with SO1861 (DAR 4) and ApoB BNA (SEQ ID NO: 2) ₃ -dSPT ₄ -ApoB; FIG. 24D), and apoB gene expression was assessed (FIGS. 18B and 20A). (GalNAc) ₃ -dSPT ₄ apoB gene silencing efficacy of ApoB over that of the absence of covalently linked saponins (GalNAc) ₃ ApoB conjugate about 100 times higher.

Example 1C trivalent GalNAc conjugated to SO1861, or a range of concentrations of the conjugate trivalent GalNAc- (L-SO 1861) in the presence of fixed doses of OKT-9 anti-CD 71 monoclonal antibody

Trivalent GalNAc (FIGS. 1A-B and 8A-G) was produced and SO1861-EMCH was conjugated to trivalent GalNAc (labile in a manner similar to that described for underivatized SO1861 in FIGS. 2A-B and 4, where SPT and SPT001 are synonymous with saponin SO 1861), DAR was 1, (trivalent GalNAc-SO1861; (GalNAc) ₃ -SPT 001); fig. 19C). Again, SO1861-EMCH refers to SO1861 in which the aldehyde group is derivatized by conversion to a hydrazone bond by reaction with N- ε -maleimidocaaproic acid hydrazide (EMCH), thereby providing SO1861-Ald-EMCH. General description of the preparation of conjugates also referred toSee the 'trivalent GalNAc-SO1861 and GalNAc-SO1861 synthesis (FIGS. 2A-B, 4)' section below.

Primary hepatocytes (ASGPR 1) highly expressing ASGPR1 were treated with a range of concentrations of trivalent GalNAc-SO1861 (trivalent GalNAc-SPT 001) in the absence or presence of 10pm CD 71-saporin (CD 71-targeting monoclonal antibody OKT-9 conjugated with the protein toxin saporin; fig. 19D) ^{High height} ) And Huh7 hepatocyte line (ASGPR 1 ^+/- Expression). Cell treatment in the absence of CD 71-saporin indicated that trivalent GalNAc-SO1861 as a single compound was not toxic up to at least 1000nM (FIGS. 19A, 19B; relative cell viability). Cell viability of cells treated with the combination of anti-CD 71 MoAb-saporin and low nM dose of trivalent GalNAc conjugated to SO1861 (DAR 1; trivalent GalNAc-SPT 001) was highly effective in killing primary hepatocytes compared to cell viability of Huh7 cells treated with the same dose of anti-CD 71 MoAb-saporin in combination with the same dose of trivalent GalNAc-SPT001 (fig. 19A and 19B).

All of these demonstrate that low doses of trivalent GalNAc-SO1861 in combination with low concentrations of CD 71-saporin are effective in inducing cell killing in ASGPR1 expressing cells, while neither compound results in death in the absence of the other compound. Thus, trivalent GalNAc-SO1861 effectively induces endosomal escape of protein toxins in ASGPR 1-expressing cells. Thus, low nM trivalent GalNAc-SPT001 enhances cytoplasmic delivery of low pM ADC anti-CD 71 MoAb-saporin in primary hepatocytes; the hepatocyte line with low ASGPR1 expression (Huh 7) did not react at low nM concentrations of trivalent GalNAc-SPT 001.

EXAMPLE 1D trivalent GalNAc+trivalent GalNAc-L-SO1861 conjugated to an ApoB antisense oligonucleotide, or a series of concentrations of conjugated trivalent GalNAc- (L-SO 1861) -ApoB antisense oligonucleotide (sapon-oligonucleotide-ASGPR ligand conjugate; FIGS. 22A-C, trivalent GalNAc-ApoBBNA-SPT 001)

DAR 1 (trivalent GalNAc-SO1861; galNAc) 3-SPT001; FIG. 19C) was produced and SO1861-EMCH was conjugated with trivalent GalNAc as described in example 1B. In addition, covalent conjugates of trivalent GalNAc coupled to both the ApoB antisense BNA oligonucleotide ApoBBNA (SEQ ID NO: 2) and SO1861-EMCH were prepared (FIG. 20D). In addition, conjugates of trivalent GalNAc and ApoB antisense BNA oligonucleotide ApoBBNA (SEQ ID NO: 2) were prepared (FIG. 20C). For an overview of the preparation of conjugates, see also the following 'synthesis of trivalent GalNAc-SO1861 and GalNAc-SO1861 (FIGS. 2A-B, 4)' sections and synthesis of 'trivalent GalNAc-BNA oligonucleotides (FIGS. 5A, 5B; FIGS. 6A-B, FIG. 7)' portion and 'Synthesis of trivalent GalNAc-S-ApoB (or HSP 27) BNA oligonucleotide' portion and 'Synthesis of trivalent GalNAc-L-ApoB (or HSP 27) BNA oligonucleotide' and 'Synthesis of dendrite (-L-SO 1861) 4-trivalent GalNAc and dendrite (-L-SO 1861) 8-trivalent GalNAc' portion and 'Synthesis of dendrite (-L-SO 1861) 4-trivalent GalNAc' and 'dendrite (-L-SO 1861) 4-L-BNA oligonucleotide-trivalent GalNAc and dendrite (-L-SO 1861) 8-L-BNA oligonucleotide-trivalent GalNAc' portion and 'dendrite (-L-SO 1861) 4-L-ApoB BNAc' portion (FIG. 19E; FIG. 20E; FIG. 23, FIG. 24).

Primary hepatocytes (ASGPR 1) highly expressing ASGPR1 were treated with a range of concentrations of trivalent GalNAc-ApoBBNA (trivalent GalNAc-ApoBBNA) in the absence or presence of 300nM of trivalent GalNAc-SO1861 (trivalent GalNAc-SPT 001) ^{High height} ) And Huh7 hepatocyte line (low ASGPR1 expression) (fig. 20A and 20B). Cell treatment in the absence of trivalent GalNAc-SPT001 showed that trivalent GalNAc-SPT001 was highly effective (about 100-fold more potent) in reducing apoB mRNA expression in primary hepatocytes compared to apoB mRNA expression in primary hepatocytes treated with trivalent GalNAc-ApoBBNA alone (FIG. 20A). Since Huh7 cells expressed very low levels of ASGPR1, none of trivalent GalNAc-ApoBBNA, or a combination of trivalent GalNAc-ApoBBNA and trivalent GalNAc-SPT001 induced a decrease in apoB mRNA expression in these hepatocytes.

All of these demonstrate that the combination of trivalent GalNAc-SPT001 with trivalent GalNAc-ApoBBNA is effective in improving silencing of apoB mRNA expression in ASGPR1 expressing cells under control conditions without third order GalNAc-SPT 001. Thus, trivalent GalNAc-SPT001 effectively induces endosomal escape of BNA in ASGPR 1-expressing cells.

In addition, primary hepatocytes and finesHepatocytes of the cell line Huh7 were conjugated with a range of concentrations of a saponin-oligonucleotide-ASGPR ligand conjugate (i.e., trivalent GalNAc conjugated with SO1861 (DAR 4 in dendrites with four SO1861 molecules) and ApoB BNA (SEQ ID NO: 2) ((GalNAc) 3-dSPT4-ApoB (FIG. 20E), or trivalent GalNAc-ApoBBNA-dendrites (SO 1861) ₄ ) Exposure, and apoB mRNA expression was assessed (fig. 20A and B). apoB mRNA expression silencing efficacy of (GalNAc) 3-dSPT4-ApoB was about 100-fold higher than that of the trivalent GalNAc-ApoBBNA conjugate lacking covalently linked SO 1861.

Primary hepatocytes (ASGPR 1) highly expressing ASGPR1 were treated with a range of concentrations of trivalent GalNAc-ApoBBNA (trivalent GalNAc-ApoBBNA) in the absence or presence of 300nM of trivalent GalNAc-SO1861 (trivalent GalNAc-SPT 001) ^{High height} ) And Huh7 hepatocyte line (low ASGPR1 expression) (fig. 21A and 21B), and apoB protein expression was assessed and compared. Cell treatment in the absence of trivalent GalNAc-SPT001 showed that trivalent GalNAc-SPT001 was highly effective (more than 1000-fold higher) in reducing apoB protein expression in primary hepatocytes compared to apoB protein expression in primary hepatocytes treated with trivalent GalNAc-ApoBBNA alone (FIG. 21A). Since Huh7 cells expressed very low levels of ASGPR1, trivalent GalNAc-ApoBBNA, or a combination of trivalent GalNAc-ApoBBNA and trivalent GalNAc-SPT001 did not induce significant reduction of apoB protein expression in these hepatocytes (fig. 21B).

All of these demonstrate that the combination of trivalent GalNAc-SPT001 with trivalent GalNAc-ApoBBNA is effective in improving silencing of apoB protein expression in ASGPR 1-highly expressing cells. Thus, trivalent GalNAc-SPT001 effectively induces endosomal escape of BNA in ASGPR 1-expressing cells.

Thus, low nM trivalent GalNAc-ApoBBNA+trivalent GalNAc-SPT001 can enhance apoBBNA endosomal escape and cytoplasmic delivery in primary hepatocytes, resulting in apoB mRNA silencing; low nM (GalNAc) 3-dSPT4-ApoB can enhance apoBBNA endosomal escape and cytoplasmic delivery in primary hepatocytes, resulting in apoB mRNA silencing; hepatocytes with very low ASGPR1 expression were non-responsive to both therapies; low nM trivalent GalNAc-apobbna+trivalent GalNAc-SPT001 can enhance ApoBBNA endosomal escape and cytoplasmic delivery in primary hepatocytes, resulting in apoB protein attenuation; low nM (GalNAc) 3-dSPT4-ApoB enhances apoBBNA endosomal escape and cytoplasmic delivery in primary hepatocytes, resulting in apoB protein attenuation; when apoB expression was considered, cells with very low ASGPR1 expression were non-responsive to both therapies.

EXAMPLE 2 trivalent GalNAc-L/S-BNA+SO1861-EMCH

HSP27BNA was conjugated with trivalent GalNAc (FIG. 5A, FIG. 5B, FIG. 6, FIG. 7), and HepG2 (ASGPR 1) was treated with a concentration range of trivalent GalNAc-L-HSP27BNA (labile conjugation, FIG. 5A-B, FIG. 6A-B) or a combination of trivalent GalNAc-S-HSP27BNA (stable conjugation, FIG. 7) with a fixed concentration of SO1861-EMCH of 4000nM ⁺ ) Cells or Huh7 (ASGPR 1) ^+/- ). HSP27 gene silencing in HepG2 and Huh7 cells was determined. Only in combination with 4000nm so1861-EMCH, effective gene silencing was observed in HEPG2 and Huh7 cells in the following cases: low concentrations of trivalent-GalNAc-L-HSP 27BNA (HepG 2: ic50=0.1 nM; huh7: ic50=3 nM) or trivalent-GalNAc-S-HSP 27BNA (HepG 2: ic50=0.1 nM; huh7: ic50=3 nM), while equivalent concentrations of trivalent-GalNAc-L-HSP 27BNA (HepG 2/Huh7: IC 50) >2000 nM) or trivalent-GalNAc-S-HSP 27BNA (HepG 2/Huh7: IC 50)>2000 nM) showed no gene silencing activity in HepG2 and Huh7 cell lines (fig. 11).

Next, apoBBNA was conjugated with trivalent GalNAc in a similar manner (FIGS. 5A-B, 6A-B, 7), and HepG2 (ASGPR 1) was treated as follows ⁺ ) Cells or Huh7 (ASGPR 1) ^+/- ): trivalent GalNAc-L-ApoBBNA (unstable conjugation, FIG. 5A-B; FIG. 6A-B, FIG. 12, FIG. 13) or trivalent GalNAc-S-ApoBBNA (stable conjugation, FIG. 7, FIG. 14, FIG. 15) or trivalent GalNAc-L-ApoB in a concentration range _Scrambling BNA or trivalent GalNAc-S-ApoB _Scrambling BNA (where scrambled ApoBBNA is an antisense scrambling oligonucleotide sequence that does not bind apoB mRNA; off-target control BNA) in combination with a fixed concentration of SO1861-EMCH of 4000 nM. HepG2 (ASGPR 1 was identified ⁺ ) Cells or Huh7 (ASGPR 1) ^+/- ) ApoB gene silencing in (a) is disclosed. Only in combination with 4000nm so1861-EMCH, effective gene silencing was observed in HepG2 cells in the following cases: low concentrations of trivalent GalNAc-L-ApoBBNA (ic50=10 nM) or trivalent GalNAc-S-ApoBBNA (ic50=10 nM), whereas equivalent concentrations are givenTrivalent GalNAc-L-ApoBBNA (IC 50>2000 nM) or trivalent GalNAc-S-ApoBBNA (IC 50)>2000 nM) showed no gene silencing activity in HepG2 cells (fig. 12, fig. 14). These treatments did not affect the viability of these cell lines (fig. 13, 15). 'L' refers to an 'unstable' linker, which indicates that the linker is cleaved in the cell (i.e., at a pH that is apparent as in endosomes and lysosomes). In contrast, 'S' refers to a 'stable' linker, which indicates that the linker is not cleaved in the cell (i.e., at a pH that is apparent as in endosomes and lysosomes). Thus, a conjugate comprising an labile bond between any two molecules that form or are comprised by the conjugate will be cleaved at a position in the labile linker, thereby dissociating the two linked or bound molecules. One example is cleavage of hydrazone bonds under acidic conditions as evident in endosomes and lysosomes of mammalian cells (e.g., human cells).

All of these demonstrate that SO1861-EMCH can enhance endosomal escape of trivalent GalNAc-L-BNA or trivalent GalNAc-S-BNA and target gene silencing of two independent gene targets. Gene silencing in HepG2 cells was more efficient than Huh7 cells, indicating that higher levels of ASGPR1 expression at the plasma membrane of the cells helped to increase uptake of GalNAc-BNA conjugates.

EXAMPLE 3 trivalent GalNAc-L/S-BNA+trivalent GalNAc-L-SO1861

SO1861-EMCH (also referred to as SPT) was conjugated to trivalent GalNAc ((GalNAc) 3 or (GN) 3) (labile in a manner similar to that described for underivatized SO1861 in FIGS. 2A-B and 4), DAR1, (trivalent-GalNAc-SO 1861). HepG2 (ASGPR 1) was treated as follows ⁺ ) Cells or Huh7 (ASGPR 1) ^+/- ) And (3) cells: trivalent GalNAc-L-ApoBBNA (unstable conjugation, FIG. 16, FIG. 17), trivalent GalNAc-S-ApoBBNA (stable conjugation (FIG. 16, FIG. 17)) or scrambled (Scr.) off-target control conjugate (trivalent GalNAc-L-ApoB) in a concentration range _Scrambling BNA or trivalent GalNAc-S-ApoB _Scrambling BNA) with a fixed concentration of trivalent GalNAc-L-SO1861 of 4000nM (DAR 1). HepG2 (ASGPR 1 was identified ⁺ ) Cells and Huh7 (ASGPR 1) ^+/- ) ApoB gene silencing in cells. Only in combination with 4000nM trivalent GalNAc-L-SO1861 In HepG2 cells (ASGPR 1) under the following conditions ⁺ ) Effective gene silencing was observed: low concentrations of trivalent GalNAc-L-ApoBBNA (ic50=50 nM) or trivalent GalNAc-S-ApoBBNA (ic50=50 nM). Two combined treatments were performed on Huh7 cells (ASGPR 1 ^+/- ) Shows reduced effective gene silencing (trivalent GalNAc-L-ApoBBNA: IC50 = 700nM; trivalent GalNAc-S-ApoBBNA: IC50 = 700 nM). Equivalent concentrations of trivalent GalNAc-L-ApoBBNA (HepG 2/Huh7: IC50>1000 nM) or trivalent GalNAc-S-ApoBBNA (HepG 2/Huh7: IC50>1000 nM) showed no gene silencing activity in HepG2 and Huh7 cell lines, 4000nM trivalent GalNAc-L-SO1861 did not induce/enhance scrambled trivalent GalNAc-L-ApoB either _Scrambling BNA+(HepG2/Huh7：IC50>1000 nM) or scrambled trivalent GalNAc-S-ApoB _Scrambling BNA(HepG2/Huh7：IC50>1000 nM) (FIG. 16, FIG. 17). These treatments did not affect the viability of these cell lines (fig. 16C, fig. 16D).

All of these demonstrate that when combined with trivalent GalNAc-L-SO1861, very low concentrations of trivalent GalNAc-L-HSP27BNA or trivalent GalNAc-S-HSP27BNA effectively induce gene silencing in ASGPR1 expressing cells and again a stronger effect is seen in cells with higher ASGPR1 expression.

Materials and methods

Abbreviations (abbreviations)

AEM N- (2-aminoethyl) maleimide trifluoroacetate salt

AMPD 2-amino-2-methyl-1, 3-propanediol

BOP (benzotriazol-1-yloxy) tris (dimethylamino) hexafluorophosphate

DIPEA N, N-diisopropylethylamine

DMF N, N-dimethylformamide

EDCI. HCl 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride

EMCH. TFA N- (epsilon-maleimidocaprooic acid) hydrazide, trifluoroacetate salt

HATU 1- [ bis (dimethylamino) methylene ] -1H-1,2, 3-triazolo [4,5-b ] pyridinium 3-oxide hexafluorophosphate

min

NMM 4-methylmorpholine

r.t. retention time

TCEP tris (2-carboxyethyl) phosphine hydrochloride

Temp temperature

TFA trifluoroacetic acid

Analysis method

LC-MS method 1

The device comprises: waters IClass; binary pump: UPIBSM, SM: UPISMFTN with SO; UPCMA, PDA: UPPDATC,210-320nm, SQD: the mass range of ACQ-SQD2 ESI depends on the molecular weight of the product:

1500-2400 or 2000-3000; ELSD: gas pressure 40psi, drift tube temp:50 ℃; column: acquity C18, 50×2.1mm,1.7 μm; temperature: 60 ℃, flow rate: 0.6mL/min, linear gradient depending on polarity of the product:

^A t ₀ ＝2％A，t _5.0min ＝50％A，t _6.0min ＝98％A

^B t ₀ ＝2％A，t _5.0min ＝98％A，t _6.0min ＝98％A

post run time: 1.0min, eluent A: acetonitrile, eluent B: 10mM ammonium bicarbonate in water (ph=9.5).

LC-MS method 2

The device comprises: waters IClass; binary pump: UPIBSM, SM: UPISMFTN with SO; UPCMA, PDA: UPPDATC,210-320nm, SQD: the mass range of ACQ-SQD2 ESI depends on the molecular weight of the product: 100-800 parts of yang/yin or 2000-3000 parts of yin; ELSD: gas pressure 40psi, drift tube temp:50 ℃; column: waters XSelectTM CSH C18, 50×2.1mm,2.5 μm, temperature: 25 ℃, flow rate: 0.5mL/min, gradient: t is t _0min ＝5％A，t _2.0min ＝98％A，t _2.7min =98%a, post run time: 0.3min, eluent a: acetonitrile, eluent B: 10mM ammonium bicarbonate in water (ph=9.5).

LC-MS method 3

The device comprises: waters IClass; binary pump: UPIBSM, SM: UPISMFTN with SO; UPCMA, PDA: UPPDATC,210-320nm, SQD: ACQ-SQD2 ESI, mass SpectrumDepending on the molecular weight of the product, 105-800, 500-1200 or 1500-2500; ELSD: gas pressure 40psi, drift tube temp:50 ℃; column: waters XSelect ^TM CSH C18, 50X 2.1mm,2.5 μm, temperature: 40 ℃, flow rate: 0.5mL/min, gradient: t is t _0min ＝5％A，t _2.0min ＝98％A，t _2.7min =98%a, post run time: 0.3min, eluent a: 0.1% formic acid in acetonitrile, eluent B: 0.1% formic acid in water.

LC-MS method 4

The device comprises: waters IClass; binary pump: UPIBSM, SM: UPISMFTN with SO; UPCMA, PDA: UPPDATC,210-320nm, SQD: the mass range of ACQ-SQD2 ESI depends on the molecular weight of the product: 100-800 parts of yang/yin or 2000-3000 parts of yin; ELSD: gas pressure 40psi, drift tube temp:50 ℃, column: waters Acquity Shield RP18, 50×2.1mm,1.7 μm, temperature: 25 ℃, flow rate: 0.5mL/min, gradient: t is t _0min ＝5％A，t _2.0min ＝98％A，t _2.7min =98%a, post run time: 0.3min, eluent a: acetonitrile, eluent B: 10mM ammonium bicarbonate in water (ph=9.5).

Preparation method

Preparation MP-LC method 1

Instrument type: reveleris ^TM Preparing MPLC; column: waters XSelect ^TM CSH C18 (145X 25mm,10 μm); flow rate: 40mL/min; column temperature: room temperature; eluent A:10 mM ammonium bicarbonate in water (ph=9.0); eluent B:99% acetonitrile + 1%10mm ammonium bicarbonate in water; gradient:

^A t _0min ＝5％B，t _1min ＝5％B，t _2min ＝10％B，t _17min ＝50％B，t _18min ＝100％B，t _23min ＝100％B

^A t _0min ＝5％B，t _1min ＝5％B，t _2min ＝20％B，t _17min ＝60％B，t _18min ＝100％B，t _23min ＝100％B；

and (3) detecting UV: 210. 235, 254nm and ELSD.

Preparation MP-LC method 2

Instrument type: reveleris ^TM Preparing MPLC; column: phenomenex LUNAC18 (3) (150X 25mm,10 μm); flow rate: 40mL/min; column temperature: room temperature; eluent A: 0.1% (v/v) formic acid in water, eluent B: 0.1% (v/v) formic acid in acetonitrile; gradient:

^A t _0min ＝5％B，t _1min ＝5％B，t _2min ＝20％B，t _17min ＝60％B，t _18min ＝

100％B，t _23min ＝100％B

^B t _0min ＝2％B，t _1min ＝2％B，t _2min ＝2％B，t _17min ＝30％B，t _18min ＝

100％B，t _23min ＝100％B

^C t _0min ＝5％B，t _1min ＝5％B，t _2min ＝10％B，t _17min ＝50％B，t _18min ＝100％B，t _23min ＝100％B

^D t _0min ＝5％B，t _1min ＝5％B，t _2min ＝5％B，t _17min ＝40％B，t _18min ＝100％B，t _23min ＝100％B；

and (3) detecting UV: 210. 235, 254nm and ELSD.

Preparation LC-MS method 3

MS instrument type: agilent Technologies G6130B Quadrapol; HPLC instrument type: agilent Technologies 1290 preparative LC; column: waters XSelect ^TM CSH (C18, 150X 19mm,10 μm); flow rate: 25ml/min; column temperature: room temperature; eluent A:100% acetonitrile; eluent B: 10mM ammonium bicarbonate in water, ph=9.0; gradient:

^A t ₀ ＝20％A，t _2.5min ＝20％A，t _11min ＝60％A，t _13min ＝100％A，t _17min ＝100％A

^B t ₀ ＝5％A，t _2.5min ＝5％A，t _11min ＝40％A，t _13min ＝100％A，t _17min ＝100％A；

And (3) detection: DAD (210 nm); and (3) detection: MSD (ESI pos/neg) mass range: 100-800; DAD-based fraction collection.

Preparation LC-MS method 4

MS instrument type: agilent Technologies G6130B Quadrapol; HPLC instrument type: agilent Technologies 1290 preparative LC; column: waters XBridge Protein (C4, 150X 19mm,10 μm); flow rate: 25ml/min; column temperature: room temperature; eluent A:100% acetonitrile; eluent B: 10mM ammonium bicarbonate in water, ph=9.0; gradient:

^A t ₀ ＝2％A，t _2.5min ＝2％A，t _11min ＝30％A，t _13min ＝100％A，t _17min ＝100％A

^B t ₀ ＝10％A，t _2.5min ＝10％A，t _11min ＝50％A，t _13min ＝100％A，t _17min ＝100％A

^C t ₀ ＝5％A，t _2.5min ＝5％A，t _11min ＝40％A，t _13min ＝100％A，t _17min ＝100％A；

and (3) detection: DAD (210 nm); and (3) detection: MSD (ESI pos/neg) mass range: 100-800; DAD-based fraction collection

Flash chromatography

Grace Reveleris C-815 is fast; solvent delivery system: a 3 piston pump with an automatic starting function, 4 independent channels, at most 4 solvents are operated at a time, and when the solvents are exhausted, the pipelines are automatically switched; maximum pump flow rate 250mL/min; maximum pressure 50 bar (725 psi); and (3) detection: UV 200-400nm, up to 4 UV signal combinations and scanning of the entire UV range, ELSD; column dimensions: 4-330g on the instrument, 750g to 3000g luer with optional stent.

Synthesis of SO1861-EMCH (SO 1861-EMCH is also referred to as SO1861-Ald-EMCH and SPT-EMCH)

To SO1861 (121 mg,0.065 mmol) and EMCH. TFA (110 mg,0.325 mmol) were added methanol (extra dry, 3.00 mL) and TFA (0.020mL, 0.260 mmol). The reaction mixture was stirred at room temperature. After 1.5 hours, the reaction mixture was subjected to preparative MP-LC. Fractions corresponding to the product were immediately combined together, frozen and lyophilized overnight to give the title compound as a white fluffy solid (120 mg, 90%). The purity based on LC-MS was 96%.

Synthesis of trivalent GalNAc-azides

Intermediate 1:

1-azido-17, 17-bis ((3- (tert-butoxy) -3-oxopropoxy) methyl) -15-oxo-3,6,9,12,19-pentaoxa-16-azabehenyl-22-oic acid tert-butyl ester

To di-tert-butyl 3,3' - ((2-amino-2- ((3- (tert-butoxy) -3-oxopropoxy) methyl) propane-1, 3-diyl) bis (oxy)) dipropionate (1.27 g,2.51 mmol) was added a solution of N-hydroxysuccinimide ester of 3-azido (peg 4) propionic acid (977 mg,2.51 mol) in DMF (10 mL). Next, DIPEA (657. Mu.L, 3.77 mmol) was added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was evaporated in vacuo and the residue was dissolved in ethyl acetate (100 mL). The resulting solution was washed with a 0.5N potassium hydrogen sulfate solution (2X 100 mL) and brine (100 mL), and dried over Na ₂ SO ₄ Dried, filtered and evaporated in vacuo. The residue was purified by flash chromatography (DCM-10% methanol in DCM (v/v) gradient 100:0 l to 0:100) to give the title compound (1.27 g, 65%) as a colorless oil. The purity based on LC-MS was 100% (ELSD).

LRMS(m/z)：780[M+1] ¹⁺

LC-MS r.t.(min)：2.10 ²

Intermediate 2:

1-azido-17, 17-bis ((2-carboxyethoxy) methyl) -15-oxo-3,6,9,12,19-pentaoxa-16-azabehenyl-22-oic acid

To a solution of tert-butyl 1-azido-17, 17-bis ((3- (tert-butoxy) -3-oxopropoxy) methyl) -15-oxo-3,6,9,12,19-pentaoxa-16-azabehenyl-22-carboxylate (1.27 g,1.63 mmol) in DCM (5.0 mL) was added TFA (5.0 mL,65 mmol). The reaction mixture was stirred at room temperature. After 1.5 hours, the reaction mixture was evaporated in vacuo and co-evaporated with toluene (3×10 mL) and DCM (3×10 mL) to give the crude title product as a colourless oil.

LRMS(m/z)：611[M+1] ¹⁺

Intermediate 3:

(10- (1-azido-3, 6,9, 12-tetraoxapentadecane-15-amido) -10- (13, 13-dimethyl-5, 11-dioxo-2, 12-dioxa-6, 10-diazatetradecyl) -5, 15-dioxo-8, 12-dioxa-4, 16-diazanonadecane-1, 19-diyl) dicarbamic acid di-tert-butyl ester

1-azido-17, 17-bis ((2-carboxyethoxy) methyl) -15-oxo-3,6,9,12,19-pentaoxa-16-azabehenyl-22-acid (997 mg,1.63 mmol), oxyma Pure (1.04 g,7.35 mmol) and EDCI.HCl (1.17 g,6.12 mmol) were dissolved in DMF (10.0 mL). Next, DIPEA (1.99 mL,11.4 mmol) was added followed by a solution of N-BOC-1, 3-propanediamine (1.07 g,6.12 mmol) in DMF (10.0 mL) directly. The reaction mixture was stirred at room temperature overnight. The reaction mixture was evaporated in vacuo and the residue was dissolved in ethyl acetate (100 mL). The resulting solution was washed with 0.5N potassium hydrogen sulfate solution (100 mL), saturated sodium hydrogen carbonate solution (2X 100 mL) and brine (100 mL), and dried over Na ₂ SO ₄ Dried, filtered and evaporated in vacuo. The residue was purified by flash chromatography (DCM-10% methanol in DCM (v/v) gradient 0:100 l to 100:0, hold at 100:0 until eluting the product) to give the title compound as a pale yellow viscous oil (1.16 g, 66%). LC-MS 99% (ELSD).

LRMS(m/z)：1080[M+1] ¹⁺

LC-MS r.t.(min)：1.51 ³

Intermediate 4:

synthesis of 3,3' - ((2- ((3- ((3-aminopropyl) amino) -3-oxopropoxy) methyl) -2- (1-azido-3, 6,9, 12-tetraoxapentadecane-15-amido) propane-1, 3-diyl) bis (oxy)) bis (N- (3-aminopropyl) propionamide) tris (2, 2-trifluoroacetate)

To a solution of di-tert-butyl (10- (1-azido-3, 6,9, 12-tetraoxapentadecane-15-amido) -10- (13, 13-dimethyl-5, 11-dioxo-2, 12-dioxa-6, 10-diazatetradecyl) -5, 15-dioxo-8, 12-dioxa-4, 16-diazanonadecane-1, 19-diyl) dicarbamate (1.16 g,1.08 mmol) in DCM (10 mL) was added TFA (10 mL,131 mmol). The reaction mixture was stirred at room temperature. After 2 hours, the reaction mixture was evaporated in vacuo, co-evaporated with toluene (3×10 mL) and DCM (3×10 mL) to give the crude title product as a pale yellow viscous oil.

LRMS(m/z)：260[M+3] ³⁺ ，390[M+2] ²⁺ ，780[M+1] ¹⁺ ，

Intermediate 5:

(2R, 3R,4R,5R, 6R) -5-acetamido-2- (acetoxymethyl) -6- ((5- ((2, 5-dioxopyrrolidin-1-yl) oxy) -5-oxopentyl) oxy) tetrahydro-2H-pyran-3, 4-diyldiacetate

5- (((2R, 3R,4R,5R, 6R) -3-acetamido-4, 5-diacetoxy-6- (acetoxymethyl) tetrahydro-2H-pyran-2-yl) oxy) pentanoic acid (according to J.Am. Chem Soc. [ American society of chemistry)]Obtained as 2014,136,16958-16961, 3.00g,6.70 mmol) and N-hydroxysuccinimide (926 mg,8.05 mmol) were dissolved in DCM (50 mL). Next, EDCI. HCl (1.54 g,8.05 mmol) and 4- (dimethylamino) pyridine (82 mg,0.67 mmol) were added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with DCM, and the resulting solution was washed with 0.5N potassium hydrogen sulfate solution (150 mL), saturated sodium bicarbonate solution (150 mL) and brine (150 mL), and dried over Na ₂ SO ₄ Dried, filtered and evaporated in vacuo to give the title compound as a white foam (3.60 g, 99%). The purity based on LC-MS was 99% (ELSD).

LRMS(m/z)：545[M+1] ¹⁺

LC-MS r.t.(min)：1.07 ³

Intermediate 6:

[ (3R, 6R) -3, 4-bis (acetyloxy) -6- {4- [ (3- {3- [2- (1-azido-3, 6,9, 12-tetraoxapentadecane-15-amido) -3- (2- { [3- (5- { [ (2R, 5R) -4, 5-bis (acetyloxy) -6- [ (acetyloxy) methyl ] -3-acetamidooxy-2-yl ] oxy } pentanamido) propyl ] carbamoyl } ethoxy) -2- [ (2- { [3- (5- { [ (2R, 5R) -4, 5-bis (acetyloxy) -6- [ (acetyloxy) methyl ] -3-acetamidooxy-2-yl ] oxy } pentanamido) propyl ] carbamoyl } ethoxy) methyl ] propoxy ] propionyl } propyl) carbamoyl ] butoxy } -5-acetamidooxy-2-yl ] methyl acetate

3,3' - ((2- ((3- ((3-aminopropyl) amino) -3-oxopropoxy) methyl) -2- (1-azido-3, 6,9, 12-tetraoxapentadecane-15-amido) propane-1, 3-diyl) bis (oxy)) bis (N- (3-aminopropyl) propanamide) (1.21 g,1.08 mmol) was dissolved in a mixture of DMF (10 mL) and DIPEA (1.69 mL,9.70 mmol). Next, (2 r,3r,4r,5r,6 r) -5-acetamido-2- (acetoxymethyl) -6- ((5- ((2, 5-dioxopyrrolidin-1-yl) oxy) -5-oxopentyl) oxy) tetrahydro-2H-pyran-3, 4-diyldiacetate (2.20 g,4.04 mmol) was added and the reaction mixture stirred at room temperature over the weekend. Next, the reaction mixture was evaporated in vacuo and the residue was purified by flash chromatography (DCM-30% methanol in DCM (v/v) gradient 0:100 l to 100:0) to give the title compound as a pale yellow foam (1.84 g, 83%). LC-MS 95% (ELSD).

LRMS(m/z)：2068[M+1] ¹⁺

LC-MS r.t.(min)：1.18 ³

Intermediate 7:

trivalent GalNAc-azides

[ (3R, 6R) -3, 4-bis (acetoxy) -6- {4- [ (3- {3- [2- (1-azido-3, 6,9, 12-tetraoxapentadecane-15-amido) -3- (2- { [3- (5- { [ (2R, 5R) -4, 5-bis (acetoxy) -6- [ (acetoxy) methyl)]-3-acetamido oxacyclohex-2-yl ]Oxy } pentanamido) propyl]Carbamoyl } ethoxy) -2- [ (2- { [3- (5- { [ (2R, 5R) -4, 5-bis (acetoxy) -6- [ (acetoxy) methyl)]-3-acetamido oxacyclohex-2-yl]Oxy } pentanamido) propyl]Carbamoyl } ethoxy) methyl]Propoxy group]Propionamido } propyl) carbamoyl]Butoxy } -5-acetamido oxazin-2-yl]Methyl acetate (300 mg,0.145 mmol) was dissolved in a mixture of triethylamine (2.00 mL,14.4 mmol), methanol (2.00 mL) and water (2.00 mL)And the reaction mixture was stirred at room temperature. After 2 hours, the reaction mixture was evaporated in vacuo. The residue was purified by preparative MP-LC. Will correspond to the product ^2B Fractions were immediately combined together, frozen and lyophilized overnight to give the title compound as a white solid (164 mg, 67%). Purity based on LC-MS was 97%.

LRMS(m/z)：1688[M-1] ^1-

LC-MS r.t.(min)：1.99 ^1A

Synthesis of trivalent GalNAc-SO1861 and GalNAc-SO1861 (FIG. 2, FIG. 4)

Intermediate 8:

SO1861-NH ₂

SO 1861-azide (6.89 mg, 3.20. Mu. Mol) was dissolved in a mixture of 32mM potassium carbonate (150. Mu.L, 4.80. Mu. Mol) and acetonitrile (150. Mu.L). After that, a 1.0M solution of trimethylphosphine in THF (32. Mu.L, 32. Mu. Mol) was directly added, and the resulting mixture was shaken for 1min and allowed to stand at room temperature. After 30min, the reaction mixture was subjected to preparative MP-LC. Fractions corresponding to the product were immediately combined together, frozen and lyophilized overnight to give the title compound (5.30 mg, 78%) as a white fluffy solid. The purity based on LC-MS was 94%.

LRMS(m/z)：1062[M-2] ^2-

LC-MS r.t.(min)：2.51 ^1B

Intermediate 9:

SO1861-DBCO

to SO 1861-amine (48.6 mg, 22.9. Mu. Mol) and DBCO-NHS (13.0 mg, 32.4. Mu. Mol) were added a solution of DIPEA (5.98. Mu.L, 34.3. Mu. Mol) and DMF (2.0 mL). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 4 hours, the reaction mixture was diluted with a solution of 50mM sodium bicarbonate (1.00 mL, 50. Mu. Mol). The resulting mixture was shaken for 1min and allowed to stand at room temperature. After 30min, the reaction mixture was subjected to preparative MP-LC. Will correspond to the product ^1B Fractions were immediately combined, frozen and lyophilized overnight to give the title compound as a white fluffy solid (16.9 mg, 31%). The purity based on LC-MS was 94%.

LRMS(m/z)：2412[M-1] ^1-

LC-MS r.t.(min)：2.45 ^1B

Synthesis of SO 1861-L-trivalent GalNAc

SO1861-DBCO (7.50 mg, 3.11. Mu. Mol) and trivalent GalNAc-azide (5.31 mg, 3.14. Mu. Mol) were dissolved in a mixture of water/acetonitrile (2:1, v/v,0.90 mL). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^3B Fractions were immediately combined, frozen and lyophilized overnight to give the title compound as a white fluffy solid (9.60 mg, 75%). Purity based on LC-MS was 99%.

LRMS(m/z)：2050[M-2] ^2-

LC-MS r.t.(min)：2.04 ^1B

Synthesis of SO1861-L-GalNAc

SO1861-DBCO (1.75 mg, 0.73. Mu. Mol) and GalNAc-PEG 3-azide (0.82 mg, 2.18. Mu. Mol) were dissolved in a mixture of water/acetonitrile (1:1, v/v,1.00 mL). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^3B Fractions were immediately combined, frozen and lyophilized overnight to give the title compound as a white fluffy solid (1.63 mg, 81%). The purity based on LC-MS was 100%.

LRMS(m/z)：2790[M-1] ^1-

LC-MS r.t.(min)：2.15 ^1B

Synthesis of trivalent GalNAc-BNA oligonucleotides (FIGS. 6, 7)

Intermediate 10:

4- { 2-azatricyclo [10.4.0.04,9] hexadeca-1 (12), 4 (9), 5,7,13,15-hexen-10-yn-2-yl } -N- [2- (2- {2- [2- ({ 4- [ (E) - { [6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamide ] imino } methyl ] phenyl } carboxamide) ethoxy ] ethoxy } ethoxy) ethyl ] -4-oxobutanamide

4- { 2-azatricyclo [10.4.0.04,9]]Hexadeca-1 (12), 4 (9), 5,7,13,15-hexen-10-yn-2-yl } -N- {2- [2- (2- {2- [ (4-formylphenyl) carboxamido-de]Ethoxy } ethoxy) ethoxy]Ethyl } -4-oxobutanamide (25.0 mg, 40.9. Mu. Mol) and EMCH. TFA (20.8 mg, 61.3. Mu. Mol) were dissolved in methanol (extra dry, 2.00 mL). Next, TFA (9.39. Mu.L, 123. Mu. Mol) was added. The reaction mixture was shaken for 1min and allowed to stand overnight in the room. The reaction mixture was subjected to preparative MP-LC. Will correspond to the product ^1B Fractions were immediately combined, frozen and lyophilized overnight to give the title compound (16.2 mg, 48%) as a white solid. Purity based on LC-MS was 91%.

LRMS(m/z)：410.2[M+2] ²⁺

LC-MS r.t.(min)：1.41 ²

Intermediate 11:

trivalent GalNAc-L-maleimide

Trivalent GalNAc-azide (20.3 mg, 12.0. Mu. Mol) and 4- { 2-azatricyclo [10.4.0.04,9 ]]Hexadeca-1 (12), 4 (9), 5,7,13,15-hexen-10-yn-2-yl } -N- [2- (2- {2- [2- ({ 4- [ (E) - { [6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamide group)]Imino } methyl group]Phenyl } carboxamide) ethoxy]Ethoxy } ethoxy) ethyl]-4-oxobutanamide (11.8 mg, 14.4. Mu. Mol) is dissolved in a mixture of water/acetonitrile (2:1, v/v,0.90 mL). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative MP-LC. Will correspond to the product ^2C Fractions were immediately combined together, frozen and lyophilized overnight to give the title compound (25.6 mg, 85%) as a white solid. The purity based on LC-MS was 88%.

LRMS(m/z)：2101[M-405] ^1- ，2304[M-202] ^1- ，2507[M-1] ^1-

LC-MS r.t.(min)：1.90 ^1B (isomer induced double peaks)

Intermediate 12:

trivalent GalNAc-S-maleimide

Trivalent GalNAc-azide (20.3 mg, 12.0. Mu. Mol) and DBCO-maleimide (10.3 mg, 24.0. Mu. Mol) were dissolved in a mixture of water/acetonitrile (2:1, v/v,0.90 mL). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative MP-LC. Will correspond to the product ^2D Fractions were immediately combined together, frozen and lyophilized overnight to give the title compound (22.2 mg, 87%) as a white solid. Purity was 85% based on LC-MS. Contains 10% hydrolyzed maleimide.

LRMS(m/z)：2115[M-1] ^1-

LC-MS r.t.(min)：1.60 ^1B (isomer induced double peaks)

Synthesis of trivalent GalNAc-S-ApoB (or HSP 27) BNA oligonucleotides

To ApoB BNA oligo disulfide (5.00 mg, 0.686. Mu. Mol) was added a solution of 20mM ammonium bicarbonate with 2.5mM TCEP (1.00 mL, 2.5. Mu. Mol). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 1 hour, the reaction mixture was filtered (5000 Xg for 30min, 2X 0.50 mL) by using a centrifugal filter with a molecular weight cut-off of 3000 Da. Next, the residue solution was washed twice with a solution of 20mM ammonium bicarbonate with 2.5mM TCEP (0.50 mL), each time filtered under the same conditions as described above. As follows, the residue solution was diluted with 20mM ammonium bicarbonate/acetonitrile (3:1, v/v,1.00 mL) and the resulting solution was directly added to trivalent GalNAc-S-maleimide (3.02 mg, 1.43. Mu. Mol). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 1.5 hours, the reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^4A Fractions were immediately combined, frozen and lyophilized overnight to give the title compound (6.75 mg, quantitative) as a white fluffy solid. The purity based on LC-MS was 94% (very broad peak). ApoB BNA sequence is shown in SEQ ID NO. 2.

LRMS(m/z)：2318[M-4] ^4-

LC-MS r.t.(min)：1.65 ^1A

Synthesis of trivalent GalNAc-S-ApoB (or HSP 27) scrambled BNA oligonucleotides

To ApoB scrambled BNA oligo disulphide (5.00 mg, 0.688. Mu. Mol) was added a solution of 20mM ammonium bicarbonate with 2.5mM TCEP (1.00 mL, 2.5. Mu. Mol). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 1 hour, the reaction mixture was filtered by using a centrifugal filter with a molecular weight cut-off of 3000Da (5000 Xg for 30min,2X 0.50 mL). Next, the residue solution was washed twice with a solution of 20mM ammonium bicarbonate with 2.5mM TCEP (0.50 mL), each time filtered under the same conditions as described above. As follows, the residue solution was diluted with 20mM ammonium bicarbonate/acetonitrile (3:1, v/v,1.00 mL), and the resulting solution was directly added to trivalent GalNAc-S-maleimide (3.09 mg, 1.46. Mu. Mol). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 1.5 hours, the reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^4A Fractions were immediately combined, frozen and lyophilized overnight to give the title compound as a white fluffy solid (5.91 mg, 93%). The purity based on LC-MS was 88% (very broad peak).

LRMS(m/z)：2311[M-4] ^4-

LC-MS r.t.(min)：1.60 ^1A

Synthesis of trivalent GalNAc-L-ApoB (or HSP 27) BNA oligonucleotides

To ApoB BNA oligo disulfide (5.00 mg, 0.686. Mu. Mol) was added a solution of 20mM ammonium bicarbonate with 2.5mM TCEP (1.00 mL, 2.5. Mu. Mol). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 1 hour, the reaction mixture was filtered (5000 Xg for 30min, 2X 0.50 mL) by using a centrifugal filter with a molecular weight cut-off of 3000 Da. Next, the residue solution was washed twice with a solution of 20mM ammonium bicarbonate with 2.5mM TCEP (0.50 mL), each time filtered under the same conditions as described above. As follows, the residue solution was diluted with 20mM ammonium bicarbonate/acetonitrile (3:1, v/v,1.00 mL), and the resulting solution was directly added to trivalent GalNAc-L-maleimide (3.63 mg, 1.45. Mu. Mol). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 1.5 hours, the reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^4A Fractions were immediately combined, frozen and lyophilized overnight to give the title compound (6.68 mg, quantitative) as a white fluffy solid. The purity based on LC-MS was 99% (very broad peak).

LRMS(m/z)：2416[M-4] ^4-

LC-MS r.t.(min)：1.97 ^1A

Synthesis of trivalent GalNAc-L-ApoB (or HSP 27) scrambled BNA oligonucleotides

To ApoB scrambled BNA oligo disulphide (5.00 mg, 0.688. Mu. Mol) was added a solution of 20mM ammonium bicarbonate with 2.5mM TCEP (1.00 mL, 2.5. Mu. Mol). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 1 hour, the reaction mixture was filtered (5000 Xg for 30min, 2X 0.50 mL) by using a centrifugal filter with a molecular weight cut-off of 3000 Da. Next, the residue solution was washed twice with a solution of 20mM ammonium bicarbonate with 2.5mM TCEP (0.50 mL), each time filtered under the same conditions as described above. As follows, the residue solution was diluted with 20mM ammonium bicarbonate/acetonitrile (3:1, v/v,1.00 mL) and the resulting solution was directly added to trivalent GalNAc-L-maleimide (3.68 mg, 1.47. Mu. Mol). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 1.5 hours, the reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^4A Fractions were immediately combined together, frozen and lyophilized overnight to give the title compound as a white fluffy solid (4.71 mg, 71%). The purity based on LC-MS was 96% (very broad peak).

LRMS(m/z)：2409[M-4] ^4-

LC-MS r.t.(min)：1.93 ^1A

Dendrite (-L-SO 1861) ₄ Trivalent GalNAc (FIG. 23D-E) and dendrite (-L-SO 1861) ₈ Synthesis of trivalent GalNAc

Intermediate 13:

trivalent GalNAc-carbamates

Trivalent GalNAc-azide ((molecule 21) is intermediate 7 shown in FIG. 8G) (36.5 mg, 21.6. Mu. Mol) was dissolved in a solution of potassium carbonate (5.97 mg, 43.2. Mu. Mol) in water (1.00 mL) and acetonitrile (1.00 mL). Next, a 1.0M solution of trimethylphosphine in THF (216. Mu.L, 216. Mu. Mol) was added, and the resulting mixture was shaken for 1min and allowed to stand at room temperature. After 45min, the reaction mixture was evaporated in vacuo and the residue was dissolved in water/acetonitrile (9:1, v/v,1 mL). The resulting solution was directly subjected to preparative MP-LC. Will correspond to the product ^2B Fractions were immediately pooled together, frozen and lyophilized overnight to give the title as a white solidCompound (36.1 mg, 98%). The purity based on LC-MS was 100%.

LRMS(m/z)：1662[M-1] ^1-

LC-MS r.t.(min)：1.62 ^1A

Intermediate 14:

trivalent GalNAc-DBCO

Trivalent GalNAc-amine formate (17.4 mg, 10.2. Mu. Mol) and DBCO-NHS (6.14 mg, 15.3. Mu. Mol) were dissolved in NMM (2.24. Mu.L, 20.3. Mu. Mol) in DMF (0.50 mL). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 2 hours, the reaction mixture was evaporated in vacuo and the residue was dissolved in water/acetonitrile (8:2, v/v,1 mL). The resulting solution was directly subjected to preparative MP-LC. Will correspond to the product ^2C Fractions were immediately combined together, frozen and lyophilized overnight to give the title compound (14.2 mg, 72%) as a white solid. The purity based on LC-MS was 96%.

LRMS(m/z)：1950[M-1] ¹

LC-MS r.t.(min)：1.86 ^1B

Intermediate 15:

dendrite (-L-SO 1861) ₈ Azide compounds

Dendritic (-L-SO 1861) ₈ -amine (19.6 mg, 1.08. Mu. Mol) and 2, 5-dioxopyrrolidin-1-yl-1-azido-3, 6,9, 12-tetraoxapentadecane-15-oic acid ester (4.17 mg, 10.8. Mu. Mol) were dissolved in DMF (1.50 mL). Next, DIPEA (1.87. Mu.L, 10.8. Mu. Mol) was added, and the mixture was shaken for 1min and allowed to stand at room temperature overnight. The reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^4B Fractions were immediately combined, frozen and lyophilized overnight to give the title compound (11.7 mg, 59%) as a white fluffy solid. The purity based on LC-MS was 92% (very broad peak).

LRMS(m/z)：2316[M-8] ^8- ，2647[M-7] ^7-

LC-MS r.t.(min)：4.29 ^1A

Dendrite (-L-SO 1861) ₄ Synthesis of trivalent GalNAc

Dendritic (-L-SO 1861) ₄ Azide (2.5)0mg, 0.266. Mu. Mol) and trivalent GalNAc-DBCO (1.56 mg, 0.799. Mu. Mol) were dissolved in a mixture of water/acetonitrile (3:1, v/v,1.00 mL). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^4B Fractions were immediately combined, frozen and lyophilized overnight to give the title compound (2.74 mg, 91%) as a white fluffy solid. The purity based on LC-MS was 86% (very broad peak).

LRMS(m/z)：2832[M-4] ^4-

LC-MS r.t.(min)：4.07 ^1A

Dendrite (-L-SO 1861) ₈ Synthesis of trivalent GalNAc

Dendritic (-L-SO 1861) ₈ Azide (2.50 mg, 0.135. Mu. Mol) and trivalent GalNAc-DBCO (0.79 mg, 0.405. Mu. Mol) are dissolved in a mixture of water/acetonitrile (3:1, v/v,1.00 mL). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^4B Fractions were immediately combined, frozen and lyophilized overnight to give the title compound (2.03 mg, 74%) as a white fluffy solid. The purity based on LC-MS was 100% (very broad peak).

LRMS(m/z)：2559[M-8] ^8- ，2925[M-7] ^7-

LC-MS r.t.(min)：4.18 ^1A

Dendrite (-L-SO 1861) ₄ -L-BNA oligonucleotide-trivalent GalNAc and dendrite (-L-SO 1861) ₈ Synthesis of L-BNA oligonucleotide-trivalent GalNAc

Intermediate 16:

trivalent GalNAc-thioacetate

Trivalent GalNAc-amine formate (18.7 mg, 11.0. Mu. Mol) and 4-nitrophenyl 3- (acetylthio) propionate (5.90 mg, 21.9. Mu. Mol) were dissolved in a solution of NMM (2.41. Mu.L, 21.9. Mu. Mol) in DMF (0.50 mL). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 2 hours, the reaction mixture was evaporated in vacuo and the residue was dissolved in a mixture of 0.1% formic acid in water and 0.1% formic acid in acetonitrile (9:1, v/v,1 mL) Is a kind of medium. The resulting solution was directly subjected to preparative MP-LC. Will correspond to the product ^2B Fractions were immediately combined, frozen and lyophilized overnight to give the title compound as a white solid (13.0 mg, 66%). The purity based on LC-MS was 100%.

LRMS(m/z)：1749[M-43] ^1- ，1792[M-1] ¹

LC-MS r.t.(min)：1.24 ^1B

Intermediate 17:

DBCO-TCO-trivalent GalNAc

Trivalent GalNAc-thioacetate (13.0 mg, 7.25. Mu. Mol) was dissolved in methanol (0.50 mL). Next, 1M sodium hydroxide solution (7.98. Mu.L, 7.98. Mu. Mol) was added. The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 30min, the reaction mixture was added to a solution of freshly prepared trifunctional linker (Ls-t) (i.e., as an example of a saponin moiety linker Ls) (7.39 mg, 6.13. Mu. Mol) in 20mM ammonium bicarbonate/acetonitrile (3:1, v/v,2.00 mL).

The trifunctional linker has IUPAC name: 5,8,11,18,21,24,27-heptaoxa-2,14,30-triaza-triacontanoic acid, 14- [16- (11, 12-didehydrodibenzo [ b, f ] azepin-5 (6H) -yl) -13, 16-dioxo-3, 6, 9-trioxa-12-azahexadecan-1-yl ] -33- (2, 5-dihydro-2, 5-dioxo-1H-pyrrol-1-yl) -15, 31-dioxo-, (1R, 4E) -4-cycloocten-1-yl ester. The trifunctional linker has the following formula (XXI):

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the resulting mixture was shaken for 1min and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative MP-LC. Will correspond to the product ^2A Fractions were immediately combined together, frozen and lyophilized overnight to give the title compound as a white solid (3.83 mg, 21%). The purity based on LC-MS was 89%.

LRMS(m/z)：2409[M-546] ^1- ，2955[M-1] ^1-

LC-MS r.t.(min)：2.66 ^1B

Intermediate 18:

Methyltetrazine-L-ApoB BNA oligonucleotides

To ApoB BNA oligo disulfide (5.00 mg, 0.686. Mu. Mol) was added a solution of 20mM ammonium bicarbonate with 2.5mM TCEP (1.00 mL, 2.5. Mu. Mol). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 1 hour, the reaction mixture was filtered (5000 Xg for 30min, 2X 0.50 mL) by using a centrifugal filter with a molecular weight cut-off of 3000 Da. Next, the residue solution was washed twice with a solution of 20mM ammonium bicarbonate with 2.5mM TCEP (0.50 mL), each time filtered under the same conditions as described above. As follows, the residue solution was diluted with 20mM ammonium bicarbonate (1.50 mL) and the resulting mixture was added directly to a solution of (E) -1- (4- ((2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hydrazino) methyl) benzamide) -N- (4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) benzyl) -3,6,9, 12-tetraoxapentadecane-15-amide (1.36 mg,1.73 μmol) in acetonitrile (0.5 mL). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 30min, the reaction mixture was frozen and lyophilized overnight to give the crude title product as a pink fluffy solid. To the crude product was added a solution of 20mM ammonium bicarbonate (1.50 mL) and the resulting suspension was filtered through a 0.45 μm syringe filter. The filtrate was lyophilized overnight to give the title product as a pink fluffy solid (5.44 mg, quantitative). The purity based on LC-MS was 90% (very broad peak).

LRMS(m/z)：2648[M-3] ^3-

LC-MS r.t.(min)：0.62 ⁴

Intermediate 19:

Methyltetrazine-L-ApoB scrambled BNA oligonucleotides

To ApoB scrambled BNA oligo disulphide (5.00 mg, 0.688. Mu. Mol) was added a solution of 20mM ammonium bicarbonate with 2.5mM TCEP (1.00 mL, 2.5. Mu. Mol). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 1 hour, the reaction mixture was filtered (5000 Xg for 30min, 2X 0.50 mL) by using a centrifugal filter with a molecular weight cut-off of 3000 Da. Next, the residue solution was washed twice with a solution of 20mM ammonium bicarbonate with 2.5mM TCEP (0.50 mL), each time filtered under the same conditions as described above. As follows, the residue solution was diluted with 20mM ammonium bicarbonate (1.50 mL) and the resulting mixture was added directly to a solution of (E) -1- (4- ((2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hydrazino) methyl) benzamide) -N- (4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) benzyl) -3,6,9, 12-tetraoxapentadecane-15-amide (1.34 mg,1.70 μmol) in acetonitrile (0.5 mL). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 30min, the reaction mixture was frozen and lyophilized overnight to give the crude title product as a pink fluffy solid. To the crude product was added a solution of 20mM ammonium bicarbonate (1.50 mL) and the resulting suspension was filtered through a 0.45 μm syringe filter. The filtrate was lyophilized overnight to give the title product as a pink fluffy solid (5.48 mg, quantitative). The purity based on LC-MS was 84% (very broad peak).

LRMS(m/z)：2639[M-3] ^3-

LC-MS r.t.(min)：0.58 ⁴

Intermediate 20:

DBCO-L-ApoB BNA oligonucleotide-trivalent GalNAc

To methyltetrazine-L-ApoB BNA oligonucleotide (5.44 mg, 0.684. Mu. Mol) and DBCO-TCO-trivalent GalNAc (2.03 mg, 0.687. Mu. Mol) was added 20mM ammonium bicarbonate/acetonitrile (3:1, v/v,1.00 mL). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^4C Fractions were immediately combined, frozen and lyophilized overnight to give the title compound as a white fluffy solid (2.87 mg, 39%). LC-MS shows a plurality of broad peaks with correct m/z values.

LRMS(m/z)：2175[M-5] ^5- ,2718[M-6] ^4-

LC-MS r.t.(min)：2.60-3.00 ^1A

Intermediate 21:

DBCO-L-ApoB scrambled BNA oligonucleotide-trivalent GalNAc

To methyl tetrazine-L-ApoB scrambled BNA oligonucleotide (5.48 mg, 0.692. Mu. Mol) and DBCO-TCO-trivalent GalNAc (1.80 mg, 0.609. Mu. Mol) was added 20mM ammonium bicarbonateAcetonitrile (3:1, v/v,1.00 mL). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^4C Fractions were immediately combined, frozen and lyophilized overnight to give the title compound as a white fluffy solid (2.15 mg, 33%). LC-MS shows a plurality of broad peaks with correct m/z values.

LRMS(m/z)：2169[M-5] ^5- ，2711[M-6] ^4-

LC-MS r.t.(min)：2.58-3.20 ^1A

Dendrite (-L-SO 1861) ₄ Synthesis of L-ApoB BNA oligonucleotide-trivalent GalNAc (molecule 34, FIG. 24D)

Trivalent GalNAc (1.49 mg, 0.137. Mu. Mol; molecule 31, FIG. 24C) and dendrites (-L-SO 1861) toward DBCO-L-ApoB BNA oligonucleotide ₄ Azide (1.33 mg, 0.142. Mu. Mol; molecule 19, FIG. 23D; dendrite (-L-SO 1861) ₄ Azide is a dendrimer (SPT 001) ₄ -NH2 (molecule 15, fig. 23C), wherein the amine dendron was treated with azido-PEG 4-NHs ester (molecule 18) with DIPEA as base in DMF was added 20mM ammonium bicarbonate/acetonitrile (3:1, v/v,0.5 mL) to make the dendron (-L-SO 1861) ₄ L-ApoB BNA oligonucleotide-trivalent GalNAc (molecule 34; 'trivalent GalNAc-ApoB BNA-SPT001 conjugate') was synthesized (FIG. 24D). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 3 hours, the reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^4B Fractions were immediately combined, frozen and lyophilized overnight to give the title compound as a white fluffy solid (0.96 mg, 35%). The purity based on LC-MS was 93% (very broad peak).

LRMS(m/z)：2025[M-10] ^10- ，2250[M-9] ^9- ，2532[M-8] ^8- ，2893[M-7] ^7-

LC-MS r.t.(min)：3.68 ^1A

The DBCO-L-ApoB BNA oligonucleotide-trivalent GalNAc (molecule 31) was synthesized by conjugating the methyltetrazine-L-ApoB BNA oligonucleotide (molecule 30) with the DBCO-TCO-trivalent GalNAc (molecule 29) (FIGS. 24A-B and 24C), wherein the DBCO-TCO-trivalent GalNAc (molecule 29) was synthesized with molecule 27 and molecule 28 (trifunctional linker), wherein molecule 27 was GalNAc-thioacetate (FIGS. 24A-B) obtained from the reaction between molecule 22, formate of trivalent GalNAc-amine, and molecule 26 (4-nitrophenyl 3- (acetylthio) propionate).

ApoB BNA, trivalent GalNAc and SPT001 (SO 1861) were conjugated into a single covalent conjugate using a trifunctional linker (FIG. 24D).

Using a method similar to the synthesis of dendrite (-L-SO 1861) 4-L-ApoB BNA oligonucleotide-trivalent GalNAc (molecule 34; 'trivalent GalNAc-ApoB BNA-SPT001 conjugate'), a conjugate was synthesized for control test purposes, in which ApoB BNA was replaced with BNA having a scrambled ApoB nucleic acid sequence.

The conjugate dendrite (-L-SO 1861) 8-L-ApoB BNA oligonucleotide-trivalent GalNAc was synthesized using a similar method to the synthesis of dendrite (-L-SO 1861) 4-L-ApoB BNA oligonucleotide-trivalent GalNAc (molecule 34; 'trivalent GalNAc-ApoB BNA-SPT001 conjugate'), wherein the ApoB BNA oligonucleotide was either ApoB BNA for silencing mRNA/protein expression or BNA with a scrambled ApoB nucleic acid sequence.

SO1861-L-ApoB BNA oligonucleotide-trivalent GalNAc Synthesis (trivalent GalNAc-ApoB BNA-SPT001 conjugate, FIGS. 22A-C)

Referring to fig. 22A-C, a conjugate comprising trivalent GalNAc, saponin SO1861 and ApoB BNA was synthesized using a trivalent linker TFL having the following molecular structure:

conjugate SO1861-L-ApoB BNA oligonucleotide-trivalent GalNAc synthesis (trivalent GalNAc-ApoB BNA-SPT001 conjugate) was formed.

Dendrite (-L-SO 1861) ₈ -L-ApoB BNA oligonucleotide-trivalent GalNAc synthesis

To DBCO-L-ApoB BNA oligonucleotide-trivalent GalNAc (1.38 mg, 0.127. Mu. Mol) and dendrite (-L-SO 1861) ₈ To the azide (2.51 mg, 0.135. Mu. Mol) was added 20mM ammonium bicarbonate/acetonitrile (3:1, v/v,0.5 mL). The reaction mixture was shaken for 1min and allowed to stand at room temperature.After 4 hours, the reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^4B Fractions were immediately combined, frozen and lyophilized overnight to give the title compound as a white fluffy solid (0.75 mg, 20%). The purity based on LC-MS was 99% (very broad peak).

LRMS(m/z)：2099[M-14] ^14- ，2261[M-13] ^13- ，2450[M-12] ^12- ，2672[M-11] ^11- ，2940[M-10] ^10-

LC-MS r.t.(min)：3.90 ^1A

Dendrite (-L-SO 1861) ₄ -L-ApoB-scrambled BNA oligonucleotide-trivalent GalNAc synthesis

BNA oligonucleotide-trivalent GalNAc (1.09 mg, 0.100. Mu. Mol) and dendrite (-L-SO 1861) were scrambled to DBCO-L-ApoB ₄ To the azide (0.96 mg, 0.102. Mu. Mol) was added 20mM ammonium bicarbonate/acetonitrile (3:1, v/v,0.5 mL). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 3 hours, the reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^4B Fractions were immediately combined together, frozen and lyophilized overnight to give the title compound as a white fluffy solid (0.45 mg, 22%). LC-MS shows a plurality of broad peaks with correct m/z values.

LRMS(m/z)：2023[M-10] ^10- ，2248[M-9] ^9- ，2528[M-8] ^8- ，2890[M-7] ^7-

LC-MS r.t.(min)：3.60-4.00 ^1A

Synthesis of anti-CD 71-saporin

Custom CD71 mab-saporin conjugates were produced and purchased from advanced targeting systems company (Advanced Targeting Systems) (san diego, california). InVivoMab grade CD71 monoclonal antibody was purchased from BioXcell company (anti-human CD71, clone OKT-9).

HSP27BNA, apoB and ApoB _Scrambling Oligonucleotide sequences

HSP27(5’-GGCacagccagtgGCG-3’)[SEQ ID NO:1](modified from Zhang et al (2011) [ Y Zhang, Z Qu, S Kim, V Shi, B Liao1, P Kraft, R Bandaru, Y Wu, LM Greenberger and ID Horak, down-modulation of cancer targets using Locked Nucleic Acid (LNA) -B ]ased antisense oligonucleotides without transfection [ use of Locked Nucleic Acid (LNA) -based antisense oligonucleotides to down-regulate cancer targets without transfection]Gene Therapy](2011)18,326-333]The antisense BNA (HSP 27) of (B) is BNA [. About. ^NC ) Having the oligonucleotide nucleic acid sequence 5'-GGCacagccagtgGCG-3'), apoB (5'-GCCTCagtctgcttcGCACC-3') [ SEQ ID NO:2]And ApoB _Scrambling (5’-GGCCTctctacaccgCTCGT-3’)[SEQ ID NO:3]BNA oligonucleotides were ordered from biosynthesis company (Bio-Synthesis Inc.) (Lewis ville, texas), containing a 5' -thiol C6 linker.

Human primary hepatocyte treatment

Cryopreserved primary human hepatocytes (cytobiotechnology limited (Cytes Biotechnologies s.l.), spanish) were thawed in hepatocyte thawing medium (cytobiotechnology limited, spanish). The cells were then resuspended in hepatocyte plating medium (cell biotechnology limited, spain). Cells were then seeded onto collagen I coated plates at a density of 215.600 cells/well or 66.600 cells/well for 48 or 96 well plates (Greiner bione). Cells were pre-incubated at 37 ℃ for 4-6 hours before starting the treatment to allow cells to attach to the cell culture plates. The plating medium was replaced with 315 μl or 108 μl of maintenance medium (cell biotechnology Co., ltd., spain) followed by the addition of conjugate from a 10-fold concentrated stock solution in PBS. Plates were incubated at 37 ℃ for 72 hours and harvested for gene expression and cell viability analysis.

RNA isolation and Gene expression analysis from human cell cultures

RNA from cells was isolated and analyzed according to standard protocols (Biorad). The qPCR primers used are indicated in table A2.

The primers used in table a2.Qpcr are shown below:

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cell viability assay (MTS)

After addition of the compound, the cells were incubated at 37℃for 72 hours and then passed through a cell filter according to the manufacturer's instructions (CellTiterAQueuus single solution cell proliferation assay, promega), MTS assay performed by Promega, determines cell viability. Briefly, MTS solution was diluted 20-fold in DMEM without phenol Red (PAN-Biotech GmbH) supplemented with 10% FBS. Cells were washed once with 200 μl PBS per well, and then 100 μl/well diluted MTS solution was added per well. Plates were incubated at 37℃for about 20-30 minutes. Subsequently, OD at 492nm was measured on a Multiskan FC plate reader from Siemens technologies (Thermo Scientific). For quantification, the background signal of the 'medium only' wells was subtracted from all other wells, and then the relative percent cell viability percentage was calculated by dividing the background correction signal of the composite treated wells by the background correction signal of the untreated wells (x 100 is percent).

FACS analysis

For each cell line, cells were seeded at appropriate density in DMEM (PAN-Biotech GmbH) supplemented with 10% fetal bovine serum (PAN-Biotech GmbH) and 1% penicillin/streptomycin (PAN-Biotech GmbH) in T75 flasks. Cultures were incubated for 72-96 hours (5% CO2, 37 ℃) until 90% confluence was achieved. Next, cells were trypsinized (Tryple Express, ji Buke Semer technologies (Gibco Thermo Scientific)) to obtain a single cell suspension, transferred to a 15mL falcon tube and centrifuged (1,400 rpm,3 min). The supernatant was discarded while the cell pellet was submerged. 500.000 cells were transferred to round bottom FACS tubes and plated with 3mL cold DPBS (Mg-free ²⁺ And Ca ^{2 +} 2% fbs). Cells were centrifuged at 1800rpm at 4℃for 3min and resuspended in 200. Mu.L cold DPBS (Mg-free) ²⁺ And Ca ²⁺ 2% fbs) or 200 μl of antibodiesSolution (containing a solution of cold DPBS (Mg-free) in 195. Mu.L ²⁺ And Ca ²⁺ 5 μl of antibody in 2% fbs). Transferrin receptors were stained with PE anti-human CD71 (accession number 334106, biolegend (bioleged)), and PE mouse IgG2a, kappa isotype control FC (accession number 400212, biolegend) was used as its matched isotype control. ASGPR1 receptor was stained with PE anti-human ASGPR1 (accession No. 130-122-963, miltenyi) and PE mouse IgG1, isotype control (accession No. 130-113-762, meitenji company) was used as its matched isotype control. The samples were incubated at 4℃for 30min. Thereafter using cold DPBS (Mg-free ²⁺ And Ca ²⁺ Cells were washed 2 times with 2% fbs and used at room temperature in DPBS (Mg-free ²⁺ And Ca ²⁺ 2% pfa solution in 2% fbs) for 20min. Cells were washed 1 time with cold DPBS and resuspended in 1000 μl cold DPBS for FACS analysis. Samples were analyzed using the Cube 8 flow cytometer system (cisseneck) from cisseneck and FCS Express 7 study software. The results of FACS analysis are summarized in table A3.

Table A3. Levels of expression of cell membrane receptors for ASGPR1 and CD71 in HepG2 and Huh7 cells

Example 4

Materials and methods

Abbreviations (abbreviations)

AEM N- (2-aminoethyl) maleimide trifluoroacetate salt

AMPD 2-amino-2-methyl-1, 3-propanediol

BOP (benzotriazol-1-yloxy) tris (dimethylamino) hexafluorophosphate

DIPEA N, N-diisopropylethylamine

DMF N, N-dimethylformamide

DTME dithiobismaleimide ethane

DTT dithiothreitol

EDCI. HCl 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride

EDTA ethylenediamine tetraacetic acid

EMCH. TFA N- (epsilon-maleimidocaprooic acid) hydrazide, trifluoroacetate salt

HATU 1- [ bis (dimethylamino) methylene ] -1H-1,2, 3-triazolo [4,5-b ] pyridinium 3-oxide hexafluorophosphate

min

NMM 4-methylmorpholine

r.t. retention time

SEC size exclusion chromatography

TBEU (tris (hydroxymethyl) aminomethane) -borate-EDTA-urea

TCEP tris (2-carboxyethyl) phosphine hydrochloride

Temp temperature

TFA trifluoroacetic acid

TFL trifunctional linkers

Analysis method

LC-MS method 1

The device comprises: waters IClass; binary pump: UPIBSM, SM: UPISMFTN with SO; UPCMA, PDA: UPPDATC,210-320nm, SQD: the mass range of ACQ-SQD2 ESI depends on the molecular weight of the product: 1500-2400 or 2000-3000; ELSD: gas pressure 40psi, drift tube temp:50 ℃; column: acquity C18, 50×2.1mm,1.7 μm; temperature: 60 ℃, flow rate: 0.6mL/min, linear gradient depending on polarity of the product:

^A t ₀ ＝2％A，t _5.0min ＝50％A，t _6.0min ＝98％A

^B t ₀ ＝2％A，t _5.0min ＝98％A，t _6.0min ＝98％A

post run time: 1.0min, eluent A: acetonitrile, eluent B: 10mM ammonium bicarbonate in water (ph=9.5).

LC-MS method 2

The device comprises: waters IClass; binary pump: UPIBSM, SM: UPISMFTN with SO; UPCMA, PDA: UPPDATC,210-320nm, SQD: the mass range of ACQ-SQD2 ESI depends on the molecular weight of the product: 100-800 parts of yang/yin or 2000-3000 parts of yin; ELSD: air flowBody pressure 40psi, drift tube temp:50 ℃; column: waters XSelect ^TM CSH C18, 50X 2.1mm,2.5 μm, temperature: 25 ℃, flow rate: 0.5mL/min, gradient: t is t _0min ＝5％A，t _2.0min ＝98％A，t _2.7min =98%a, post run time: 0.3min, eluent a: acetonitrile, eluent B: 10mM ammonium bicarbonate in water (ph=9.5).

LC-MS method 3

The device comprises: waters IClass; binary pump: UPIBSM, SM: UPISMFTN with SO; UPCMA, PDA: UPPDATC,210-320nm, SQD: ACQ-SQD2 ESI, the mass range depends on the molecular weight of the product, positive/negative 105-800, 500-1200 or 1500-2500; ELSD: gas pressure 40psi, drift tube temp:50 ℃; column: waters XSelect ^TM CSH C18, 50X 2.1mm,2.5 μm, temperature: 40 ℃, flow rate: 0.5mL/min, gradient: t is t _0min ＝5％A，t _2.0min ＝98％A，t _2.7min =98%a, post run time: 0.3min, eluent a: 0.1% formic acid in acetonitrile, eluent B: 0.1% formic acid in water.

LC-MS method 4

The device comprises: waters IClass; binary pump: UPIBSM, SM: UPISMFTN with SO; UPCMA, PDA: UPPDATC,210-320nm, SQD: the mass range of ACQ-SQD2 ESI depends on the molecular weight of the product: 100-800 parts of yang/yin or 2000-3000 parts of yin; ELSD: gas pressure 40psi, drift tube temp:50 ℃, column: waters Acquity Shield RP18, 50×2.1mm,1.7 μm, temperature: 25 ℃, flow rate: 0.5mL/min, gradient: t is t _0min ＝5％A，t _2.0min ＝98％A，t _2.7min =98%a, post run time: 0.3min, eluent a: acetonitrile, eluent B: 10mM ammonium bicarbonate in water (ph=9.5).

Preparation method

Preparation MP-LC method 1

Instrument type: reveleris ^TM Preparing MPLC; column: waters XSelect ^TM CSH C18 (145X 25mm,10 μm); flow rate: 40mL/min; column temperature: room temperature; eluent A:10 mM ammonium bicarbonate in water (ph=9.0); eluent B:99% acetonitrile + 1%10mm ammonium bicarbonate in water; gradient:

^A t _0min ＝5％B，t _1min ＝5％B，t _2min ＝10％B，t _17min ＝50％B，t _18min ＝

100％B，t _23min ＝100％B

^A t _0min ＝5％B，t _1min ＝5％B，t _2min ＝20％B，t _17min ＝60％B，t _18min ＝

100％B，t _23min ＝100％B；

and (3) detecting UV: 210. 235, 254nm and ELSD.

Preparation MP-LC method 2

Instrument type: reveleris ^TM Preparing MPLC; column: phenomenex LUNAC18 (3) (150X 25mm,10 μm); flow rate: 40mL/min; column temperature: room temperature; eluent A: 0.1% (v/v) formic acid in water, eluent B: 0.1% (v/v) formic acid in acetonitrile; gradient:

^A t _0min ＝5％B，t _1min ＝5％B，t _2min ＝20％B，t _17min ＝60％B，t _18min ＝100％B，t _23min ＝100％B

^B t _0min ＝2％B，t _1min ＝2％B，t _2min ＝2％B，t _17min ＝30％B，t _18min ＝100％B，t _23min ＝100％B

^C t _0min ＝5％B，t _1min ＝5％B，t _2min ＝10％B，t _17min ＝50％B，t _18min ＝100％B，t _23min ＝100％B

^D t _0min ＝5％B，t _1min ＝5％B，t _2min ＝5％B，t _17min ＝40％B，t _18min ＝100％B，t _23min ＝100％B；

and (3) detecting UV: 210. 235, 254nm and ELSD.

Preparation LC-MS method 3

MS instrument type: agilent Technologies G6130B Quadrarupole; HPLC instrument type: agilent Technologies 1290 preparative LC; column: waters XSelect ^TM CSH (C18, 150X 19mm,10 μm); flow rate: 25ml/min; column temperature: room temperature; eluent A:100% acetonitrile; eluent B: 10mM ammonium bicarbonate in water, ph=9.0; gradient:

^A t ₀ ＝20％A，t _2.5min ＝20％A，t _11min ＝60％A，t _13min ＝100％A，t _17min ＝100％A

^B t ₀ ＝5％A，t _2.5min ＝5％A，t _11min ＝40％A，t _13min ＝100％A，t17min＝100％A；

and (3) detection: DAD (210 nm); and (3) detection: MSD (ESI pos/neg) mass range: 100-800; DAD-based fraction collection.

Preparation LC-MS method 4

MS instrument type: agilent Technologies G6130B Quadrapol; HPLC instrument type: agilent Technologies 1290 preparative LC; column: waters XBridge Protein (C4, 150X 19mm,10 μm); flow rate: 25ml/min; column temperature: room temperature; eluent A:100% acetonitrile; eluent B: 10mM ammonium bicarbonate in water, ph=9.0; gradient:

^A t ₀ ＝2％A，t _2.5min ＝2％A，t _11mi n＝30％A，t _13min ＝100％A，t _17min ＝100％A

^B t ₀ ＝10％A，t _2.5min ＝10％A，t _11min ＝50％A，t _13min ＝100％A，t _17mi n＝100％A

^C t ₀ ＝5％A，t _2.5min ＝5％A， _t11min ＝40％A，t _13min ＝100％A，t17min＝100％A；

and (3) detection: DAD (210 nm); and (3) detection: MSD (ESI pos/neg) mass range: 100-800; DAD-based fraction collection.

Flash chromatography

Grace RevelerisC-815 is fast; solvent delivery system: a 3 piston pump with an automatic starting function, 4 independent channels, at most 4 solvents are operated at a time, and when the solvents are exhausted, the pipelines are automatically switched; maximum pump flow rate 250mL/min; maximum pressure 50 bar (725 psi); and (3) detection: UV 200-400nm, up to 4 UV signal combinations and scanning of the entire UV range, ELSD; column dimensions: 4-330g on the instrument, 750g to 3000g luer with optional stent.

UV-visible spectrophotometry

Protein concentration was determined using a Thermo Nanodrop 2000 spectrometer and an extinction coefficient ε 280 ((mg/ml) -1 cm-1); the concentration of the oligonucleotides was determined using an extinction coefficient ε260,000M-1 cm-1.

Ellman analysis was performed using a Perkin Elmer lambda 25 spectrophotometer and the molar extinction coefficient epsilon 41214150M-1cm-1 of TNB. For SAMSA-fluorescein, the experimentally determined molar extinction coefficients epsilon 495= 58,700M-1cm-1 and Rz 280:495=0.428 were used.

Size Exclusion Chromatography (SEC) analysis

Conjugates were analyzed by SEC using the Akta Purifier 10 system and Biosep SEC-s3000 column, eluting with DPBS: IPA (85:15). The purity of the conjugate is determined by integration of the conjugate peak uv signal over time with the integration of the integrated area of the impurity/aggregate forming peak.

SDS-PAGE and Western blotting

Native proteins and conjugates were analyzed against protein ladder by SDS-PAGE under heat denatured, non-reducing and reducing conditions using 4-12% bis-tris gels and MES as running buffers (200V, about 40 min). Samples were prepared at 0.5mg/ml, containing LDS sample buffer and MOPS running buffer as diluent. For the reduced samples, DTT was added to a final concentration of 50mM. The samples were heat treated at 90-95℃for 2 minutes and 5. Mu.g (10. Mu.l) was added to each well. Protein ladder (10 μl) was loaded without pretreatment. The wells were filled with 1 XLDS sample buffer (10. Mu.l). After gel running, wash three times with DI water (100 ml) with shaking (15 min, 200 rpm). Coomassie staining was performed by shaking incubation (60 min, 200 rpm) of the gel with pagebue protein stain (30 ml). Excess staining solution was removed, the gel was rinsed twice with DI water (100 ml) and decolorized with DI water (100 ml) (60 min, 200 rpm). The resulting gel was imaged and then further processed using ImageJ (Rasband, w.s., imageJ, national institute of health (u.s.national Institutes of Health), bescenda, maryland, usa).

TBEU-PAGE

Native proteins, conjugates and BNA standards were analyzed for oligonucleotide ladder by TBEU-PAGE under heat-denatured, non-reducing and reducing conditions using 15% TBE-urea gel and TBE as running buffer (180V, about 60 minutes). Sample preparation to 0.5mg/ml, BNA standard preparation to 20. Mu.g/ml, each containing TBE urea sample buffer and purified H as diluent ₂ O. The samples and standards were heat treated at 70℃for 3 minutes and 10. Mu.l of each well was added, corresponding to 5. Mu.g of protein and conjugate samples per lane and 0.2. Mu.g of BNA. The oligonucleotide ladder reconstituted to 0.1. Mu.g/band/ml in TE pH 7.5 (2. Mu.l) was loaded without pretreatment. After gel running, it was stained with freshly prepared ethidium bromide solution (1. Mu.g/ml) while shaking (40 min, 200 rpm). The resulting gel was visualized by UV epi-illumination (254 nm), imaged and then treated with ImageJ (Rasband, w.s., imageJ, national institute of health (u.s.national Institutes of Health), bescens da, maryland, usa).

MALDI-TOF-MS

MALDI-TOF spectra were recorded on a MALDI mass spectrometer (Bruker Ultrafex III). Typically, nanomolar to micromolar range samples dissolved in MilliQ water were spotted on targets (MTP 384 target plate polished steel T F, bruker Daltons) by dry drop method using super DHB (99%, fluka) or sinapic acid (SA, 99%, sigma-aldrich) as a matrix dissolved in acetonitrile (MADLI-TOF-MS test, sigma)/0.1% tfa (7:3 v/v). PepMix (peptide calibration standard, bruker Daltons) or protein mass (protein calibration standard, sigma-aldrich) was used as the calibration standard.

Trifunctional linker- (L-hydrazone-SO 1861) - (L-BNA oligonucleotide) - (trivalent-GalNAc) Synthesis (FIG. 35)

Trifunctional linkers (TFLs) have IUPAC names: 5,8,11,18,21,24,27-heptaoxa-2,14,30-triaza-triacontanoic acid, 14- [16- (11, 12-didehydrodibenzo [ b, f ] azepin-5 (6H) -yl) -13, 16-dioxo-3, 6, 9-trioxa-12-azahexadecan-1-yl ] -33- (2, 5-dihydro-2, 5-dioxo-1H-pyrrol-1-yl) -15, 31-dioxo-, (1R, 4E) -4-cycloocten-1-yl ester. The trifunctional linker has the following formula (XXI):

intermediate 1:

SO 1861-L-hydrazone-azide (molecule 3)

To SO1861 (60 mg,0.032mmol, molecule 1) and 1-azido-3, 6,9, 12-tetraoxapentadecane-15-hydrazide (39.3 mg,0.129mmol, molecule 2) were added methanol (extra dry, 1.00 mL) and TFA (9.86 μl,0.129 mmol), and the reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative MP-LC. Fractions 1 corresponding to the product were immediately pooled together, frozen and lyophilized overnight to give the title compound as a white fluffy solid (58.4 mg, 84%). Purity based on LC-MS was 99%.

LRMS(m/z)：2150[M-H] ^1-

LC-MS r.t.(min)：1.10 ^3B

Intermediate 2:

trifunctional linkers- (L-hydrazone-SO 1861) - (TCO) - (maleimide) (molecule 5)

A solution of TFL- (DBCO) - (TCO) - (maleimide) (15 mg, 12.5. Mu. Mol, molecule 4) in DMF (2.0 mL) was added to SO 1861-L-hydrazone-azide (26.8 mg, 12.5. Mu. Mol, molecule 3). The reaction mixture was shaken for 30 minutes and allowed to stand at room temperature. After 30 minutes, the reaction mixture was subjected to preparative MP-LC. Will correspond to the product ^1C Fractions were immediately pooled together, frozen and lyophilized overnight to give the title compound as a white fluffy solid (31.4 mg, 75%). LC-MS basedThe purity was 96%.

MS(m/z)：3354[M-H] ^1-

Intermediate 3:

trivalent GalNAc-thioacetate (molecule 8)

Trivalent GalNAc-amine formate (18.7 mg, 11.0. Mu. Mol, molecule 6) and 4-nitrophenyl 3- (acetylthio) propionate (5.90 mg, 21.9. Mu. Mol, molecule 7) were dissolved in a solution of NMM (2.41. Mu.L, 21.9. Mu. Mol) in DMF (0.50 mL). Trivalent GalNAc carbamates were obtained based on trivalent gallium azide as shown in fig. 8G as intermediate 7. In fig. 35B, formic acid of molecule 6 is not shown for simplicity. The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 2 hours, the reaction mixture was evaporated in vacuo and the residue was dissolved in a mixture of 0.1% formic acid in water and 0.1% formic acid in acetonitrile (9:1, v/v,1 mL). The resulting solution was directly subjected to preparative MP-LC. Will correspond to the product ^2B Fractions were immediately combined, frozen and lyophilized overnight to give the title compound as a white solid (13.0 mg, 66%). The purity based on LC-MS was 100%.

LRMS(m/z)：1749[M-43] ^1- ，1792[M-H] ¹

LC-MS r.t.(min)：1.24 ^1B

Intermediate 4：

Trifunctional linker- (L-SO 1861) - (TCO) - (trivalent GalNAc) (molecule 9)

Trivalent GalNAc-thioacetate (13.0 mg, 7.25. Mu. Mol, molecule 8) was dissolved in methanol (0.50 mL). Next, 1M sodium hydroxide solution (7.98. Mu.L, 7.98. Mu. Mol) was added. The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 30 minutes, the reaction mixture was added to a solution of freshly prepared TFL- (DBCO) - (TCO) - (maleimide) (20.6 mg, 6.13. Mu. Mol, molecule 5) in 20mM ammonium bicarbonate/acetonitrile (3:1, v/v,2.00 mL).

The resulting mixture was shaken for 1min and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative MP-LC. Will correspond to the product ^2A Fractions were immediately pooled together, frozen and lyophilizedNight to give the title compound as a white solid (3.83 mg, 21%). The purity based on LC-MS was 89%.

MS(m/z)：5106[M+H] ⁺

Intermediate 5:

Methyltetrazine-L-ApoB BNA oligonucleotides (molecule 12)

To ApoB#02BNA oligo disulfide (3.3 mg, 0.686. Mu. Mol, molecule 10) was added a solution of 20mM ammonium bicarbonate with 2.5mM TCEP (1.00 mL, 2.5. Mu. Mol). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 1 hour, the reaction mixture was filtered (5000 Xg for 30min, 2X 0.50 mL) by using a centrifugal filter with a molecular weight cut-off of 3000 Da. Next, the residue solution was washed twice with a solution of 20mM ammonium bicarbonate with 2.5mM TCEP (0.50 mL), each time filtered under the same conditions as described above. As follows, the residue solution was diluted with 20mM ammonium bicarbonate (1.50 mL) and the resulting mixture was added directly to a solution of (E) -1- (4- ((2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hydrazino) methyl) benzamide) -N- (4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) benzyl) -3,6,9, 12-tetraoxapentadecane-15-amide (1.36 mg,1.73 μmol, molecule 11) in acetonitrile (0.5 mL). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 30min, the reaction mixture was frozen and lyophilized overnight to give the crude title product as a pink fluffy solid. To the crude product was added a solution of 20mM ammonium bicarbonate (1.50 mL) and the resulting suspension was filtered through a 0.45 μm syringe filter. The filtrate was lyophilized overnight to give the title product as a pink fluffy solid (3.4 mg, 89%). Purity based on LC-MS was 90%.

MS(m/z)：5579[M+Na] ⁺

Trifunctional linker- (L-hydrazone-SO 1861) - (L-BNA oligonucleotide) - (trivalent GalNAc) (molecule 13)

TFL- (L-hydrazone-SO 1861) - (TCO) - (trivalent GalNAc) (16.1 mg, 3.15. Mu. Mol, molecule 9) and methyltetrazine-BNA oligonucleotide (10.2 mg, 1.83. Mu. Mol, molecule 12) were dissolved in a solution of 20mM ammonium bicarbonate/acetonitrile (3:1, v/v,1.00 mL). The reaction mixture was shaken for 1min and allowed to stand at room temperature. At 3 hoursAfter that time, the reaction mixture was frozen and lyophilized overnight. The crude product was dissolved in 20mM ammonium bicarbonate (1 mL) and the resulting solution was directly subjected to preparative LC-MS. Will correspond to the product ^1B The fractions were immediately combined together, frozen and lyophilized overnight to give the title compound (27.2 mg, 81%) as a white fluffy solid. Purity based on LC-MS was 91%.

MS(m/z)：10660[M+H] ⁺

A similar procedure was used as for the synthesis of trifunctional linkers- (L-hydrazone-SO 1861) - (L-BNA oligonucleotide) - (trivalent GalNAc) (molecule 13); a conjugate may be synthesized for control test purposes (molecule 13 a) in which ApoB BNA is replaced with BNA having a scrambled ApoB nucleic acid sequence (molecule 12 a).

Trifunctional linker- (dendrite (L-hydrazone-SO 1861) 4) - (L-BNA oligonucleotide) - (trivalent-GalNAc) Synthesis (FIG. 36)

Intermediate 6:

SO1861-EMCH (molecule 15)

To SO1861 (121 mg,0.065mmol, molecule 1) and EMCH. TFA (110 mg,0.325mmol, molecule 14) were added methanol (extra dry, 3.00 mL) and TFA (0.020mL, 0.260 mmol). The reaction mixture was stirred at room temperature. After 1.5 hours, the reaction mixture was subjected to preparative MP-LC. Will correspond to the product ¹ Fractions were immediately pooled together, frozen and lyophilized overnight to give the title compound as a white fluffy solid (120 mg, 90%). The purity based on LC-MS was 96%.

LRMS(m/z)：2069[M-1] ^1-

LC-MS r.t.(min)：1.084

Intermediate 7:

dendrite (-L-SO 1861) 4-amine (molecule 17)

N, N' - ((9S, 19S) -14- (6-aminocaproyl) -1-mercapto-9- (3-mercaptopropionamido) -3,10,18-trioxo-4,11,14,17-tetraazaditridecane-19, 23-diyl) bis (3-mercaptopropionamide) formate (2.73 mg, 3.13. Mu. Mol, molecule 16) was dissolved in a mixture of 20mM NH4HCO3 and 0.5mM TCEP/acetonitrile (3:1, v/v,3.00 mL). Next, SO1861-EMCH (29.2 mg,0.014mmol, min.) was addedSub 15) and the reaction mixture was stirred at room temperature. After 1.5 hours, the reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^3B Fractions were immediately combined, frozen and lyophilized overnight to give the title compound as a white fluffy solid (12.3 mg, 43%). Purity based on LC-MS was 97%.

LRMS(m/z)：1517[M-6] ^6- ，1821[M-5] ^5- ，2276[M-4] ^4-

LC-MS r.t.(min)：4.39 ^5A

Intermediate 8

Dendrite (-L-SO 1861) ₄ Azide (molecule 19)

Dendritic (SO 1861) ₄ -amine (6.81 mg, 0.748. Mu. Mol, molecule 17) and 2, 5-dioxopyrrolidin-1-yl-1-azido-3, 6,9, 12-tetraoxapentadecane-15-carboxylate (2.90 mg, 7.48. Mu. Mol, molecule 18) were dissolved in DMF (1.00 mL). Next, DIPEA (1.302. Mu.L, 7.48. Mu. Mol) was added, and the mixture was shaken for 1 minute and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^3C Fractions were immediately pooled together, frozen and lyophilized overnight to give the title compound as a white fluffy solid (5.86 mg, 84%). Purity based on LC-MS was 90%.

LRMS(m/z)：2344[M-4] ^4-

LC-MS r.t.(min)：4.78 ^5B

Intermediate 9:

trifunctional linker (DBCO) - (TCO) - (trivalent GalNAc) (molecule 20)

Trivalent GalNAc-thioacetate (13.0 mg, 7.25. Mu. Mol, molecule 8) was dissolved in methanol (0.50 mL). Next, 1M sodium hydroxide solution (7.98. Mu.L, 7.98. Mu. Mol) was added. The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 30 minutes, the reaction mixture was added to a solution of freshly prepared trifunctional linker (7.39 mg, 6.13. Mu. Mol, molecule 4) in 20mM ammonium bicarbonate/acetonitrile (3:1, v/v,2.00 mL). The resulting mixture was shaken for 1min and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative M P-LC. Will correspond to the product ^2A Fractions were immediately combined together, frozen and lyophilized overnight to give the title compound as a white solid (3.83 mg, 21%). The purity based on LC-MS was 89%.

LRMS(m/z)：2409[M-546] ^1- ，2955[M-1] ^1-

LC-MS r.t.(min)：2.66 ^1B

Intermediate 10:

trifunctional linker (DBCO) - (L-ApoB BNA oligonucleotide) - (trivalent GalNAc) (molecule 21)

To methyltetrazine-L-ApoB BNA oligonucleotide (5.82 mg, 0.684. Mu. Mol, molecule 12) and TFL- (DBCO) - (TCO) - (trivalent GalNAc) (2.03 mg, 0.687. Mu. Mol, molecule 20) was added 20mM ammonium bicarbonate/acetonitrile (3:1, v/v,1.00 mL). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^4C Fractions were immediately combined together, frozen and lyophilized overnight to give the title compound as a white fluffy solid (3.73 mg, 64%). LC-MS shows a plurality of broad peaks with correct m/z values.

MS(m/z)：8511[M+H] ⁺

Trifunctional linkers (dendrites (-L-hydrazone-SO 1861) ₄ ) - (L-ApoB BNA oligonucleotide) - (trivalent GalNAc Synthesis) (molecule 22)

To TFL- (DBCO) - (L-ApoB BNA oligonucleotide) - (trivalent GalNAc) (1.16 mg, 0.137. Mu. Mol; molecule 21) and dendrites (-L-SO 1861) ₄ Azide (1.39 mg, 0.142. Mu. Mol; molecule 19) was added 20mM ammonium bicarbonate/acetonitrile (3:1, v/v,0.5 mL). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 3 hours, the reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^4B The fractions were immediately combined together, frozen and lyophilized overnight to give the title compound (1.36 mg, 54%) as a white fluffy solid. The purity based on LC-MS was 92% (very broad peak).

MS(m/z)：18336[M+H] ⁺

A similar procedure was used as for the synthesis of trifunctional linkers (dendrite (-L-hydrazone-SO 1861) 4) - (L-ApoB BNA oligonucleotide) - (trivalent GalNAc synthesis) (molecule 22); a conjugate may be synthesized for control test purposes (molecule 22 a) in which ApoB BNA is replaced with BNA having a scrambled ApoB nucleic acid sequence (molecule 12 a).

Trifunctional linker- (dendrite (L-hydrazone-SO 1861) 8) - (BNA oligonucleotide) - (trivalent-GalNAc) Synthesis (FIG. 37)

Intermediate 11:

dendrite (-L-SO 1861) ₈ Amine (molecule 24)

(2S) -N- [ (1S) -1- { [2- (6-amino-N- {2- [ (2S) -2, 6-bis (3-sulfanylpropionamido) hexanamido]Caproamide group]Ethyl caproamide) ethyl group]Carbamoyl } -5- [ (2S) -2, 6-bis (3-sulfanyl-propionamido) hexanamide]Amyl group]2, 6-bis (3-sulfanyl-propionamido) caproamide formate (0.52 mg, 0.299. Mu. Mol, molecule 23) and SO1861-EMCH (29.2 mg,0.014mmol, molecule 15) were dissolved in a mixture of 20mM NH4HCO3 and 0.5mM TCEP/acetonitrile (3:1, v/v,1.00 mL) and the resulting mixture was shaken for 1min and allowed to stand at room temperature. After 30 minutes, TCEP (0.30 mg, 1.05. Mu. Mol) was added and the reaction mixture was shaken for 1 minute. The mixture was then subjected directly to preparative LC-MS. Will correspond to the product ^3B The fractions were immediately combined together, frozen and lyophilized overnight to give the title compound (2.17 mg, 40%) as a white fluffy solid. Purity based on LC-MS was 97%.

LRMS(m/z)：2282[M-8] ^8- ，2607[M-7] ^7-

LC-MS r.t.(min)：4.41 ^5A

Intermediate 12：

Dendrite (-L-SO 1861) ₈ Azide (molecule 25)

Dendritic (-L-SO 1861) ₈ -amine (19.6 mg, 1.08. Mu. Mol, molecule 24) and 2, 5-dioxopyrrolidin-1-yl-1-azido-3, 6,9, 12-tetraoxapentadecane-15-carboxylate (4.17 mg, 10.8. Mu. Mol, molecule 18) were dissolved in DMF (1.50 mL). Next, DIPEA (1.87. Mu.L, 10.8. Mu. Mol) was added, and the mixture was shaken for 1min and allowed to stand at room temperature overnight. The reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^4B Fraction(s)Immediately combined together, frozen and lyophilized overnight to give the title compound (11.7 mg, 59%) as a white fluffy solid. The purity based on LC-MS was 92% (very broad peak).

LRMS(m/z)：2316[M-8] ^8- ，2647[M-7] ^7-

LC-MS r.t.(min)：4.29 ^1A

Trifunctional linkers (dendrites (-L-hydrazone-SO 1861) ₈ ) - (L-ApoB BNA oligonucleotide) - (trivalent GalNAc Synthesis) (molecule 26)

To TFL- (DBCO) - (L-ApoB BNA oligonucleotide) - (trivalent GalNAc) (1.16 mg, 0.137. Mu. Mol; molecule 21) and dendrites (-L-SO 1861) ₈ Azide (2.72 mg, 0.142. Mu. Mol; molecule 25) was added 20mM ammonium bicarbonate/acetonitrile (3:1, v/v,0.5 mL). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 3 hours, the reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^4B Fractions were immediately combined, frozen and lyophilized overnight to give the title compound as a white fluffy solid (1.6 mg, 42%). The purity based on LC-MS was 93% (very broad peak).

MS(m/z)：27655[M+H] ⁺

Using a method similar to the synthesis of trifunctional linkers (dendrites (-L-hydrazone-SO 1861) 8) - (L-ApoB BNA oligonucleotides) - (trivalent GalNAc synthesis) (molecule 26), a conjugate can be synthesized for control test purposes (molecule 26 a), in which ApoB BNA is replaced with BNA having a scrambled ApoB nucleic acid sequence (molecule 12 a).

Trifunctional linkers- (L-semicarbazone-SO 1861) - (BNA oligonucleotide) - (trivalent-GalNAc Synthesis) (FIG. 38)

Intermediate 13:

SO 1861-L-semicarbazone-azide (molecule 28)

To SO1861 (60 mg,0.032mmol, molecule 1) and 4- (6-azidohexanoyl) piperazine-1-carbohydrazide (36.5 mg,0.129mmol, molecule 27) were added methanol (extra dry, 1.00 mL) and TFA (9.86 μl,0.129 mmol), and the reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative MP-LC. Will correspond to the productA kind of electronic device ¹ Fractions were immediately pooled together, frozen and lyophilized overnight to give the title compound as a white fluffy solid (30 mg, 44%). Purity based on LC-MS was 99%.

MS(m/z)：2126[M-1] ^1-

Intermediate 14:

trifunctional linkers- (L-semicarbazone-SO 1861) - (TCO) - (maleimide) (molecule 29)

A solution of TFL- (DBCO) - (TCO) - (maleimide) (15 mg, 12.5. Mu. Mol, molecule 4) in DMF (2.0 mL) was added to SO 1861-L-azide (26.6 mg, 12.5. Mu. Mol, molecule 28). The reaction mixture was shaken for 30 minutes and allowed to stand at room temperature. After 30 minutes, the reaction mixture was subjected to preparative MP-LC. The 1C fractions corresponding to the product were immediately pooled together, frozen and lyophilized overnight to give the title compound as a white fluffy solid (27.5 mg, 66%). The purity based on LC-MS was 96%.

MS(m/z)：3331[M-1] ^1-

Intermediate 15:

trifunctional linker- (L-semicarbazone-SO 1861) - (TCO) - (trivalent GalNAc) (molecule 30)

Trivalent GalNAc-thioacetate (13.0 mg, 7.25. Mu. Mol, molecule 8) was dissolved in methanol (0.50 mL). Next, 1M sodium hydroxide solution (7.98. Mu.L, 7.98. Mu. Mol) was added. The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 30 minutes, the reaction mixture was added to a solution of freshly prepared TFL- (L-semicarbazone-SO 1861) - (TCO) - (maleimide) (20.4 mg, 6.13. Mu. Mol, molecule 29) in 20mM ammonium bicarbonate/acetonitrile (3:1, v/v,2.00 mL).

The resulting mixture was shaken for 1min and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative MP-LC. Will correspond to the product ^2A Fractions were immediately combined, frozen and lyophilized overnight to give the title compound (16.7 mg, 54%) as a white solid. The purity based on LC-MS was 94%.

MS(m/z)：5081[M-1] ^1-

Trifunctional linkers- (L-semicarbazone-SO 1861) - (L-BNA oligonucleotides) - (trivalent GalNAc) (molecule 31)

TFL- (L-semicarbazone-SO 1861) - (TCO) - (trivalent GalNAc) (16 mg, 3.15. Mu. Mol, molecule 30) and methyltetrazine-BNA oligonucleotide (10.2 mg, 1.83. Mu. Mol, molecule 12) were dissolved in a solution of 20mM ammonium bicarbonate/acetonitrile (3:1, v/v,1.00 mL). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 3 hours, the reaction mixture was frozen and lyophilized overnight. The crude product was dissolved in 20mM ammonium bicarbonate (1 mL) and the resulting solution was directly subjected to preparative LC-MS. Will correspond to the product ^1B Fractions were immediately combined, frozen and lyophilized overnight to give the title compound (11.3 mg, 59%) as a white fluffy solid. Purity based on LC-MS was 92%.

MS(m/z)：10638[M+H] ⁺

A similar procedure was used as for the synthesis of trifunctional linkers- (L-semicarbazone-SO 1861) - (L-BNA oligonucleotide) - (trivalent GalNAc) (molecule 31); a conjugate may be synthesized for control test purposes (molecule 31 a) in which ApoB BNA is replaced with BNA having a scrambled ApoB nucleic acid sequence (molecule 12 a).

Trifunctional linker-dendrite (L-semicarbazone-SO 1861) 4- (L-BNA oligonucleotide) - (trivalent-GalNAc) Synthesis (FIG. 39)

Intermediate 16:

benzyl 4- (2- (tert-butoxycarbonyl) hydrazine-1-carbonyl) piperazine-1-carboxylate (molecule 33)

To a stirred solution of phosgene in toluene (20%, w/w,15.8mL,30.0 mmol) at 0deg.C was slowly (10 min) added a solution of 1-Cbz-piperazine (1.93 mL,10.0mmol, molecule 1) in dichloromethane (25 mL) and DIPEA (3.83 mL,22.0 mmol). The reaction mixture was stirred at room temperature. After 30 min, the reaction mixture was evaporated in vacuo and co-evaporated with dichloromethane (2×50 mL). Next, the residue was dissolved in dichloromethane (100 mL) and the resulting solution was stirred at 0 ℃. A solution of Boc hydrazine (2.33 mL,15.0 mmol) in dichloromethane (20 mL) and DiPEA (3.83 mL,22.0 mL) was added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with dichloromethane (100 mL), and the resulting solution was taken upWashed with 0.5N potassium bisulfate solution (2X 100 mL) and brine (100 mL), and dried over Na ₂ SO ₄ Dried, filtered and evaporated in vacuo. The residue was purified by flash chromatography (first by an ethyl acetate-heptane gradient, 5:95 (v/v) rising to 100% ethyl acetate followed by flushing with 10% methanol in DCM (v/v)) to give the title compound (2.42 g, 64%) as a white solid. The purity based on LC-MS was 100%.

LRMS(m/z)：279/323/401[M-99/M-55/M+23] ¹⁺

LC-MS r.t.(min)：1.27 ¹

Intermediate 17:

tert-butyl 2- (piperazine-1-carbonyl) hydrazine-1-carboxylate (molecule 34)

To benzyl 4- (2- (tert-butoxycarbonyl) hydrazine-1-carbonyl) piperazine-1-carboxylate (2.42 g,6.39mmol, molecular 33) was added methanol (50 mL). The mixture was heated to obtain a clear solution. Next, palladium (10% palladium on charcoal, 50% wet with water, 1.50 g) was added and the resulting mixture was stirred under a hydrogen atmosphere by using a balloon. After 1 hour, the reaction mixture was filtered through celite and the filtrate was evaporated in vacuo to give the title product as a white solid (1.56 g, quantitative).

LRMS(m/z)：189/245[M-55/M+1] ¹⁺

Intermediate 18:

tert-butyl 2- (4- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoyl) piperazine-1-carbonyl) hydrazine-1-carboxylate (molecule 36)

6-Maleimidohexanoic acid (720 mg,3.84mmol, mol 35), tert-butyl 2- (piperazine-1-carbonyl) hydrazine-1-carboxylate (781 mg,3.20mmol, mol 34), EDCI.HCl (730 mg,3.84 mmol) and Oxyma Pure (591 mg,4.16 mmol) were dissolved in a mixture of dichloromethane (25 mL) and DIPEA (835. Mu.L, 4.80 mmol) and the reaction mixture was stirred at room temperature. After 2 hours, the reaction mixture was evaporated in vacuo and the residue was dissolved in ethyl acetate (50 mL). The resulting solution was washed with 0.5N potassium hydrogen sulfate solution (50 mL), saturated sodium hydrogen carbonate solution (2X 50 mL) and brine (50 mL), and dried over Na ₂ SO ₄ Drying, filtering and treatingEvaporated in vacuo. The residue was purified by flash chromatography (DCM-10% methanol in DCM (v/v) gradient 100:0 l to 40:60) to give the title compound (634 mg, 45%) as a white solid. Purity based on LC-MS was 97%.

LRMS(m/z)：338/382/460[M-99/M-55/M+23] ¹⁺

LC-MS r.t.(min)：1.02 ¹

Intermediate 19:

S01861-SC-Mal (molecule 37)

Tert-butyl 2- (4- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoyl) piperazine-1-carbonyl) hydrazine-1-carboxylate (25.0 mg, 57.1. Mu. Mol, molecule 36) was dissolved in a mixture of dichloromethane (500. Mu.L) and TFA (500. Mu.L) and the reaction mixture was stirred at room temperature. After 30 min, the reaction mixture was evaporated in vacuo and co-evaporated with dichloromethane (3×5 mL) and methanol (5 mL). The residue and SO1861 (21.3 mg, 11.4. Mu. Mol, molecule 1) were dissolved in methanol (extra dry, 1.00 mL), and the resulting mixture was shaken for 1 minute and allowed to stand at room temperature. After 4 hours, the reaction mixture was subjected to preparative MP-LC. Will correspond to the product ² Fractions were immediately pooled together, frozen and lyophilized overnight to give the title compound as a white fluffy solid (13.7 mg, 55%). Purity based on LC-MS was 97%.

LRMS(m/z)：2181[M-1] ^1-

LC-MS r.t.(min)：2.13 ³

Intermediate 20:

dendrites (L-semicarbazone-SO 1861) ₄ Amine (molecule 39)

N, N' - ((9S, 19S) -14- (6-aminocaproyl) -1-mercapto-9- (3-mercaptopropionamido) -3,10,18-trioxo-4,11,14,17-tetraazaditridecane-19, 23-diyl) bis (3-mercaptopropionamide) formate (2.73 mg, 3.13. Mu. Mol, molecule 38) was dissolved in a mixture of 1M NaOH and MeOH (1:1, v/v,3.00 mL) and stirred for 30 minutes. Next, 5mL of trimethylphosphine (9.5 mg, 125. Mu. Mol) dissolved in THF was added, and the mixture was stirred for 30 minutes. Next, a solution of 20mM NH4HCO3 and acetonitrile (3:1, v/v,3 was added00 mL) of SO1861-SC-Mal (30.54 mg,0.014mmol, mol 37) and the reaction mixture was stirred at room temperature. After 1.5 hours, the reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^3B Fractions were immediately combined, frozen and lyophilized overnight to give the title compound (19 mg, 64%) as a white fluffy solid. Purity based on LC-MS was 95%.

MS(m/z)：9553[M+H] ⁺

Intermediate 21:

dendrite (-L-SO 1861) ⁴ Azide (molecule 40)

Dendrite (L-semicarbazone-SO 1861) 4-amine (7.1 mg, 0.748. Mu. Mol, molecule 39) and 2, 5-dioxopyrrolidin-1-yl-1-azido-3, 6,9, 12-tetraoxapentadecane-15-carboxylate (2.90 mg, 7.48. Mu. Mol, molecule 18) were dissolved in DMF (1.00 mL). Next, DIPEA (1.302. Mu.L, 7.48. Mu. Mol) was added, and the mixture was shaken for 1 minute and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^3C Fractions were immediately pooled together, frozen and lyophilized overnight to give the title compound as a white fluffy solid (6.4 mg, 87%). The purity based on LC-MS was 93%.

MS(m/z)：9826[M+H] ⁺

Intermediate 22

Trivalent GalNAc-thiol (molecule 41)

Trivalent GalNAc-thioacetate (16.2 mg, 9.02. Mu. Mol, molecule 8) was dissolved in methanol (500. Mu.L). Next, a 1.00M solution of sodium hydroxide (11.0. Mu.L, 11.0. Mu. Mol) was added. The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 30min, the reaction mixture was subjected to preparative MP-LC. Will correspond to the product ^1A Fractions were immediately combined, frozen and lyophilized overnight to give the title compound as a white solid (13.1 mg, 83%). Purity based on LC-MS was 98%.

LRMS(m/z)：1748[M-H] ^1-

LC-MS r.t.(min)：1.78 ^1A

Intermediate 23:

TFL- (DBCO) - (TCO) - (trivalent GalNAc) (molecule 20)

TFL- (DBCO) - (TCO) - (Mal) (15.0 mg, 12.4. Mu. Mol, molecule 4) was dissolved in acetonitrile (0.50 mL). Next, a solution of 20mM ammonium bicarbonate (1.50 mL) was added and the resulting solution was transferred directly to trivalent GalNAc-thiol (22.7 mg, 13.0. Mu. Mol, molecule 41). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 1 hour, the reaction mixture was frozen and lyophilized overnight to give the crude title product as a white solid. The purity based on LC-MS was 84%.

LRMS(m/z)：2409[M-546] ^1- ，2955[M-1] ^1-

LC-MS r.t.(min)：2.63 ^1B

Intermediate 24

TFL- (dendrite (L-semicarbazone-SO 1861) 4) - (TCO) - (trivalent GalNAc) (molecule 41)

Dendritic (L-semicarbazone-SO 1861) ₄ Azide (5.2 mg, 0.529. Mu. Mol, molecule 40) and TFL- (DBCO) - (TCO) - (trivalent GalNAc) (1.5 mg, 0.5. Mu. Mol, molecule 20) were dissolved in DMF (2.00 mL). The mixture was shaken for 10min and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^3C Fractions were immediately pooled together, frozen and lyophilized overnight to give the title compound as a white fluffy solid (6.1 mg, 90%). The purity based on LC-MS was 93%.

MS(m/z)：12781[M+H] ⁺

TFL- (-dendrites (L-semicarbazone-SO 1861) 4) - (L-BNA oligonucleotide) - (trivalent GalNAc) (molecule 42)

TFL- (dendrite (L-semicarbazone-SO 1861) 4) - (TCO) - (trivalent GalNAc) (5.2 mg, 0.41. Mu. Mol, molecule 41) and methyltetrazine-BNA oligonucleotide (2.7 mg, 0.5. Mu. Mol, molecule 12) were dissolved in a solution of 20mM ammonium bicarbonate/acetonitrile (3:1, v/v,1.00 mL). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 3 hours, the reaction mixture was frozen and lyophilized overnight. The crude product was dissolved in 20mM ammonium bicarbonate (1 mL) and the resulting solution was directly subjected to preparative LC-MS. Will correspond to the production of Object(s) ^1B Fractions were immediately combined, frozen and lyophilized overnight to give the title compound as a white fluffy solid (1.8 mg, 24%). The purity based on LC-MS was 96% (multiple (broad) peaks due to regioisomers).

MS(m/z)：18336[M+H] ⁺

Using a method similar to that for the synthesis of trifunctional linkers (dendrites (-L-semicarbazone-SO 1861) 4) - (L-ApoB BNA oligonucleotides) - (trivalent GalNAc synthesis) (molecule 42), a conjugate can be synthesized for control test purposes (molecule 42 a), in which ApoB BNA is replaced with BNA having a scrambled ApoB nucleic acid sequence (molecule 12 a).

Trifunctional linker- (dendrite (-L-semicarbazone-SO 1861) 8) -BNA oligonucleotide-trivalent-GalNAc synthesis (FIG. 40)

Intermediate 25:

dendrites (L-semicarbazone-SO 1861) ⁸ Amine (molecule 44)

(2S) -N- [ (1S) -1- { [2- (6-amino-N- {2- [ (2S) -2, 6-bis (3-sulfanylpropionamido) hexanamido]Caproamide group]Ethyl caproamide) ethyl group]Carbamoyl } -5- [ (2S) -2, 6-bis (3-sulfanyl-propionamido) hexanamide]Amyl group]2, 6-bis (3-sulfanyl-propionamido) caproamide formate (0.61 mg, 0.299. Mu. Mol, molecular 43) was dissolved in a mixture of 1M NaOH and MeOH (1:1, v/v,3.00 mL) and stirred for 30min. Next, 5mL of trimethylphosphine (2 mg, 24. Mu. Mol) dissolved in THF was added, and the mixture was stirred for 30 minutes. Next, an aqueous solution of NH in 20mM was added ₄ HCO ₃ And SO1861-SC-Mal (30.54 mg,0.014mmol, molecular 37) in acetonitrile (3:1, v/v,3.00 mL), and the reaction mixture was stirred at room temperature. After 1.5 hours, the reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^3B Fractions were immediately combined, frozen and lyophilized overnight to give the title compound as a white fluffy solid (4.6 mg, 81%). Purity based on LC-MS was 95%.

MS(m/z)：19146[M+H] ⁺

Intermediate 26:

dendrite (-L-SO 1861) ⁸ Azide (molecule 45)

Dendritic (L-semicarbazone-SO 1861) ₈ -amine (5.4 mg, 0.28. Mu. Mol, molecule 44) and 2, 5-dioxopyrrolidin-1-yl-1-azido-3, 6,9, 12-tetraoxapentadecane-15-carboxylate (1.1 mg, 2.8. Mu. Mol, molecule 18) were dissolved in DMF (1.00 mL). Next, DIPEA (1.302. Mu.L) was added, and the mixture was shaken for 1 minute and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^3C Fractions were immediately pooled together, frozen and lyophilized overnight to give the title compound as a white fluffy solid (4.35 mg, 80%). The purity based on LC-MS was 93%.

MS(m/z)：19420[M+H] ⁺

Intermediate 27:

TFL- (dendrite (L-semicarbazone-SO 1861) 8) - (TCO) - (trivalent GalNAc) (molecule 46)

Dendritic (L-semicarbazone-SO 1861) ₈ Azide (10.3 mg, 0.529. Mu. Mol, molecule 45) and TFL- (DBCO) - (TCO) - (trivalent GalNAc) (1.5 mg, 0.5. Mu. Mol, molecule 20) were dissolved in DMF (2.00 mL). The mixture was shaken for 10min and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative LC-MS. Will correspond to the product ^3C Fractions were immediately pooled together, frozen and lyophilized overnight to give the title compound as a white fluffy solid (8.75 mg, 74%). Purity based on LC-MS was 91%.

MS(m/z)：22373[M+H] ⁺

TFL- (dendrite (L-semicarbazone-SO 1861) 8) - (L-BNA oligonucleotide) - (trivalent GalNAc) (molecule 47)

TFL- (dendrite (L-semicarbazone-SO 1861) 8) - (TCO) - (trivalent GalNAc) (9.2 mg, 0.41. Mu. Mol, molecule 46) and methyltetrazine-BNA oligonucleotide (2.8 mg, 0.5. Mu. Mol, molecule 12) were dissolved in a solution of 20mM ammonium bicarbonate/acetonitrile (3:1, v/v,1.00 mL). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 3 hours, the reaction mixture was frozen and lyophilized overnight. The crude product was dissolved in 20mM ammonium bicarbonate (1 mL) and the resulting solution was directly subjected to preparative LC-MS. Will correspond to the product ^1B Fractions were immediately combined, frozen and lyophilized overnight to give the title compound as a white fluffy solid (1.26 mg, 11%). Purity based on LC-MS was 97%.

MS(m/z)：27928[M+H] ⁺

Using a method similar to that for the synthesis of trifunctional linkers (dendrites (-L-semicarbazone-SO 1861) 8) - (L-ApoB BNA oligonucleotides) - (trivalent GalNAc synthesis) (molecule 47), a conjugate can be synthesized for control test purposes (molecule 47 a), in which ApoB BNA is replaced with BNA having a scrambled ApoB nucleic acid sequence (molecule 12 a).

Trivalent linker- (L-SPT 001) - (blocked TCO) - (trivalent GalNAc) Synthesis (FIG. 41)

Intermediate 28:

n- (2-hydroxyethyl) -2- (4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) phenyl) acetamide (molecule 50)

To a solution of methyltetrazine-NHS ester (30.0 mg, 91.7. Mu. Mol, molecule 48) in DMF (1.00 mL) was added ethanolamine (11.1. Mu.L, 0.184mmol, molecule 49) and triethylamine (25.5. Mu.L, 0.183 mmol). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 1 hour, the reaction mixture was subjected to preparative MP-LC. Will correspond to the product ^1B Fractions were immediately combined, frozen and lyophilized overnight to give the title compound as a purple solid (19.2 mg, 77%). The purity based on LC-MS was 89%.

LRMS(m/z)：274[M+1] ¹⁺

LC-MS r.t.(min)：0.85 ²

TCO blocking on TFL has been performed on the following conjugates:

TFL- (L-hydrazone-SPT 001) - (blocked TCO) - (trivalent GalNAc) (molecule 51)

TFL- (dendrite (L-hydrazone-SPT 001) 4) - (blocked TCO) - (trivalent GalNAc) (molecule 52)

TFL- (dendrite- (L-hydrazone-SPT 001) 8) - (blocked TCO) - (trivalent GalNAc) (molecule 53)

TFL- (L-semicarbazone-SPT 001) - (blocked TCO) - (trivalent GalNAc) (molecule 54)

TFL- (dendrites ((L-semicarbazone-SPT 001) 4) - (blocked TCO) - (trivalent GalNAc) (molecule 55)

TFL- (dendrite (L-semicarbazone-SPT 001) 8) - (blocked TCO) - (trivalent GalNAc) (molecule 56)

TFL- (L-hydrazone-SPT 001) - (blocked TCO) - (trivalent GalNAc) (molecule 51) illustratively describes this procedure:

TFL- (L-hydrazone-SPT 001) - (blocked TCO) - (trivalent GalNAc) (molecule 51)

A solution of TLF- (DBCO) - (TCO) - (trivalent GalNAc) (36.8 mg, 12.5. Mu. Mol, molecule 20) in DMF (2.0 mL) was added to SO 1861-L-hydrazone-azide (26.8 mg, 12.5. Mu. Mol, molecule 3). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 30min, N- (2-hydroxyethyl) -2- (4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) phenyl) acetamide (4.08 mg, 14.9. Mu. Mol, molecule 50) was added. The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 30min, the reaction mixture was subjected to preparative MP-LC. Will correspond to the product ^1C The fractions were immediately combined, frozen and lyophilized overnight to give the title compound as a white fluffy solid (37.3 mg, 56%). The purity based on LC-MS was 96% (double peak caused by regioisomer).

LRMS(m/z)：2676[M-2] ^2-

LC-MS r.t.(min)：2.23 ^1B

Trivalent linker- (blocked DBCO) - (L-BNA oligonucleotide) - (trivalent GalNAc) Synthesis (FIG. 42)

Intermediate 29:

TFL- (blocked DBCO) - (TCO) - (trivalent GalNAc) (molecule 58)

A solution of 1-azido-3, 6, 9-trioxaundecan-11-ol (2.17 mg, 9.88. Mu. Mol, molecule 57) in DMF (0.50 mL) was added to TFL- (DBCO) - (TCO) - (trivalent GalNAc) (14.6 mg, 4.94. Mu. Mol, molecule 20). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative MP-LC. Will correspond to the product ^1C Fractions were immediately combined, frozen and lyophilized overnight to give the title compound as a white solid (10.00 mg, 64%). Purity based on LC-MS was 98%.

MS(m/z)：3175[M+H] ⁺

LC-MS r.t.(min)：2.33 ^1B

Trivalent linker- (blocked DBCO) - (BNA oligonucleotide) - (trivalent GalNAc) (molecule 59)

TFL- (blocked DBCO) - (TCO) - (trivalent GalNAc) (10.0 mg, 3.15. Mu. Mol, molecule 58) and methyltetrazine-BNA oligonucleotide (10.0 mg, 1.83. Mu. Mol, molecule 12) were dissolved in a solution of 20mM ammonium bicarbonate/acetonitrile (3:1, v/v,1.00 mL). The reaction mixture was shaken for 1min and allowed to stand at room temperature. After 3 hours, the reaction mixture was frozen and lyophilized overnight. The crude product was dissolved in 20mM ammonium bicarbonate (1 mL) and the resulting solution was directly subjected to preparative LC-MS. Will correspond to the product ^1B Fractions were immediately combined, frozen and lyophilized overnight to give the title compound as a white fluffy solid (11.3 mg, 72%). Purity based on LC-MS was 95%.

LRMS(m/z)：2149[M-4] ^4- ，2866[M-3] ^3-

LC-MS r.t.(min)：2.92 ^1A

Using a method similar to the synthesis of trivalent linkers- (blocked DBCO) - (BNA oligonucleotides) - (trivalent GalNAc) (molecule 59), a conjugate can be synthesized for control test purposes (molecule 59 a), in which ApoB BNA is replaced with BNA having a scrambled ApoB nucleic acid sequence (molecule 12 a).

Fig. 43A and B show an overview of all conjugate 'conjugates C1' produced by applying the trifunctional linker (TFL) method. Furthermore, conjugates comprising a single GalNAc moiety according to the present invention have been synthesized and tested.

Cell-Titer Glo (CTG) Cell viability assay

After addition of the compound, the cells were incubated at 37℃for 72 hours and then @ according to the manufacturer's instructions2.0 cell viability assay, promega) cell viability was determined by CTG assay. Briefly, the cell plates were first equilibrated to room temperature for 30 minutes. Next, 120. Mu.L of CTG solution was added to each well containing 120. Mu.L of the treatment medium. Will bePlates were briefly mixed (10 seconds, 600 rpm) and incubated at room temperature for approximately 10 minutes in the dark. Subsequently, the luminescence signal was measured on a Spectramax ID5 plate reader (molecular instruments company (Molecular Devices)). For quantification, the background signal of "medium only" wells was subtracted from all other wells, and then the relative percent cell viability of the treated cells was calculated by dividing the background corrected signal of the treated wells by the background corrected signal of the untreated wells (×100 as a percentage).

Human primary hepatocyte treatment

Cryopreserved primary human hepatocytes (cytobiotechnology limited (Cytes Biotechnologies s.l.), spanish) were thawed in hepatocyte thawing medium (cytobiotechnology limited, spanish). Cells were then resuspended in hepatocyte plating medium (cell biotechnology limited, spain) and seeded onto collagen-I coated plates in 48-well or 96-well plates (Greiner bione) at a density of 215.600 cells/well or 66.600 cells/well, respectively. Cells were pre-incubated at 37 ℃ for 4-6 hours before starting the treatment to allow cells to attach to the cell culture plates. The plating medium was replaced with 315 μl or 108 μl of maintenance medium (cell biotechnology Co., ltd., spain) followed by the addition of conjugate from a 10-fold concentrated stock solution in PBS. Plates were incubated at 37 ℃ for 72 hours and harvested for gene expression and cell viability analysis.

ApoB#02 oligonucleotide sequences

Mouse and human sequence compatible ApoB#02 (5'-GCattggtatTCA-3') [ SEQ ID NO:12 ]]Is BNA ^NC Oligonucleotides and are used by the biosynthesis company with a 5' -thiol C6 linker (wherein BNA ^NC Base uppercase) and complete phosphorothioate backbone synthesis (lewis ville, texas).

RNA isolation and Gene expression analysis from Primary mice and human hepatocytes

According to the manufacturer's instructions, use TRIThe solution (Sesamer technologies) isolated RNA from cells. Make the following stepsUse of iScript with Standard protocols ^TM The cDNA synthesis kit (Berle Corp.) was converted to cDNA. Quantitative real-time PCR assay (qRT-PCR) was used, using iTaq ^TM General->Green supersubstitre (Berle Corp.) and Light Cycler 480 (Rockreuz diagnostics Inc. (Roche Diagnostics), luo Teke Lez City (Rotkreuz), switzerland) were used to determine the level of ApoB expression and the level of a specific hepatocyte housekeeping gene using specific DNA primers listed in Table A4 (study A) and Table A7 (study B), respectively. Analysis was performed by the delta Ct method to determine apoB expression relative to 2 hepatocyte-specific housekeeping control mrnas. Each analytical reaction was performed in triplicate.

Table A4 primers used in qPCR analysis of mouse primary hepatocytes/liver tissue analysis (study A)

FACS analysis

For each cell line, cells were seeded at appropriate density in DMEM (PAN-Biotech GmbH) supplemented with 10% fetal bovine serum (PAN-Biotech GmbH) and 1% penicillin/streptomycin (PAN-Biotech GmbH) in T75 flasks. Cultures were incubated for 72-96 hours (5% CO2, 37 ℃) until 90% confluence was achieved. Next, cells were trypsinized (Tryple Express, ji Buke Semer technologies (Gibco Thermo Scientific)) to obtain a single cell suspension, transferred to a 15mL falcon tube and centrifuged (1,400 rpm,3 min). The supernatant was discarded while the cell pellet was submerged. 500.000 cells were transferred to round bottom FACS tubes and plated with 3mL cold DPBS (Mg-free ²⁺ And Ca ^{2 +} 2% fbs). Cells were centrifuged at 1800rpm at 4℃for 3min and resuspended in 200. Mu.L cold DPBS (Mg-free) ²⁺ And Ca ²⁺ 2% FBS) or 200. Mu.L antibody solution (containing a solution of 195. Mu.L cold DPBS (Mg-free) ²⁺ And Ca ²⁺ 5 μl of antibody in 2% fbs). Transferrin receptor was expressed against human CD71 using PE (accession number 334106, biological legend (bioleged)) PE mouse IgG2a, kappa isotype control FC (accession number 400212, biolegend company) was used as its matched isotype control. ASGPR1 receptor was stained with PE anti-human ASGPR1 (accession No. 130-122-963, miltenyi) and PE mouse IgG1, isotype control (accession No. 130-113-762, meitenji company) was used as its matched isotype control. The samples were incubated at 4℃for 30min. Thereafter using cold DPBS (Mg-free ²⁺ And Ca ²⁺ Cells were washed 2 times with 2% fbs and used at room temperature in DPBS (Mg-free ²⁺ And Ca ²⁺ 2% pfa solution in 2% fbs) for 20min. Cells were washed 1 time with cold DPBS and resuspended in 1000 μl cold DPBS for FACS analysis. Samples were analyzed using the Cube 8 flow cytometer system (cisseneck) from cisseneck and FCS Express 7 study software. The results of FACS analysis are summarized in table A3.

In vivo tolerability and efficacy of trivalent GalNAc-conjugates in C57BL/6J model

i. Dose administration and lifetime. Treatment groups of 8C 57BL/6J male mice (14 mice in the vehicle control group in study A) were dosed (approximately 6-7 weeks at arrival and approximately 8-9 weeks at dosing). The compounds formulated according to tables A5 and A6 in PBS or PBS alone (vehicle) were administered at 5ml/kg in the tail vein (intravenous, intravenous drip), the dose volumes corresponding to the individual body weights. Where applicable, the trivalent (GalNAc) is immediately followed by the trivalent GalNAc-ApoB conjugate ₃ SO1861 (EMCH) was co-administered at 5ml/kg in the tail vein (intravenous drip). Animals were weighed weekly and 2-3 clinical observations were made daily for the first 48 hours and at least once daily thereafter until the end of the study.

Serum, liver and kidney tissue. Blood was sampled prior to dosing (pre-dosing) and at designated post-dosing time points (e.g., 24 hours, 72 hours, 196 hours, 336 hours, 672 hours post-dosing) and serum was generated from the collected blood for biomarker analysis using standard procedures. At the relevant inter-study dosing time points, 3 animals (6 animals in the vehicle control group in study a) were sacrificed at each time point for each treatment group to collect terminal bleeding and to collect relevant organs (e.g., liver and kidney tissue) for further analysis. At the end of the study, the remaining 2 animals were sacrificed per group (4 in the control group in study a). Half of the liver and one kidney were frozen and used for tissue RNA analysis.

Table A5 Experimental design of in vivo tolerability and efficacy of trivalent GalNAc-conjugates, study A

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Table A6 Experimental design of in vivo tolerability and efficacy of trivalent GalNAc-conjugates, study B

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Biomarker RNA analysis in liver tissue

According to the manufacturer's instructions, useReagents (Semer technologies) mRNA was isolated from frozen liver tissue. Use of iScript ^TM The cDNA synthesis kit (Berle Corp.) was converted to cDNA. Quantitative real-time PCR assay (qRT-PCR) was used, using iTaq ^TM General->Green supersubstitre (Berle Corp.) and Light Cycler 480 (Roche diagnostics Co., roche Diagnostics), luo Teke Lez City (Rotkreuz), switzerland) with the specific D listed in Table A4 (study A) and Table A7 (study B), respectivelyThe NA primers determine ApoB expression levels and the level of a particular liver housekeeping gene. Analysis was performed by the delta Ct method to determine apoB expression relative to 2 liver-specific housekeeping control mrnas. Each analytical reaction was performed in triplicate.

Table A7. primers used in qPCR analysis of liver tissue (study B)

Biomarker analysis apoB protein

ApoB protein levels in mouse serum were determined using an ApoB ELISA (ab 230932, ai Bokang company (Abcam), cambridge City (Cambridge), UK) according to the manufacturer's instructions.

Biomarker analysis of cholesterol

LDL-cholesterol in mouse serum was measured photometrically according to the manufacturer's instructions using the LDL-cholesterol specific two-step AU480 assay kit from Beckman Coulter, respectively.

Biomarker analysis of cholesterol

Serum alanine Aminotransferase (ALT) levels were measured using an AU480 assay kit (photometry) from beckmann coulter, according to the manufacturer's instructions.

Results

(GalNAc) ₃ In vivo efficacy of-SO 1861-ApoB#02 in C57BL/6J mice (study A)

In study A, C57BL/6J mice were treated as described and dosed according to Table A5. Conjugates used in this study were produced as described in FIGS. 35 and 38, 41 and 42, (GalNAc) ₃ -ApoB#02、(GalNAc) ₃ -SO1861, (GalNAc) 3-SO1861-ApoB#02 (EMCH) and (GalNAc) ₃ -SO1861-ApoB#02 (sc). Clinical observations were recorded and different biomarkers of efficacy and tolerability were analyzed.

As shown in fig. 25, after 24 hours, apoB RNA levels (i.e., gene expression) were not significantly reduced in animals treated with 0.1mg/kg apob#02BNA (apob#02) compared to vehicle-dosed groups (n=5 animals), and ApoB RNA levels were reduced by approximately 15% and 21% in groups dosed with 1mg/kg or 2mg/kg apob#02BNA, respectively (all n=3 animals/group). Likewise, after 24 hours, a dose of 1mg/kg (GalNAc) of 3-ApoB#02 (corresponding to 0.54mg/kg ApoB#02 BNA) reduced ApoB expression by about 53% (n=3 animals) compared to vehicle control, whereas 0.1mg/kg (GalNAc) alone compared to vehicle ₃ Apob#02 did not show significant decrease (n=3). In contrast, when 0.1mg/kg (GalNAc) ₃ Apob#02 when co-administered with 5mg/kg (GalNAc) 3-SO1861, apoB expression had been readily reduced by about 69% after 24 hours (n=2 animals); whereas 0.01mg/kg (GalNAc) ₃ Apob#02, when co-administered with 5mg/kg (GalNAc) 3-SO1861, still showed a slight 16% decrease compared to vehicle (n=2 animals). Most notably, treatment with 1mg/kg (GalNAc) 3-SO1861-ApoB#02BNA (corresponding to 0.44mg/kg ApoB#02BNA) resulted in (GalNAc) compared to the 24 hour post-dose control ₃ -SO1861-ApoB#02BNA (EMCH) reduced by about 77% and (GalNAc) ₃ -SO1861-apob#02BNA (sc) was reduced by 90% (two groups of n=3 animals each).

After 72 hours, the highest dose of apob#02bna administered (2 mg/kg) showed a maximum decrease of 39% (n=3 animals) compared to vehicle (n=5 animals), whereas 1mg/kg (GalNAc) ₃ Apob#02 resulted in 68% decrease in ApoB expression (n=3 animals). Again, when compared to 5mg/kg (GalNAc) ₃ When SO1861 is co-administered, the dosage is 10 times lower than 0.1mg/kg (GalNAc) ₃ ApoB#02 resulted in an even 82% decrease in apoB expression level (n=3 animals), whereas 0.1mg/kg (GalNAc) alone ₃ Apob#02 showed only a 12% decrease (n=3 animals). With different 1mg/kg (GalNAc) ₃ Treatment with-SO 1861-ApoB#02BNA conjugate resulted in (GalNAc) ₃ -SO1861-ApoB#02BNA (EMCH) is reduced by about 76% and (GalNAc) ₃ -SO1861-apob#02BNA (sc) was reduced by 96% (two groups of n=3 animals each). The effect was long lasting and after 336 hours, for2mg/kg apoB#2BNA, apoB expression level was reduced by 30% (n=2 animals), and for 1mg/kg (GalNAc) ₃ Apob#02, apoB expression level reduced by 25%. Co-administration group with 10-fold reduced load (i.e., 0.1mg/kg (GalNAc) ₃ -ApoB#02+5mg/kg(GalNAc) ₃ -SO 1861) still showed a 25% decrease. With different 1mg/kg (GalNAc) ₃ Treatment with-SO 1861-ApoB#02BNA conjugate resulted in (GalNAc) ₃ -SO1861-ApoB#02BNA (EMCH) reduced by about 61% and (GalNAc) ₃ -SO1861-apob#02BNA (sc) was reduced by 65% (two groups of n=3 animals each). In conclusion, conjugation of SO1861 to the load significantly improves the efficacy of the load in vivo.

As shown in fig. 26, downregulation of apoB mRNA in the liver (as shown in fig. 25) also translates into a decrease in apoB protein in serum. After 24 hours, for 1mg/kg (GalNAc) ₃ Apob#02 (n=8 animals, 54%) and likewise for a 10-fold lower (GalNAc) ₃ -ApoB#02+5mg/kg(GalNAc) ₃ SO1861 (i.e., 0.1mg/kg (GalNAc) ₃ -ApoB#02+5mg/kg(GalNAc) ₃ -SO 1861) (n=8 animals, 56%) of the co-administered group, apoB protein levels decreased by more than 50%; most notably, for 1mg/kg (GalNAc) compared to the control (n=14 animals) ₃ -SO1861-apob#02 (EMCH) group (n=8 animals, 69% reduction) and 1mg/kg (GalNAc) ₃ -SO1861-apob#02 (sc) group (n=8 animals, 83% reduction), they decreased. After 72 hours, although all treatment groups showed apoB protein reduction, the most significant effect was observed compared to vehicle controls for: 1mg/kg (GalNAc) ₃ -SO1861-apob#02 (EMCH) group (n=5 animals, 95% reduction) and 1mg/kg (GalNAc) ₃ group-SO 1861-apob#02 (sc) (n=5 animals, 97% reduction), or when co-administered 0.1mg/kg (GalNAc) ₃ ApoB#02 and 5mg/kg (GalNAc) ₃ -SO1861, resulting in a nearly 90% decrease in apoB (n=5 animals). After 72 hours from the administration, when combined with 5mg/kg (GalNAc) ₃ -SO1861 (n=4 animals) when co-administered, even 0.01mg/kg (GalNAc) ₃ ApoB#02 also reduced ApoB protein by about 25% whereas the dose alone was 10-fold higher by 0.1mg/kg (GalNAc) ₃ Apob#02 reduced ApoB protein by 35% (n=5 animals). For all of the treatment groups analyzed,this effect is durable and apoB protein reduction is still observed at 192 and 336 hours; note that 0.1mg/kg ApoB#02 (196 hours), 0.01mg/kg (GalNAc) ₃ -ApoB+5mg/kg(GalNAc) ₃ SO1861 (196 hours and 336 hours) and 0.1mg/kg (GalNAc) ₃ -ApoB+5mg/kg(GalNAc) ₃ The level of-SO 1861 (196 hours) was not reported. At 192 hours, 1mg/kg (GalNAc) ₃ ApoB#02 showed a 47% decrease (n=2 animals), but 1mg/kg (GalNAc) ₃ -SO1861-apob#02 (EMCH) and 1mg/kg (GalNAc) 3-SO1861-apob#02 (sc) showed a decrease of 87% and 90%, respectively (n=2 animals per group). At 336 hours, 1mg/kg (GalNAc) ₃ ApoB#02 showed a 28% decrease (n=2 animals), but 1mg/kg (GalNAc) ₃ -SO1861-apob#02 (EMCH) and 1mg/kg (GalNAc) 3-SO1861-apob#02 (sc) still showed a 66% and 65% decrease, respectively (n=2 animals per group).

As shown in fig. 27, the observed reduction of serum apoB protein (as shown in fig. 26) converts well to serum LDL-cholesterol (LDL-C) levels. After 24 hours, LDL-C levels in animals treated with 0.1, 1 or 2mg/kg apob#02BNA (n=3 animals/group) were not significantly reduced compared to vehicle (n=5 animals). The dose was 1mg/kg of targeted (GalNAc) compared to vehicle and ApoB#02BNA group ₃ ApoB #02 has reduced LDL-C to an average of 2.7mg/dl (n=3 animals) within 24 hours, i.e. 53%. It has been observed that when combined with 5mg/kg (GalNAc) ₃ When SO1861 is co-administered, the dosage is 10 times lower than 0.1mg/kg (GalNAc) ₃ ApoB#02 has reduced LDL-C to an average of 2.7mg/dl (n=3 animals) within 24 hours, i.e. 53% and 1mg/kg (GalNAc) ₃ -SO1861-ApoB#02 (EMCH) and 1mg/kg (GalNAc) ₃ -SO1861-apob#02 (sc) showed a 66% decrease and 77% decrease, respectively. After 72 hours, the highest dose of apob#02bna (2 mg/kg) showed about 50% reduction in LDL-C (n=3 animals) compared to vehicle (n=5 animals), while 1mg/kg (GalNAc) ₃ ApoB#02 was 72% lower (n=3 animals) and the dose was 10-fold lower by 0.1mg/kg (GalNAc) ₃ -ApoB#02+5mg/kg(GalNAc) ₃ The case of SO1861 is even a 78% decrease (n=3 animals); 1mg/kg (GalNAc) ₃ -SO1861-ApoB#02 (EMCH) showed a decrease of 72%,while at 1mg/kg (GalNAc) ₃ in-SO 1861-apob#02 (sc), LDL-C above LOD was not measured, i.e. 100% reduction of ApoB protein compared to control (all n=3 animals). The effect was durable and after 336 hours, the ldl-C level was still reduced by about 40% for 2mg/kg apob#02bna (in all groups of n=2 animals), and for 1mg/kg (GalNAc) ₃ ApoB#02 and for 0.1mg/kg (GalNAc) ₃ -ApoB#02+5mg/kg(GalNAc) ₃ The co-administered group of SO1861 showed a 48% decrease in LDL-C levels, showing a 10-fold improvement in LDL-C reduction in the latter based on the load administered. Likewise, at 1mg/kg (GalNAc) ₃ The most significant decrease (74% decrease) was observed in the group-SO 1861-ApoB#02 (EMCH) and 1mg/kg (GalNAc) ₃ the-SO 1861-ApoB#02 (sc) group showed a 57% decrease.

Taken together, this suggests that in combination with SO1861, apob#02 has significantly increased efficacy and is effective in inducing downregulation of ApoB RNA, i.e., gene silencing in the liver of C57BL/6J mice, as well as all measured efficacy of 'downstream' serum biomarkers, such as ApoB protein and LDL-cholesterol.

(GalNAc) 3-SO1861-ApoB#02 in vivo tolerability in C57BL/6J mice (study A)

Conjugates used in this study were produced as described in FIGS. 35, 38, 41 and 42, (GalNAc) ₃ -ApoB#02、(GalNAc) ₃ -SO1861、(GalNAc) ₃ -SO1861-ApoB#02 (EMCH) and (GalNAc) ₃ -SO1861-ApoB#02(sc)。

During the course of the study, all treatments, doses and dose regimens as described in table A5 were well tolerated and did not show treatment specific adverse reaction findings or any specific clinical observations indicating intolerance of the conjugate. The treated mice gained weight comparable to mice receiving vehicle. Serum ALT levels were determined and the results are shown in fig. 28. ALT levels in vehicle mice ranged on average from 47 to 61U/L/group (n=5 animals for vehicle) during the study period. ALT levels in different dose groups increased transiently at different time points, sometimes with each animal mutated/group large, and did not show specific therapeutic or dose relationships (all groups at 24 hours and 72 hours N=3, except 0.01mg/kg (GalNAc) ₃ -ApoB#02+5mg/kg(GalNAc) ₃ -SO1861 (where both time points are n=2)). Notably, after 336 hours, ALT levels returned to baseline and were comparable to vehicle controls for all dosing groups (all groups were n=2).

Taken together, this suggests ApoB#02, (GalNAc) ₃ -ApoB#02、(GalNAc) ₃ SO1861 (GalNAc alone or in combination) ₃ -apob#02 combination) and (GalNAc) ₃ -SO1861-ApoB#02 (EMCH) and (GalNAc) ₃ -SO1861-ApoB#02 (sc) is well tolerated in the doses and dose regimen applied, in particular the administration of SO1861 containing conjugates (GalNAc) 3-SO1861-ApoB#02 (EMCH) and (GalNAc) ₃ -SO1861-ApoB#02 (sc) and (GalNAc) ₃ -SO1861 and (GalNAc) ₃ Co-administration of apob#02 did not have direct tolerability problems, but showed high efficacy in the C57BL/6J mouse model.

(GalNAc) ₃ In vitro potency of-SO 1861-ApoB#02

BNA, apoB#02 (SEQ ID NO: 12) and SO1861-EMCH or SO1861-SC-Mal with trivalent-GalNAc ((GalNAc) targeting apoB ₃ ) Conjugation to give (GalNAc) ₃ -SO1861-ApoB#02 (EMCH) and (GalNAc) ₃ -SO1861-ApoB#02 (sc) (FIGS. 35 and 38), (GalNAc) ₃ -SO1861 (FIG. 41) and (GalNAc) ₃ ApoB#02BNA (FIG. 42) and in primary human hepatocytes and HepG2 (ASGPR) ⁺ ) And Huh7 (ASGPR) ^+/- ) Cell lines were used to evaluate their effect on apoB RNA levels. In primary hepatocytes, this evaluation showed (GalNAc) ₃ -SO1861-ApoB#02 (EMCH) and (GalNAc) ₃ -SO1861-apob#02 (sc) shows IC50 = 1nM (relative apob#02BNA concentration), whereas no SO1861 is present (GalNAc) ₃ Apob#02BNA showed IC50 = 7nM (relative to apob#02BNA concentration), and apob#02BNA transfection (with RNAi-MAX) showed IC50 = 8nM. Apob#02 alone (without any transfection agent) showed IC50 = 400nM, whereas (GalNAc) ₃ SO1861 had no effect on apoB RNA levels (FIG. 29A). All of these indicate (GalNAc) ₃ -SO1861-apob#02 enhances cytoplasmic delivery of apob#02BNA in human primary hepatocytes, resulting in an improvement of about 10-fold in efficiency at low nM levels of apob#02BNA oligonucleotides.

For HepG2 cells (ASGPR 1 ⁺ ) Only (GalNAc) ₃ -SO1861-ApoB#02 (EMCH) and (GalNAc) ₃ -SO1861-apob#02 (sc) showed activity at the measured concentrations (ic50=40 nM and ic50=20 nM, respectively) (fig. 29B), whereas in Huh7 cells (ASGPR 1 ^+/- )，(GalNAc) ₃ -SO1861-apob#02 (EMCH) shows activity at ic50=100 nM, and (GalNAc) ₃ -SO1861-apob#02 (sc) showed activity at ic50=40 nM (fig. 29C).

(GalNAc) ₃ dSPT4-ApoB#02 and (GalNAc) ₃ In vitro potency of-dSPT 8-ApoB#02

BNA, apoB#02 (SEQ ID NO: 12) and SO1861-EMCH or SO1861-sc-Mal targeting apoB with trivalent-GalNAc ((GalNAc) ₃ ) Conjugation (FIGS. 1, 8) to produce (GalNAc) ₃ -SO1861-ApoB#02 (EMCH) (FIG. 35), (GalNAc) ₃ -SO1861-ApoB#02 (sc) (FIG. 38)), (GalNAc) ₃ -d(SO1861) ₄ -ApoB#02(sc)((GalNAc) ₃ -dSPT4-apob#02; FIG. 39), (GalNAc) ₃ -d(SO1861) ₈ -ApoB#02(sc)((GalNAc) ₃ -dSPT8-apob#02; FIG. 40) and (GalNAc) ₃ Apob#02BNA (fig. 42) and conjugates were tested on primary human hepatocytes (fig. 30) to evaluate their effect on ApoB RNA levels. In primary hepatocytes, this evaluation showed (GalNAc) ₃ -d (SO 1861) 8-ApoB#02 (sc) shows an IC50 of 1.5nM (relative conjugate concentration), whereas (GalNAc) ₃ -SO1861-ApoB#02(EMCH)、(GalNAc) ₃ -SO1861-ApoB#02(sc)、(GalNAc) ₃ -d(SO1861) ₄ ApoB#02 (sc) or (GalNAc) ₃ Apob#02 was less potent (IC 50 between 5 and 8 nM) (figure 30). This suggests that the increased amount of SO1861 molecules per GalNAc conjugate enhances the potency of the conjugate, thereby reducing the amount of apob#02BNA loading required to reduce apoB mRNA levels.

(GalNAc) ₃ In vivo efficacy of-SO 1861-ApoB#02 in C57BL/6J mice (study B)

In study B, C57BL/6J mice were treated as described and dosed according to Table A6. Conjugates used in this study were generated as described in FIGS. 38, 39, 40, 41, 42, (GalNAc) ₃ -SO1861-ApoB#02(sc)、(GalNAc ₎₃ -d(SO1861)4-ApoB#02(sc)、(GalNAc) ₃ -d(SO1861)8-ApoB#02(sc)、(GalNAc) ₃ ApoB#02 and (GalNAc) ₃ -SO 1861). Clinical observations were recorded and different biomarkers of efficacy and tolerability were analyzed.

As shown in FIG. 31, apoB RNA levels (i.e., gene expression) were reduced in animals treated with reference 2mg/kg ApoB#02BNA (53%, from control) after 72 hours, but in animals treated with (1 mg/kg (GalNAc), respectively ₃ -ApoB#02+0.1mg/kg(GalNAc) ₃ -SO 1861) and 1mg/kg (GalNAc) ₃ Even more reductions in the group dosed with SO1861-apob#02 (sc) (to 24% and 21% of vehicle control, respectively) showed significantly improved efficacy when SO1861 was added to the conjugate (n=3 animals per group). Furthermore, when the saponin per load was increased, the load was reduced 10 times by the dose (0.173 mg/kg (GalNAc) ₃ D (SO 1861) 4-ApoB#02 (sc) (refer, briefly, to 0.1mg/kg in FIG. 31 for simplicity), also known as (GalNAc) ₃ dSPT4-ApoB#02 (sc) or 0.265mg/kg (GalNAc) ₃ -d (SO 1861) 8-ApoB#02 (sc), also known as GalNAc ₃ dSPT8-ApoB#02 (sc)), corresponding to the same loading dose (0.044 mg/kg ApoB02 BNA) (contained in 0.1mg/kg (GalNAc) ₃ In SO1861-ApoB #02 (sc), but SO1861 was 4-fold and 8-fold more, respectively, apoB expression was reduced to 74% and 38%, respectively, for 0.1mg/kg (GalNAc) ₃ -SO1861-apob#02 (sc) which is 89% of the control, shows that the increase in saponins significantly increases the efficacy of the conjugate. Reduction of the amount of saponins (0.1 mg/kg (GalNAc) ₃ -SO1861(sc)+0.1mg/kg(GalNAc) ₃ Apob#02) resulted in lower potency (71% ApoB expression compared to vehicle). After 336 hours, the effect of down-regulation of expression was 1mg/kg (GalNAc) ₃ the-SO 1861-apob#02 (sc) dose group was still present and was 49% of vehicle control (n=3 mice).

As shown in fig. 32, downregulation of apoB RNA in the liver (as shown in fig. 31) also translates into a reduction of apoB protein in serum. The pre-dose level range for each group was 156-198 μg/ml (n=8 per group). 72 hours post-dose, apoB protein levels were reduced in animals treated with baseline 2mg/kg ApoB#02BNA (average 57 μg/ml compared to 156 for vehicle controlMu g/ml), but in each case (1 mg/kg (GalNAc) ₃ -ApoB#02+0.1mg/kg(GalNAc) ₃ -SO 1861) and 1mg/kg (GalNAc) ₃ Even more reductions in the group dosed with SO1861-apob#02 (sc) (to 18 μg/ml and 21 μg/ml, respectively, of vehicle control) showed significantly improved efficacy when SO1861 was added to the conjugate (n=8 animals per group). As with RNA levels, when increasing the saponin per load, the total amount of the total saponins was increased by administering 0.173mg/kg (GalNAc) separately ₃ -d (SO 1861) 4-ApoB#02 (sc) or 0.265mg/kg (GalNAc) ₃ -d (SO 1861) 8-ApoB#02 (sc), serum apoB protein was reduced to 89. Mu.g/ml and 50. Mu.g/ml, respectively, with respect to 0.1mg/kg (GalNAc) ₃ -SO1861-apob#02 (sc) which was 141 μg/ml, indicated that the increase in saponin significantly increased the efficacy of the conjugate in reducing ApoB protein. Reduction of the amount of saponins (0.1 mg/kg (GalNAc) ₃ -SO1861(sc)+0.1mg/kg(GalNAc) ₃ ApoB#02) resulted in lower potency (122. Mu.g/ml apoB protein. After 336 hours, the protein was reduced by 1mg/kg (GalNAc) ₃ -SO1861-apob#02 (sc) dose group was still present and was 59 μg/ml compared to 143 μg/ml in vehicle control (n=5 mice in all groups); at 672 hours, apoB protein levels varied between 100-152 μg/ml in all groups (n=2 mice).

As shown in fig. 33, the observed reduction of serum apoB protein (as shown in fig. 32) converts well to serum LDL-cholesterol (LDL-C) levels. The LDL-C level was reduced (2 mg/dl compared to vehicle control of 4 mg/dl) in animals treated with baseline 2mg/kg ApoB#02BNA 72 hours post-dose, but at 1mg/kg (GalNAc) respectively ₃ -ApoB#02+0.1mg/kg(GalNAc) ₃ -SO 1861) and 1mg/kg (GalNAc) ₃ Even more reductions (to 0.7mg/dl and 1.3mg/dl μg/ml, respectively) in the group to which SO1861-apob#02 (sc) was administered showed significantly improved efficacy when SO1861 was added to the conjugate (n=3 animals per group). As with RNA and apoB protein levels, when increasing the saponin per load, the total amount of the saponin per load was increased by administering 0.173mg/kg (GalNAc) respectively ₃ -d (SO 1861) 4-ApoB#02 (sc) or 0.265mg/kg (GalNAc) ₃ -d (SO 1861) 8-ApoB#02 (sc), LDL-C was reduced to 2.3mg/dl and 1.3mg/dl, respectively, with respect to 0.1mg/kg (GalNAc) ₃ -SO1861-ApoB#02 (sc) which is 43mg/dl, indicating that an increase in saponins significantly increases the efficacy of the conjugates in reducing LDL-C. Reduction of the amount of saponins (0.1 mg/kg (GalNAc) ₃ -SO1861(sc)+0.1mg/kg(GalNAc) ₃ ApoB#02) resulted in lower potency (3.7 mg/dl LDL-C). After 336 hours, the LDL-C was reduced at 1mg/kg (GalNAc) ₃ -SO1861-ApoB#02 (sc) dose group (2 mg/dl; n=3 animals) and dose group (1 mg/kg (GalNAc) ₃ -ApoB#02+0.1mg/kg(GalNAc) ₃ -SO 1861), where it is 3.3mg/dl (n=3 animals), is still present. Persistence of the display effect; at 672 hours, LDL-C levels varied between 3-4mg/dl for all groups (n=2 mice).

Taken together, this suggests that in combination with SO1861, apob#02 has significantly increased efficacy and is effective in inducing downregulation of ApoB RNA, i.e., gene silencing in the liver of C57BL/6J mice, as well as all measured efficacy of 'downstream' serum biomarkers, such as ApoB protein and LDL-cholesterol.

(GalNAc) ₃ -SO1861-ApoB#02 in vivo tolerability in C57BL/6J mice (study B)

Conjugates used in this study were generated as described in FIGS. 38, 39, 40, 41, 42, (GalNAc) ₃ -SO1861-ApoB#02(sc)、(GalNAc) ₃ -d(SO1861)4-ApoB#02(sc)、(GalNAc) ₃ -d(SO1861)8-ApoB#02(sc)、(GalNAc) ₃ ApoB#02 and (GalNAc) ₃ -SO1861)。

During the course of the study, all treatments, doses and dose regimens as described in table A6 were well tolerated and did not show treatment specific adverse reaction findings or any specific clinical observations or general organ morphology indicating intolerance of the conjugate. The treated mice gained weight comparable to mice receiving vehicle. Serum ALT levels were determined and the results are shown in fig. 34. At 72 hours, 0.173mg/kg (GalNAc) ₃ ALT levels in the d (SO 1861) 4-ApoB#02 (sc) group appeared to increase briefly, but returned to vehicle control levels at 336 hours post-dose; also, it was at 0.265mg/kg (GalNAc) ₃ Increased in the group-d (SO 1861) 4-apob#02 (sc), but recovered to vehicle levels at a later point in time after dosing. Notably, 1 mg/kg%GalNAc) ₃ -SO1861-apob#02 (sc) showed an increase up to 336 hours after dosing, but was measured as 166U/L at 672 hours (n=2 mice).

Taken together, the data indicate that the saponin conjugates have good tolerability in the doses and dosage regimens employed, particularly with the administration of SO 1861-containing conjugates (GalNAc) ₃ -SO1861-ApoB#02(sc)、(GalNAc) ₃ -d (SO 1861) 4-ApoB#02 (sc) and (GalNAc) ₃ -d (SO 1861) 8-ApoB#02 (sc), and coadministration (GalNAc) ₃ -SO1861 and (GalNAc) ₃ ApoB#02 has no direct tolerability problem and shows high efficacy in the C57BL/6J mouse model.

Sequence listing

<110> sapromil technologies inc (Sapreme Technologies b.v.)

<120> conjugates of saponins, oligonucleotides and GALNAC

<130> P6096514PCT1

<150> PCTNL2021050384

<151> 2021-06-18

<150> PCTNL2021050549

<151> 2021-09-09

<160> 20

<170> PatentIn version 3.5

<210> 1

<211> 16

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> HSP27 antisense BNA oligonucleotides

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ggcacagcca gtggcg 16

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<213> Artificial sequence (Artificial Sequence)

<220>

<223> APOB BNA oligonucleotides

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gcctcagtct gcttcgcacc 20

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<213> Artificial sequence (Artificial Sequence)

<220>

<223> APOB scrambled BNA oligonucleotides

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ggcctctcta caccgctcgt 20

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<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> HSP27 Forward primer

<400> 4

gcagtccaac gagatcacca 20

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

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<223> HSP27 reverse primer

<400> 5

taaggcttta cttggcggca 20

<210> 6

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<213> Artificial sequence (Artificial Sequence)

<220>

<223> ApoB Forward primer

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agggtccggg aatctgatga 20

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<213> Artificial sequence (Artificial Sequence)

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<223> ApoB reverse primer

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<210> 8

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> HBMS Forward primer

<400> 8

cacccacaca cagcctactt 20

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> HBMS reverse primer

<400> 9

gtacccacgc gaatcactct 20

<210> 10

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> GUSB forward primer

<400> 10

gaaaatacgt ggttggagag ct 22

<210> 11

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> GUSB reverse primer

<400> 11

ccgagtgaag atcccctttt ta 22

<210> 12

<211> 13

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> ApoB#02 oligonucleotides

<400> 12

gcattggtat tca 13

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<213> Artificial sequence (Artificial Sequence)

<220>

<223> ApoB#02 Forward primer

<400> 13

gctaacacta agaaccagaa gatc 24

<210> 14

<211> 20

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<213> Artificial sequence (Artificial Sequence)

<220>

<223> ApoB#02 reverse primer

<400> 14

tgtccgtcta aggatcctgc 20

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<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Mm PPIA Forward primer

<400> 15

gcggcaggtc catctacg 18

<210> 16

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Mm PPIA reverse primer

<400> 16

gccatccagc cattcagtc 19

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<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Mm SDHA Forward primer

<400> 17

gaggaagcac accctctcat 20

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Mm SDHA reverse primer

<400> 18

ggagcggata gcaggaggta 20

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Mm RPS17 Forward primer

<400> 19

gtgcgaggag atcgccatta 20

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Mm RPS17 reverse primer

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atccgcttca tcagatgcgt 20

Claims (21)

1. An oligonucleotide conjugate comprising at least one saponin covalently linked to a ligand of an asialoglycoprotein receptor (ASGPR), wherein the ligand of ASGPR comprises at least one N-acetylgalactosamine (GalNAc) moiety, preferably three or four GalNAc moieties, more preferably three GalNAc moieties, more preferably the ligand of ASGPR comprises (GalNAc) ₃ Tris or (GalNAc) ₃ Tris, and also covalently linked to an oligonucleotide, wherein said at least one saponin is a monosaccharide or disaccharide pentacyclic triterpene saponin of the 12, 13-dehydrooleanane type, preferably having an aldehyde function at the C-23 position of the aglycone core structure of said saponin,

wherein the oligonucleotide conjugate comprises 1-16 saponin moieties, preferably 1-8 saponin moieties, more preferably 1 saponin moiety, 4 saponin moieties or 8 saponin moieties.

2. The oligonucleotide conjugate according to claim 1, wherein the at least one GalNAc moiety, preferably three GalNAc moieties, the at least one saponin, preferably 1-16 saponin moieties, more preferably 1-8 saponin moieties such as 1, 4 or 8 saponin moieties, and the oligonucleotide are covalently bound via a trifunctional linker, preferably wherein each of the one or more GalNAc moieties, the one or more saponin moieties and the oligonucleotide are covalently bound to a separate arm of the trifunctional linker.

3. The oligonucleotide conjugate according to claim 1 or 2, comprising one saponin moiety, or 4 saponin moieties, preferably 4 saponin moieties, covalently bound to a dendron, preferably a G2 dendron such as e.g. N, N' - ((9S, 19S) -14- (6-aminocaproyl) -1-mercapto-9- (3-mercaptopropionylamino) -3,10,18-trioxo-4,11,14,17-tetraazaditridecane-19, 23-diyl) bis (3-mercaptopropionamide), or 8 saponin moieties, preferably 8 saponin moieties, covalently bound to a dendron, preferably a G3 dendron such as e.g. (2S) -N- [ (1S) -1- { [2- (6-amino-N- {2- [ (2S) -2, 6-bis (3-sulfanylpropionylamino) hexanylamino ] ethyl } hexanylamino) carbamoyl { 5S) -2, 6-bis (3-sulfanylpropionylamino } -2-alkyl ] hexanamide.

4. The oligonucleotide conjugate according to any one of claims 1-3, wherein said oligonucleotide conjugate comprises a saponin moiety, and wherein said oligonucleotide conjugate is according to molecule (EE):

，

wherein molecule (EE) is via a trifunctional linker according to formula (XXI):

covalent conjugation products obtained with covalent conjugation of the following (1), (2) and (3):

(1) A saponin derivative according to molecule (AA):

，

wherein the method comprises the steps of

Represents a saponin moiety according to formula (SM):

，

wherein R is ¹ And R is ² Independently selected from the group consisting of hydrogen, monosaccharides, linear oligosaccharides and branched oligosaccharides, and wherein said saponin moiety according to formula (SM) is based on a saponin comprising an aldehyde group at the C-23 position,

and

(2) GalNAc conjugate according to molecule (FF):

，

wherein the method comprises the steps of

Represents a tri-GalNAc conjugate according to molecule (DD 1) or according to molecule (DD 2):

(DD1)，

wherein y1, y2 and y3 are each independently an integer selected from 0-20, preferably 1-15, more preferably 2-12, even more preferably 2-10, even more preferably 2-8, most preferably 2 and 3, and preferably y1, y2 and y3 are the same, and y4 is an integer selected from 1-100, preferably 2-80, more preferably 3-70, even more preferably 4-60, even more preferably 4-50, even more preferably 4-40, even more preferably 4-30, even more preferably 4-20, even more preferably 4-6, most preferably 4-5, such as 4;

wherein x1, x2 and x3 are each independently an integer selected from 0 to 20, preferably 1 to 15, more preferably 2 to 12, even more preferably 2 to 10, even more preferably 2 to 8, most preferably 2 and 3, and preferably x1, x2 and x3 are the same, and x4 is an integer selected from 1 to 50, preferably 2 to 40, more preferably 3 to 30, even more preferably 4 to 20, even more preferably 5 to 15, most preferably 8 to 12, such as 9,

And preferably represents a tri-GalNAc conjugate according to molecule (DD 3) or according to molecule (DD 4):

and

(3) The oligonucleotide having a linker according to molecule (GG):

，

wherein molecule (GG) represents the conjugation product of a conjugation reaction between linker (E) -1- (4- ((2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoyl) hydrazono) methyl) benzoylamino) -N- (4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) benzyl) -3,6,9, 12-tetraoxapentadecane-15-amide and said oligonucleotide-linker molecule according to molecule (HH):

5. the oligonucleotide conjugate of any one of claims 1-3, comprising four saponin moieties, and wherein the oligonucleotide conjugate is according to molecule (PP):

，

wherein the molecule (PP) is a saponin-GalNAc conjugate according to the molecule (LL):

a covalent conjugation product obtained by covalent conjugation with:

the oligonucleotide having a linker according to molecule (GG):

，

wherein the molecule (GG) represents the conjugation product of a conjugation reaction between a linker (E) -1- (4- ((2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoyl) hydrazono) methyl) benzoylamino) -N- (4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) benzyl) -3,6,9, 12-tetraoxapentadecane-15-amide and the oligonucleotide-linker molecule according to molecule (HH):

，

Wherein molecule (LL) is prepared by reacting a saponin derivative according to molecule (JJ):

a covalent conjugation product obtained by covalent conjugation with:

conjugate of trifunctional linker according to molecule (MM) and GalNAc:

，

wherein the molecule (JJ) is the conjugation product of N, N' - ((9 s,19 s) -14- (6-aminocaproyl) -1-mercapto-9- (3-mercaptopropionamido) -3,10,18-trioxo-4,11,14,17-tetraazaditridec-19, 23-diyl) bis (3-mercaptopropionamide) conjugated to: first with a saponin derivative according to molecule (KK):

，

wherein the method comprises the steps of

Represents a saponin moiety according to formula (SM):

，

wherein R is ¹ And R is ² Independently selected from the group consisting of hydrogen, monosaccharides, linear oligosaccharides and branched oligosaccharides according to the invention, and wherein said saponin moiety according to formula (SM) is based on a saponin comprising an aldehyde group at the C-23 position,

and then with 2, 5-dioxopyrrolidin-1-yl-1-azido-3, 6,9, 12-tetraoxapentadecane-15-oic acid ester,

and wherein the molecule (MM) is a conjugate of: a trifunctional linker according to formula (XXI):

and GalNAc conjugates according to molecule (NN):

，

wherein the method comprises the steps of

Represents a molecule (DD 1) or a molecule (DD 2) according to claim 4, preferably a molecule (DD 3) or a molecule (DD 4) according to claim 4, more preferably a tri-GalNAc conjugate of a molecule (DD 3) according to claim 4.

6. The oligonucleotide conjugate of any one of claims 1-3, comprising eight saponin moieties, and wherein the oligonucleotide conjugate is according to molecule (SS):

，

wherein the molecule (SS) is prepared by reacting a saponin-GalNAc conjugate with the molecule (RR):

a covalent conjugation product obtained by covalent conjugation with: an oligonucleotide having a linker according to molecule (GG):

，

wherein molecule (GG) represents the conjugation product of the conjugation reaction of linker (E) -1- (4- ((2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoyl) hydrazono) methyl) benzoylamino) -N- (4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) benzyl) -3,6,9, 12-tetraoxapentadecane-15-amide and an oligonucleotide-linker molecule according to molecule (HH):

，

wherein the molecule (RR) is obtained by reacting a saponin derivative according to the molecule (QQ):

a covalent conjugation product obtained by covalent conjugation with:

conjugate of trifunctional linker according to molecule (MM) and GalNAc:

，

wherein the molecule (QQ) is the conjugation product of (2S) -N- [ (1S) -1- { [2- (6-amino-N- {2- [ (2S) -2, 6-bis (3-sulfanylpropionamido) hexanamido ] ethyl } hexanamido) ethyl ] carbamoyl } -5- [ (2S) -2, 6-bis (3-sulfanylpropionamido) hexanamido ] pentyl ] -2, 6-bis (3-sulfanylpropionamido) hexanamide formate conjugated to: first

(a) And a saponin derivative according to molecule (KK):

，

wherein the method comprises the steps of

Represents a saponin moiety according to formula (SM):

，

wherein R is ¹ And R is ² Independently selected from the group consisting of hydrogen, monosaccharides, linear oligosaccharides and branched oligosaccharides according to the invention, and wherein the saponin moiety according to formula (SM) is based on a soap comprising an aldehyde group at the C-23 positionThe glycoside is used as a carrier of the pharmaceutical composition,

and then

(b) With 2, 5-dioxopyrrolidin-1-yl-1-azido-3, 6,9, 12-tetraoxapentadecane-15-oic acid ester,

and wherein the molecule (MM) is a conjugate of: a trifunctional linker according to formula (XXI):

and GalNAc conjugates according to molecule (NN):

，

wherein the method comprises the steps of

Represents a molecule (DD 1) or a molecule (DD 2) according to claim 4, preferably a molecule (DD 3) or a molecule (DD 4) according to claim 4, more preferably a tri-GalNAc conjugate of a molecule (DD 3) according to claim 4.

7. The oligonucleotide conjugate of any one of claims 1-6, wherein the conjugate comprises a saponin that is any one or more of:

a) A saponin selected from any one or more of list a:

-a mixture of saponins of quillaja saponaria (Quillaja Saponaria), or saponins isolated from quillaja saponaria, such as Quil-A, QS-17-api, QS-17-xyl, QS-21A, QS-21B, QS-7-xyl;

-a mixture of saponins of carnation (Gypsophila elegans), or saponins isolated from carnation;

-a mixture of saponinium album saponins, or saponins isolated from saponinium album;

-a mixture of sapogenins of saponaria (Saponaria officinalis), or saponins isolated from saponaria; and

-a saponaria saponins mixture, or saponins isolated from saponaria barks, such as Quil-A, QS-17-api, QS-17-xyl, QS-21A, QS-21B, QS-7-xyl; or (b)

b) A saponin comprising a silk diabolo sapogenin core structure selected from list B:

SA1641, carnation saponin A, NP-017772, NP-017774, NP-017777, NP-017778, NP-018109, NP-017888, NP-017889, NP-018108, SO1658 and phytolaccagenin; or (b)

c) A saponin comprising a sapogenin core structure selected from list C:

AG1856, AG1, AG2, agrostemmoside E, GE1741, silk bamboo saponin 1 (Gyp 1), NP-017674, NP-017810, NP-003881, NP-017676, NP-017677, NP-017705, NP-017706, NP-017773, NP-017775, SA1657, saponaria oside B, SO1542, SO1584, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862, SO1904, QS-7api, QS-17, QS-18, QS-21A-apio, QS-21A-xylo, QS-21B-apio and QS-21B-xylo; or (b)

d) A sapogenin core structure comprising a 12, 13-dehydrooleanane type selected from list D, having no aldehyde group at the C-23 position of the aglycone:

escin Ia, escin salt, alpha-hederagenin, AMA-1, AMR, AS6.2, AS64R, assam saponin F, dipsacus asperosaponin B, esculentoside A, lonicera macranthoides saponin A, NP-005236, NP-012672, primula acid 1, saikosaponin A, saikosaponin D, tea seed saponin I and tea seed saponin J;

preferably, the saponin is any one or more of the saponins selected from list A, B or C, more preferably, the saponins are selected from list B or C,

even more preferably, the saponins are selected from list C.

8. The oligonucleotide conjugate according to any one of claims 1-7, wherein the saponin is any one or more of: AG1856, GE1741, saponins isolated from Quil-A, QS-17, QS-21, QS-7, SA1641, saponins isolated from soapbark, saponaria oside B, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862 and SO1904; preferably, wherein the saponin is any one or more of the following: QS-21, SO1832, SO1861, SA1641 and GE1741; more preferably wherein the saponin is QS-21, SO1832 or SO1861; most preferably SO1861.

9. The oligonucleotide conjugate according to any one of claims 1-8, wherein the saponin is a saponin isolated from saponaria, preferably wherein the saponin is any one or more of the following: saponarioside B, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862 and SO1904; more preferably, wherein the saponin is any one or more of the following: SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862 and SO1904; even more preferably, wherein the saponin is any one or more of SO1832, SO1861 and SO 1862; even more preferably, wherein the saponins are SO1832 and SO1861; most preferably SO1861.

10. The oligonucleotide conjugate according to any one of claims 1-9, wherein the saponin is SO1861, and wherein the oligonucleotide conjugate is provided by conjugation to provide the oligonucleotide conjugate by: SO1861 saponin derivative according to molecule (VII) a:

with the oligonucleotide and with at least one GalNAc moiety.

11. The oligonucleotide conjugate according to any one of claims 1-10, wherein the oligonucleotide comprised by the conjugate is defined as a nucleic acid of no longer than 150nt, preferably wherein the oligonucleotide has a size of 5-150nt, preferably 8-100nt, most preferably 10-50nt.

12. A second pharmaceutical composition comprising the oligonucleotide conjugate according to any one of claims 1-11, and optionally a pharmaceutically acceptable excipient and/or optionally a pharmaceutically acceptable diluent.

13. The second pharmaceutical composition according to claim 12 or the oligonucleotide conjugate according to any one of claims 1-11 for use as a medicament.

14. The second pharmaceutical composition according to claim 12 or the oligonucleotide conjugate according to any one of claims 31-11 for use in the treatment or prevention of a disease or health problem in which the expression product involves any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AT3, GO, CC5, X gene of HBV, S gene of HBV, AAT, miR-122, hepatitis B virus HbsAg, LDHA, CEBPA and LDH; and/or for use in the treatment or prevention of diseases or health problems involving any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AT3, GO, CC5, X gene of HBV, S gene of HBV, AAT, miR-122, hepatitis B virus HbsAg, LDHA, CEBPA and LDH.

15. The second pharmaceutical composition according to claim 12 or the oligonucleotide conjugate according to any one of claims 1-11 for use in the treatment or prevention of a disease or health problem in which the expression product involves any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AAT, miR-122, hepatitis B virus HbsAg, LDHA and CEBPA; and/or for use in the treatment or prevention of diseases or health problems involving any one or more of the following genes: HSP27, apoB, TTR, PCSK, TMPRSS6, ALAS1, AAT, miR-122, hepatitis B virus HbsAg, LDHA and CEBPA.

16. The second pharmaceutical composition according to claim 12 or the oligonucleotide conjugate according to any one of claims 1-11 for use according to claim 14 or 15, wherein the use is the treatment or prevention of a disease or health problem in which the expression product relates to any one or more of the following genes: HSP27 and apoB, preferably apoB; and/or for the treatment or prevention of diseases or health problems involving any one or more of the following genes: HSP27 and apoB, preferably apoB.

17. The second pharmaceutical composition according to claim 12 or the oligonucleotide conjugate according to any one of claims 1-11 for use according to any one of claims 14-16, wherein the use is in the treatment or prevention of cancer, infectious disease, viral infection, hypercholesterolemia, cardiovascular disease, primary hyperoxaluria, hemophilia a, hemophilia B, AAT-associated liver disease, acute hepatic porphyria, TTR-mediated amyloidosis, hereditary TTR amyloidosis (hATTR), complement-mediated disease, hepatitis b infection, hepatitis c infection, alpha 1-antitrypsin deficiency, beta-thalassemia or autoimmune disease.

18. The second pharmaceutical composition according to claim 12 or the oligonucleotide conjugate according to any one of claims 1-11 for use according to any one of claims 14-17, wherein the use is the treatment or prevention of cancer such as endometrial cancer, breast cancer, lung cancer or hepatocellular carcinoma; and/or cardiovascular diseases such as hypercholesterolemia, preferably hypercholesterolemia.

19. The second pharmaceutical composition according to claim 12 or the oligonucleotide conjugate according to any one of claims 1-11 for use in reducing LDL-cholesterol in a subject, preferably a human subject.

20. An in vitro or ex vivo method for transferring an oligonucleotide conjugate according to any one of claims 1-11 from outside a cell into said cell, preferably subsequently transferring an oligonucleotide comprised by said oligonucleotide conjugate into the cytoplasm of said cell, said method comprising the steps of:

a) Providing a cell expressing ASGPR on its surface, preferably selected from the group consisting of hepatocytes, virus-infected cells and tumor cells, and providing the oligonucleotide conjugate of any one of claims 1-11 for transfer into the provided cell;

b) Contacting the cell of step a) with the oligonucleotide conjugate of step a) in vitro or ex vivo, thereby effecting transfer of the oligonucleotide conjugate from outside the cell into the cell, and preferably subsequently effecting transfer of the oligonucleotide contained in the oligonucleotide conjugate into the cytoplasm of the cell.

21. The oligonucleotide conjugate according to any one of claims 1-11, wherein the saponin comprised by the oligonucleotide conjugate is isolated from a plant.