Saponin, oligonucleotide, and GalNAc complexes

Abstract

The present invention relates to a complex comprising a saponin covalently linked to a ligand of ASGPR and comprising an oligonucleotide covalently linked to the saponin and the ligand of ASGPR, the ligand comprising at least one GalNAc moiety.In addition, the present invention relates to a pharmaceutical composition comprising the complex of the present invention.Furthermore, the present invention relates to a pharmaceutical composition of the present invention for use as a medicament.The present invention also relates to a pharmaceutical composition of the present invention for use in the treatment or prevention of diseases or health problems involving the expression products of genes apoB, HSP17, TTR, PCSK9, ALAS1, AT3, GO, CC5, HBV X gene, HBV S gene, AAT, and LDH, as well as for use in the treatment or prevention of cancer, infectious diseases, viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, AAT-related liver disease, acute hepatic porphyria, TTR amyloidosis, complement-related disease, hepatitis B infection, or autoimmune disease. The present invention also relates to in vitro or ex vivo methods for transferring the oligonucleotide complexes of the invention from the outside of a cell to the inside of said cell.

Description

[Technical Field]

[0001] The present invention relates to an oligonucleotide conjugate comprising a saponin covalently linked to a ligand of ASGPR (ASGPR ligand) and comprising a covalently linked oligonucleotide, i.e., a (saponin, oligonucleotide, ASGPR ligand) conjugate comprising a saponin covalently linked to a ligand of ASGPR, wherein the ligand comprises at least one GalNAc, and wherein the saponin-oligonucleotide-ASGPR ligand conjugate further comprises a covalently linked oligonucleotide, such as a siRNA or antisense oligonucleotide (referred to as the oligonucleotide conjugate of the present invention). Furthermore, the present invention relates to a pharmaceutical composition of the present invention comprising the saponin-oligonucleotide-ASGPR ligand conjugate for use as a medicine. The present invention also relates to pharmaceutical compositions of the present invention, for use in the treatment or prevention of diseases or health problems involving, for example, the expression products of the genes HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AT3, GO, CC5, HBV X gene, HBV S gene, AAT, miR-122, Hepatitis B virus HbsAg, LDHA, CEBPA, and LDH, as well as for use in the treatment or prevention of, for example, cancer, infectious disease, viral infection, hypercholesterolemia, cardiovascular disease, primary hyperoxaluria, hemophilia A, hemophilia B, AAT-associated liver disease, acute hepatic porphyria, TTR amyloidosis, hereditary TTR amyloidosis (hATTR), complement-related disease, hepatitis B infection, hepatitis C infection, α1-antitrypsin deficiency, β-thalassemia, or autoimmune disease. The present invention also relates to methods for producing the oligonucleotide conjugates of the present invention. Finally, the present invention relates to in vitro or ex vivo methods for transferring the saponin-oligonucleotide-ASGPR ligand complex of the present invention (also referred to as the oligonucleotide complex of the present invention) from the outside of a cell to the inside of said cell. [Background technology]

[0002] The ability to inhibit protein function is an essential part of drug discovery, whether in humans or pathogens. Traditional small molecule drugs and antibodies are strongly limited to a subset of available targets. Small molecules primarily bind effectively to cofactor or transfer factor sites, sites that are designed to accept small molecules. Antibodies can bind to a wider variety of proteins but are usually limited to extracellular targets.

[0003] Oligonucleotide therapeutics is a relatively young and rapidly evolving field that aims to overcome many of the problems encountered with small molecule or antibody drugs by directly manipulating genetic transcription and translation pathways. The potency and versatility of oligonucleotides, particularly their promise to silence genes encoding proteins that are "undruggable" by classical small molecule drugs, make them attractive drug candidates. The first oligonucleotide drugs were based on antisense technology, whereby single-stranded nucleic acid molecules would bind with sequence specificity to their complementary mRNA targets, thereby triggering double-strand degradation by the RNase H system.

[0004] In 1998, Andrew Fire and Craig Mello published a landmark paper identifying double-stranded RNA (dsRNA) as the causative agent of post-transcriptional gene silencing (PTGS) in Caenorhabditis elegans, a phenomenon they termed RNA interference (RNAi). The discovery of RNAi kicked off a revolution in biology, explaining the puzzling observation of gene silencing in plants and fungi and ultimately demonstrating that non-coding RNAs are central regulators of gene expression in multicellular organisms. Shortly thereafter, it was discovered that small dsRNAs (typically 15–30 base pairs (bp)) can catalytically induce RNAi silencing in mammalian cells without eliciting a nonspecific interferon response. Targeting the RNAi pathway with small dsRNAs, such as small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs), has several theoretical advantages over antisense: it is significantly more efficient (catalytic) and provides longer-lasting inhibition of gene expression. This may be reflected in lower doses and lower costs, along with less frequent dosing. Lower exposure may also mean fewer toxicity issues with siRNA.

[0005] However, before siRNA can reach its target in vivo, it faces several significant obstacles that block its pathway to the RNA-induced silencing complex (RISC) machinery. Once in the bloodstream, siRNA is susceptible to degradation by endogenous nucleases and renal excretion due to its small size and highly anionic nature. In addition, before reaching its target cells, siRNA must pass through endothelial tight junctions in blood vessels and diffuse through the extracellular matrix. Due to its multiple negative charges, siRNA does not immediately bind to or cross the cell membrane; once inside the cell, it must escape from endosomes to interact with its intracellular protein targets.

[0006] Therefore, the success of oligonucleotide (e.g., siRNA) therapy is highly dependent on the effective delivery of the drug from outside the cell into the cytosol. As a result, considerable attention has been focused on developing delivery systems that improve oligonucleotide (e.g., siRNA and antisense oligonucleotide) delivery. In addition to viral delivery, the main methods used to improve siRNA (or other oligonucleotide) delivery into cells use liposomes, cell membrane-penetrating peptides (CPPs) and their mimic molecules, or nanoparticles (Non-Patent Document 1). Safety concerns, such as the possibility of insertional mutagenesis and immunogenicity, are thought to limit the future of viral approaches.

[0007] Liposome is the most commonly used delivery vector for oligonucleotides such as siRNA, where oligonucleotides are encapsulated in lipid bilayer.Some problems are encountered in the delivery of oligonucleotides by liposome system, such as oxygen radical mediated toxicity (typical of cationic liposomes), cytotoxicity, effect on gene regulation and inflammatory response.In addition, the in vivo delivery using lipid appears to be predominantly targeted to liver and spleen.

[0008] Cell membrane-penetrating peptides (CCPs), also called protein transduction domains, are short peptides (usually <30 amino acid residues in length) with the unusual property of being able to cross cell membranes. By covalently linking to oligonucleotides or by forming noncovalent complexes with them, CPPs can enhance the cellular uptake of oligonucleotides. The field of CPP-mediated oligonucleotide delivery is extremely complex because it combines, in a single drug, the challenges posed by two far-from-mature fields: oligonucleotide technology and peptide technology. In addition to issues related to oligonucleotides, typical challenges encountered with CPPs relate to the (lack of) in vivo stability of the peptide chain, often requiring the use of unnatural peptide derivatives that can be complex to synthesize and / or may exhibit reduced cell membrane-penetrating activity.

[0009] Despite improved cellular delivery, overcoming endosomal trapping remains one of the major challenges for the design of efficient CPPs and other transport systems (Non-Patent Document 1).

[0010] Nanoparticles and nanocarriers are nanoscale oligonucleotide delivery systems, typically composed of a polymer complexed with an oligonucleotide, a biological stabilizer, and a cell-targeting ligand. An exemplary siRNA nanocarrier system is known as the siRNA dynamic polyconjugate. This system is based on an amphiphilic polymer linked to polyethylene glycol (PEG) as a biological stabilizer and a hepatocyte-targeting ligand, where the siRNA is covalently attached to the polymer via a disulfide bond. The targeting and PEG moieties are attached to the polymer via maleamic acid linkages, forming negatively charged nanoparticles that do not bind to serum proteins. After internalization by endocytosis, the maleamic acid bond is rapidly hydrolyzed by endosomal acidification, exposing the cationic amine groups of the polymer and inducing endosomal escape via the proton sponge effect. Nanocarriers are also known in the form of cationic polymers such as polyethyleneimine (PEI) (optionally combined with cyclodextrin), which can form electrostatic complexes with oligonucleotides such as siRNA, providing protection from degradation and aiding internalization. However, toxicity at higher concentrations resulting from membrane disruption and apoptosis induction may limit the application of cationic polymers as therapeutic drug delivery agents. Besides the complex preparation of nanoparticles and nanocarriers, their long-term stability is a major barrier to their commercially viable use.

[0011] Oligonucleotides can be used to regulate gene expression through a range of processes, including RNAi, target degradation by RNase H-mediated cleavage, splicing regulation, non-coding RNA inhibition, gene activation and programmed gene editing.Oligonucleotides are nucleic acid polymers that have the potential to treat or manage a wide range of diseases.Although the majority of oligonucleotide therapeutics focus on gene silencing, other strategies, including splicing regulation and gene activation, are being pursued, expanding the scope of possible targets from those that are generally accessible to conventional pharmaceutical modalities.

[0012] Examples of pharmaceutical oligonucleotides are single-stranded antisense oligonucleotides and double-stranded small interfering RNAs (siRNAs), which share a common underlying principle. Oligonucleotides bind to target RNAs through Watson-Crick base pairing, and the resulting duplex directs the degradation of the target messenger RNA (mRNA). Oligonucleotides can regulate the expression of corresponding RNAs in the cytoplasm (in the case of siRNAs) or nucleus (in the case of antisense oligonucleotides). Antisense oligonucleotides (ASOs) are small (~18–30 nucleotides), synthetic, single-stranded nucleic acid polymers of diverse chemistries that can be used to regulate gene expression by various mechanisms. ASOs can be subdivided into two major categories: RNase H-competent and steric-blocked. Stereoblocked oligonucleotides are ASOs designed to bind to target transcripts with high affinity but do not induce target transcript degradation because they lack RNase H competence. Stereoblocked oligonucleotides can mask specific sequences on target transcripts, thereby interfering with transcript-RNA and / or RNA-protein interactions. siRNA molecules are the effector molecules of RNAi and classically 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 of the siRNA (the guide or antisense strand) is complementary to the target transcript, while the other strand is called the passenger or sense strand. The siRNA acts to guide the Argonaute 2 protein (AGO2) as part of the RNA-induced silencing complex (RISC) to the complementary target transcript. Perfect complementarity between the siRNA and the target transcript results in cleavage of the guide strand at opposite positions 10-11 of the target (i.e., slicer activity) catalyzed by AGO2, resulting in gene silencing. siRNA approaches include Dicer substrate siRNAs, internally split small siRNAs, self-delivered siRNAs (asymmetric and hydrophobic), single-stranded siRNAs, and bivalent siRNAs.Stereoblocking ASOs competitively inhibit miRNAs by directly binding to small RNA species within the RISC complex. Such ASOs are known as anti-miRNA oligonucleotides, anti-miRs, or antagomirs. The first anti-miRNA drug to enter clinical trials was miravirsen (SPC3649), an ASO designed to treat chronic hepatitis C virus (HCV) infection by targeting the liver-specific miRNA miR-122. Another class of oligonucleotide therapeutics regulates RNA function by binding to splice sites on pre-mRNA, resulting in the skipping of mutation-containing exons in diseases such as muscular dystrophy.

[0013] Further examples of oligonucleotides for clinical applications are microRNAs (miRNAs), which are endogenous RNAi triggers implicated in, for example, cancer, cell cycle progression, infectious diseases, immunity, diabetes, metabolism, skeletal muscle formation, and muscular dystrophy. miRNA hairpins embedded in long primary miRNA transcripts are sequentially processed by two RNase III family enzymes, DICER1 (Dicer) and DROSHA, which release the hairpin and then cleave the loop sequence, respectively. The resulting double-stranded RNA (similar to siRNA) is loaded onto Argonaute proteins (e.g., AGO2), where one strand is discarded to generate a mature single-stranded miRNA species. Like siRNAs, miRNAs guide RISC to target sequences, where they initiate gene silencing. In contrast to siRNAs, miRNAs typically bind via partial complementarity and induce silencing through a Dicer-independent mechanism.

[0014] Therapeutic oligonucleotides are generally 15 to 30 nucleotides in length and are designed to be complementary to specific regions of messenger RNA (mRNA) or regulatory RNAs that encode proteins implicated in disease. After parenteral administration, the oligonucleotides enter cells and bind to either complementary RNA. Once an oligonucleotide drug binds to its complementary mRNA or pre-mRNA, a series of events ensues. The outcome depends in part on the nature of the targeted sequence and can include destruction of the mRNA by means of enzymatic cleavage (which is useful when the mRNA is mutated and encodes a pathogenic protein), alteration of the pre-mRNA splicing pattern (which is useful when the "default" splicing pattern generates a pathogenic product), or alteration of the function of the regulatory RNA.

[0015] An example of a drug molecule containing an oligonucleotide is an siRNA-GalNAc conjugate, in which the siRNA moiety targets the PCSK9 enzyme. This enzyme binds to and degrades the low-density lipoprotein receptor (when bound to low-density lipoprotein), making it a target for the treatment of cardiovascular disease. Another example is a GalNAc conjugate containing an antisense oligonucleotide against apolipoprotein(a), which is expressed in the liver. The gene DMD is also a target for oligonucleotide-based drug molecules. Duchenne muscular dystrophy is a uniformly fatal disease caused by mutations in the gene DMD, which encodes dystrophin. Spinal muscular atrophy is an autosomal recessive disease caused by mutations in SMN1, which result in loss of SMN1 protein function. The target of oligonucleotide-based therapy is the SMN2 gene.

[0016] Examples of oligonucleotide drugs that use a sequence-driven cleavage mechanism to reduce the levels of disease-related mRNA and its protein product include mipomersen and inclisiran, which use this cleavage mechanism to alter cholesterol dynamics. Each of these drugs targets a different gene product, each of which is important in hypercholesterolemia. The common factor in each mechanism is the activation of an endogenous enzyme upon hybridization of the oligonucleotide drug to its target, resulting in cleavage of the targeted mRNA at the site of hybridization. Mipomersen is a single-stranded oligonucleotide with a sequence complementary to a portion of the RNA encoding apolipoprotein B (apoB), a component of low-density lipoprotein (LDL) cholesterol produced in the liver. When mipomersen hybridizes to apolipoprotein B pre-mRNA, the presence of the DNA-RNA heteroduplex attracts and activates RNase H, which cleaves the mRNA in the heteroduplex. Cleavage inactivates apolipoprotein B mRNA, thereby reducing the amount of apolipoprotein B produced. As a result, the excretion of very low-density lipoprotein cholesterol from the liver is reduced, ultimately resulting in a decrease in circulating LDL cholesterol levels. Inclisiran induces the cleavage of mRNA encoding proprotein convertase subtilisin-kexin type 9 (PCSK9), an enzyme that negatively regulates LDL receptor (LDLR) levels. Individuals with naturally occurring genetic variants that reduce PCSK9 activity have increased LDLR levels, reduced LDL cholesterol levels, and reduced cardiovascular risk compared to individuals without these variants. Inclisiran cleaves and inactivates PCSK9 mRNA, which reduces PCSK9 levels, thereby increasing both LDLR levels and LDL cholesterol clearance and reducing circulating LDL cholesterol levels. Inclisiran is a double-stranded small interfering RNA (siRNA). One of the RNA strands of inclisiran is complementary to a portion of PCSK9 mRNA.Once inclisiran enters the cell, the complementary strand (or guide strand) is loaded onto the RNA-induced silencing complex (RISC), a protein complex that presents the strand to the intracellular environment. Once a near-perfect complementary sequence (on an mRNA molecule) hybridizes with a portion of the guide strand, an enzyme that is part of the RISC cleaves the mRNA. The mRNA cleavage product cannot be translated, thereby reducing PCSK9 protein levels.

[0017] Oligonucleotide-based drugs that trigger RNase H-mediated and RISC-mediated cleavage (inotersen and patisiran, respectively) are used to treat transthyretin (TTR) amyloidosis. In this form of amyloidosis, mutations in TTR induce misfolding of the protein product, which leads to the formation of amyloid deposits in multiple tissues, including peripheral neurons and the heart.

[0018] FDA-approved oligonucleotide therapeutics targeting the liver include mipomersen (targeting the apoB gene to treat homozygous familial hypercholesterolemia), defibrotide (hepatic veno-occlusive disease), patisiran TTR (hereditary transthyretin amyloidosis, polyneuropathy), inotersen TTR (hereditary transthyretin amyloidosis, polyneuropathy), and givosiran. Givosiran is a GalNAc conjugate for the treatment of acute hepatic porphyria. The target gene is ALAS1. Oligonucleotide drug candidates in clinical development that target genes in the liver include, for example: miravirsen (hepatitis C infection), which targets miR-122; RG-101 (hepatitis C infection), which targets miR-122; AB-729 (hepatitis B infection), which targets hepatitis B virus HBsAg; ARO-AAT (α1-antitrypsin deficiency), which targets AAT; SLN124 (β-thalassemia), which targets the gene TMPRSS6; DCR-PHXC (primary hyperoxaluria), which targets LDHA; and MTL-CEPBA (hepatocellular carcinoma), which targets CEBPA.

[0019] Despite considerable progress, two major hurdles—drug safety and delivery—prevent the widespread application of oligonucleotide therapeutics. The inability to deliver oligonucleotide drug candidates to organs and cells expressing disease-related RNAs has proven to be the most significant obstacle to the successful development of oligonucleotide therapeutics. Oligonucleotides are significantly larger than traditional small-molecule drug candidates. Their size, coupled with their highly anionic nature, makes it difficult for them to diffuse through the cell membrane to reach the cytoplasmic and nuclear compartments. Oligonucleotides are typically large, hydrophilic polyanions (single-stranded ASOs are ~4–10 kDa, and double-stranded siRNAs are ~14 kDa), a property that means they do not readily cross the plasma membrane. Additionally, difficulties arise when considering the sufficient release and delivery of oligonucleotides in the cytosol and / or nucleus. This requires the escape of oligonucleotides from the endolysosomal system prior to lysosomal degradation or re-excretion by exocytosis to reach the correct intracellular site of action. Current oligonucleotide cellular delivery strategies are inefficient, leading to renal excretion of the drug or delivery of the majority of the drug to cells and tissues that are not the therapeutic target. Despite improved oligonucleotide (e.g., siRNA) delivery to cells achieved by some of the aforementioned delivery systems, endosomal escape remains a major barrier to the application of oligonucleotide-based therapeutics, such as RNAi-based therapeutics. Only a small fraction of endocytosed oligonucleotides escape into the cytoplasmic space where they can exert their intended function, while the majority remain trapped in the endocytic compartment and are inactive. Recent literature suggests a passive siRNA escape rate of <0.01% (Non-Patent Document 2). Furthermore, it has been shown that the ability of cells to functionally internalize oligonucleotides to produce targeted effects (e.g., knockdown) appears to be independent of the degree to which cells internalize bulk oligonucleotides.These observations have led to the hypothesis of distinct "productive" and "non-productive" free uptake pathways, although the molecular mechanisms that distinguish these pathways remain elusive (Non-Patent Document 2).

[0020] Overall, regardless of the underlying mechanism (improved endosomal escape, increased "productive" pathway uptake, or another mechanism), there remains a significant need for improved delivery systems for oligonucleotides that result in increased efficacy (e.g., targeted knockdown), less toxicity, and / or fewer off-target effects.

[0021] One emerging strategy involves conjugating antisense oligonucleotides (ASOs) to receptor ligands to increase oligonucleotide potency and distribution to selected tissues. For example, conjugation of triantennary N-acetylgalactosamine (GalNAc) to oligonucleotide therapeutics results in 10- to 30-fold increased potency in isolated hepatocytes and in vivo in the liver. GalNAc is found on damaged glycoproteins that have lost terminal sialic acid residues from their pendant oligosaccharides. The liver produces very high levels (10 per cell) of GalNAc on the surface of hepatocytes. 5 ~10 6 Liver cells function to clear these proteins from the systemic circulation by expressing the trimeric asialoglycoprotein receptor (ASGPR) at a pH on the order of 1000 (or 1000). The ASGPR specifically binds GalNAc at neutral pH for endocytosis of circulating macromolecules from the blood and releases GalNAc at acidic pH (~5-6) for cargo unloading in early endosomes. The liberated ASGPR is then recycled back to the cell surface for reuse. Approximately one-third of RNAi drugs currently in clinical trials are single-molecule, chemically modified RNAi triggers conjugated to multivalent GalNAc ligands that target ASGPR. The favorable physiology of the liver, the unique properties of the ASGPR, the non-toxic nature of the GalNAc ligand, and the simplicity of the GalNAc-siRNA complex make this an attractive approach for systemic RNAi delivery to hepatocytes.

[0022] Targeted delivery of oligonucleotide therapeutics (oligonucleotide bioconjugates) to hepatocytes via uptake mediated by the asialoglycoprotein receptor (ASGPR) promotes interaction between the conjugate and its corresponding cell surface receptor protein, leading to subsequent internalization by receptor-mediated endocytosis. Interaction of the bioconjugate with the cell-type-associated receptor thereby enables targeted delivery to specific tissues or cell types within tissues. ASGPR binds and internalizes, for example, triantennary N-acetylgalactosamine (GalNAc) conjugated to therapeutic antisense oligonucleotides, which are internalized and then released inside the cell, where they can hybridize to their corresponding pre-messenger RNA (mRNA) and induce RNA-DNA heteroduplex breaks. Alternatively, they can be complexed with small interfering RNA (siRNA) directed against, for example, disease-related genes. Following release of the siRNA from the endosomal or lysosomal compartment, the now cytoplasmic siRNA can load onto the RNA-induced silencing complex (RISC). The loaded RISC can then scan all expressed RNA for sequence complementarity. When a complementary sequence is detected by hybridization, an enzyme within the RISC cleaves the targeted mRNA, thereby reducing the expression of the disease-related protein. RNAi stands for RNA interference. This receptor-mediated uptake allows for lower dosing than that required for therapeutic delivery of unconjugated oligonucleotides. Both single-stranded and double-stranded oligonucleotides can be delivered to liver cells using GalNAc conjugates. For example, drugs for the treatment of diseases such as hemophilia A and hemophilia B, as well as the siRNA-GalNAc conjugates inclisiran targeting the gene PCSK9 and givosiran targeting the gene ALAS1, have been developed based on oligonucleotide-GalNAc conjugates.

[0023] Despite technological advances, achieving efficient oligonucleotide delivery remains a major limitation in technology applications. Oligonucleotide-based drug platforms employ approaches aimed at improving oligonucleotide delivery, such as chemical modification of oligonucleotides, bioconjugation, and the use of nanocarriers for oligonucleotide delivery, but delivery challenges still require improvement. There remains a need for improved delivery systems that further increase the efficacy, reduce toxicity, and / or mitigate off-target effects of ASGPR-targeting oligonucleotide systems (e.g., GalNAc-oligonucleotide conjugates). [Prior art documents] [Non-patent literature]

[0024] [Non-Patent Document 1] Gooding, Matt et al., Chemical Biology & Drug Design, Vol. 80.6 (2012): pp. 787-809 [Non-patent document 2] Setten, Ryan L., John J. Rossi, and Si-ping Han, Nature Reviews Drug Discovery, Vol. 18.6 (2019): pp. 421-446 Summary of the Invention

[0025] One embodiment of the present invention relates to an oligonucleotide conjugate comprising at least one saponin covalently linked to a ligand of the asialoglycoprotein receptor (ASGPR), wherein the ligand of the ASGPR comprises at least one N-acetylgalactosamine (GalNAc) moiety, preferably three or four GalNAc moieties, and more preferably the ligand of the ASGPR comprises or consists of (GalNAc)3Tris, and further covalently linked to an oligonucleotide, wherein the at least one saponin is selected from monodesmosidic triterpenoid saponins and bisdesmosidic triterpenoid saponins.

[0026] 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, and more preferably the ligand of ASGPR comprises or consists of (GalNAc)3Tris) and further covalently linked to an oligonucleotide, wherein the at least one saponin is a 12,13-dehydrooleanane-type monodesmosidic or bisdesmosidic pentacyclic triterpene saponin, preferably having an aldehyde functional group at position C23 of the saponin aglycone core structure, and wherein the oligonucleotide conjugate comprises 1 to 16 saponin moieties, preferably 1 to 8 saponin moieties, more preferably 1 saponin moiety, 4 saponin moieties, or 8 saponin moieties.

[0027] One embodiment is an oligonucleotide conjugate of the invention, wherein at least one GalNAc moiety, preferably three GalNAc moieties, at least one saponin, preferably 1 to 16 saponin moieties, more preferably 1 to 8 saponin moieties, e.g., 1, 4, or 8 saponin moieties, and the oligonucleotide are covalently linked via a trifunctional linker, preferably wherein each of the GalNAc moiety, saponin or saponin moiety, and oligonucleotide is covalently linked to a separate arm of the trifunctional linker.

[0028] An embodiment is an oligonucleotide conjugate of the invention comprising one saponin moiety, or four saponin moieties, preferably four saponin moieties, covalently attached to a dendron, preferably a G2 dendron, such as, for example, N,N'-((9S,19S)-14-(6-aminohexanoyl)-1-mercapto-9-(3-mercaptopropanamide)-3,10,18-trioxo-4,11,14,17-tetraazatricosane-19,23-diyl)bis(3-mercaptopropanamide), or eight saponin moieties. and preferably eight saponin moieties covalently attached to a dendron, preferably a G3 dendron, such as, for example, (2S)-N-[(1S)-1-{[2-(6-amino-N-{2-[(2S)-2,6-bis[(2S)-2,6-bis(3-sulfanylpropanamido)hexanamido]hexanamido]ethyl}hexanamido)ethyl]carbamoyl}-5-[(2S)-2,6-bis(3-sulfanylpropanamido)hexanamido]pentyl]-2,6-bis(3-sulfanylpropanamido)hexanamide.

[0029] In one embodiment, the oligonucleotide conjugate of the present invention is provided, wherein at least one saponin moiety is linked via a hydrazone bond or a semicarbazone bond. Preferably, at least one saponin is covalently attached to the oligonucleotide conjugate via a semicarbazone bond. Such a hydrazone bond or semicarbazone bond (with a linker) is formed, for example, via an aldehyde group at the C23 atom of the quillaia acid aglycone core structure of the saponin or the gypsogenin aglycone core structure of the saponin.

[0030] The following oligonucleotide complexes of the present invention are preferred: the oligonucleotides contained in the complexes are defined as nucleic acids not longer than 150 nt, preferably wherein the oligonucleotides have a size of 5 to 150 nt, preferably 8 to 100 nt, and most preferably 10 to 50 nt.

[0031] One aspect of the present invention relates to a pharmaceutical composition comprising an oligonucleotide conjugate of the present invention and optionally a pharmaceutically acceptable excipient and / or optionally a pharmaceutically acceptable diluent.

[0032] Certain aspects of the present invention relate to an oligonucleotide conjugate of the present invention or to a pharmaceutical composition comprising an oligonucleotide conjugate of the present invention for use as a medicament.

[0033] Certain aspects of the present invention relate to pharmaceutical compositions comprising the oligonucleotide conjugates of the present invention for use in the treatment or prevention of diseases or health problems involving expression products of any one or more of the genes HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AT3, GO, CC5, HBV X gene, HBV S gene, 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 genes HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AT3, GO, CC5, HBV X gene, HBV S gene, AAT, miR-122, Hepatitis B virus HBsAg, LDHA, CEBPA, and LDH.

[0034] Certain aspects of the present invention relate to pharmaceutical compositions comprising the oligonucleotide conjugates of the invention for use in the treatment or prevention of diseases or health problems involving expression products of any one or more of the genes HSP27, apoB, TTR, PCSK9, 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 genes HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AAT, miR-122, Hepatitis B virus HbsAg, LDHA, and CEBPA.

[0035] Certain aspects of the present invention relate to pharmaceutical compositions comprising the oligonucleotide conjugates of the present invention for use in the treatment or prevention of diseases or health problems involving the expression products of any one or more of the 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 genes HSP27 and apoB, preferably apoB.

[0036] Certain embodiments of the present invention relate to pharmaceutical compositions comprising the oligonucleotide conjugates of the present invention for use in the treatment or prevention of cancer, infectious diseases, viral infections, hypercholesterolemia, cardiovascular diseases, primary hyperoxaluria, hemophilia A, hemophilia B, AAT-associated liver disease, acute hepatic porphyria, TTR amyloidosis, hereditary TTR amyloidosis (hATTR), complement-related diseases, hepatitis B infection, hepatitis C infection, α1-antitrypsin deficiency, β-thalassemia, or autoimmune diseases.

[0037] Certain aspects of the present invention relate to pharmaceutical compositions comprising an oligonucleotide conjugate of the present invention for use in the treatment or prevention of cancer, such as endometrial cancer, breast cancer, lung cancer, or hepatocellular carcinoma, and / or cardiovascular disease, such as hypercholesterolemia, preferably hypercholesterolemia.

[0038] One aspect of the present invention relates to an in vitro or ex vivo method for transferring an oligonucleotide complex of the present invention from the outside of a cell to the inside of said cell, preferably followed by transferring the oligonucleotide contained in the oligonucleotide complex of the present invention into the cytosol and / or nucleus of said cell, comprising the steps of: a) providing a cell expressing an ASGPR, preferably ASGPR1, on its surface, the cell preferably being selected from a liver cell, a virus-infected mammalian cell, and a mammalian tumor cell, wherein preferably said cell is a human cell; b) providing an oligonucleotide complex of the invention for transfection into the cells provided in step a); c) contacting the cells of step a) in vitro or ex vivo with the oligonucleotide complex of step b), preferably in a liquid medium, thereby resulting in the transfer of the oligonucleotide complex from outside the cells into said cells, and optionally and preferably thereby resulting in the subsequent transfer of the oligonucleotides contained in the oligonucleotide complex into the cytosol and / or nucleus of said cells.

[0039] One aspect of the present invention relates to an in vitro or ex vivo method for transferring an oligonucleotide complex of the present invention from the outside of a cell to the inside of said cell, and preferably subsequently transferring the oligonucleotide contained in said oligonucleotide complex into the cytosol of said cell, comprising the steps of: a) providing a cell expressing ASGPR on its surface, the cell preferably being selected from a liver cell, a virus-infected cell, and a tumor cell; and providing an oligonucleotide complex of the invention for transfection into the provided cell; b) contacting the cells of step a) in vitro or ex vivo with the oligonucleotide complex of step a), thereby resulting in the transfer of the oligonucleotide complex from outside the cells into said cells, and preferably thereby resulting in the subsequent transfer of the oligonucleotides contained in the oligonucleotide complex into the cytosol of said cells.

[0040] Preferably the cells are human cells. Preferably the cells are liver cells.

[0041] One embodiment of the present invention relates to a saponin conjugate comprising at least one saponin covalently linked to a ligand of the asialoglycoprotein receptor (ASGPR), wherein the ligand of the ASGPR comprises at least one N-acetylgalactosamine (GalNAc) moiety, preferably three or four GalNAc moieties, and more preferably the ligand of the ASGPR comprises or consists of (GalNAc)3Tris, and wherein the at least one saponin is selected from monodesmosidic triterpenoid saponins and bisdesmosidic triterpenoid saponins.

[0042] One aspect of the present invention relates to a pharmaceutical combination comprising: - a first pharmaceutical composition comprising a saponin complex of the present invention, and optionally a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent; and - a second pharmaceutical composition comprising a second complex of an effector molecule and a ligand of ASGPR (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 or consists of (GalNAc)3Tris) or a third complex of an effector molecule and a binding molecule comprising a binding site for a cell surface molecule, and optionally comprising a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent.

[0043] One aspect of the present invention relates to a pharmaceutical composition comprising: - a saponin complex according to the invention, - a second complex of an effector molecule and a ligand of ASGPR (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 or consists of (GalNAc)3Tris) or a third complex of an effector molecule and a binding molecule comprising a binding site for a cell surface molecule, and optionally a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent.

[0044] One aspect of the present invention relates to a pharmaceutical combination of the present invention comprising a saponin complex of the present invention or to a pharmaceutical composition of the present invention comprising a saponin complex of the present invention, for use as a medicament.

[0045] One aspect of the present invention relates to 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, for use in the treatment or prevention of a disease or health problem involving the expression product of any one or more of the genes HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AT3, GO, CC5, HBV X gene, HBV S gene, AAT, miR-122, Hepatitis B virus HbsAg, LDHA, CEBPA, and LDH.

[0046] One aspect of the present invention relates to an in vitro or ex vivo method for transferring a second or third complex of the present invention from the outside of a cell to the inside of the cell, and preferably thereafter transferring an effector molecule contained in the second or third complex of the present invention into the cytosol of the cell, comprising the steps of: a) providing a cell expressing ASGPR on its surface, and when the third complex is to be transferred into a cell expressing a cell surface molecule comprising a binding molecule for binding the third complex to the cell surface molecule, the cell is preferably selected from a liver cell, a virus-infected cell, and a tumor cell; b) providing any one of the second complexes or the third complex of the invention for transfection into the cells provided in step a); c) providing a saponin complex of the present invention; d) contacting the cells of step a) in vitro or ex vivo with the second or third complex of step b) and the saponin complex of step c), thereby resulting in the transfer of the second or third complex from outside the cells into the cells, preferably thereby resulting in the subsequent transfer of the second or third complex into the cytosol of the cells, or preferably thereby resulting in the subsequent transfer of at least the effector molecule comprised in the second or third complex into the cytosol of the cells.

[0047] One aspect of the present invention relates to a method for providing an oligonucleotide conjugate of the present invention, comprising the steps of: a) providing at least one saponin moiety comprising a covalently attached first linker, wherein the first linker comprises at least one first reactive group for covalent attachment 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 attached third linker, wherein the third linker comprises a third reactive group for covalent attachment to a fourth reactive group on the fourth linker or an eighth reactive group on the seventh linker; c) providing at least one GalNAc moiety comprising a covalently attached fifth linker, wherein the fifth linker comprises a fifth reactive group for covalent attachment to a sixth reactive group on a sixth linker or a ninth reactive group on a seventh linker; and (d1) linking the first linker to the second linker by forming a covalent bond between the first reactive group and the second reactive group, linking the third linker to the fourth linker by forming a covalent bond between the third reactive group and the fourth reactive group, linking the fifth linker to the sixth linker by forming a covalent bond between the fifth reactive group and the sixth reactive group, and covalently linking the second linker, the fourth linker, and the sixth linker together, thereby providing an oligonucleotide; or (d2) 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 an oligonucleotide conjugate; Either one.

[0048] An embodiment is a method for providing an oligonucleotide conjugate of the invention, wherein the seventh linker is a trifunctional linker, for example, a trifunctional linker represented by formula (XXI):

[0049] [ka]

[0050] The following methods for providing an oligonucleotide conjugate of the invention are preferred: at least one saponin moiety is 1 to 16 saponin moieties, preferably 1 to 8 saponin moieties, for example 1, 4, or 8 saponin moieties.

[0051] The following method for providing an oligonucleotide conjugate of the invention is preferred: the saponin is SO1861, SO1832, QS-21, or a functional derivative of any of them, preferably SO1861 or SO1832.

[0052] The following methods for providing oligonucleotide conjugates of the invention are preferred: the saponin moiety is covalently linked via a hydrazone or semicarbazone bond.

[0053] The following methods for providing an oligonucleotide conjugate of the invention are preferred: at least one GalNAc moiety is 1 to 4 GalNAc moieties, preferably 1 or 3 GalNAc moieties.

[0054] In one embodiment, the oligonucleotide conjugate of the present invention, wherein the saponin contained in the oligonucleotide conjugate is isolated from a plant. Preferably, the saponin is isolated from a part of a plant, such as a root, or a part of a tree, such as bark. Preferably, the saponin is isolated from a root derived from a plant.

[0055] definition The term "GalNAc" has its usual scientific meaning and refers herein to N-acetylgalactosamine and its IUPAC designation 2-(acetylamino)-2-deoxy-D-galactose.

[0056] The term "(GalNAc)3Tris" has its customary scientific meaning, e.g., in the field of siRNA-based therapeutics, and herein refers to a moiety comprising three GalNAc units, each separately covalently bound via at least one linker to the hydroxyl groups of tris(hydroxymethyl)aminomethane (Tris) (IUPAC name: 2-amino-2-(hydroxymethyl)propane-1,3-diol). (GalNAc)3Tris can exist as a molecule containing a free amine or can be further functionalized via the remaining amine linkage sites to form conjugates containing (GalNAc)3Tris moieties, e.g., as described herein.

[0057] The term "oligonucleotide" has its usual scientific meaning and refers herein to a single strand of two or more nucleotides. That is, an oligonucleotide is a short oligomer composed of ribonucleotides or deoxyribonucleotides. Examples include RNA and DNA, as well as any modified RNA or DNA, such as locked nucleic acid (LNA) or 2'-O,4'-C-aminoethylene or 2'-O,4'-C-aminomethylene bridged nucleic acid (BNA). NC A nucleic acid is a strand of nucleic acid that contains nucleotide analogs, such as bridged nucleic acids (BNAs), also known as bridged nucleic acids (BNAs), where the nucleotides are ribonucleotides or deoxyribonucleotides.

[0058] As used herein, the terms "nucleic acid," "oligonucleotide," and "polynucleotide" are synonymous and should be interpreted to encompass any polymeric molecule made from units, where the units include nucleobases (or simply "bases," e.g., classical nucleobases such as adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U), or any known non-classical, 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 polymeric molecule to undergo hydrogen-bonding-based nucleobase pairing (e.g., Watson-Crick base pairing) with naturally occurring nucleic acids, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), under appropriate hybridization conditions. These naturally occurring nucleic acids should be understood to be polymeric molecules made from units that are nucleotides.

[0059] Thus, from a chemical perspective, the term nucleic acid according to this definition can be interpreted to encompass polymeric molecules that are chemically DNA or RNA, as well as nucleic acid analogs, also known as xenonucleic acids (XNAs) or artificial nucleic acids, which are polymeric molecules in which one or more (or all) of the units are modified nucleotides or functional equivalents of nucleotides. Nucleic acid analogs are well known in the art and are being actively used in research and medicine due to various properties, such as improved specificity and / or affinity, higher binding strength to their targets, and / or increased stability in vivo. Representative examples of nucleic acid analogs include, but are not limited to, locked nucleic acids (LNAs), also known as bridged nucleic acids (BNAs), phosphorodiamidate morpholino oligomers (PMOs, also known as morpholinos), peptide nucleic acids (PNAs), glycol nucleic acids (GNAs), threose nucleic acids (TNAs), hexitol nucleic acids (HNAs), 2'-deoxy-2'-fluoroarabinonucleic acid (FANA or FNA), 2'-deoxy-2'-fluororibonucleic acid (2'-FRNA or FRNA), altritol nucleic acid (ANA), cyclohexene nucleic acid (CeNA), and the like.

[0060] According to the general rule, the length of a nucleic acid is expressed herein by the number of units that make up a single strand of nucleic acid. Because each unit corresponds to exactly one nucleic acid base that can undergo one base pairing event, regardless of whether the nucleic acid in question is a single-stranded (ss) or double-stranded (ds) nucleic acid, the length is often expressed in terms of so-called "base pairs" or "bp". In naturally occurring nucleic acids, 1 bp corresponds to 1 nucleotide and is abbreviated as 1 nt. For example, a single-stranded nucleic acid made up of 1000 nucleotides (or a double-stranded nucleic acid made up of two complementary strands, each made up of 1000 nucleotides) is described as having a length of 1000 base pairs or 1000 bp, and this length can also be expressed as 1000 nt or 1 kilobase, abbreviated as 1 kb. 2 kilobases or 2 kb is equivalent to a length of 2000 base pairs, which corresponds to 2000 nucleotides of single-stranded RNA or DNA. To avoid confusion, however, in light of the fact that nucleic acids as defined herein can comprise or consist of units that are not only chemically nucleotides but also their functional equivalents, the length of a nucleic acid will preferentially be expressed herein by "bp" or "kb" rather than the designation "nt" which is equally common in the art.

[0061] In advantageous embodiments, the nucleic acids disclosed herein are no longer than 1 kb, preferably no longer than 500 bp, and most preferably no longer than 250 bp.

[0062] In a particularly advantageous embodiment, the nucleic acid is an oligonucleotide (or simply oligo), defined as a nucleic acid not longer than 100 bp, i.e., any polymer molecule made up of not more than 100 units according to the definition provided above, wherein each unit contains a nucleic acid base or a functional equivalent thereof. This allows the oligonucleotide to undergo hydrogen bond-based nucleic acid base pairing with DNA or RNA under appropriate hybridization conditions. It will be readily understood that within the scope of the above definition, the oligonucleotides disclosed herein can contain or consist of units that are not only nucleotides but also their synthetic equivalents. In other words, from a chemical point of view, the term oligonucleotide as used herein can be understood to include or consist of RNA, DNA, or nucleic acid analogs, such as, but not limited to, LNA (BNA), PMO (morpholino), PNA, GNA, TNA, HNA, FANA, FRNA, ANA, CeNA, and / or the like.

[0063] The term " gene targeting mediated by RNAi " has its usual scientific meaning, and herein refers to the in vivo, ex vivo or in vitro approach of influencing the function of gene in cells by introducing oligonucleotides such as small double-stranded RNA molecules into said cells, and the oligonucleotides are targeted to the mRNA involved in the transcription of gene.For example, small double-stranded RNA molecules can effectively trigger the RNAi silencing of specific genes.

[0064] The term "bridged nucleic acid" or for short "BNA" or "locked nucleic acid" or for short "LNA" or 2'-O,4'-C-aminoethylene or 2'-O,4'-C-aminomethylene bridged nucleic acid (BNA NC) has its usual scientific meaning and refers herein to modified RNA nucleotides. BNAs are also referred to as "conformationally locked RNA molecules" or "inaccessible RNA molecules." BNA monomers can contain five-, six-, or even seven-membered bridge structures with "locked" C3'-endo sugar puckering. Bridges are synthetically incorporated at the 2',4' positions of the ribose to provide 2',4'-BNA monomers. BNA monomers can be incorporated onto oligonucleotide polymer structures using standard phosphoramidite chemistry known in the art. BNAs are structurally rigid oligonucleotides with increased binding affinity and stability. The term "BNA" refers to BNA NC or 2',4'-BNA NC The term "2'-O,4'-aminoethylene-bridged nucleic acid" also refers to an oligonucleotide containing one or more nucleotide building blocks having a six-membered bridge structure with an NO linkage and an (NH) or (N-Me) residue.

[0065] The term antisense oligonucleotide has its usual scientific meaning and may be referred to herein in shorthand as "AON" or "ASO".

[0066] The term "BNA-based antisense oligonucleotide" or abbreviated "BNAAON" has its usual scientific meaning and refers herein to a single antisense nucleotide in which at least one of the nucleotides is a BNA.

[0067] The term "proteinaceous" has its usual scientific meaning and, as used herein, refers to a molecule that is protein-like. It means that the molecule possesses, to some extent, physicochemical properties characteristic of a protein, is made of a protein, relates to a protein, contains a protein, is associated with a protein, consists of a protein, resembles a protein, or is a protein. For example, the term "proteinaceous" as used in "proteinaceous molecule" refers to the presence of at least a portion of a molecule that resembles or is a protein, where "protein" should be understood to include chains of amino acid residues at least two residues in length, and therefore includes peptides, polypeptides, and proteins, as well as assemblies of proteins or protein domains. In a proteinaceous molecule, at least two amino acid residues are linked via an amide bond, e.g., a peptide bond. In a proteinaceous molecule, 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, a proteinaceous molecule is a molecule containing at least two amino acid residues, preferably between 2 and about 2,000 amino acid residues. In one embodiment, a proteinaceous molecule is a molecule containing 2 to 20 amino acids (typical for peptides). In one embodiment, the proteinaceous molecule is a molecule comprising 21 to 1,000 amino acids (typically a polypeptide, a protein, a protein domain, e.g., an antibody, a Fab, a scFv, one or more Vh domains, a ligand of a receptor, e.g., EGF). Preferably, the amino acid residues are (typically) linked via peptide bonds. According to the present invention, said amino acid residues are or comprise (modified) (non-)natural amino acid residues.

[0068] The terms "effector molecule" or "effector moiety," when referring to an effector molecule, e.g., as part of a covalent complex, have their usual scientific meaning, and herein refer to a molecule that can selectively bind to one or more of a target molecule's proteins, peptides, carbohydrates, sugars such as glycans, (phospho)lipids, nucleic acids such as DNA, RNA, or enzymes, thereby controlling the biological activity of such one or more target molecules. The effector molecule can be, for example, a molecule selected from one or more of a small molecule, e.g., a drug molecule, a toxin, e.g., a protein toxin, an oligonucleotide, e.g., BNA, xenonucleic acid, or siRNA, an enzyme, a peptide, a protein, 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 a small molecule, such as a drug molecule, a toxin, such as a protein toxin, an oligonucleotide, such as BNA, xenonucleic acid or siRNA, an enzyme, a peptide, a protein, or any combination thereof, which can selectively bind to any one or more of a target molecule, such as a protein, peptide, carbohydrate, sugar, such as glycan, (phospho)lipid, nucleic acid, such as DNA, RNA, or enzyme, and regulates the biological activity of such one or more target molecules by binding to the target molecule. Typically, an effector molecule can exert a biological effect inside a cell, such as a mammalian cell, such as a human cell, for example, in the cytosol of the cell. Exemplary effector molecules, therefore, are drug molecules, plasmid DNA, toxins, such as toxins contained in antibody-drug conjugates (ADCs), oligonucleotides, such as siRNA, BNAs, and nucleic acids contained in antibody-oligonucleotide conjugates (AOCs). For example, an effector molecule can act as a ligand that can increase or decrease (intracellular) enzyme 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 the conjugates of the invention.

[0069] The term "health problem" has its usual scientific meaning, and herein refers to a condition of a subject's body, such as a human patient, or any part thereof, organ, muscle, vein, artery, skin, limb, blood, cell, etc., that is not optimal compared to the condition of that body or part thereof in a healthy subject, thereby interfering with the proper function and / or well-being of the subject, e.g., impairing the normal function of the human body.

[0070] The term "GalNAc-decorated oligonucleotide drug" has its usual scientific meaning, herein referring 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.

[0071] The term "locked nucleic acid", or "LNA" for short, a bridged nucleic acid, has its usual scientific meaning and refers herein to an oligonucleotide containing one or more nucleotide building blocks in which an additional methylene bridge locks the ribose moiety in either the C3'-endo conformation (beta-D-LNA) or the C2'-endo (alpha-L-LNA) conformation, as known in the art.

[0072] The term "chemically modified metabolically stable siRNA" has its usual scientific meaning, and herein refers to an siRNA molecule that contains chemical modifications compared to RNA oligonucleotides composed of naturally occurring nucleotides such that the modified siRNA molecule is more resistant to metabolic degradation, digestion, enzymatic lysis, etc., i.e., more stable.

[0073] The term "saponin" has its usual scientific meaning and refers herein to a group of amphiphilic glycosides containing one or more hydrophilic glycone moieties combined with a lipophilic aglycone core that is a sapogenin. Saponins can be naturally occurring or synthetic (i.e., non-naturally occurring). The term "saponin" encompasses naturally occurring saponins, derivatives of naturally occurring saponins, and saponins synthesized de novo by chemical and / or biotechnological synthetic routes.

[0074] The term "saponin derivative" (also known as "modified saponin") has its customary scientific meaning, and herein refers 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 include derivatization of an aldehyde group on the aglycone core, a carboxyl group on the sugar chain, or an acetoxy group on the sugar chain. Typically, a saponin derivative has no natural counterpart, i.e., it is not naturally produced, for example, by a plant or tree. The term "saponin derivative" encompasses derivatives obtained by derivatization of a naturally occurring saponin, as well as derivatives synthesized de novo by chemical and / or biotechnological synthetic routes that result in a compound corresponding to a naturally occurring saponin that has been derivatized by one or more chemical modifications.

[0075] The term "aglycone core structure" has its usual scientific meaning and refers herein to the aglycone core of a saponin without one or two carbohydrate antennae or sugar chains (glycans) attached thereto. For example, quillaic acid is the aglycone glycoside core structure of SO1861, QS-7, and QS-21.

[0076] The term "glycan" has its usual scientific meaning and refers herein to either a glycan, a carbohydrate antenna, a single sugar moiety (monosaccharide), or a chain containing multiple sugar moieties (oligosaccharide, polysaccharide). A glycosyl chain may consist of only sugar moieties or may also contain additional moieties, such as any one of the following: 4E-methoxycinnamic acid, 4Z-methoxycinnamic acid, and 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), e.g., present on QS-21.

[0077] The terms "SO1861" and "SO1862" refer to the same saponin from Saponaria officinalis in its deprotonated or api form, respectively. The molecular mass is 1862 daltons, which is the formal mass including the proton on the glucuronic acid. At neutral pH, the molecule is deprotonated. When the mass is measured using mass spectrometry in negative ion mode, the measured mass is 1861 daltons. Similarly, the terms "SO1831" and "SO1832" refer to the same saponin from Saponaria officinalis in its deprotonated or api form, respectively. The molecular mass is 1832 daltons, which is the formal mass including the proton on the glucuronic acid. At neutral pH, the molecule is deprotonated. When the mass is measured using mass spectrometry in negative ion mode, the measured mass is 1831 daltons.

[0078] The term "Api / Xyl-" or "Api- or Xyl-" in the context of glycan names has its usual scientific meaning, and herein refers to glycans that either contain an apiose (Api) moiety or a xylose (Xyl) moiety.

[0079] The term "antibody-drug conjugate" or "ADC" has its customary scientific meaning and refers herein to any conjugate of an antibody, e.g., an IgG, Fab, scFv, immunoglobulin, immunoglobulin fragment, one or more Vh domains, etc., and any molecule, e.g., a drug substance, toxin, oligonucleotide, enzyme, small molecule drug compound, etc., that can exert a therapeutic effect when contacted with cells of a subject, such as a human patient.

[0080] The term "antibody-oligonucleotide conjugate" or "AOC" has its customary scientific meaning and refers herein to any oligonucleotide molecule, such as an antibody, e.g., an IgG, Fab, scFv, immunoglobulin, immunoglobulin fragment, one or more Vh domains, that can exert a therapeutic effect when contacted with cells of a subject, such as a human patient, e.g., any conjugate with an oligonucleotide selected from a natural or synthetic single nucleic acid, including: DNA, modified DNA, RNA, modified RNA, synthetic nucleic acids presented as single- or double-stranded molecules, e.g., BNA, allele-specific oligonucleotides (ASO), short- or small-interfering RNA (siRNA, silencing RNA), antisense DNA, antisense RNA, etc.

[0081] The term "conjugate" has its usual scientific meaning and refers herein to at least a first molecule covalently linked to at least a second molecule, thereby forming a covalently coupled assembly comprising or consisting of the first and second molecules. Exemplary conjugates are (GalNAc)3Tris-siRNA, ADC, AOC, and SO1861-EMCH (EMCH linked to the aldehyde group of the aglycone glycoside core structure of saponin).

[0082] The term "moiety" has its usual scientific meaning, and herein refers to a molecule that is bound, linked, or conjugated to an additional molecule, linker, molecular assembly, etc., thereby forming part of a larger molecule, complex, or molecular assembly. Typically, a moiety is a first molecule that is covalently bound to a second molecule, initially involving one or more chemical groups present on the first molecule and one or more chemical groups present on the second molecule. For example, saporin is a typical effector molecule. As part of an antibody-drug conjugate, saporin is a typical effector moiety on an ADC. As part of an antibody-oligonucleotide conjugate, BNA or siRNA are typical effector moieties on an AOC.

[0083] For example, the term "S" as used in antibody-saponin conjugates or constructs that include a linker represents a "stable linker" that remains intact in endosomes and the cytosol.

[0084] For example, the term "L" used in antibody-saponin conjugates or constructs that include a linker represents a "labile linker" that is cleaved under the slightly acidic conditions of the endosome.

[0085] The terms first, second, third, and the like are used in the specification and claims to distinguish, for example, between similar elements, compositions, components of compositions, or separate method steps, and not necessarily to describe a sequential or chronological order. The terms are interchangeable under appropriate circumstances, and embodiments of the invention may operate in other sequences than those described or illustrated herein, unless otherwise specified.

[0086] The embodiments of the invention described herein may be combined and operated in conjunction unless otherwise specified.

[0087] Furthermore, although various embodiments may be referred to as "preferably" or "eg" or "for example" or "specifically" and the like, these should not be construed as limiting the scope of the invention, but rather as exemplary ways in which the invention may be implemented.

[0088] The term "comprising" as used in the claims should not be construed as being limited to the subsequently listed elements, method steps, or composition components, for example. It does not exclude other elements, method steps, or components in a certain composition. It needs to be interpreted as specifying the presence of the referenced claimed feature, object, (method) step, or component, but does not prohibit the presence or addition of one or more other features, objects, steps, or components, or groups thereof. Thus, the scope of the phrase "a method comprising steps A and B" should not be limited to a method consisting of only steps A and B; rather, for the present invention, the only recited steps of the method are A and B, and the claims should be interpreted to encompass equivalents of those method steps. Thus, the scope of the phrase "a composition comprising components A and B" should not be limited to a composition consisting of only components A and B; rather, for the present invention, the only recited components of the composition are A and B, and the claims should be interpreted to encompass equivalents of those components.

[0089] In addition, the reference to an element or component by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element or component is present, unless the context clearly requires that there is only one of the element or component. The indefinite article "a" or "an" therefore normally means "at least one."

[0090] The term "DAR" generally stands for drug-antibody ratio and refers to the average drug-to-antibody ratio for a given preparation of antibody-drug conjugate (ADC), and here refers to the ratio of the number of conjugated SO1861 moieties or SPT001 or generally saponin moieties, or conjugated payload, e.g., AON, e.g., ApoBBNA, to conjugate molecules. [Brief explanation of the drawings]

[0091] [Figure 1A] Figure 1A-C: Synthesis of trivalent GalNAc. [Figure 1B] Figure 1A-C: Synthesis of trivalent GalNAc. [Figure 1C] Figure 1A-C: Synthesis of trivalent GalNAc. [Figure 2A] Figure 2A-B: Synthesis of SO1861-DBCO. [Figure 2B] Figure 2A-B: Synthesis of SO1861-DBCO. [Figure 3] Figure 3: Synthesis of monovalent GalNAc-SO1861. [Figure 4] Figure 4: Synthesis of trivalent GalNAc-SO1861. [Figure 5A] Figure 5A-C: Synthesis of monovalent GalNAc-BNAs. [Figure 5B] Figure 5A-C: Synthesis of monovalent GalNAc-BNAs. [Figure 5C] Figure 5A-C: Synthesis of monovalent GalNAc-BNAs. [Figure 6A] Figure 6A-B: Synthesis of trivalent GalNAc-BNA. [Figure 6B] Figure 6A-B: Synthesis of trivalent GalNAc-BNA. [Figure 7] Figure 7: Synthesis of trivalent GalNAc-BNAs. [Figure 8A] Figure 8A-G: Synthesis of trivalent GalNAc. [Figure 8B] Figure 8A-G: Synthesis of trivalent GalNAc. [Figure 8C] Figure 8A-G: Synthesis of trivalent GalNAc. [Figure 8D] Figure 8A-G: Synthesis of trivalent GalNAc. [Figure 8E] Figure 8A-G: Synthesis of trivalent GalNAc. [Figure 8F] Figure 8A-G: Synthesis of trivalent GalNAc. [Figure 8G] Figure 8A-G: Synthesis of trivalent GalNAc. [Figure 9] Figures 9A and 9B: General toxicity (MTS) of GalNAc-S01861, S01861-EMCH, and trivalent GalNAc-S01861 against HepG2 (A) and Huh7 (B) cell lines. The legend next to Figure 9B also applies to Figure 9A. [Figure 10] Figures 10A and 10B: HepG2 (A) and Huh7 (B) cell line cell killing assay (MTS). The legend presented beside Figure 10B also applies to Figure 10A. [Figure 11] Figures 11A and 11B: Gene expression analysis in HepG2 (A) and Huh7 (B) cell lines. [Figure 12] Figures 12A and 12B: Gene expression analysis in HepG2 (A) and Huh7 (B) cell lines. The legend presented beside Figure 12B also applies to Figure 12A. [Figure 13] Figures 13A and 13B: Cell viability assays in HepG2 (A) and Huh7 (B) cell lines. The legend presented beside Figure 13B also applies to Figure 13A. [Figure 14] Figures 14A and 14B: Gene expression analysis in HepG2 (A) and Huh7 (B) cell lines. The legend presented beside Figure 14B also applies to Figure 14A. [Figure 15] Figures 15A and 15B: Cell viability assays in HepG2 (A) and Huh7 (B) cell lines. The legend presented beside Figure 15B also applies to Figure 15A. [Figure 16A] Figures 16A and 16B: Gene expression analysis in HepG2 (A) and Huh7 (B) cell lines. The legend presented next to Figure 16B also applies to Figure 16A. [Figure 16B]Figures 16C and 16D: Cell viability assays in HepG2 (C) and Huh7 (D) cell lines. The legend presented beside Figure 16D also applies to Figure 16C. [Figure 17] Figures 17A and 17B: Gene expression analysis in HepG2 (A) and Huh7 (B) cell lines. The legend presented next to Figure 17B also applies to Figure 17A. [Figure 18] Figure 18A: Relative cell viability of primary human hepatocytes (compared to untreated cells ("Untr"), which is set at 100%) by contacting the cells with a concentration series of trivalent GalNAc conjugated to SO1861 ((GN)3-SPT), a combination of 10 pM monoclonal antibody anti-CD71 conjugated to saporin (CD71-SPRN) and a concentration series of SO1861 linked to the aglycone aldehyde group by EMCH (SPT-EMCH), a concentration series of 10 pM CD71-SPRN and (GN)3-SPT, and a concentration series of 10 pM CD71-SPRN in combination with a dendron having four SO1861 moieties covalently attached thereto (DAR=4, i.e., a ratio of (GN)3:dendron of 1:1 and a ratio of dendron:SO1861 of 1:4) ((GN)3-dSPT4). Figure 18B: Relative apoB expression in primary human hepatocytes (compared to untreated cells ("Untr"), set at 100%) by contacting the cells with a concentration series of trivalent GalNAc complexed to an antisense oligonucleotide targeting apoB mRNA ((GalNAc)3-ApoB), a combination of 300 nM trivalent GalNAc complexed to the saponin SO1861 ((GalNAc)3-SPT001) and a concentration series of (GalNAc)3-ApoB, or a conjugate of trivalent GalNAc complexed to a dendron having four SO1861 moieties covalently attached thereto (DAR=4) and complexed to an antisense oligonucleotide targeting apoB ((GalNAc)3-dSPT4-ApoB). SO1861 is also referred to herein and in the claims as "SPT001" and "dSPT." [Figure 19A] Figures 19A and 19B: Cell viability (both compared to untreated cells ("Untr"), which is set at 100%) of primary human hepatocytes (A) and the Huh7 hepatocyte cell line (B) by contacting cells with a concentration series of trivalent GalNAc conjugated to SO1861 (trivalent GalNAc-SPT001), 10 pM monoclonal antibody anti-CD71 conjugated to saporin (CD71-saporin) in combination with a concentration series of the covalently conjugated trivalent GalNAc-SPT001. [Figure 19B] Figure 19C: Illustration of a covalent trivalent GalNAc-SPT001 conjugate (DAR=1 for saponin SO1861). The "S" in the illustration is SO1861 in SPT001. Figure 19D: Illustration of an anti-CD71 antibody-saporin conjugate (OKT-9), where "T" is the toxin saporin covalently linked to the antibody heavy chain. [Figure 19C] Figure 19E: Illustration of a covalent trivalent (GalNAc)3-dSPT4 (or GN3-dSPT4) conjugate (DAR=4 for saponin SO1861). The "S" in the illustration is SO1861 for SPT001. dSPT4 describes a dendron with four SO1861 molecules conjugated as described elsewhere. [Figure 20A]Figure 20A and B: Relative apoB expression in primary human hepatocytes (A) and the Huh7 hepatocyte cell line (B). By contacting cells with: a concentration series of trivalent GalNAc complexed to an antisense oligonucleotide to target apoB to silence ApoB mRNA and ApoB protein expression (trivalent GalNAc-ApoBBNA), a combination of 300 nM trivalent GalNAc complexed to SO1861 (trivalent GalNAc-SPT001) (DAR=1 for the bound SPT001 (saponin SO1861)) and a concentration series of trivalent GalNAc-ApoBBNA, or a concentration series of trivalent GalNAc complexed to four SO1861 moieties covalently attached thereto (DAR=4) and complexed to an antisense oligonucleotide to target apoB to silence ApoB mRNA and ApoB protein expression (trivalent GalNAc-ApoBBNA-SPT001, where four SO1861 molecules were conjugated to the GalNAc / oligonucleotide complex by a single dendron moiety). [Figure 20B] Figure 20C: Illustration of a covalent trivalent GalNAc-ApoBBNA conjugate. "A" in the illustration is the antisense oligonucleotide ApoB (ApoBBNA). Figure 20D: Illustration of trivalent GalNAc-ApoBBNA-SPT001. "S" in the illustration is SPT001, also known as SO1861. "E" in the illustration is an effector moiety antisense oligonucleotide targeting apoB (ApoBBNA). [Figure 20C] Figure 20E: Illustration of (GalNAc)3-dSPT4-ApoB conjugated to a dendron having four SO1861 moieties covalently attached thereto (DAR=4). The "S" in the illustration is SPT001, also referred to as SO1861. The "E" in the illustration is an effector moiety antisense oligonucleotide targeting apoB (ApoBBNA). [Figure 21]Figure 21A and B: Absolute apoB protein expression in primary human hepatocytes (A) and the Huh7 hepatocyte cell line (B) under the influence of contacting the cells with: a concentration series of trivalent GalNAc-ApoBBNA, a concentration series combination of 300 nM trivalent GalNAc-SPT001 and trivalent GalNAc-ApoBBNA, or a concentration series of trivalent GalNAc-ApoBBNA-SPT001, where four SO1861 molecules were conjugated to the GalNAc / oligonucleotide conjugate by a single dendron moiety (hence also referred to as (GalNAc)3-dSPT4-ApoB). [Figure 22A] Figure 22A: Synthesis of saponin-oligonucleotide-ASGPR ligand conjugate, i.e., trivalent GalNAc-ApoBBNA-SPT001. Also referred to as (GalNAc)3-SO1861-ApoB. [Figure 22B] Figure 22B: Synthesis of saponin-oligonucleotide-ASGPR ligand conjugate, i.e., trivalent GalNAc-ApoBBNA-SPT001. Also referred to as (GalNAc)3-SO1861-ApoB. [Figure 22C] Figure 22C: Synthesis of saponin-oligonucleotide-ASGPR ligand conjugate, i.e., trivalent GalNAc-ApoBBNA-SPT001. Also referred to as (GalNAc)3-SO1861-ApoB. [Figure 23A] Figure 23A: Synthesis of SO1861-OEG-NHS (SPT001-L-NHS, molecule 12, where "L" refers to the labile cleavable linker). [Figure 23B] Figure 23B: Synthesis of dendron (SPT001)4-NH2 (molecule 15 from molecules 13 and 14). [Figure 23C] Figure 23C: Dendron (SPT001) 4-azide synthesis (molecule 19 from molecules 15 and 18). [Figure 23D] Figures 23D-E: Dendron (SPT001) 4-trivalent GalNAc synthesis (molecule 24 from molecules 19 and 23). Molecule 21 shown in Figure 23D is intermediate 7 shown in Figure 8G. [Figure 23E] Figure 23D-E: Dendron (SPT001) 4-trivalent GalNAc synthesis (molecule 24 from molecules 19 and 23). [Figure 24A] Figure 24A-B: Synthesis of DBCO-TCO-trivalent GalNAc (molecule 29) from molecule 27 and molecule 28 (trifunctional linker), where molecule 27 is a GalNAc-thioacetate obtained from the reaction between molecule 22, the formate salt of trivalent GalNAc-amine, and molecule 26 (4-nitrophenyl 3-(acetylthio)propanate). [Figure 24B] Figure 24A-B: Synthesis of DBCO-TCO-trivalent GalNAc (molecule 29) from molecule 27 and molecule 28 (trifunctional linker), where molecule 27 is a GalNAc-thioacetate obtained from the reaction between molecule 22, the formate salt of trivalent GalNAc-amine, and molecule 26 (4-nitrophenyl 3-(acetylthio)propanate). [Figure 24C] Figure 24C: Synthesis of DBCO-L-ApoBBNA oligo-trivalent GalNAc (molecule 31) by conjugating methyltetrazine-L-ApoBBNA oligo (molecule 30) to DBCO-TCO-trivalent GalNAc (molecule 29). [Figure 24D] Figure 24D: Synthesis of dendron (-L-SO1861)4-L-ApoBBNA oligo-trivalent GalNAc (molecule 34, "trivalent GalNAc-ApoBBNA-SPT001 conjugate") by conjugating molecule 31 (DBCO-L-ApoBBNA oligo-trivalent GalNAc) and molecule 19 (dendron (SPT001)4-azide). [Figure 25] Figure 25: ApoB expression analysis in liver tissue of C57BL / 6J mice. [Figure 26]Figure 26: Serum apoB protein analysis in C57BL / 6J mice. Levels not reported for 0.1 mg / kg ApoB#02 (196 hr), 0.01 mg / kg (GalNAc)3-ApoB + 5 mg / kg (GalNAc)3-S01861 (196 hr and 336 hr), and 0.1 mg / kg (GalNAc)3-ApoB + 5 mg / kg (GalNAc)3-S01861 (196 hr). [Figure 27] Figure 27: Serum LDL cholesterol (LDL-C) analysis in C57BL / 6J mice. [Figure 28] Figure 28: Serum ALT analysis in C57BL / 6J mice. [Figure 29A] Figure 29A-C: ApoB expression analysis and cell viability assay in primary human hepatocyte HepG2 and Huh7 cells for ApoB#02 complex. [Figure 29B] Figure 29A-C: ApoB expression analysis and cell viability assay in primary human hepatocyte HepG2 and Huh7 cells for ApoB#02 complex. [Figure 29C] Figure 29A-C: ApoB expression analysis and cell viability assay in primary human hepatocyte HepG2 and Huh7 cells for ApoB#02 complex. [Figure 30] FIG. 30: ApoB expression analysis and cell viability assay in primary human hepatocytes for ApoB#02 complex. [Figure 31] Figure 31: ApoB expression analysis in liver tissue of C57BL / 6J mice. [Figure 32] Figure 32: Serum ApoB protein analysis in C57BL / 6J mice. [Figure 33] Figure 33: Serum LDL cholesterol (LDL-C) analysis in C57BL / 6J mice. [Figure 34] Figure 34: Serum ALT analysis in C57BL / 6J mice. [Figure 35A] Figure 35A-D: Trifunctional linker-(L-hydrazone-SO1861)-(L-BNA oligo)-(trivalent GalNAc) synthesis. [Figure 35B] Figure 35A-D: Trifunctional linker-(L-hydrazone-SO1861)-(L-BNA oligo)-(trivalent GalNAc) synthesis. [Figure 35C] Figure 35A-D: Trifunctional linker-(L-hydrazone-SO1861)-(L-BNA oligo)-(trivalent GalNAc) synthesis. [Figure 35D] Figure 35A-D: Trifunctional linker-(L-hydrazone-SO1861)-(L-BNA oligo)-(trivalent GalNAc) synthesis. [Figure 36A] Figure 36A-E: Trifunctional linker-(dendron(-L-hydrazone-SO1861)4)-(L-BNA oligo)-(trivalent GalNAc) synthesis. [Figure 36B] Figure 36A-E: Trifunctional linker-(dendron(-L-hydrazone-SO1861)4)-(L-BNA oligo)-(trivalent GalNAc) synthesis. [Figure 36C] Figure 36A-E: Trifunctional linker-(dendron(-L-hydrazone-SO1861)4)-(L-BNA oligo)-(trivalent GalNAc) synthesis. [Figure 36D] Figure 36A-E: Trifunctional linker-(dendron(-L-hydrazone-SO1861)4)-(L-BNA oligo)-(trivalent GalNAc) synthesis. [Figure 36E] Figures 36A-E: Trifunctional linker-(dendron(-L-hydrazone-SO1861)4)-(L-BNA oligo)-(trivalent GalNAc) synthesis. Note that the drawing in Figure 36E encompasses two panels labeled Figure 36E and Figure 36E (continued). These two panels together demonstrate the conjugation of molecule 21 with molecule 19, thereby forming conjugated molecule 22. [Figure 36F] Figures 36A-E: Trifunctional linker-(dendron(-L-hydrazone-SO1861)4)-(L-BNA oligo)-(trivalent GalNAc) synthesis. Note that the drawing in Figure 36E encompasses two panels labeled Figure 36E and Figure 36E (continued). These two panels together demonstrate the conjugation of molecule 21 with molecule 19, thereby forming conjugated molecule 22. [Figure 37A] Figure 37A-C: Trifunctional linker-(dendron(-L-hydrazone-SO1861)8)-(BNA oligo)-(trivalent GalNAc) synthesis. [Figure 37B] Figures 37A-C: Trifunctional linker-(dendron(-L-hydrazone-SO1861)8)-(BNA oligo)-(trivalent GalNAc) synthesis. Note that the drawing in Figure 37B encompasses two panels labeled Figure 37B and Figure 37B (continued). These two panels together demonstrate the conjugation of molecule 24 with molecule 18, thereby forming conjugated molecule 25 (intermediate 12). [Figure 37C] Figures 37A-C: Trifunctional linker-(dendron(-L-hydrazone-SO1861)8)-(BNA oligo)-(trivalent GalNAc) synthesis. Note that the drawing in Figure 37B encompasses two panels labeled Figure 37B and Figure 37B (continued). These two panels together demonstrate the conjugation of molecule 24 with molecule 18, thereby forming conjugated molecule 25 (intermediate 12). [Figure 37D] Figures 37A-C: Trifunctional linker-(dendron(-L-hydrazone-SO1861)8)-(BNA oligo)-(trivalent GalNAc) synthesis. Note that the drawing in Figure 37C encompasses two panels labeled Figure 37C and Figure 37C (continued). These two panels together demonstrate the conjugation of molecule 25 with molecule 21, thereby forming conjugated molecule 26. [Figure 37E] Figures 37A-C: Trifunctional linker-(dendron(-L-hydrazone-SO1861)8)-(BNA oligo)-(trivalent GalNAc) synthesis. Note that the drawing in Figure 37B encompasses two panels labeled Figure 37B and Figure 37B (continued). These two panels together demonstrate the conjugation of molecule 24 with molecule 18, thereby forming conjugated molecule 25 (intermediate 12). [Figure 38A] Figure 38A-C: Trifunctional linker-(L-semicarbazone-SO1861)-(BNA oligo)-(trivalent GalNAc) synthesis. [Figure 38B]Figure 38A-C: Trifunctional linker-(L-semicarbazone-SO1861)-(BNA oligo)-(trivalent GalNAc) synthesis. [Figure 38C] Figure 38A-C: Trifunctional linker-(L-semicarbazone-SO1861)-(BNA oligo)-(trivalent GalNAc) synthesis. [Figure 39A] Figure 39A-F: Trifunctional linker-(dendron(L-semicarbazone-SO1861)4)-(L-BNA oligo)-(trivalent GalNAc) synthesis. [Figure 39B] Figure 39A-F: Trifunctional linker-(dendron(L-semicarbazone-SO1861)4)-(L-BNA oligo)-(trivalent GalNAc) synthesis. [Figure 39C] Figure 39A-F: Trifunctional linker-(dendron(L-semicarbazone-SO1861)4)-(L-BNA oligo)-(trivalent GalNAc) synthesis. [Figure 39D] Figure 39A-F: Trifunctional linker-(dendron(L-semicarbazone-SO1861)4)-(L-BNA oligo)-(trivalent GalNAc) synthesis. [Figure 39E] Figures 39A-F: Trifunctional linker-(dendron(L-semicarbazone-SO1861)4)-(L-BNA oligo)-(trivalent GalNAc) synthesis. Note that the drawing in Figure 39E encompasses two panels labeled Figure 39E and Figure 39E (continued). These two panels together demonstrate the conjugation of molecule 40 with molecule 20, thereby forming conjugated intermediate 24. [Figure 39F] Figures 39A-F: Trifunctional linker-(dendron(L-semicarbazone-SO1861)4)-(L-BNA oligo)-(trivalent GalNAc) synthesis. Note that the drawing in Figure 39E encompasses two panels labeled Figure 39E and Figure 39E (continued). These two panels together demonstrate the conjugation of molecule 40 with molecule 20, thereby forming conjugated intermediate 24. [Figure 39G] Figure 39A-F: Trifunctional linker-(dendron(L-semicarbazone-SO1861)4)-(L-BNA oligo)-(trivalent GalNAc) synthesis. [Figure 40A] Figures 40A-D: Trifunctional linker-(dendron(L-semicarbazone-SO1861)8)-(L-BNA oligo)-(trivalent GalNAc) synthesis. Note that the drawing in Figure 40A encompasses two panels labeled Figure 40A and Figure 40A (continued). These two panels together demonstrate the conjugation of molecule 43 with molecule 37, thereby forming conjugate (molecule 44) intermediate 25. [Figure 40B] Figures 40A-D: Trifunctional linker-(dendron(L-semicarbazone-SO1861)8)-(L-BNA oligo)-(trivalent GalNAc) synthesis. Note that the drawing in Figure 40A encompasses two panels labeled Figure 40A and Figure 40A (continued). These two panels together demonstrate the conjugation of molecule 43 with molecule 37, thereby forming conjugate (molecule 44) intermediate 25. [Figure 40C] Figures 40A-D: Trifunctional linker-(dendron(L-semicarbazone-SO1861)8)-(L-BNA oligo)-(trivalent GalNAc) synthesis. Note that the drawing in Figure 40B encompasses two panels labeled Figure 40B and Figure 40B (continued). These two panels together demonstrate the conjugation of molecule 44 with molecule 18, thereby forming conjugated molecule 45 (intermediate 26). [Figure 40D] Figures 40A-D: Trifunctional linker-(dendron(L-semicarbazone-SO1861)8)-(L-BNA oligo)-(trivalent GalNAc) synthesis. Note that the drawing in Figure 40B encompasses two panels labeled Figure 40B and Figure 40B (continued). These two panels together demonstrate the conjugation of molecule 44 with molecule 18, thereby forming conjugated molecule 45 (intermediate 26). [Figure 40E] Figures 40A-D: Trifunctional linker-(dendron(L-semicarbazone-SO1861)8)-(L-BNA oligo)-(trivalent GalNAc) synthesis. Note that the drawing in Figure 40C encompasses two panels labeled Figure 40C and Figure 40C (continued). These two panels together demonstrate the conjugation of molecule 45 with molecule 20, thereby forming conjugated molecule 46 (intermediate 27). [Figure 40F]Figures 40A-D: Trifunctional linker-(dendron(L-semicarbazone-SO1861)8)-(L-BNA oligo)-(trivalent GalNAc) synthesis. Note that the drawing in Figure 40C encompasses two panels labeled Figure 40C and Figure 40C (continued). These two panels together demonstrate the conjugation of molecule 45 with molecule 20, thereby forming conjugated molecule 46 (intermediate 27). [Figure 40G] Figure 40A-D: Trifunctional linker-(dendron(L-semicarbazone-SO1861)8)-(L-BNA oligo)-(trivalent GalNAc) synthesis. [Figure 41] Figure 41: Synthesis of trivalent linker-(L-SPT001)-(blocked TCO)-(trivalent GalNAc) synthesis. [Figure 42A] Figure 42A-B: Trivalent linker-(blocked DBCO)-(L-BNA oligo)-(trivalent GalNAc) synthesis. [Figure 42B] Figure 42A-B: Trivalent linker-(blocked DBCO)-(L-BNA oligo)-(trivalent GalNAc) synthesis. [Figure 43A]Figures 43A-B: Overview of conjugates that can be obtained by applying the TFL (trifunctional linker) methodology. (A) General scheme for the synthesis of a family of oligonucleotide conjugates "Conjugate C1" of the invention by covalently conjugating GalNAc, a saponin, here SO1861, and an oligonucleotide, here BNA, to separate arms of a trifunctional linker. (B) Family of conjugates of Conjugate C1 synthesized according to the general scheme in (A), with indicated option R1 for the conjugated saponin and indicated option R2 for the conjugated BNA. The drawings in Figures 43A and B together illustrate the conjugation of a trifunctional linker, labeled molecule 4, with a targeting ligand, labeled molecule 41, an effector, labeled molecule 12, and one of the enhancers, labeled molecules 3, 19, 25, 28, 40, and 45, or one of the control blocking agents, labeled molecules 5, 12a, and 50, thereby forming a complex having the general complex structure "complex C1" illustrated in Figure 43B, which includes groups R1 and R2, where R1 is any one of the groups R1 illustrated under complex C1 in the complex, and R2 on complex C1 is any one of the groups R2 illustrated under complex C1 in Figure 43B. [Figure 43B]Figures 43A-B: Overview of conjugates that can be obtained by applying the TFL (trifunctional linker) methodology. (A) General scheme for the synthesis of a family of oligonucleotide conjugates "Conjugate C1" of the invention by covalently conjugating GalNAc, a saponin, here SO1861, and an oligonucleotide, here BNA, to separate arms of a trifunctional linker. (B) Family of conjugates of Conjugate C1 synthesized according to the general scheme in (A), with indicated option R1 for the conjugated saponin and indicated option R2 for the conjugated BNA. The drawings in Figures 43A and B together illustrate the conjugation of a trifunctional linker, labeled molecule 4, with a targeting ligand, labeled molecule 41, an effector, labeled molecule 12, and one of the enhancers, labeled molecules 3, 19, 25, 28, 40, and 45, or one of the control blocking agents, labeled molecules 5, 12a, and 50, thereby forming a complex having the general complex structure "complex C1" illustrated in Figure 43B, which includes groups R1 and R2, where R1 is any one of the groups R1 illustrated under complex C1 in the complex, and R2 on complex C1 is any one of the groups R2 illustrated under complex C1 in Figure 43B. DETAILED DESCRIPTION OF THE INVENTION

[0092] While the present invention will be described with reference to specific embodiments, the invention is not limited thereto but only by the claims.

[0093] A first aspect of the present invention relates to a saponin conjugate comprising at least one saponin covalently linked to a ligand of the asialoglycoprotein receptor (ASGPR), wherein the ligand of the ASGPR comprises at least one N-acetylgalactosamine (GalNAc) moiety, preferably three or four GalNAc moieties, more preferably the ligand of the ASGPR comprises or consists of (GalNAc)3Tris, wherein the at least one saponin is selected from monodesmosidic triterpenoid saponins and bisdesmosidic triterpenoid saponins. Preferably, the ASGPR is ASGPR1. One embodiment of the present invention relates to an oligonucleotide conjugate comprising at least one saponin covalently linked to a ligand of the asialoglycoprotein receptor (ASGPR) (wherein the ASGPR ligand comprises at least one N-acetylgalactosamine (GalNAc) moiety, preferably three or four GalNAc moieties, and more preferably the ASGPR ligand comprises or consists of (GalNAc)3Tris) and further covalently linked to an oligonucleotide, wherein the at least one saponin is selected from monodesmosidic triterpenoid saponins and bisdesmosidic triterpenoid saponins. Typically, the ASGPR is ASGPR1. The triterpene saponin is typically and preferably a 12,13-dehydrooleanane-type triterpenoid saponin, for example, a triterpenoid saponin belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position C23, or a bisdesmosidic triterpene saponin. Preferred are monodesmosidic or bisdesmosidic pentacyclic triterpene saponins of the 12,13-dehydrooleanane type, preferably having an aldehyde functional group at position C23.The following oligonucleotide complexes of the invention are preferred: the oligonucleotides contained in the complexes are defined as nucleic acids not longer than 150 nt, preferably wherein the oligonucleotides have a size of 5 to 150 nt, preferably 8 to 100 nt, and most preferably 10 to 50 nt.

[0094] Surprisingly, the inventors have established that the efficacy of gene silencing oligonucleotides (ASOs, AONs, siRNAs, BNAs) is improved by at least a factor of 10, i.e., at least a factor of 100 or even more than a factor of 1000, when the therapeutic oligonucleotide is contacted with mammalian cells bearing an ASGPR, such as human cells, such as human liver cells, in the presence of a saponin, such as SO1861, covalently complexed to a ligand of the liver cell-specific receptor ASGPR1, e.g., trivalent GalNAc (GN3) (i.e., a complex comprising three GalNAc moieties), resulting in increased mRNA and / or protein expression for the target gene targeted by the therapeutic oligonucleotide. For example, combining saponin, provided as a complex containing GalNAc separate from the oligonucleotide or as a saponin moiety covalently linked to a complex of an ASGPR ligand and an oligonucleotide, with a therapeutic oligonucleotide such as siRNA or ASO for treating cancer (for example, by silencing HSP27 expression) or treating excessively high plasma levels of (LDL) cholesterol by targeting the apoB gene, widens the therapeutic window of the therapeutic oligonucleotide.When target cells are contacted with an oligonucleotide coupled to an ASGPR1 ligand, in the presence of an ASGPR1-targeting ligand containing a covalently linked saponin, effective gene silencing (any reasonable degree of silencing) is achieved at a lower oligonucleotide dose, compared to the gene silencing obtained in the absence of the (ASGPR1-targeting) saponin.In other words, the present inventors have established that when receptor-bearing cells in vitro or in vivo are contacted with the oligonucleotide complex of the present invention, near-complete (defined as at least 95% or higher) RNA expression inhibition is achieved. In addition, the inventors have also achieved near complete inhibition of protein expression, for example apoB protein, which subsequently leads to near complete clearance of LDL cholesterol in serum.This surprisingly strong degree and efficacy of gene silencing under the influence of the oligonucleotide conjugates of the present invention cannot be achieved when the saponin is not part of a complex containing a payload (an effector moiety, here an oligonucleotide) and a ligand of ASGPR, here ASGPR1, such as GN3. The improved efficacy of gene silencing after contacting target (liver) cells expressing ASGPR1 with a conjugate containing the saponin of the present invention is evident when evaluating mRNA expression and / or protein expression related to the target gene, as well as indirectly when evaluating LDL cholesterol levels. This is because LDL particles contain one copy of the apoB protein (more specifically, the apoB100 protein), the expression of which is silenced by contacting cells with the oligonucleotide conjugates of the present invention. Improved gene silencing is achieved at a fixed oligonucleotide dose in the presence of an ASGPR1-targeting ligand containing a covalently linked saponin, compared to the efficacy of gene silencing obtained in the absence of saponin when contacting target cells with the same dose of oligonucleotide coupled to a ligand of ASGPR1. The inventors have found that combining a conjugate of a therapeutic oligonucleotide and a ligand of ASGPR1, such as (GalNAc)3 (also referred to as GN3 or (GN)3), with a conjugate of saponin and a ligand of ASGPR1, such as (GalNAc)3, when the combination is contacted with ASGPR1-bearing cells, such as human liver cells, resulted in a surprisingly high improvement in the efficacy of gene silencing, compared to the efficacy obtained in the absence of the saponin conjugate.The inventors have also found that covalently coupling a saponin to a therapeutic oligonucleotide and a ligand of ASGPR1, such as (GalNAc)3, when the combination is contacted with ASGPR1-bearing cells, such as human liver cells, resulted in a surprisingly high improvement in the efficacy of gene silencing, compared to the efficacy obtained in the absence of the saponin on the GalNAc-oligonucleotide conjugate.

[0095] Triterpene glycoside-type saponins have the ability to stimulate the delivery of effector molecules from the outside of target cells to the inside of the cells and then to the cytosol of the cells. Saponins promote receptor-mediated uptake of certain effector molecules, such as protein toxins contained on ADCs. Endocytosis of the toxin as part of an ADC delivers the toxin to the endosomes and / or lysosomes of the target cells. Without wishing to be bound by any theory, the subsequent release of the ADC, or at least the toxin, from the endosome or lysosome into the cytosol of the cell is stimulated or mediated by the triterpenoid saponins used in the present invention. When target cells are contacted with a toxin at a dose too low to exert an intracellular effect, co-administration of a toxin and free saponin, for example as part of an ADC, such that the target cells are contacted with both the ADC and the saponin can result in efficient toxin-mediated killing of target cells at the same toxin dose that is ineffective when contacted with cells in the absence of saponin. Therefore, under the influence of free saponin, the efficacy of the toxin is improved. For example, when the tumor cell receptor targeted by the ADC is not truly (exclusively) tumor cell-specific, but is also expressed, for example, in healthy cells, elsewhere in the patient's body, or in cells present in the same organ as the target cells, the toxin dose required for efficient and sufficient target cell killing may still be accompanied by unwanted off-target effects. Furthermore, the required dose of free saponin needed to achieve a sufficient degree of toxin activation (delivery of the toxin to the target cell cytosol, the saponin threshold for mediating on-target delivery) may be at a level that induces unwanted off-target effects. Because free saponin is administered systemically and is not targeted when the target (tumor) cells to be treated with the toxin are considered, a relatively high saponin dose must be administered to the patient in need of toxin treatment to achieve a local saponin dose at the target cell level.Furthermore, one of the further problems and disadvantages encountered when free saponin is co-administered with, for example, an ADC is the difficulty of optimizing the synchronization of the activity of the ADC + SPT001 (free saponin) at the correct time and place in the target cells of the patient. The inventors now provide a one-component complex solution that offers a solution to this problem.

[0096] The inventors have now surprisingly found that when saponin is conjugated to a cell targeting moiety, such as a receptor ligand (e.g., EGF or a cytokine), an antibody, or a binding fragment or domain thereof, the saponin dose required to enhance the effect of the toxin on tumor cells can be reduced by a factor of 10, or even by a factor of about 100 to 1000. In tumor cells, a series of cell surface receptors are now known for their specific expression on the surface of such abnormal cells. Saponins have been successfully coupled by the inventors to antibodies and ligands, such as trastuzumab, cetuximab, etc., for binding to receptors typically concentrated on tumor cells. Such targeted saponin conjugates can indeed, for example, improve the cytotoxic effect of the toxin inside the target cell, requiring an effective dose of the conjugate that is at least 10-fold, for example 100- to 1000-fold lower, compared to the dose required for the same saponin in free form, i.e., without the tumor cell targeting moiety.

[0097] The inventors have now further discovered that target cell-specific delivery and subsequent endocytosis of (targeted) saponins is also possible for non-abnormal or abnormal cells, for example not involved in cancer (tumor cells), autoimmune diseases, virus-infected cells, etc. Thus, surprisingly, the inventors have found that ASGPR provides a suitable target receptor on cells bearing this receptor, such as liver cells, for the entry of saponins equipped with ligands for ASGPR, specifically ASGPR1 (see for example Figure 3; saponin SO1861 (also referred to as "SPT001") complexed with a single GalNAc moiety). Known ligands such as monovalent GalNAc and trivalent GalNAc(GN)3 have been successfully coupled to one or multiple (2, 4, 8, etc.) saponin molecules to provide saponin conjugates containing an ASGPR ligand and at least one saponin moiety, preferably a bisdesmosidic pentacyclic triterpene saponin of the 12,13-dehydrooleanane type bearing an aldehyde functional group at position C23.

[0098] Contacting cells expressing an ASGPR such as ASGPR1, such as liver cells, with a saponin complex comprising a ligand for ASGPR (see, e.g., Figures 4 and 19C) surprisingly results in demonstrable saponin uptake when such cells are co-targeted with, for example, a gene silencing nucleic acid and a target cell surface molecule, e.g., CD71, a complex comprising a binding molecule for ASGPR, resulting in improved saponin-dependent gene silencing in the target cell, or a complex comprising such a binding molecule and a toxin to the target cell, resulting in saponin-dependent cytotoxicity. Contacting cells expressing an ASGPR such as ASGPR1, such as liver cells, with a saponin complex containing an ASGPR ligand surprisingly results in demonstrable saponin uptake when the cells are contacted with such a saponin complex containing a saponin, a ligand of ASGPR1, such as GalNAc or (GN)3, and further containing an oligonucleotide for silencing mRNA expression and / or protein expression in the cells, i.e., an oligonucleotide complex of the present invention comprising at least one saponin, a ligand of ASGPR1, and an oligonucleotide covalently linked together, for example, via a trifunctional linker. Providing a saponin with an ASGPR ligand results in an effect in the target cell that is exerted by a co-administered effector molecule, such as a targeted oligonucleotide or toxin (e.g., an anti-CD71 antibody or ASGPR ligand coupled to a nucleic acid or toxin), or by an oligonucleotide that is also complexed with the saponin and the ASGPR1 ligand on an oligonucleotide complex of the present invention (see also Figures 22A-C and 24D). When target cells are contacted with the effector molecule alone in the absence of targeted saponin, e.g., free oligonucleotide or oligonucleotide complexed with, e.g., trivalent GalNAc, such effects are evident to a much lesser extent, if at all.Therefore, when a target cell containing an ASGPR on its surface is considered, and such a target cell is contacted with both a saponin-ASGPR ligand of the present invention and a (targeted) effector molecule, or an oligonucleotide conjugate comprising a saponin for improving endosomal escape of the effector moiety from endosomes and / or lysosomes into the cytosol and / or ultimately the nucleus of the target cell, GalNAc as a ligand for ASGPR1, and an oligonucleotide as the effector moiety, the saponin conjugate of the present invention and the oligonucleotide conjugate of the present invention improve the efficacy of the effector molecule, such as an oligonucleotide. This improves the therapeutic window of the effector molecule. Under the influence of co-administration of the saponin conjugate or the influence of the oligonucleotide conjugate, a higher therapeutic effect is obtained at the same dose, or a similar therapeutic effect is obtained at a lower dose under the influence of co-administration of the saponin conjugate or the influence of the oligonucleotide conjugate, where the oligonucleotide is the effector moiety. The inventors have also found that at least a 10-fold, for example about 100- to 1000-fold lower dose of saponin or oligonucleotide conjugate is required to achieve a certain level of therapeutic effect in target ASGPR-expressing cells, e.g., liver cells, induced by a co-administered ((ASGPR1)-targeted) effector molecule, e.g., an ASO, siRNA, BNA, toxin, e.g., a protein toxin, or by an oligonucleotide included as an effector moiety on an oligonucleotide conjugate, when compared to the dose of saponin required to achieve a similar effect when the saponin does not comprise a ligand for ASGPR.The inventors have also found that when at least one saponin moiety, e.g., a SO1861 moiety or a SO1832 moiety, is covalently linked to an oligonucleotide conjugate containing at least one GalNAc, e.g., a trivalent GalNAc, at least a 10-fold, e.g., about 100- to 1000-fold lower dose of the oligonucleotide is required to achieve a certain level of therapeutic effect in target ASGPR-expressing cells, e.g., liver cells, induced by an ((ASGPR1)-targeted) oligonucleotide as an effector molecule such as an ASO, siRNA, or BNA administered to the cells.

[0099] Thus, surprisingly, the present inventors have now provided an improved therapeutic method using ASGPR as a target receptor for saponin-mediated delivery of an effector molecule, such as a therapeutic oligonucleotide, in the cytosol of ASGPR-expressing cells, resulting in a similar therapeutic effect induced by the effector molecule at a lower dose than would be possible in the absence of a saponin conjugate of the present invention or in the absence of a covalently linked saponin on an oligonucleotide conjugate. Thus, by providing a saponin-ASGPR ligand conjugate of the present invention and an oligonucleotide conjugate of the present invention (e.g., a saponin, an oligonucleotide, and (GalNAc)3 covalently linked together on a single conjugate), the present invention provides improved therapeutic uses of the stimulatory effect of saponin, for example, when delivery of an effector molecule to the cytosol or nucleus of a liver cell is considered. The advantages achieved by the present inventors are particularly the following: a lower dose required for the effector molecule, e.g., an oligonucleotide (siRNA or ASO). By co-administering a saponin with an (ASGPR1)-targeted) effector molecule, or by administering an oligonucleotide conjugate containing a GalNAc moiety for targeting ASGPR1, comprising: an oligonucleotide (e.g., an siRNA or ASO for silencing mRNA expression for a selected gene and / or for silencing protein expression by targeting a selected gene) and at least one saponin for stimulating endosomal escape of the oligonucleotide once the oligonucleotide conjugate is endocytosed by binding of an ASGPR1 ligand on the oligonucleotide conjugate to cellular ASGPR1 and delivered to endosomes / lysosomes of ASGPR1-bearing cells, such as liver cells; and by requiring a lower dose of saponin. By providing the saponin as a covalent conjugate (oligonucleotide conjugate of the invention) of a ligand for ASGPR, such as trivalent GalNAc, and one or more saponin moieties (saponin conjugates of the invention) and an effector moiety, such as a therapeutic oligonucleotide (e.g., siRNA or ASO).Surprisingly, the ASGPR ligand can be the same on both the saponin conjugate and the targeted effector molecule conjugate, and under the influence of saponin stimulation, an efficient effect mediated by the effector molecule can be achieved in target cells. That is, a saponin conjugate and an effector molecule conjugate, both of which contain a covalently linked ASGPR ligand, such as trivalent GalNAc, can be co-administered to target cells, such as liver cells, that expose ASGPR on their surface. As a result, both conjugates target the same receptor and use the same pathway of endocytosis into the cell, efficiently achieving the desired therapeutic effect of the effector molecule. This surprising finding is highly beneficial for liver cell-targeted therapies, such as those involving cytosolic delivery of nucleic acids (ASOs, BNAs, siRNAs, to name a few). Because liver cells express ASGPRs, such as ASGPR1, which are specific to liver cells, there are virtually no additional liver cell receptors that are sufficiently specific for use in liver cell-targeted therapies.

[0100] The present invention therefore opens new avenues for improving liver cell-directed therapy, where effector molecules such as ASOs and siRNAs must be delivered and located intracellularly for their mechanism of action. The targeted saponins provided as the saponin conjugates and oligonucleotide conjugates of the present invention broaden the therapeutic window of such effector molecules (oligonucleotides, e.g., siRNAs, ASOs). At the same time, by providing saponins with liver cell-targeting binding molecules, the therapeutic window of saponins is also improved according to the present invention. Thus, the present inventors have achieved efficient oligonucleotide delivery, thereby resolving a major technical application limitation evident in the field of therapeutic oligonucleotides. For oligonucleotide-based drug platforms, the present inventors provide a solution that improves oligonucleotide delivery based on the bioconjugation of the present invention for improved delivery of oligonucleotides. The improved delivery system of the present invention results in increased efficacy of oligonucleotides. This may help reduce the toxicity of ASGPR-targeted oligonucleotide systems (e.g., GalNAc-oligonucleotide conjugates) and / or reduce off-target effects.

[0101] The present inventors have synthesized non-proteinaceous conjugates composed of two or three biologically active small molecules, compared to the molecular size (molecular weight) of antibodies used in antibody-based (antibody-drug) conjugates, such as IgG. These conjugates of the present invention remain biologically active when the biological activities of each of the two or three small molecules contained in the conjugate, the saponin conjugate of the present invention, and the oligonucleotide conjugate of the present invention are considered. Although the conjugates of the present invention are relatively small, the two or three small molecules covalently conjugated together on a single molecule can still exert biological activities related to the properties of each of the small molecules. That is, saponin enhances endosomal and lysosomal escape of oligonucleotides taken up by cells. That is, ASGPR-bearing cells take up the saponin conjugates and oligonucleotide conjugates containing GalNAc of the present invention. That is, when ASGPR-bearing cells are contacted with the oligonucleotide conjugates of the present invention, a gene silencing effect is evident. The saponin and GalNAc-containing conjugates of the present invention provide endosomal escape-enhancing activity for oligonucleotides when co-administered to the same cells as the oligonucleotide and GalNAc-containing conjugates. Both saponin and oligonucleotide are mediated by the binding of GalNAc to ASGPR on target cells and are taken up by those cells.Under the influence of saponin in endosome / lysosome, oligonucleotides delivered to the cytosol to a higher degree exert their intracellular biological activity, which is expressed as the silencing of genes targeted by oligonucleotides (for example, targeting genes HSP27 and apoB).The oligonucleotide complex of the present invention provides endosomal escape enhancement activity to the oligonucleotides contained in the oligonucleotide complex.Both saponin and oligonucleotide are mediated by the binding of GalNAc to ASGPR on target cells and are taken up by those cells as part of the oligonucleotide complex of the present invention.Under the influence of saponin in endosomes / lysosomes, oligonucleotides delivered to the cytosol to a higher degree exert their intracellular biological activity, which is expressed as the silencing of genes targeted by the oligonucleotide (for example, targeting the gene HSP27 or apoB).Thus, although relatively small molecules are complexed together into a single complex, at least one saponin (for example, 1, 4, or 8 saponin moieties), a GalNAc moiety or a cluster of GalNAc moieties (for example, (GN)3), and each of the oligonucleotides still exhibits biological activity, compared to the biological activity measured when cells are contacted with, for example, free saponin (for example, in combination with a molecule that exerts its activity in the cytosol), GalNAc-oligonucleotide, or free oligonucleotide.Although saponin, GalNAc, and oligonucleotide are relatively small molecules, complexing these molecules together does not interfere with their biological activity.

[0102] Introduction to Saponin Complexes The present invention provides conjugates of a saponin and a ligand of the 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 invention, each GalNAc moiety is attached to the remainder of the ASGPR ligand, or to the saponin moiety in cases where the ASGPR ligand consists of a single GalNAc moiety, via a covalent bond to the oxygen at position "1" as indicated in formula (I):

[0103] [ka]

[0104] Formula (II) S As shown in Figure 1, the ASGPR ligand preferably comprises a saponin moiety S linked to a saponin moiety linker L S It consists of a single GalNAc moiety linked via

[0105] [ka]

[0106] The ASGPR ligand may contain more than one GalNAc moiety, for example, 2, 3, 4, 5, or 6 GalNAc moieties, preferably 3 or 4 GalNAc moieties, more preferably 3 GalNAc moieties. In such cases, it is preferred that the GalNAc moieties are each separately covalently linked to the central bridging moiety B via the oxygen at position "1," which effectively forms a bridge between the GalNAc moiety and the saponin moiety, preferably via the saponin moiety linker L. S The GalNAc moiety is represented by formula (III) S can be directly attached to the bridging moiety B as shown in:

[0107] [ka]

[0108] where n is an integer greater than or equal to 2, and L S is a saponin moiety linker and S is a saponin moiety. More preferably, the GalNAc moiety is linked to bridging moiety B by a GalNAc linker L GAL via formula (IV) S are combined as shown in:

[0109] [ka]

[0110] where n is an integer greater than or equal to 2, and L S is the saponin moiety linker and S is the saponin moiety. GAL can be independently selected, but one skilled in the art will appreciate that L GAL It will be appreciated that in the case where each of the following represents the same moiety, synthesis of the saponin conjugate is simplified.

[0111] Saponin Complex Linker L S Saponin moiety linker L S is a compound represented by the formula (II) S or to GalNAc of formula (III) S and (IV) S The identity and size of the linker is not particularly limited and may be any moiety suitable for covalently linking the saponin to the bridging moiety B of formula (II) S or to GalNAc of formula (III) S and (IV) S to the bridging moiety B by means of "conventional" chemical functional groups (e.g., ester bonds) and by means of linkers E containing, for example, more than 10 or more than 20 carbon atoms. S Suitable linkers for linking molecules together, L S and related coupling reactions are described in Handbook of Bioconjugate Techniques, Hermanson, Greg T., Academic Press, 2013.

[0112] Those skilled in the art will appreciate that the saponin moiety linker L S typically comprises at least a first precursor L covalently linked to a GalNAc or bridging moiety B. S1 and a second precursor L covalently linked to the saponin moiety. S2 This principle is based on the formula (II): S is illustrated in the following reaction scheme:

[0113] [ka]

[0114] In the formula, L Sis the saponin moiety linker, and L S1 is the saponin moiety linker L S is a precursor of L, which is covalently linked to GalNAc or to a bridging moiety B S2 is the saponin moiety linker L S is a precursor of , which is covalently bound to a saponin moiety, where S is the saponin moiety.

[0115] Formula (III) S The corresponding reaction scheme for the compound is as follows:

[0116] [ka]

[0117] In the formula, L S is the saponin moiety linker, and L S1 is the saponin moiety linker L S is a precursor of L, which is covalently linked to GalNAc S2 is the saponin moiety linker L S wherein n is an integer greater than or equal to 2, B is a bridging moiety, and S is a saponin moiety.

[0118] Formula (IV) S The corresponding reaction scheme for the compound is as follows:

[0119] [ka]

[0120] In the formula, L S is the saponin moiety linker, and L S1 is the saponin moiety linker L S is a precursor of L, which is covalently linked to GalNAc S2 is the saponin moiety linker L Sis a precursor of the formula (I), which is covalently linked to a saponin moiety, 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.

[0121] Saponin moiety linker L S comprises at least a first precursor L covalently linked to a GalNAc or bridging moiety B S1 and a second precursor L covalently linked to the saponin moiety. S2 wherein the coupling reaction is, for example, an azide-alkyne cycloaddition, a thiol-maleimide coupling, a Staudinger reaction, a nucleophilic ring-opening of a strained heterocyclic electrophile (e.g., an aziridine, an epoxide, a cyclic sulfate, an aziridinium ion, an episulfonium ion), a non-aldol carbonyl reaction (e.g., urea, thiourea, hydrazone, oxime ether, amide, or heteroaromatic ring formation), or an addition to a carbon-carbon double bond (e.g., epoxidation, aziridination, dihydroxylation, sulfentyl halide addition, nitrosyl halide addition, or Michael addition), preferably wherein the coupling reaction is an azide-alkyne cycloaddition, a thiol-maleimide coupling, a Staudinger reaction, a nucleophilic ring-opening of a strained heterocyclic electrophile, and more preferably wherein the coupling reaction is an azide-alkyne cycloaddition or a thiol-maleimide coupling.

[0122] According to some embodiments of the present invention, the saponin moiety linker L S comprises a semicarbazone and / or a hydrazone and / or a 1,2,3-triazole, preferably a hydrazone and a 1,2,3-triazole or at least a semicarbazone. Whenever reference is made to a 1,2,3-triazole in the context of the linker of this application, this preferably means a 1H-1,2,3-triazole.

[0123] 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 well-known "click" chemistry tool in the field of bioconjugation and is described, for example, in Hermanson, Greg T., Bioconjugate Techniques, Academic Press, 2013. Such semicarbazones are the result of, for example, coupling between a terminal semicarbazide and an aldehyde-containing compound (e.g., an aldehyde-containing saponin).

[0124] Such 1,2,3-triazoles are the result, for example, of coupling between azide and alkyne-containing compounds. This azide / alkyne coupling is a commonly known "click" chemistry tool in the field of bioconjugation and is described, for example, in Hermanson, Greg T., Bioconjugate Techniques, Academic Press, 2013.

[0125] As a result, the saponin moiety linker L S is a first precursor L covalently linked to a GalNAc or bridging moiety B S1 and a second precursor L covalently linked to the saponin moiety. S2 is the result of a coupling reaction between the first precursor L S1 contains an azide, and the second precursor L S2 It is preferred that R 1 comprises an alkyne, preferably a hydrazone resulting from hydrazide / aldehyde coupling to an aldehyde on the saponin moiety, or preferably a semicarbazone resulting from semicarbazide / aldehyde coupling to an aldehyde on the saponin moiety.

[0126] Formula (V) S Precursor L present on the compound S1 Some embodiments of the structure are the following azides:

[0127] [ka]

[0128] 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.

[0129] Formula (VII) S or (VIII) S Precursor L present on the compound S1 Some embodiments of the structure are the following azides:

[0130] [ka]

[0131] In the formula, c represents an integer equal to or greater than 0, preferably c represents an integer in the range of 5 to 15, and more preferably c represents 9.

[0132] Formula (VI) S Precursor L present on the compound S2 Some embodiments of the structure are the following hydrazones:

[0133] [ka]

[0134] In the formula, a represents an integer greater than or equal to 0, preferably a represents an integer in the range of 2 to 6, more preferably a represents 4, and wherein L S2a represents an alkyne-containing moiety. In light of the present disclosure, one of skill in the art will appreciate that the hydrazone depicted in formula (XIX) results from the reaction of a hydrazide with an aldehyde of the saponin moiety.

[0135] L S2apreferably contains less than 20 carbon atoms, more preferably L S2a represents a moiety according to formula (XX):

[0136] [ka]

[0137] Thus, in some embodiments, the saponin complex has formula (II) as described herein. S , (III) S or (IV) S wherein the saponin moiety linker L S is represented by formula (VI) S with a compound of formula (V) S , (VII) S or (VIII) S wherein the first precursor L S1 is an azide, for example an azide of formula (XVII) or formula (XVIII), wherein the second precursor L S2 is a compound of formula (XIX), wherein the alkyne-containing moiety L S2a , for example, alkynes of formula (XX) and the hydrazones resulting from reaction of a hydrazide with an aldehyde of the saponin moiety.

[0138] Saponin ComplexSaponin One aspect of the present invention relates to a saponin conjugate comprising at least one saponin covalently linked to a ligand of the asialoglycoprotein receptor (ASGPR), wherein the ligand of the ASGPR comprises at least one N-acetylgalactosamine (GalNAc) moiety, preferably three or four GalNAc moieties, more preferably three GalNAc moieties (also referred to as triGalNAc and (GN)3), and more preferably the ligand of the ASGPR comprises or consists of (GalNAc)3Tris, wherein the at least one saponin is selected from monodesmosidic triterpenoid saponins and bisdesmosidic triterpenoid saponins.

[0139] One embodiment of the present invention relates to an oligonucleotide conjugate comprising at least one saponin, preferably 1 to 32, more preferably 1 to 16, and most preferably 1 to 8 saponin moieties, e.g., 1, 4, or 8 saponin moieties, covalently linked to a ligand of the asialoglycoprotein receptor (ASGPR), wherein the ligand of the ASGPR comprises at least one N-acetylgalactosamine (GalNAc) moiety, preferably 3 or 4 GalNAc moieties, more preferably 3 GalNAc moieties, and more preferably the ligand of the ASGPR comprises or consists of (GalNAc)3Tris, and further covalently linked to an oligonucleotide, wherein the at least one saponin is selected from monodesmosidic triterpenoid saponins and bisdesmosidic triterpenoid saponins. The saponin is selected for its endosomal escape-enhancing activity, and the intracellular effect of a molecule such as a proteinaceous toxin (e.g., saporin, dianthin) or a gene silencing oligonucleotide such as HSP27BNA is evaluated when contacted with a target cell in the presence or absence of the saponin. For saponins used in the complexes of the present invention, the presence of the saponin in the endosome of the cell typically increases the biological activity (e.g., gene silencing, toxicity) in the cytosol of the cell by at least a factor of 10 when the toxin or oligonucleotide is initially co-located with the saponin in the endosome.

[0140] An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the saponin is a pentacyclic triterpene saponin of the 12,13-dehydrooleanane type, preferably having an aldehyde functional group at position C23 of the aglycone core structure of the saponin.

[0141] One embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the saponin is a 12,13-dehydrooleanane-type monodesmosidic or bisdesmosidic pentacyclic triterpene saponin, preferably having an aldehyde functional group at position C23 of the aglycone core structure of the saponin.

[0142] 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-hydroxyoleanolic acid, 16 alpha-hydroxyoleanolic acid, Hederagenin (23-hydroxyoleanolic acid), 16alpha,23-dihydroxyoleanolic acid, gypsogenin, Quillaja acid, Protoessigenin-21(2-methylbut-2-enoate)-22-acetate, 23-oxo-valintgenol C-21,22-bis(2-methylbut-2-enoate), 23-oxo-valintgenol C-21(2-methylbut-2-enoate)-16,22-diacetate, digitogenin, 3,16,28-trihydroxyoleanan-12-ene, Dipsogenic acid, and their derivatives.

[0143] Preferably, the saponin comprises an aglycone core structure selected from quillaic acid and gypsogenin or a derivative thereof, and more preferably, the aglycone core structure of the saponin is quillaic acid or a derivative thereof. Such preferred aglycones (or derivatives thereof) comprise an aldehyde group or a derivative thereof at the C23 atom, as described elsewhere herein. Without wishing to be bound by any theory, such an aldehyde group contributes to the endosomal escape-enhancing activity of triterpene glycoside-type saponins of group C, particularly those containing aglycones selected from quillaic acid and gypsogenin.

[0144] An 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 that is located at the C3 atom or C4 of the aglycone core structure of the at least one saponin. 28 atom, preferably C3 atom, and / or wherein at least one saponin comprises a second sugar chain, which is attached to the C atom of the aglycone core structure of at least one saponin. 28 Preferably, the saponin comprises a first and a second sugar chain. Therefore, when the saponin conjugate of the present invention or the saponin contained in the oligonucleotide conjugate of the present invention has two glycans (sugar chains), the first sugar chain is attached at position C3 of the aglycone core structure, and the second sugar chain is attached at position C4 of the aglycone core structure. 28 are combined at

[0145] An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein: Saponins comprise a sugar chain attached to an aglycone core structure selected from Group A below: 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-, and their derivatives, or Saponins comprise a sugar chain attached to an aglycone core structure selected from Group B below: Glc-, Gal-, Rha-(1→2)-[Xyl-(1→4)]-Rha-, Rha-(1→2)-[Ara-(1→3)-Xyl-(1→4)]-Rha-, Ara-, Xyl-, Xyl-(1→4)-Rha-(1→2)-[R1-(→4)]-Fuc-, where R1 is 4E-methoxycinnamic acid; Xyl-(1→4)-Rha-(1→2)-[R2-(→4)]-Fuc-, where R2 is 4Z-methoxycinnamic acid; Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-4-OAc-Fuc-, Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-3,4-di-OAc-Fuc-, Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R3-(→4)]-3-OAc-Fuc-, where 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→3)-(Ara- or Xyl-)(1→4)-(Rha- or Fuc-)(1→2)-[4-OAc-(Rha- or Fuc-)(1→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→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R4-(→4)]-Fuc-, where R4 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)-[R5-(→4)]-Fuc-, where 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→3)-Xyl-(1→4)-Rha-(1→2)-[3,4-di-OAc-Qui-(1→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→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R6-(→4)]-Fuc-, where R6 is 5-O-[5-O-Rha-(1→2)-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid; Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R7-(→4)]-Fuc-, where R7 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid; Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R8-(→4)]-Fuc-, where R8 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)-[R9-(→4)]-Fuc-, where R9 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)-[R10-(→4)]-Fuc-, where R10 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)-[R11-(→3)]-Fuc-, where 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-(→3)]-Fuc-, where R12 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid; Glc-(1→3)-[Glc-(1→6)]-Gal-, and their derivatives, or The saponin is a bisdesmosidic triterpene glycoside (a bisdesmosidic triterpenoid saponin, preferably a pentacyclic saponin, more preferably a 12,13-dehydrooleanane-type saponin, preferably having an aldehyde functional group at position C23 of the aglycone core structure of the saponin), comprising a first sugar chain selected from Group A attached to the aglycone core structure, and a second sugar chain selected from Group B attached to the aglycone core structure.

[0146] Therefore, when the saponin conjugate of the present invention or the saponin contained in the oligonucleotide conjugate of the present invention has two glycans (sugar chains), the first sugar chain is attached at position C3 of the aglycone core structure, and the second sugar chain is attached at position C4 of the aglycone core structure. 28 are combined at

[0147] One 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 quillaic acid, gypsogenin, and derivatives thereof, preferably the aglycone core structure of the saponin is quillaic acid or gypsogenin, more preferably quillaic acid.

[0148] One embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the saponin is a bisdesmosidic saponin comprising at least 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)-[Xyl-(1→3)]-GlcA, and / or wherein the branched second sugar chain of at least four sugar residues comprises a terminal fucose residue and / or a terminal rhamnose residue.

[0149] One embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the saponin comprises a first glycan at position C3 of the saponin aglycone core structure and / or a second glycan at position C28 of the saponin aglycone core structure, preferably wherein the first glycan is a carbohydrate substituent at the C3 beta-OH group of the saponin aglycone core structure and / or wherein the second glycan is a carbohydrate substituent at the C28-OH group of the saponin aglycone core structure.

[0150] Without wishing to be bound by any theory, the presence of an aldehyde group or a derivative thereof on the aglycone core structure (also referred to herein as the "aglycone") is beneficial to the ability of saponins to stimulate and / or enhance the endosomal escape of (effector) molecules, such as oligonucleotides, when such saponins are co-localized intracellularly with these (effector) molecules, such as oligonucleotides, in the endosomes of said cells. Thus, saponin conjugates of the invention or oligonucleotide conjugates of the invention comprising saponins having an aglycone with an aldehyde group are preferred. In quillaic acid and gypsogenin, the aldehyde group is located at the C of the aglycone. 23 It's in the atom.

[0151] In one 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 bark saponin, dipsacoside B, saikosaponin A, saikosaponin D, macrantoidin A, esculentoside A, phytolaccagenin, essinate, AS6.2, NP-005236, AMA-1, AMR, alpha-hederin, NP-012672, NP-017777, NP-017778, NP-017774, NP-018110, NP-017772, NP-018109, NP-017888, NP-017889, NP-018108, SA1641, AEX55, NP-017674, NP-017810, AG1, NP-003881, N P-017676, NP-017677, NP-017706, NP-017705, NP-017773, NP-017775, SA1657, AG2, SO1861, GE1 741, SO1542, SO1584, SO1658, SO1674, SO1832, SO1862, SO1904, QS-7, QS1861, QS-7api, QS1862, Q S-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, assamsaponin F, digitonin, primulic acid 1, and AS64R, their stereoisomers, their derivatives, and combinations thereof. Preferably, the saponin is selected from the group consisting of QS-21, QS-21 derivatives, SO1861, SO1861 derivatives, SO1832, SO1832 derivatives, SA1641, SA1641 derivatives, GE1741, GE1741 derivatives, and combinations thereof. More preferably, the saponin is selected from the group consisting of QS-21 derivatives, SO1861 derivatives, and combinations thereof. Most preferably, the saponin is a SO1861 derivative. More preferably, the saponin is selected from the group consisting of QS-21, QS-21 derivatives, SO1861, SO1861 derivatives, SA1641, SA1641 derivatives, GE1741, GE1741 derivatives, and combinations thereof.More preferably, the saponin is selected from the group consisting of a QS-21 derivative, a SO1861 derivative, 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 a similar degree as their underivatized native counterparts.

[0152] Preferred embodiments are oligonucleotide conjugates of the invention and / or saponin conjugates of the invention, wherein the saponin contained in the conjugate is any one or more of the following: a) A saponin selected from any one or more of List A below: Quillaja saponaria saponin mixtures or saponins isolated from Quillaja saponaria, such as Quil-A, QS-17-api, QS-17-xyl, QS-21, QS-21A, QS-21B, QS-7-xyl, - Gypsophila elegans saponin mixture, or saponins isolated from Gypsophila elegans; - Saponinum albumensis saponin mixture or saponins isolated from Saponinum albumensis, - Saponaria officinalis saponin mixture or saponins isolated from Saponaria officinalis, and Quillaja bark saponin mixtures or saponins isolated from quillaja bark, such as Quil-A, QS-17-api, QS-17-xyl, QS-21, QS-21A, QS-21B, QS-7-xyl, or b) A saponin containing a gypsogenin aglycone core structure selected from List B below: SA1641, gypsoside A, NP-017772, NP-017774, NP-017777, NP-017778, NP-018109, NP-017888, NP-017889, NP-018108, SO1658, and phytolaccagenin, or c) A saponin containing a quillaric acid aglycone core structure selected from List C below: AG1856, AG1, AG2, agrostemoside E, GE1741, gypsophila saponin 1 (Gyp1), NP-017674, NP-017810, NP-003881, NP-017676, NP-017677, NP-017705, NP-017706, NP-017773, NP-017775, SA1657, saponariosides B, SO1542, SO1584, SO1674, SO1700, SO1730, SO1772, 10SO1832, SO1861, SO1862, SO1904, QS-7, QS-7api, QS-17, QS-18, QS-21A-apio, QS-21A-xylo, QS-21B-apio, and QS-21B-xylo; or d) Saponins containing a 12,13-dehydrooleanane type aglycone core structure without an aldehyde group at C23 of the aglycone selected from List D below: Escin Ia, escinate, alpha-hederin, AMA-1, AMR, AS6.2, AS64R, assamsaponin F, dipsacoside B, esculentoside A, macranthoidine A, NP-005236, NP-012672, primulic 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 lists A, B or C, more preferably a saponin selected from list B or C, even more preferably a saponin selected from list C.

[0153] Preferred are oligonucleotide conjugates of the present invention and / or saponin conjugates of the present invention, wherein the saponin is any one or more of the following: AG1856, GE1741, a saponin isolated from Quillaja saponaria, Quil-A, QS-17, QS-21, QS-7, SA1641, a saponin isolated from Saponaria officinalis, saponarioside B, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862, and SO1904. Preferably, the saponin herein is any one or more of QS-21, SO1832, SO1861, SA1641, and GE1741, more preferably, the saponin herein is QS-21, SO1832, or SO1861, most preferably SO1861.

[0154] More specific embodiments are oligonucleotide conjugates of the invention and / or saponin conjugates of the invention, wherein the saponin is a saponin isolated from Saponaria officinalis, preferably wherein the saponin is any one or more of saponariosides B, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862, and SO1904, more preferably wherein the saponin is any one or more of 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 saponin is SO1832 and SO1861; most preferably SO1861.

[0155] Such triterpene glycoside-type saponins can enhance endosomal escape of (effector) molecules, such as oligonucleotides contained in the oligonucleotide complexes of the present invention, present in the endosomes (or lysosomes) of cells when the saponins co-localize with such (effector) molecules inside the cell. The present inventors have established that the endosomal escape-enhancing activity of these saponins is at least about 5-fold more potent, i.e., at least about 10-fold more potent, for example 10- to 1000-fold more potent, or about 100- to 1000-fold more potent, when the saponins are contained in the saponin complexes of the present invention or when the saponins are contacted with cells. Free saponin can stimulate delivery of an (effector) molecule in the cytosol of a cell when such cells are contacted with the (effector) molecule and saponin at concentrations of saponin that are 100-1000 times higher than the concentrations of the same saponin contained in a saponin complex of the invention or contained in an oligonucleotide complex of the invention required to achieve the same degree of delivery of an (effector) molecule, such as an oligonucleotide selected from an ASO or siRNA, from outside the cell to the inside of the endosome, and ultimately in the cytosol and / or nucleus of said cell. Saponins that exhibit such endosomal escape-enhancing activity, and saponins that share a high structural similarity to saponins with established ability to enhance cytosolic delivery of (effector) molecules, are listed in Table A1.When the saponin is part of a saponin conjugate of the invention or part of an oligonucleotide conjugate of the invention, the targeted delivery of the saponin onto a target cell, preferably a liver cell, by binding of a ligand of an ASGPR such as ASGPR1, for example 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 to the endosome of said cell, is therefore at least 5 to 10 times more effective (potent), for example about 100 to 1000 times more effective (potent), compared to contacting the same cell with free, untargeted saponin that does not have a ligand of an ASGPR to bind to the ASGPR of the target cell.

[0156] Typically, in the saponin conjugates and oligonucleotide conjugates of the present invention, once the conjugates are taken up by ASGPR-bearing cells and transported into endosomes, the covalently bound saponin on the conjugate is cleaved (released) from the conjugate. In the endosome, the chemical conditions and / or pH are such that the covalent bond between the saponin and the remainder of the conjugate is broken, and the saponin is released in a free form within the endosome. Preferably, the cleaved saponin retains its native chemical structure. For example, when an aldehyde group of the saponin is involved in the covalent coupling of the saponin to a GalNAc moiety (typically via a linker) and / or to an oligonucleotide (typically via a linker), it is preferred that the saponin released from the conjugate within the endosome again contains this aldehyde group formed under the influence of the decoupling chemistry. Without wishing to be bound by any theory, it is believed that saponins, in their free form (preferably with a freely accessible aldehyde group, preferably with an aldehyde functional group at position C23 of the saponin aglycone core structure), exert optimal endosomal escape-enhancing activity in endosomes. Saponins are cleaved from conjugates to which they are attached via a hydrazone or semicarbazone bond, for example, under the influence of the acidic pH of the endosomes of cells, e.g., mammalian cells, e.g., human cells, e.g., tumor cells, liver cells. Thus, it is preferred that the saponin is covalently attached to the conjugates of the present invention via a cleavable bond, preferably a cleavable covalent bond that is cleaved inside the endosomes of a cell, e.g., a bond that is cleaved due to the acidic conditions of the endosome.

[0157] [Table 1]

[0158] [Table 2]

[0159]

Table 3

[0160]

Table 4

[0161]

Table 5

[0162]

Table 6

[0163]

Table 7

[0164]

Table 8

[0165]

Table 9

[0166]

Table 10

[0167]

Table 11

[0168]

Table 12

[0169]

Table 13

[0170] The saponin in the saponin complex of the present invention and / or the saponin in the oligonucleotide complex of the present invention is preferably derived from a plant. The saponin contained in the saponin complex of the present invention and / or the saponin contained in the oligonucleotide complex of the present invention is preferably isolated from a plant. The term "plant" should be understood to include plants and trees. For example, saponin is isolated from plant roots or tree bark. For example, saponin is isolated from plant roots. Examples of such plants from which saponin is derived (isolated) include Quillaja saponaria, Gypsophila paniculata L., Saponaria officinalis, such as Saponaria officinalis L., and Gypsophila elegans, such as Gypsophila elegans M. Beebe. Preferably, the saponin complex and / or oligonucleotide complex comprises a single type of saponin, preferably a single type of saponin derived from plant material such as a plant root, for example SO1861, SO1862, or SO1832 from Saponaria officinalis (e.g., Saponaria officinalis L., preferably Saponaria officinalis L.) (root), or QS-21, QS-7, or QS-17 from, for example, Quillaja saponaria (root).

[0171] Suitable sources for isolating saponins according to the invention, i.e., those exhibiting endosomal escape-enhancing activity, are Quillaja saponaria, Gypsophila paniculate L., Saponaria officinalis, and Gypsophila elegans, as well as Quillaja bark. Suitable saponins for the saponin derivatives of the invention and for the saponin complexes of the invention are therefore, for example: Quillaja saponaria saponins, saponins isolated from Quillaja saponaria, such as Quil-A, QS-17-api, QS-17-xyl, QS-21, QS-21A, QS-21B, QS-7-xyl, Saponinum album, saponin isolated from Saponinum album, Saponaria officinalis saponin, a saponin isolated from Saponaria officinalis (preferred); Quillaja bark saponin, saponins isolated from Quillaja bark saponin, such as Quil-A, QS-17-api, QS-17-xyl, QS-21, QS-21A, QS-21B, QS-7-xyl.

[0172] Additionally, not only QS-21, but also the individual saponins present in QS-21, i.e., the saponins depicted as saponins in Scheme Q, are suitable saponins for the saponin complexes of the present invention:

[0173] [ka]

[0174] Therefore, in all embodiments of the present invention, the saponin, preferably a single type of saponin, is preferably a saponin isolated from plant material, for example from the root of a plant.

[0175] In one embodiment, the oligonucleotide conjugate of the present invention, wherein the saponin contained in the oligonucleotide conjugate is isolated from a plant. Preferably, the saponin is isolated from a part of a plant, such as a root, or a part of a tree, such as bark. Preferably, the saponin is isolated from a root derived from a plant.

[0176] In some embodiments, the saponin or oligonucleotide conjugate comprises at least one saponin, wherein the saponin is a derivative of: i. The aglycone core structure of the saponin contains an aldehyde group that is derivatized; ii. The sugar chain of the saponin, preferably a sugar chain selected from Group A, contains a derivatized carboxyl group; iii. The sugar chain of the saponin, preferably a sugar chain selected from Group B, contains a derivatized acetoxy (Me(CO)O-) group; or iv. Any combination of derivatizations (i), (ii), and / or (iii) is present.

[0177] Exemplary derivatizations (i) of the aldehyde group of the aglycone core structure include: reduction to an alcohol such as a primary alcohol, conversion to a hydrazone (functional group) or semicarbazone (functional group), conversion to a hemiacetal, conversion to an acetal, oxidation to a carboxyl, conversion to an imine, conversion to an oxime, conversion to a β-hydroxyketone, or conversion to an enone, preferably reduction to an alcohol such as a primary alcohol, or conversion to a hydrazone (functional group) or semicarbazone (functional group). Once present in the endosomes of a cell that takes up the saponin or oligonucleotide conjugate of the invention, the derivatized saponin of the conjugate of the invention still exerts its endosomal escape-enhancing activity.

[0178] In some embodiments, the saponin is a derivative, wherein the aglycone core structure comprises an aldehyde group that is derivatized by: - Reduction to alcohols, such as primary alcohols, or - Formula R b HC=NNHR a hydrazones of formula R b HC=NNH(CO)R a to hydrazones, In the formula, R a is a group containing less than 20 carbon atoms, preferably less than 10 carbon atoms, and R b is the remainder of the saponin. Preferably, R a is an N-alkylated maleimide. In a specific embodiment, the aldehyde group is converted to a hydrazone bond by reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), N-[β-maleimidopropionic acid] hydrazide (BMPH), or N-[κ-maleimidoundecanoic acid] hydrazide (KMUH).

[0179] Exemplary derivatizations of the carboxyl groups of the glycans (ii) include: reduction to an alcohol, such as a primary alcohol, conversion to an amide, conversion to an ester, conversion to an amine, e.g., a primary or secondary amine, or decarboxylation, preferably reduction to an alcohol, such as a primary alcohol, conversion to an amide, or conversion to an ester, more preferably conversion to an amide.

[0180] Preferably the carboxyl group that is derivatized is part of a glucuronic acid moiety and preferably the sugar chain is selected from Group A.

[0181] In some embodiments, the saponin is a derivative, wherein the sugar chain, preferably a sugar chain selected from Group A, comprises a carboxyl group, preferably a carboxyl group of a glucuronic acid moiety, and is derivatized by: - Reduction to alcohols, such as primary alcohols, - Formula R a NH(CO)R b to an amide, or - Formula R a O(CO)R b Conversion of the compound to an ester, In the formula, R a is a group containing less than 20 carbon atoms, preferably less than 10 carbon atoms, and R b is the remainder of the saponin. Preferably, R a is an N-alkylated maleimide or diol. In specific embodiments, the carboxyl group is derivatized by conversion to an amide bond by reaction with 2-amino-2-methyl-1,3-propanediol (AMPD) or N-(2-aminoethyl)maleimide (AEM).

[0182] Exemplary derivatizations of acetoxy groups on sugar chains (iii) include: conversion to an alcohol, e.g., a secondary alcohol, by deacetylation, optionally followed by conversion of the resulting alcohol to an ether, ester, or ketone, preferably conversion to an alcohol, e.g., a secondary alcohol, by deacetylation.

[0183] Preferably, the acetoxy group to be derivatized is part of a sugar chain selected from Group B.

[0184] In some embodiments, the saponin is a derivative, wherein the sugar chain, preferably a sugar chain selected from Group B, comprises an acetoxy group that is derivatized by: - conversion to an alcohol, e.g. a secondary alcohol, by deacetylation, or - conversion by deacetylation to an alcohol, for example a secondary alcohol, and then conversion of the resulting alcohol to an alcohol of formula R a OR b ether, formula R a (CO)OR b or an ester of formula R a (CO)R b to a ketone, In the formula, R a is a group containing less than 20 carbon atoms, preferably less than 10 carbon atoms, and R b is the remainder of the saponin. Preferably, R a is an N-alkylated maleimide or diol.

[0185] Thus, in some embodiments, the saponin is a derivative of: ii. The aglycone core structure contains an aldehyde group that is derivatized with: - reduction to alcohol, - conversion to a hydrazone bond by reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), - conversion to a hydrazone bond by reaction with N-[β-maleimidopropionic acid] hydrazide (BMPH), or - conversion to a hydrazone bond by reaction with N-[κ-maleimidoundecanoic acid]hydrazide (KMUH), iii. The sugar chain selected from Group A contains 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 2-amino-2-methyl-1,3-propanediol (AMPD) or N-(2-aminoethyl)maleimide (AEM); iv. The glycan selected from Group B contains an acetoxy group (Me(CO)O-) that has been derivatized by conversion to a hydroxyl group (HO-); or v. Any combination of derivatizations (i), (ii), and / or (iii) is present.

[0186] An 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 contains an aglycone core structure that includes an aldehyde group that is derivatized; ii. The saponin derivative comprises a sugar chain, preferably a sugar chain selected from Group A, wherein the sugar chain comprises a carboxyl group that is derivatized; or iii. The saponin derivative comprises a sugar chain, preferably a sugar chain selected from Group B, wherein the sugar chain comprises a derivatized acetoxy (Me(CO)O-) group; or iv. Saponin derivatives include any combination of derivatizations i, ii, and iii, preferably any combination of two of derivatizations i, ii, and iii.

[0187] In one 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-21, QS-21A, QS-21A-api, QS-21A-xyl, QS-21B, QS-21B-api, QS-21B-xyl, QS-7-xyl, Q S-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, Quillaja saponin, Saponinum albumensis, QS-18, Quil-A, Gyp1, gypsoside A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, SO1904, their stereoisomers, their derivatives, and combinations thereof. Preferably, the saponin is selected from the group consisting of QS-21, QS-21 derivatives, SO1861, SO1861 derivatives, SO1832, SA1641, SA1641 derivatives, GE1741, GE1741 derivatives, and combinations thereof. More preferably, the saponin is selected from the group consisting of QS-21 derivatives, SO1861 derivatives, and combinations thereof. Most preferably, the saponin is a SO1861 derivative. Most preferably, the saponin is selected from the group consisting of a QS-21 derivative, a SO1861 derivative, SO1861, and combinations thereof. Even more preferably, the saponin is a SO1861 derivative or SO1861 or a SO1832 derivative or SO1832.

[0188] An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the saponin is a saponin derivative of quillaia acid saponin or gypsogenin saponin according to the invention, represented by molecule 1:

[0189] [ka]

[0190] During the ceremony, A1 represents hydrogen, a monosaccharide, or a linear or branched oligosaccharide, preferably A1 represents a sugar chain selected from Group A, more preferably A1 represents a sugar chain selected from Group A, and A1 comprises or consists of a glucuronic acid moiety; A2 represents hydrogen, a monosaccharide, or a linear or branched oligosaccharide, preferably A2 represents a sugar chain selected from Group B, more preferably A2 represents a sugar chain selected from Group B, A2 contains at least one acetoxy (Me(CO)O-) group, for example 1, 2, 3, or 4 acetoxy groups; wherein at least one of A1 and A2 is not hydrogen, and preferably both A1 and A2 are oligosaccharide chains; R is the hydrogen of gypsogenin or the hydroxyl of quillaic acid; Here, the saponin derivative corresponds to the saponin represented by molecule 1 in which at least one, preferably one or two, more preferably one of the following derivatizations is present: i. Position C of Quillaia acid or gypsogenin 23 wherein the aldehyde group in ii. A1 represents a glycan selected from Group A, and when A1 comprises or consists of a glucuronic acid moiety, the carboxyl group of the glucuronic acid moiety of A1 is derivatized; and iii. When A2 represents a glycan selected from Group B, and A2 contains at least one acetoxy group, one or more, preferably all, of the acetoxy groups of one sugar moiety or of two or more sugar moieties of A2 are derivatized.

[0191] An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein A1 represents a glycan selected from Group A and comprises or consists of a glucuronic acid moiety, wherein the carboxyl group of the glucuronic acid moiety of A1 is derivatized, and / or wherein A2 represents a glycan selected from Group B and A2 comprises at least one acetoxy group, wherein at least one acetoxy group of A2 is derivatized.

[0192] An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the saponin represented by molecule 1 is a bisdesmosidic triterpene saponin.

[0193] An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the saponin derivative corresponds to the saponin represented by molecule 1 in which at least one, preferably one or two, more preferably one of the following derivatizations is present: i. Position C of Quillaia acid or gypsogenin 23 where the aldehyde group is derivatized with: - reduction to alcohol, - conversion to a hydrazone bond by reaction with N-ε-maleimidocaproic hydrazide (EMCH), thereby providing saponin-Ald-EMCH, e.g., SO1861-Ald-EMCH or QS-21-Ald-EMCH, wherein the maleimide group of EMCH is optionally derivatized by formation of a thioether bond with mercaptoethanol; - conversion to a hydrazone bond by reaction with N-[β-maleimidopropionic acid] hydrazide (BMPH), wherein the maleimide group of BMPH is optionally derivatized by formation of a thioether bond with mercaptoethanol, or - conversion to a hydrazone bond by reaction with N-[κ-maleimidoundecanoic acid]hydrazide (KMUH), wherein the maleimide group of KMUH is optionally derivatized by formation of a thioether bond with mercaptoethanol; ii. A1 represents a sugar chain selected from Group A, and when A1 comprises or consists of a glucuronic acid moiety, the carboxyl group of the glucuronic acid moiety of A1 is derivatized 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 saponin-Glu-AMPD, e.g., QS-21-Glu-AMPD or SO1861-Glu-AMPD, or saponin-Glu-AEM, e.g., QS-21-Glu-AEM or SO1861-Glu-AEM; and iii. When A2 represents a glycan selected from Group B, and A2 contains at least one acetoxy group, one or more, preferably all, of the acetoxy groups of one or more sugar moieties of A2 are derivatized by conversion to a hydroxyl group (HO-) by deacetylation.

[0194] An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein A1 is Gal-(1→2)-[Xyl-(1→3)]-GlcA and / or A2 is Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc, and preferably the saponin represented by molecule 1 is 3-O-beta-D-galactosidase. Pyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)]-beta-D-glucuronopyranosylquilayic acid 28-O-beta-D-glucopyranosyl-(1→3)-beta-D-xylopyranosyl-(1→4)-alpha-L-rhamnopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)-4OAc-beta-D-quinovopyranosyl-(1→4)]-beta-D-fucopyranoside. More preferably, the saponin is any one or more of SO1861, SO1832, GE1741, SA1641, and QS-21, or a derivative thereof, and most preferably SO1861 or a derivative thereof, or a SO1832 derivative or SO1832.

[0195] An 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 contains an aglycone core structure containing an aldehyde group that is derivatized by: - reduction to alcohol, - conversion to a hydrazone bond by reaction with N-ε-maleimidocaproic hydrazide (EMCH), thereby providing saponin-Ald-EMCH, e.g., SO1861-Ald-EMCH or QS-21-Ald-EMCH, wherein the maleimide group of EMCH is optionally derivatized by formation of a thioether bond with mercaptoethanol; - conversion to a hydrazone bond by reaction with N-[β-maleimidopropionic acid] hydrazide (BMPH), where the maleimide group of BMPH is optionally derivatized by formation of a thioether bond with mercaptoethanol, or - conversion to a hydrazone bond by reaction with N-[κ-maleimidoundecanoic acid]hydrazide (KMUH), wherein the maleimide group of KMUH is optionally derivatized by formation of a thioether bond with mercaptoethanol; ii. the saponin derivative comprises a sugar chain, preferably a sugar chain selected from Group A, wherein the sugar chain comprises a carboxyl group, preferably the carboxyl group of a glucuronic acid moiety, which 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), thereby providing saponin-Glu-AMPD, e.g., QS-21-Glu-AMPD or SO1861-Glu-AMPD, or saponin-Glu-AEM, e.g., QS-21-Glu-AEM or SO1861-Glu-AEM; or iii. The saponin derivative comprises a sugar chain, preferably a sugar chain selected from Group B, wherein the sugar chain comprises an acetoxy (Me(CO)O-) group that has been derivatized by conversion to a hydroxyl group (HO-) by deacetylation; or iv. Saponin derivatives include any combination of derivatizations i, ii, and iii, preferably any combination of two of derivatizations i, ii, and iii. Preferably, the saponin derivative comprises an aglycone core structure, wherein the aglycone core structure comprises an aldehyde group that has been derivatized by conversion to a hydrazone bond by reaction with EMCH, wherein the maleimide group of EMCH is optionally derivatized by formation of a thioether bond with mercaptoethanol.

[0196] An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the saponin is a saponin derivative, wherein: i. Saponin derivatives include an aglycone core structure containing an aldehyde group that has been derivatized by conversion to a hydrazone bond by reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), thereby providing saponin-Ald-EMCH, e.g., SO1861-Ald-EMCH or QS-21-Ald-EMCH; ii. the saponin derivative comprises a sugar chain, preferably a sugar chain selected from Group A, wherein the sugar chain comprises 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 saponin-Glu-AEM, e.g., QS-21-Glu-AEM or SO1861-Glu-AEM; or iii. Saponin derivatives include combinations of derivatizations i and ii.

[0197] An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the saponin derivative comprises an 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, wherein the sugar chain comprises a carboxyl group, preferably the 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).

[0198] 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:

[0199] [ka]

[0200] Or, where the saponin derivative is represented by molecule 3:

[0201] [ka]

[0202] One embodiment is a saponin complex or an oligonucleotide complex of the present invention, wherein at least one saponin and an ASGPR ligand are covalently linked directly or via at least one linker. One embodiment is an oligonucleotide complex of the present invention, wherein at least one saponin and an ASGPR ligand 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 an ASGPR ligand and an oligonucleotide are covalently linked directly or via at least one linker. Preferably, at least one saponin, an ASGPR ligand, and an oligonucleotide are linked via at least one linker.

[0203] One embodiment is a saponin conjugate of the invention, wherein the GalNAc moiety is attached to the saponin S, preferably via a saponin linker Ls, by a conjugate of formula (II) S are combined as shown:

[0204] [ka]

[0205] One embodiment is a saponin conjugate of the invention, wherein the GalNAc moieties are each separately covalently linked to a central bridging moiety B via an oxygen at position "1" of the GalNAc moiety, which ties the bridge between the GalNAc moieties and the saponin moiety preferably via a saponin moiety linker L S via formula (III) S Effectively forming as shown in:

[0206] [ka]

[0207] wherein n is an integer greater than or equal to 2, preferably n is 3, and L S is the saponin moiety linker and S is the saponin moiety.

[0208] One embodiment is a saponin conjugate of the invention, wherein the GalNAc moiety is linked to the bridging moiety B by a GalNAc linker L GAL via formula (IV) S are combined as shown in:

[0209] [ka]

[0210] wherein n is an integer greater than or equal to 2, preferably n is 3, and L S is the saponin moiety linker and S is the saponin moiety.

[0211] One embodiment is a saponin conjugate of the invention, wherein the saponin moiety linker Ls binds the saponin to formula (II) S or to GalNAc as shown in formula (III) S and (IV) S represents any chemical moiety suitable for covalently bonding to the bridging moiety B as follows:

[0212] One embodiment is a saponin conjugate of the invention, wherein the saponin moiety linker L S comprises at least a first precursor L covalently linked to a GalNAc or bridging moiety B S1 and a second precursor L covalently linked to the saponin moiety. S2 where L is the result of a coupling reaction between S1 is a saponin moiety linker L covalently linked to GalNAc or to a bridging moiety B S It is a precursor of L S2 is the saponin moiety linker L covalently attached to the saponin moiety S It is a precursor to

[0213] One embodiment is a saponin conjugate of the invention, wherein the saponin moiety linker L S comprises at least a first precursor L covalently linked to a GalNAc or bridging moiety B S1 and a second precursor L covalently linked to the saponin moiety. S2 wherein the coupling reaction is selected from the group consisting of azide-alkyne cycloaddition, thiol-maleimide coupling, Staudinger reaction, nucleophilic ring-opening of a strained heterocyclic electrophile, non-aldol carbonyl reaction, and addition to a carbon-carbon double bond; preferably, wherein the coupling reaction is azide-alkyne cycloaddition, thiol-maleimide coupling, Staudinger reaction, nucleophilic ring-opening of a strained heterocyclic electrophile; more preferably, wherein the coupling reaction is azide-alkyne cycloaddition or thiol-maleimide coupling.

[0214] One embodiment is a saponin conjugate of the invention, wherein the saponin moiety linker L S is a first precursor L covalently linked to a GalNAc or bridging moiety B S1 and a second precursor L covalently linked to the saponin moiety. S2 is the result of a coupling reaction between the first precursor L S1 contains an azide, and the second precursor L S2 comprises an alkyne, preferably a hydrazone resulting from hydrazide / aldehyde coupling to the aldehyde of the saponin moiety.

[0215] One embodiment is a saponin complex of the invention, wherein precursor L S1 is the following azide of formula (XVII):

[0216] [ka]

[0217] 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 where precursor L S1 The structure of includes the following azide of formula (XVIII):

[0218] [ka]

[0219] In the formula, c represents an integer equal to or greater than 0, preferably c represents an integer in the range of 5 to 15, and more preferably c represents 9.

[0220] One embodiment is a saponin complex of the invention, wherein precursor L S2 The structure includes the following hydrazone having the formula (XIX):

[0221] [ka]

[0222] In the formula, a represents an integer greater than or equal to 0, preferably a represents an integer in the range of 2 to 6, more preferably a represents 4, and wherein L S2a represents an alkyne-containing moiety.

[0223] One embodiment is a saponin complex of the invention, wherein L S2a contains less than 20 carbon atoms, preferably L S2a represents a moiety according to formula (XX):

[0224] [ka]

[0225] One embodiment is a saponin complex of the invention, wherein the bridging moiety is a compound of formula (XV):

[0226] [ka]

[0227] wherein the oxygen atom of the compound of formula (XV) is linked to GalNAc or to a GalNAc linker L GAL and the nitrogen atom of the compound of formula (XV) is linked to a saponin moiety linker L S is combined with

[0228] One embodiment is a saponin complex of the invention, wherein L GAL represents any chemical moiety suitable for covalently linking GalNAc to bridging moiety B.

[0229] One embodiment is a saponin complex of the invention, wherein L GAL contains 2 to 25 carbon atoms, preferably 7 to 15 carbon atoms, more preferably 11 carbon atoms, wherein L GAL contains at least one, and preferably two, amide moieties.

[0230] One embodiment is a saponin complex of the invention, wherein L GAL is a compound according to formula (XVI):

[0231] [ka]

[0232] One embodiment is an oligonucleotide conjugate of the present invention, in which at least one GalNAc moiety, at least one saponin, and an oligonucleotide are covalently linked via a trifunctional linker, preferably, each of the GalNAc moiety, saponin, and oligonucleotide is covalently linked to a separate arm of the trifunctional linker. A preferred oligonucleotide conjugate of the present invention is the following oligonucleotide conjugate: at least one GalNAc moiety, preferably three GalNAc moieties, at least one saponin, preferably 1 to 16 saponin moieties, more preferably 1 to 8 saponin moieties, e.g., 1, 4, or 8 saponin moieties, and an oligonucleotide are covalently linked via a trifunctional linker, preferably, each of the GalNAc moiety, saponin / saponin moiety, and oligonucleotide is covalently linked to a separate arm of the trifunctional linker. An example of a trifunctional linker suitable for incorporation into an oligonucleotide conjugate of the present invention is a trifunctional linker represented by formula (XXI):

[0233] [ka]

[0234] One embodiment is an oligonucleotide conjugate of the invention, wherein a trifunctional linker, e.g., a trifunctional linker represented by Formula (XXI), is covalently attached to one or more saponin moieties, preferably 1 to 16 saponin moieties, more preferably 1 to 8 saponin moieties, e.g., 1, 4, or 8 saponin moieties, via a first arm of the linker; covalently attached to at least one GalNAc moiety, preferably 1 to 4 GalNAc moieties, more preferably 3 GalNAc moieties, via a second arm of the linker; and covalently attached to an oligonucleotide, preferably an AON, e.g., a BNA or siRNA, via the third arm of the trifunctional linker.

[0235] An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the ligand of the ASGPR is (GalNAc)3Tris, represented by molecule (DD3) or molecule (DD4):

[0236] [ka]

[0237] Alternatively, where the ligand of the ASGPR is monoGalNAc, represented by molecule II':

[0238] [ka]

[0239] One embodiment is a saponin conjugate of the invention, wherein at least one saponin is covalently attached to a molecule (DD3) or molecule (Moelcule) (DD4) or molecule II' of the invention via a linker represented by molecule III':

[0240] [ka]

[0241] where the hydrazide moiety of molecule III' forms a covalent hydrazone bond with the aldehyde group on the saponin, and the dibenzocyclooctyne group forms a covalent bond with the azide group of molecule (DD3) or (ir) molecule (DD4) or molecule II'.

[0242] An embodiment is an oligonucleotide conjugate of the invention, wherein at least one saponin is covalently attached to a ligand of an ASGPR via at least one cleavable linker, and / or wherein at least one saponin is covalently attached to an oligonucleotide via at least one cleavable linker.

[0243] An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein at least one saponin is covalently attached to a ligand of an ASGPR via at least one cleavable linker.

[0244] An embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the cleavable linker is susceptible to cleavage under acidic conditions, reductive conditions, enzymatic conditions, and / or light-induced conditions, preferably the cleavable linker comprises a cleavable bond selected from: hydrazone and hydrazide bonds that are susceptible to cleavage under acidic conditions, and / or bonds that are susceptible to proteolysis, for example proteolysis by cathepsin B, and / or bonds that are susceptible to cleavage under reductive conditions, such as disulfide bonds.

[0245] One embodiment is a saponin conjugate of the invention or an oligonucleotide conjugate of the invention, wherein the cleavable linker is cleaved in vivo, for example, under acidic conditions present in the endosomes and / or lysosomes of mammalian cells, preferably human cells. Preferably, the cleavable linker is cleaved in vivo at a pH between 4.0 and 6.5, more preferably at a pH of ≦5.5.

[0246] One 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 therebetween, e.g., 7, 9, 12 saponin moieties, preferably 1, 4, or 8 saponin moieties. Saponins SO1861 or SO1832, or functional derivatives thereof, are preferred; for example, QS-21, or a functional derivative thereof, are also suitable saponins.

[0247] In some embodiments, the saponin conjugates of the invention or the oligonucleotide conjugates of the invention comprise one saponin moiety, in some embodiments, the saponin conjugates of the invention or the oligonucleotide conjugates of the invention comprise four or eight saponin moieties.

[0248] One of the many benefits of the conjugates of the present invention, and the application of the saponin and oligonucleotide conjugates of the present invention, is the possibility to select the number of saponin moieties on the conjugate that is suitable for achieving improved intracellular oligonucleotide activity (e.g., gene silencing activity) when compared to the activity of the oligonucleotide when taken up by cells that have not been co-contacted with the saponin along with the oligonucleotide. The inventors demonstrate that the incorporation of a single saponin moiety on the conjugates of the present invention is already sufficient to achieve improved intracellular oligonucleotide activity, measured as gene silencing (see the Examples section). In a further example, it is demonstrated that gene silencing is further improved by including, for example, four or eight saponin moieties on the conjugates of the present invention. Saponin and oligonucleotide conjugates offer the freedom to couple any number between 1 and 64 or even more, for example, 128 saponin moieties, to GalNAc moieties, preferably three GalNAc moieties, in the saponin conjugate, or to a GalNAc moiety and oligonucleotide in the oligonucleotide conjugate. As mentioned above, the present inventors have surprisingly found that combining saponin and GalNAc or combining saponin, GalNAc, and an oligonucleotide does not interfere with the biological activity of any of the components of the complex. Furthermore, increasing the number of saponin moieties on the complex can improve oligonucleotide activity. Without wishing to be bound by any theory, an increased number of saponin moieties increases the endosomal escape-enhancing activity of the saponin once it is taken up by cells and imported into endosomes. Oligonucleotides administered as part of an oligonucleotide complex present in endosomes together with saponin, or separately as GalNAc-oligonucleotide complexes, are more efficiently imported from endosomes into the cytosol as a result of the improved endosomal escape activity mediated by the saponin, resulting in increased gene silencing activity.For example, the possibility of incorporating more than one saponin moiety onto a complex allows for the custom design of complexes when optimal gene silencing by a selected oligonucleotide is considered. Oligonucleotides that import relatively inefficiently from endosomes into the cytosol, e.g., 1-2% efficiency, will benefit from the presence of a single saponin moiety on the complex (a 1:1 ratio on the oligonucleotide complex), whereas oligonucleotides that are released less efficiently from endosomes into the cytosol may be better imported into the cytosol by applying more than a single saponin moiety onto the complex, e.g., 2-100 moieties, 2-64 moieties, 2-34 moieties, 2-16 moieties, 4-12 moieties, or 4-8 moieties. A preferred method for incorporating more than one saponin moiety is the incorporation of a dendron onto the complex of the invention, such as a G2, G3, or G4 dendron, for example, to link 4, 8, or 16 saponin moieties together onto the complex. In the Examples section, examples of improved gene silencing (degree of silencing at a selected time point, duration of silencing of target gene) are provided for oligonucleotide complexes containing, for example, a single saponin moiety, four saponin moieties, and eight saponin moieties.The inventors have established that by contacting cells with the oligonucleotide complexes of the present invention, the degree of gene silencing at a certain time point is improved, and the duration of gene silencing is also extended when compared to gene silencing in cells contacted with oligonucleotides in the absence of saponin.

[0249] In the oligonucleotide conjugates of the invention, it is suitable (and preferred) that the conjugates contain one or three GalNAc moieties, preferably three GalNAc moieties.

[0250] According to the present invention, the saponin typically comprises one or more moieties of SO1861 or SO1832 (also referred to as SO1831), e.g., 1 to 32 moieties of SO1861 or SO1832, 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, and most preferably 1, 4, or 8 moieties, e.g., 1, 4, or 8 SO1861 moieties or 1, 4, or 8 SO1832 moieties. The present inventors have established that when considering endosomal escape-enhancing activity against oligonucleotides (or protein toxins) within endosomes, SO1832 is approximately equally active as SO1861. As outlined herein above, the number of saponin moieties in the complexes of the present invention can be optimized when considering the degree and duration of gene silencing by contacting cells with the oligonucleotide. For oligonucleotide conjugates of the invention, this also applies to the number of GalNAc moieties on the conjugates of the invention: typically, optimal gene silencing (degree and / or duration) is achieved when one or three GalNAc moieties are included in the conjugates of the invention.

[0251] Saponin SO1832 consists of a quillaric acid aglycone core bearing the carbohydrate substituent Gal-(1→2)-[Xyl-(1→3)]-GlcA- at the C3 beta-OH group and the carbohydrate substituent Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc- at the C28-OH group (see also Table A1). The chemical formula is C 82 H 128 O 45 and has an exact mass of 1832.77 Daltons. The saponin structure of SO1832 follows the molecule (SO1832):

[0252] [ka]

[0253] Those skilled in the art will appreciate that the saponin included in the saponin complexes of the present invention may be a saponin having formula (II) as described herein. S , (III) S or (IV) S wherein the saponin moiety linker L S a first precursor L covalently linked to a GalNAc or bridging moiety; S1 and a second precursor L covalently linked to the saponin moiety. S2 and the second precursor L S2 contains a hydrazone resulting from hydrazide / aldehyde coupling to an aldehyde on the saponin moiety, or a semicarbazone functionality resulting from semicarbazide / aldehyde coupling to an aldehyde on the saponin moiety, as described herein, this means that the aldehyde group is already occupied by a linker; consequently, suitable saponin derivatives are those in which the saponin has been derivatized with (ii) a carboxyl group on the sugar chain, as described herein, and / or (iii) an acetoxy group on the sugar chain, as described herein.

[0254] Introduction to effector molecule complexes Certain pharmaceutical combinations and certain pharmaceutical compositions of the present invention comprise a second complex of an effector molecule, referred to herein as an "effector molecule complex," and a ligand of the asialoglycoprotein receptor (ASGPR). When the effector molecule is attached to the remainder of the effector molecule complex, it is referred to herein as an "effector moiety." The ASGPR ligand comprises at least one N-acetylgalactosamine (GalNAc) moiety, preferably three GalNAc moieties (GN)3. According to a preferred embodiment of the present invention, each GalNAc moiety is attached to the remainder of the ASGPR ligand, or to the effector moiety in cases where the ASGPR ligand consists of a single GalNAc moiety, via a covalent bond to the oxygen at position "1" as indicated in formula (I):

[0255] [ka]

[0256] Formula (II) E As shown in Figure 1, the ASGPR ligand is preferably linked to an effector moiety linker L E It consists of a single GalNAc moiety linked to the effector moiety E via

[0257] [ka]

[0258] The ASGPR ligand may contain more than one GalNAc moiety, for example, 2, 3, 4, 5, or 6 GalNAc moieties, preferably 3 or 4 GalNAc moieties, and most preferably 3 moieties. In such cases, it is preferred that the GalNAc moieties are each separately covalently linked to a central bridging moiety B via an oxygen at position "1," which allows the bridge between the GalNAc moiety and the effector moiety to be preferably linked to an effector moiety linker L. E The GalNAc moiety is effectively formed via the formula (III) E can be directly attached to the bridging moiety B as shown in:

[0259] [ka]

[0260] where n is an integer greater than or equal to 2, and L E is an effector moiety linker and E is an effector moiety. More preferably, the GalNAc moiety is linked to bridging moiety B by a GalNAc linker L GAL via formula (IV) E are combined as shown in:

[0261] [ka]

[0262] where n is an integer greater than or equal to 2, and L E is the effector moiety linker and E is the effector moiety. GAL can be independently selected, but one skilled in the art will appreciate that L GAL It will be appreciated that in the case where each of represents the same moiety, synthesis of the conjugate is simplified.

[0263] Effector molecule complex linker L E Effector part linker L E is a compound having an effector moiety of formula (II) E or to GalNAc of formula (III) E and (IV) E The linker represents any chemical moiety suitable for covalently linking the effector molecule to the bridging moiety B of formula (II). The identity and size of the linker are not particularly limited and may be any suitable chemical moiety for covalently linking the effector molecule to the bridging moiety B of formula (II). E or to GalNAc of formula (III) E and (IV) E to the bridging moiety B by means of a "conventional" chemical functional group (e.g., an ether bond) and typically includes, for example, a linker E L Suitable effector moiety linkers L E and related coupling reactions are described in Handbook of Bioconjugate Techniques, Hermanson, Greg T., Academic Press, 2013.

[0264] Those skilled in the art will appreciate that the effector moiety linker L E typically comprises at least a first precursor L covalently linked to a GalNAc or bridging moiety B E1 and a second precursor L covalently linked to an effector moiety. E2This principle is illustrated in the following reaction scheme for a compound of formula (II):

[0265] [ka]

[0266] In the formula, L E is the effector moiety linker, and L E1 is the effector moiety linker L E is a precursor of L, which is covalently linked to GalNAc E2 is the effector moiety linker L E which is covalently linked to an effector moiety, where E is the effector moiety.

[0267] The corresponding reaction scheme for compounds of formula (III) is as follows:

[0268] [ka]

[0269] In the formula, L E is the effector moiety linker, and L E1 is the effector moiety linker L E is a precursor of L, which is covalently linked to GalNAc E2 is the effector moiety linker L E which is covalently linked to an effector moiety, where n is an integer greater than or equal to 2, and E is an effector moiety.

[0270] Formula (IV) E The corresponding reaction scheme for the compound is as follows:

[0271] [ka]

[0272] In the formula, L E is the effector moiety linker, and L E1 is the effector moiety linker L E is a precursor of L, which is covalently linked to GalNAc E2 is the effector moiety linker L E is a precursor of the formula: GAL is the GalNAc linker and B is the bridging moiety.

[0273] Effector part linker L E comprises at least a first precursor L covalently linked to a GalNAc or bridging moiety E1 and a second precursor L covalently linked to an effector moiety. E2 wherein the coupling reaction is, for example, an azide-alkyne cycloaddition, a thiol-maleimide coupling, a Staudinger reaction, a nucleophilic ring-opening of a strained heterocyclic electrophile (e.g., an aziridine, an epoxide, a cyclic sulfate, an aziridinium ion, an episulfonium ion), a non-aldol carbonyl reaction (e.g., urea, thiourea, hydrazone, oxime ether, amide, or heteroaromatic ring formation), or an addition to a carbon-carbon double bond (e.g., epoxidation, aziridination, dihydroxylation, sulfentyl halide addition, nitrosyl halide addition, or Michael addition), preferably wherein the coupling reaction is an azide-alkyne cycloaddition, a thiol-maleimide coupling, a Staudinger reaction, a nucleophilic ring-opening of a strained heterocyclic electrophile, more preferably wherein the coupling reaction is an azide-alkyne cycloaddition or a thiol-maleimide coupling.

[0274] According to some embodiments of the present invention, the effector moiety linker L Econtains a succinimide thioether moiety. Such succinimide thioethers are the result of thiol-maleimide coupling between, for example, an N-substituted maleimide and a thiol- or sulfhydryl-containing compound. This thiol-maleimide coupling is a commonly known "click" chemistry tool in the field of bioconjugation and is described, for example, in Hermanson, Greg T., Bioconjugate Techniques, Academic Press, 2013, p. 289. Consequently, the effector moiety linker L E is a first precursor L covalently linked to a GalNAc or bridging moiety E1 and a second precursor L covalently linked to an effector moiety. E2 is the result of a coupling reaction between the first precursor L E1 contains an N-substituted maleimide, and the second precursor L E2 Preferably, comprises a thiol or a precursor thereof. A suitable thiol precursor is a disulfide, which can be cleaved (e.g., in situ) by reduction to the corresponding thiol.

[0275] Formula (V) E , (VII) E or (VIII) E Precursor L present on the compound E1 A preferred structure of is the following terminally N-substituted maleimide:

[0276] [ka]

[0277] wherein X represents any linker suitable for covalently linking a terminal N-substituted maleimide to GalNAc or the bridging moiety B. Those skilled in the art will appreciate that X may be the result of a coupling reaction between a first moiety covalently linked to GalNAc or the bridging moiety and a second moiety covalently linked to the maleimide. X may comprise a hydrazone and / or a 1,2,3-triazole. Whenever reference is made to 1,2,3-triazole in the context of the linkers herein, this preferably refers to 1H-1,2,3-triazole.

[0278] Formula (V) E , (VII) E or (VIII) E Precursor L present on the compound E1 Some embodiments of the structure are the following terminally N-substituted maleimides:

[0279] [ka]

[0280] 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 each represent 2; and wherein L E1a represents a hydrazone and / or a 1,2,3-triazole, preferably a 1,2,3-triazole, where L E1b represents a hydrazone and / or a 1,2,3-triazole, preferably a hydrazone;

[0281] [ka]

[0282] [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 to 15, more preferably b represents 2 and c represents 9, and wherein L E1a represents a hydrazone and / or a 1,2,3-triazole, preferably a 1,2,3-triazole, where L E1b represents a hydrazone and / or a 1,2,3-triazole, preferably a hydrazone; and

[0283] [ka]

[0284] [wherein c represents an integer greater than or equal to 0, preferably c represents an integer in the range of 5 to 15, more preferably c represents 9, and wherein L E1c represents a hydrazone and / or a 1,2,3-triazole, preferably a 1,2,3-triazole].

[0285] L E1a , L E1b , and L E1c each preferably contains less than 20 carbon atoms, and 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 formula (XIII), and L E1b is a hydrazone of formula (XIV), and L E1c is a 1,2,3-triazole of formula (XV):

[0286] [ka]

[0287] Thus, in some embodiments, the effector molecule conjugate has formula (II) as described herein. E , (III) E or (IV) E wherein the effector moiety linker L E is represented by formula (VI) E and a compound of formula (V) E , (VII) E or (VIII) E wherein the first precursor L is the result of a coupling reaction between the compound E1 is a terminally N-substituted maleimide of formula (X), (XI), or (XII), wherein L E1a is, for example, a 1,2,3-triazole of formula (XIII), and L E1b is, for example, a hydrazone of formula (XIV), and L E1c is for example a 1,2,3-triazole of formula (XV):

[0288] Effector molecule complex Effector part One aspect of the present invention relates to a pharmaceutical combination comprising: - a first pharmaceutical composition comprising a saponin complex of the present invention and optionally a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent; and - a second pharmaceutical composition comprising a second complex of an effector molecule and a ligand of ASGPR (wherein the ligand of ASGPR preferably comprises at least one GalNAc moiety, preferably three or four GalNAc moieties, and more preferably the ligand of ASGPR comprises or consists of (GalNAc)3Tris), or a third complex of an effector molecule and a binding molecule comprising a binding site for a cell surface molecule, and optionally comprising a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent.

[0289] One aspect of the present invention relates to a pharmaceutical composition comprising: - a saponin complex according to the invention, - a second complex of an effector molecule and a ligand of ASGPR (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 or consists of (GalNAc)3Tris) or a third complex of an effector molecule and a binding molecule comprising a binding site for a cell surface molecule, and optionally a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent.

[0290] One embodiment is a pharmaceutical combination of the invention comprising a saponin complex of the invention or a pharmaceutical composition of the invention comprising a saponin complex of the invention, wherein the effector molecule comprises or consists of at least one of a small molecule, such as a drug molecule, a toxin, such as a protein toxin, an oligonucleotide, such as an AON, such as a BNA, xenonucleic acid, or siRNA, an enzyme, a peptide, a protein, or any combination thereof; preferably, the effector molecule is a toxin, enzyme, or oligonucleotide.

[0291] One 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 ligand of the ASGPR comprises or is (GalNAc)3Tris, and / or wherein the ligand of the ASGPR and the effector molecule are conjugated via a covalent bond, preferably via at least one linker.

[0292] One embodiment is a pharmaceutical combination of the invention comprising a saponin complex of the invention, or a pharmaceutical composition of the invention comprising a saponin complex 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 shaped 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 oligonucleotides (BNA-AONs).

[0293] An embodiment is a pharmaceutical combination of the invention comprising a saponin complex of the invention or a pharmaceutical composition of the invention comprising a saponin complex of the invention, wherein the effector molecule is an oligonucleotide selected from any one or more of the following: an anti-miRNA, a BNA-AON, or an siRNA, e.g., a BNA-based siRNA, selected from a chemically modified siRNA, a metabolically stable siRNA, and a chemically modified metabolically stable siRNA.

[0294] One 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 effector molecule is an oligonucleotide capable of silencing any one of the genes apolipoprotein B (apoB), HSP27, transthyretin (TTR), proprotein convertase subtilisin / kexin type 9 (PCSK9), delta-aminolevulinic acid synthase 1 (ALAS1), antithrombin 3 (AT3), glycolate oxidase (GO), complement component C5 (CC5), hepatitis B virus (HBV) X gene, HBV S gene, alpha-1 antitrypsin (AAT), and lactate dehydrogenase (LDH) when present inside a mammalian cell, and / or an oligonucleotide capable of targeting an aberrant miRNA when present inside a mammalian cell, for example. One 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 effector molecule is an oligonucleotide that, when present inside a mammalian cell, silences any one of the genes HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AT3, GO, CC5, HBV X gene, HBV S gene, AAT, miR-122, Hepatitis B virus HbsAg, LDHA, CEBPA, and LDH. The oligonucleotides can be used to target abnormal miRNAs, for example, when present inside mammalian cells, and / or can be used in the treatment or prevention of diseases or health problems involving any one or more of the genes HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AT3, GO, CC5, HBV X gene, HBV S gene, AAT, miR-122, Hepatitis B virus HbsAg, LDHA, CEBPA, and LDH. Preferred target genes are HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AAT, miR-122, Hepatitis B virus HbsAg, LDHA, and CEBPA. The genes HPS27 and apoB are more preferred.

[0295] One 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 effector molecule is an oligonucleotide capable of targeting, e.g., when present inside a mammalian cell, an mRNA involved in the expression of any one of the proteins apoB, HSP27, TTR, PCSK9, ALAS1, AT3, GO, CC5, an expression product of the HBV X gene, an expression product of the HBV S gene, AAT, and LDH, or is capable of antagonizing or restoring miRNA function, e.g., inhibiting the repression of oncogenic miRNA (onco-miR) or onco-miR expression, e.g., when present inside a mammalian cell. One 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 effector molecule is an oligonucleotide capable of targeting, e.g., when present inside a mammalian cell, an mRNA involved in expression of any one of the proteins HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AT3, GO, CC5, HBV X gene, HBV S gene, 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, e.g., inhibiting repression of oncogenic miRNA (onco-miR) or onco-miR expression, e.g., when present inside a mammalian cell.

[0296] One embodiment is a pharmaceutical combination of the invention comprising a saponin complex of the invention or a pharmaceutical composition of the invention comprising a saponin complex of the invention, wherein the effector molecule is or comprises a toxin, which toxin comprises or consists of at least one molecule selected from any one or more of the following: a peptide, a protein, an enzyme such as urease and Cre recombinase, a proteinaceous toxin, a ribosome-inactivating protein, and / or a bacterial toxin, a plant toxin, more preferably any one or more of the following: a viral toxin such as apoptin, a bacterial toxin such as shiga toxin, shiga-like toxin, Pseudomonas aeruginosa exotoxin (PE) or exotoxin A of PE, a full-length or truncated diphtheria toxin, a hydroxybenzoate ... toxin (DT), cholera toxin, fungal toxins such as alpha-sarcin, plant toxins including ribosome-inactivating proteins and type 2 ribosome-inactivating protein A chains, such as giantin, e.g., giantin-30 or giantin-32, saporin, e.g., saporin-S3 or saporin-S6, bouganin or a deimmunized derivative of bouganin, debouganin, Shiga-like toxin A, pokeweed antiviral protein, ricin, ricin A chain, modeccin, modeccin A chain, abrin, abrin A chain, volkensin, volkensin A chain, viscumin, viscumin A chain, or animal or human toxins such as frog RNase, or granzyme B or angiogenin from humans, or a fragment or derivative of any of these.Preferably, the protein toxin is dianthin and / or saporin, and / or at least one of a ribosome-targeting toxin, an elongation factor-targeting toxin, a tubulin-targeting toxin, a DNA-targeting toxin, and an RNA-targeting toxin, more preferably, any one or more of the following: emtansin, pasudotox, maytansinoid derivative DM1, maytansinoid derivative DM4, monomethyl auristatin E (MMAE, vedotin), monomethyl auristatin F (MMAF, mafodotin), calicheamicin, N-acetyl-gamma calicheamicin, pyrrolobenzodiazepine (PBD) dimer, benzodiazepine, CC-1065 analog, dextran, benzodiazepine ... duocarmycin, doxorubicin, paclitaxel, docetaxel, cisplatin, cyclophosphamide, etoposide, docetaxel, 5-fluorouracil (5-FU), mitoxantrone, tubulysin, indolinobenzodiazepine, AZ13599185, cryptophycin, rhizoxin, methotrexate, anthracycline, camptothecin analog, SN38, DX8951f, exatecan mesylate, truncated form of Pseudomonas aeruginosa exotoxin (PE38), duocarmycin derivatives, amanitin, α-amanitin, splicostatin, thilanstatin, ozogamicin, tesirin, amberstatin 269, and soravtansine, or derivatives thereof.

[0297] Complex cross-linked part B Formula (III) S or (IV) S and the saponin complex of formula (III) E or (IV) E The linking moiety B present in the effector molecule complex of isoform is a linker L of two or more, preferably three or four, more preferably three GalNAc moieties and a saponin moiety. S or an effector moiety linker L E represents any moiety suitable for covalently linking

[0298] According to a preferred embodiment, the bridging moiety B is a compound of formula (XV):

[0299] [ka]

[0300] In the formula, the oxygen atom of the compound of formula (XV) is attached to GalNAc (formula (III) S or (III) E (corresponding to the compound shown in Figure 1) or GalNAc linker L GAL to (formula (IV) S or (IV) E (corresponding to the compound of formula (XV)), and the nitrogen atom of the compound of formula (XV) is linked to the saponin moiety linker L S or an effector moiety linker L E is combined with

[0301] Those skilled in the art will recognize that the bridging moiety B is a compound of formula (III) S , (III) E , (IV) S or (IV) E It will be appreciated that these compounds correspond to effector molecule complexes in which the ASGPR ligand consists of (GalNAc)3Tris.

[0302] Complex Linker L GAL GalNAc Linker L GAL is represented by formula (IV) S or (IV) E The linker represents any chemical moiety suitable for covalently linking GalNAc to the bridging moiety of the formula (I). The identity and size of the linker are not particularly limited.

[0303] According to some embodiments, the GalNAc linker L GAL Each of the GalNAc linkers L contains 2 to 25 carbon atoms, preferably 7 to 15 carbon atoms, and more preferably 11 carbon atoms. GAL contains at least one, preferably two, amide moieties. Particularly preferred GalNAc linkers L GALis a compound according to formula (XVI):

[0304] [ka]

[0305] One embodiment is a pharmaceutical combination of the invention comprising a saponin complex of the invention or a pharmaceutical composition of the invention comprising a saponin complex of the invention, wherein the binding molecule comprising a binding site for a cell surface molecule comprised in the third complex is a ligand of the cell surface molecule, or an antibody comprising a binding site for the cell surface molecule, or at least one domain or fragment thereof comprising the binding site.

[0306] One 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 third conjugate is any one or more of the following: an antibody-toxin conjugate, a receptor ligand-toxin conjugate, an antibody-drug conjugate, a receptor ligand-drug conjugate, an antibody-nucleic acid conjugate, or a receptor ligand-nucleic acid conjugate.

[0307] One 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 in the third conjugate is selected from the group consisting of the following cell surface molecules: CD71, CD63, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin alpha V beta 3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, integrin A, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, integrin A, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, integrin B ... It can bind to any one of IL-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-1, IL-2, IL-1, IL-2, IL-3, IL-4 ...1, IL-2, IL-1, IL-1, IL-2, IL-1, IL-1, IL-2, IL-1, IL-1, IL-2, IL-1, IL-1, IL-1, IL-2, IL-1, IL-1, IL-1, IL-1, IL-1, IL-1, IL-2, IL-1, IL-1, IL-1, IL-1, IL-1

[0308] One embodiment is a pharmaceutical combination of the invention comprising a saponin complex of the invention or a pharmaceutical composition of the invention comprising a saponin complex of the invention, wherein the binding molecule comprising a binding site for a cell surface molecule included in the third complex is or comprises any one of the following: an antibody, preferably a monoclonal antibody, such as a human monoclonal antibody, IgG, a molecule comprising or consisting of a single domain antibody, at least one V HH Domain, preferably Camel V H , variable heavy chain neoantigen receptor (V NAR ) domains, Fab, scFv, Fv, dAb, F(ab)2, and Fcab fragments.

[0309] Certain aspects of the present invention relate to pharmaceutical combinations of the present invention comprising a saponin conjugate of the present invention or to pharmaceutical compositions of the present invention comprising a saponin conjugate of the present invention for use as a medicament. Certain aspects of the present invention relate to oligonucleotide conjugates of the present invention for use as a medicament.

[0310] One aspect of the present invention relates to a pharmaceutical combination of the invention comprising a saponin complex of the invention, or a pharmaceutical composition of the invention comprising a saponin complex of the invention, for use in the treatment or prevention of a disease or health problem involving the expression product of any one or more of the genes apoB, TTR, PCSK9, ALAS1, AT3, GO, CC5, HBV X gene, HBV S gene, AAT, and LDH. One aspect of the present invention relates to 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, for use in the treatment or prevention of a disease or health problem involving expression products of any one or more of the genes HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AT3, GO, CC5, HBV X gene, HBV S gene, 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, more preferably HSP27, apoB.

[0311] One embodiment is a pharmaceutical combination of the invention comprising a saponin complex of the invention, or a pharmaceutical composition of the invention comprising a saponin complex of the invention, for use in the treatment or prevention of cancer, an infectious disease, a viral infection, hypercholesterolemia, cardiovascular disease, primary hyperoxaluria, hemophilia A, hemophilia B, AAT-related liver disease, acute hepatic porphyria, TTR amyloidosis, hereditary TTR amyloidosis (hATTR), complement-related disease, hepatitis B infection, hepatitis C infection, alpha 1-antitrypsin deficiency, beta-thalassemia, or an autoimmune disease, or a pharmaceutical combination of the invention comprising a saponin complex of the invention or a pharmaceutical composition of the invention for use in the invention.

[0312] One embodiment is a pharmaceutical combination comprising a saponin complex of the invention for use in the invention, or a pharmaceutical composition comprising a saponin complex of the invention for use in the invention or a pharmaceutical composition comprising an oligonucleotide complex of the invention, wherein the saponin is a saponin derivative according to the invention, preferably a QS-21 derivative, or a SO1861 derivative, or a SO1832 derivative.

[0313] One aspect of the present invention relates to an in vitro or ex vivo method for transferring a second or third complex of the present invention from the outside of a cell to the inside of said cell, and preferably thereafter transferring an effector molecule comprised in the second or third complex according to the present invention into the cytosol of said cell, comprising the steps of: a) providing a cell, which expresses an ASGPR on its surface, and which expresses a cell surface molecule comprising a binding molecule for binding to said cell surface molecule when the third complex is to be transferred into the cell, said cell being preferably selected from a liver cell, a virus-infected cell, and a tumor cell; b) providing the second or third complex of the invention for transfection into the cells provided in step a); c) providing a saponin complex of the present invention; d) contacting the cells of step a) in vitro or ex vivo with the second or third complex of step b) and the saponin complex of step c), thereby resulting in the transfer of the second or third complex from outside the cells into said cells, preferably thereby resulting in the subsequent transfer of the second or third complex into the cytosol of said cells, or preferably thereby resulting in the subsequent transfer of at least the effector molecule comprised in the second or third complex into the cytosol of said cells.

[0314] One embodiment is an oligonucleotide conjugate of the invention, wherein the oligonucleotide is one of a BNA, a xenonucleic acid, a siRNA, or an antisense oligonucleotide.

[0315] In one 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 shaped 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 oligonucleotides (BNA-AONs).

[0316] An embodiment is an oligonucleotide conjugate of the invention, wherein the oligonucleotide is selected from any one or more of the following: an anti-miRNA, a BNA-AON, or an siRNA, e.g., a BNA-based siRNA, selected from a chemically modified siRNA, a metabolically stable siRNA, and a chemically modified metabolically stable siRNA.

[0317] One embodiment is an oligonucleotide conjugate of the invention, wherein the oligonucleotide is capable of silencing any one of the genes apolipoprotein B (apoB), HSP27, transthyretin (TTR), proprotein convertase subtilisin / kexin type 9 (PCSK9), TMPRSS6, delta-aminolevulinic acid synthase 1 (ALAS1), antithrombin 3 (AT3), glycolate oxidase (GO), complement component C5 (CC5), hepatitis B virus (HBV) X gene, HBV S gene, alpha-1 antitrypsin (AAT), miR-122, hepatitis B virus HbsAg, LDHA, CEBPA, and lactate dehydrogenase (LDH), e.g., when present inside a mammalian cell, preferably a human cell, and / or is capable of targeting an aberrant miRNA, e.g., when present inside a mammalian cell. Preferably, the gene is any one of the genes HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AAT, miR-122, hepatitis B virus HbsAg, LDHA, and CEBPA for expressing the proteins HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AAT, miR-122, hepatitis B virus HbsAg, LDHA, and CEBPA.

[0318] One embodiment is an oligonucleotide conjugate of the invention, wherein the oligonucleotide is an oligonucleotide capable of silencing any one of the genes apolipoprotein B (apoB) and HSP27, for example, when present inside a mammalian cell, preferably when present inside a human cell.

[0319] One embodiment is an oligonucleotide conjugate of the invention, wherein the oligonucleotide is capable of targeting, e.g., when present inside a mammalian cell, preferably a human cell, an mRNA involved in the expression of any one of the proteins apoB, HSP27, TTR, PCSK9, TMPRSS6, ALAS1, AT3, GO, CC5, HBV X gene expression product, HBV S gene expression product, AAT, miR-122, Hepatitis B virus HBsAg, LDHA, CEBPA, and LDH, or is capable of antagonizing or restoring miRNA function, e.g., inhibiting repression of oncogenic miRNA (onco-miR) or onco-miR expression, e.g., when present inside a mammalian cell, preferably a human cell. Preferably, the protein is a liver protein selected from the following: HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AAT, miR-122, Hepatitis B virus HBsAg, LDHA, and CEBPA.

[0320] One embodiment is an oligonucleotide conjugate of the invention, wherein the oligonucleotide is capable of targeting an mRNA involved in the expression of any one of the proteins apoB and HSP27, for example, when present inside a mammalian cell, preferably when present inside a human cell.

[0321] One aspect of the invention relates to a method for providing an oligonucleotide conjugate of the invention, comprising the steps of: (a) providing at least one saponin moiety comprising a covalently attached first linker, wherein the first linker comprises at least one first reactive group for covalent attachment to a second reactive group on a second linker or to a seventh reactive group on a seventh linker; (b) providing an oligonucleotide comprising a covalently attached third linker, wherein the third linker comprises a third reactive group for covalent attachment to a fourth reactive group on the fourth linker or to an eighth reactive group on the seventh linker; (c) providing at least one GalNAc moiety comprising a covalently attached fifth linker, wherein the fifth linker comprises a fifth reactive group for covalent attachment to a sixth reactive group on a sixth linker or to a ninth reactive group on a seventh linker; and (d1) linking the first linker to the second linker by forming a covalent bond between the first reactive group and the second reactive group, linking the third linker to the fourth linker by forming a covalent bond between the third reactive group and the fourth reactive group, linking the fifth linker to the sixth linker by forming a covalent bond between the fifth reactive group and the sixth reactive group, and covalently linking the second linker, the fourth linker, and the sixth linker together, thereby providing an oligonucleotide; or (d2) 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 an oligonucleotide conjugate; Either one.

[0322] An embodiment is a method for providing an oligonucleotide conjugate of the invention, wherein the seventh linker is a trifunctional linker, for example, a trifunctional linker represented by formula (XXI):

[0323] [ka]

[0324] The following methods for providing an oligonucleotide conjugate of the invention are preferred: at least one saponin moiety is 1 to 16 saponin moieties, preferably 1 to 8 saponin moieties, such as 1, 4, or 8 saponin moieties.

[0325] The following method for providing an oligonucleotide conjugate of the invention is preferred: the saponin is SO1861, SO1832, QS-21, or a functional derivative of any of them, preferably SO1861 or SO1832.

[0326] The following method for providing an oligonucleotide conjugate of the invention is preferred: the saponin moiety is covalently linked via a hydrazone or semicarbazone bond.

[0327] The following methods for providing an oligonucleotide conjugate of the invention are preferred: at least one GalNAc moiety is 1 to 4 GalNAc moieties, preferably 1 or 3 GalNAc moieties.

[0328] One aspect of the present invention relates to a pharmaceutical composition comprising an oligonucleotide conjugate of the present invention and optionally a pharmaceutically acceptable excipient and / or optionally a pharmaceutically acceptable diluent.

[0329] An aspect of the present invention relates to a pharmaceutical composition comprising an oligonucleotide conjugate of the present invention and optionally a pharmaceutically acceptable excipient and / or optionally a pharmaceutically acceptable diluent, for use as a medicament.An aspect of the present invention relates to an oligonucleotide conjugate of the present invention for use as a medicament.

[0330] Certain aspects of the present invention relate to pharmaceutical compositions comprising the oligonucleotide conjugates of the invention and, optionally, a pharmaceutically acceptable excipient and / or an optional pharmaceutically acceptable diluent, or to oligonucleotide conjugates of the invention for use in the treatment or prevention of diseases or health problems involving expression products of any one or more of the genes HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AT3, GO, CC5, HBV X gene, HBV S gene, 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 genes HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AT3, GO, CC5, HBV X gene, HBV S gene, AAT, miR-122, Hepatitis B virus HBsAg, LDHA, CEBPA, and LDH. For example, the disease is cancer associated with the (over)expression of HSP27 in tumor cells. Administering an oligonucleotide complex of the present invention, including an oligonucleotide for silencing the HSP27 gene, to a patient suffering from a tumor whose tumor growth is associated with the (over)expression of HSP27 results in silencing of the HSP27 gene, thereby halting tumor growth. For example, a health problem associated with apoB expression is a health problem related to elevated levels of LDL cholesterol in the blood of a human subject, such as a patient at risk of atherosclerosis and / or hypercholesterolemia and / or a patient suffering from atherosclerosis and / or hypercholesterolemia. Administering an oligonucleotide of the present invention, including an oligonucleotide for silencing the apoB gene, to the human subject or patient inhibits or reduces apoB expression, thereby resulting in a reduction in LDL (cholesterol) levels, since apoB is a protein component of LDL particles.Decreased levels of circulating LDL are associated with less circulating LDL cholesterol and thereby a reduced risk of LDL cholesterol-related health and disease symptoms in: patients suffering from atherosclerosis and / or hypercholesterolemia, or subjects at risk of developing atherosclerosis and / or hypercholesterolemia under the influence of higher-than-normal blood levels of LDL cholesterol.

[0331] Certain aspects of the present invention relate to pharmaceutical compositions comprising the oligonucleotide conjugates of the invention or to the oligonucleotide conjugates of the invention for use in the treatment or prevention of diseases or health problems involving expression products of any one or more of the genes HSP27, apoB, TTR, PCSK9, 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 genes HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AAT, miR-122, Hepatitis B virus HbsAg, LDHA, and CEBPA.

[0332] An embodiment is a pharmaceutical composition for use in accordance with the invention comprising an oligonucleotide conjugate of the invention, or an oligonucleotide conjugate of the invention for use in accordance with the invention, wherein said use is in the treatment or prevention of a disease or health problem involving the expression product of any one or more of the genes HSP27 and apoB, preferably apoB, and / or for use in the treatment or prevention of a disease or health problem involving any one or more of the genes HSP27 and apoB, preferably apoB.

[0333] An embodiment is a pharmaceutical composition comprising an oligonucleotide conjugate of the invention for use in accordance with the invention, or an oligonucleotide conjugate of the invention for use in accordance with the invention, wherein said use is in the treatment or prevention of cancer, an infectious disease, a viral infection, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, AAT-associated liver disease, acute hepatic porphyria, TTR amyloidosis, hereditary TTR amyloidosis (hATTR), complement-related disease, hepatitis B infection, or an autoimmune disease.

[0334] An embodiment is a pharmaceutical composition for use in accordance with the invention comprising an oligonucleotide conjugate of the invention, or an oligonucleotide conjugate of the invention for use in accordance with the invention, wherein said use is in the treatment or prevention of cancer, e.g., endometrial cancer, breast cancer, lung cancer, and hypercholesterolemia, preferably hypercholesterolemia.

[0335] Certain embodiments of the present invention relate to pharmaceutical compositions comprising the oligonucleotide conjugates of the invention or to the oligonucleotide conjugates of the invention for use in the treatment or prevention of: cancer, an infectious disease, a viral infection, hypercholesterolemia, cardiovascular disease, primary hyperoxaluria, hemophilia A, hemophilia B, AAT-associated liver disease, acute hepatic porphyria, TTR amyloidosis, hereditary TTR amyloidosis (hATTR), complement-related disease, hepatitis B infection, hepatitis C infection, α1-antitrypsin deficiency, β-thalassemia, or an autoimmune disease.

[0336] Certain aspects of the present invention relate to pharmaceutical compositions comprising the oligonucleotide conjugates of the invention or to the oligonucleotide conjugates of the invention for use in the treatment or prevention of: cancer, such as endometrial cancer, breast cancer, lung cancer, or hepatocellular carcinoma, and / or cardiovascular disease, such as atherosclerosis and / or hypercholesterolemia, preferably atherosclerosis and / or hypercholesterolemia.

[0337] For example, the disease is cancer associated with the (over)expression of HSP27 in tumor cells. Administering an oligonucleotide complex of the present invention containing an oligonucleotide for silencing the HSP27 gene to a patient suffering from a tumor whose tumor growth is associated with the (over)expression of HSP27 results in silencing of the HSP27 gene, thereby halting tumor growth. For example, a health problem associated with apoB expression is a health problem related to elevated levels of LDL cholesterol in the blood of a human subject, such as a patient at risk of atherosclerosis and / or hypercholesterolemia and / or a patient suffering from atherosclerosis and / or hypercholesterolemia. Administering an oligonucleotide complex of the present invention containing an oligonucleotide for silencing the apoB gene to the human subject or patient inhibits or reduces apoB expression, thereby resulting in a reduction of LDL (cholesterol), since apoB is a protein component of LDL particles. Decreased levels of circulating LDL are associated with less circulating LDL cholesterol and thereby a reduced risk of LDL cholesterol-related health and disease symptoms in: patients suffering from atherosclerosis and / or hypercholesterolemia, or subjects at risk of developing atherosclerosis and / or hypercholesterolemia under the influence of higher-than-normal blood levels of LDL cholesterol.

[0338] Certain embodiments of the present invention relate to pharmaceutical compositions comprising the oligonucleotide conjugates of the present invention or oligonucleotide conjugates of the present invention for use in lowering LDL cholesterol in a subject. Certain embodiments of the present invention relate to pharmaceutical compositions comprising the oligonucleotide conjugates of the present invention or oligonucleotide conjugates of the present invention for use in methods for lowering LDL cholesterol levels in the blood of a human subject. Certain embodiments of the present invention relate to pharmaceutical compositions comprising the oligonucleotide conjugates of the present invention or oligonucleotide conjugates of the present invention for use in methods for treating or preventing cardiovascular disease. Typically, the treatment or prevention is in a human patient suffering from cardiovascular disease or a human subject at risk of developing cardiovascular disease. Typically, cardiovascular disease is associated with elevated blood levels of LDL cholesterol compared to the upper limit of the range of normal LDL cholesterol levels.

[0339] One aspect of the present invention relates to an in vitro or ex vivo method for transferring an oligonucleotide complex of the present invention from the outside of a cell to the inside of said cell, and preferably subsequently transferring the oligonucleotide contained in the oligonucleotide complex of the present invention into the cytosol and / or nucleus of said cell, comprising the steps of: a) providing a cell expressing an ASGPR, preferably ASGPR1, on its surface, the cell preferably being selected from a liver cell, a virus-infected mammalian cell, and a mammalian tumor cell, wherein preferably said cell is a human cell; b) providing an oligonucleotide complex of the invention for transfection into the cells provided in step a); c) contacting the cells of step a) in vitro or ex vivo with the oligonucleotide complex of step b), preferably in a liquid medium, thereby resulting in the transfer of the oligonucleotide complex from outside the cells into said cells, and optionally and preferably thereby resulting in the subsequent transfer of the oligonucleotides contained in the oligonucleotide complex into the cytosol and / or nucleus of said cells.

[0340] Surprisingly, the inventors have found that: a glycosyl group consisting of an aglycone core structure, preferably a triterpene aglycone core structure as defined herein linked to quillaric acid and a first sugar chain "R 1 " and the second glycan "R 2 and at least one of:

[0341] [ka]

[0342] In the formula, R 1 and R 2 are independently selected from hydrogen, monosaccharides, linear oligosaccharides, and branched oligosaccharides; X=O, P, or S; Y=NR 3 R 4 where R 3 and R 4 independently represent H, unsubstituted C1-C10 straight chain, branched, or cyclic alkyl, unsubstituted C2-C10 straight chain, branched, or cyclic alkenyl, or unsubstituted C2-C10 straight chain or branched alkynyl, or a covalently bonded linker, preferably R 3 and R 4 is H, or Y=

[0343] [ka]

[0344] wherein n and m are each independently an integer selected from 1, 2, or 3; Z=CH2, O, S, P, or NR 5 and In the formula, R 5is H, unsubstituted C1-C10 straight chain, branched, or cyclic alkyl, unsubstituted C2-C10 straight chain, branched, or cyclic alkenyl, unsubstituted C2-C10 straight chain or branched alkynyl, or a covalently bonded linker, or a maleimide moiety according to formula (II1)a:

[0345] [ka]

[0346] or an azide moiety according to formula (II1)b:

[0347] [ka]

[0348] wherein o is an integer selected from 0 to 10, preferably 2 to 7, and more preferably 4 to 6. has reduced toxicity when considering cell viability of cells contacted with the saponin derivative, has activity when considering, for example, the cytotoxicity of the toxin or enhancement of BNA-mediated gene silencing when the aldehyde functional group is converted to a semicarbazone functional group according to formula (I1) (without wishing to be bound by any theory, this relates to similar or improved endosomal escape-enhancing activity of the modified saponin (i.e., saponin derivative)), and / or has reduced hemolytic activity when compared to the toxicity, activity, and hemolytic activity of the unmodified saponin. That is, a saponin derivative has at least one, preferably two, and more preferably all three of the following: (i) reduced toxicity when considering cell viability of cells contacted with the saponin derivatives; (ii) improved activity when the aldehyde functionality is converted to a semicarbazone functionality according to formula (I1), e.g., enhanced BNA-mediated gene silencing (without wishing to be bound by any theory, related to similar or improved endosomal escape-enhancing activity of the modified saponin); and / or (iii) Reduced hemolytic activity. The toxicity, activity, and hemolytic activity of the saponin derivative are then compared with those of the unmodified saponin on which the saponin derivative is based.Therefore, the present inventors provide saponin derivatives with improved therapeutic window.Because the saponin derivatives have lower cytotoxicity than that determined for their naturally occurring counterparts, lower hemolytic activity than that determined for the naturally occurring counterparts of the saponin derivatives, and the ratio between the IC50 value of cytotoxicity and, for example, the IC50 value of toxin enhancement or the IC50 value of gene silencing is similar or increased, and / or the ratio between the IC50 value of saponin hemolytic activity and, for example, the IC50 value of toxin enhancement or the IC50 value of gene silencing is similar or increased.The term "endosomal escape enhancing activity" can be abbreviated as "activity".

[0349] In addition, the present inventors have surprisingly established (tumor) cell killing by contacting such cells with a saponin conjugate based on a saponin derivative containing a semicarbazone functional group together with an ADC, such as an antibody-protein toxin conjugate, despite moderate to low expression of the cell surface receptor targeted by the cell surface molecule-binding molecule (e.g., an antibody) contained in the saponin conjugate, and / or despite moderate to low expression of the cell surface receptor targeted by the ADC. The saponin derivative and saponin conjugate contain a semicarbazone functional group. For example, in a comparative example, when cells were contacted with a saponin conjugate containing the same cell surface molecule-binding molecule (e.g., an antibody) but containing a hydrazone functional group (=NN(H)—C(O)—) instead of the semicarbazone functional group, such cell killing could not be established, or could only be established to a lower degree. The present inventors have thereby provided saponin derivatives and saponin conjugates that are more potent when activation or enhancement of an effector moiety, such as that contained in an ADC or AOC, is considered.

[0350] Furthermore, the present inventors have found that saponin derivatives containing semicarbazone functional groups hydrolyze more rapidly and in greater amounts to the corresponding native saponins containing a "free" aldehyde functional group under acidic conditions, such as those present in the endosomes and / or lysosomes of mammalian cells, compared to art-known saponin derivatives containing a hydrazone functional group (=NN(H)-C(O)-). This has the advantage that, for example, a lower amount of a saponin derivative according to the present invention may be administered to a patient in need of enhancing a gene silencing oligonucleotide, such as an ADC or AOC or BNA coupled to one or more GalNAc moieties, to obtain the same amount of a native saponin containing a "free" aldehyde to act as an endosomal escape enhancer for a targeted toxin or targeted oligonucleotide, compared to the required amount of a saponin derivative containing a hydrazone functional group (=NN(H)-C(O)-). Without wishing to be bound by any theory, the faster and more effective release of saponins containing an aldehyde functional group from saponin derivatives containing a semicarbazone functional group, when compared to the release of saponins containing an aldehyde functional group from saponin derivatives containing a hydrazone functional group, is believed by the inventors to underlie the improved activity of saponin conjugates containing saponin derivatives containing a semicarbazone functional group, such as conjugates comprising a saponin, an oligonucleotide, and a cell surface receptor ligand, for example, one to three GalNAc moieties (for targeting (liver) cells expressing ASGPR), wherein the activity is enhanced effector moiety activity, such as a gene silencing oligonucleotide, inside the cytosol or nucleus of the targeted cell due to improved endosomal escape.

[0351] Surprisingly, conversion (derivatization) of the aldehyde group at C23 of the saponin aglycone to a semicarbazone functional group according to formula (I1) results in reduced cytotoxicity when such saponin derivatives are contacted with cells, i.e., various cell types. Therefore, it is beneficial to provide a series of saponin derivatives with reduced cytotoxicity, where the reduced cytotoxicity is relative to the cytotoxicity determined for the unmodified, naturally occurring saponin counterpart. Saponin derivatives can be formed from such naturally occurring saponins, such as SO1861, the equally active SO1832, and QS-21 (isoforms), preferably SO1861 or SO1832. When reduced cytotoxicity is considered, saponin derivatives containing a semicarbazone functional group are equally suitable when a saponin with reduced cytotoxicity is to be provided.

[0352] The present inventors have therefore provided saponin derivatives that have improved therapeutic windows compared to the corresponding underivatized saponins when it comes to cytotoxicity and / or hemolytic activity, as well as when it comes to enhancing (gene silencing) oligonucleotides, for example. Such saponin derivatives containing semicarbazone functional groups are particularly suitable for application in therapeutic regimens involving GalNAc-oligonucleotide conjugates, for example, in combination with saponin derivatives, or oligonucleotide conjugates of the invention comprising a covalently linked saponin together with a GalNAc moiety and an oligonucleotide, for example, for the prevention or treatment of cancer, hypercholesterolemia, cardiovascular disease, or elevated levels of LDL cholesterol in (human) subjects in need thereof above normal levels in healthy subjects. The safety of such saponin derivatives is improved when cytotoxicity and / or hemolytic activity is suspected, particularly when such saponin derivatives are administered to patients in need of treatment, for example, with an ADC or AOC, or with a conjugate comprising at least one GalNAc moiety and an oligonucleotide, for example, a BNA for silencing genes such as HSP27 and apoB, or when a saponin derivative containing a semicarbazone functional group is part of an oligonucleotide conjugate of the invention.

[0353] One embodiment is an oligonucleotide conjugate of the invention that comprises one saponin moiety.

[0354] One embodiment is an oligonucleotide conjugate of the present invention, in which at least one GalNAc moiety, at least one saponin, and an oligonucleotide are covalently linked via a trifunctional linker, preferably, each of the GalNAc moiety, saponin, and oligonucleotide is covalently linked to a separate arm of the trifunctional linker. A preferred oligonucleotide conjugate of the present invention is the following: at least one GalNAc moiety, preferably three GalNAc moieties, at least one saponin, preferably 1 to 16 saponin moieties, more preferably 1 to 8 saponin moieties, e.g., 1, 4, or 8 saponin moieties, and an oligonucleotide are covalently linked via a trifunctional linker, preferably, each of the GalNAc moiety, saponin / saponin moiety, and oligonucleotide is covalently linked to a separate arm of the trifunctional linker. An example of a trifunctional linker suitable for incorporation into an oligonucleotide conjugate of the present invention is a trifunctional linker represented by formula (XXI):

[0355] [ka]

[0356] One embodiment is an oligonucleotide conjugate of the invention, wherein a trifunctional linker, e.g., a trifunctional linker represented by Formula (XXI), is covalently attached to one saponin moiety or more saponin moieties, preferably 1 to 16 saponin moieties, more preferably 1 to 8 saponin moieties, e.g., 1, 4, or 8 saponin moieties, via a first arm of the linker; covalently attached to at least one GalNAc moiety, preferably 1 to 4 GalNAc moieties, more preferably 3 GalNAc moieties, via a second arm of the linker; and covalently attached to an oligonucleotide, preferably an AON, e.g., a BNA or siRNA, via the third arm of the trifunctional linker. The following oligonucleotide conjugates of the invention are preferred: at least one GalNAc moiety, preferably three GalNAc moieties, at least one saponin, preferably 1 to 16 saponin moieties, more preferably 1 to 8 saponin moieties, e.g., 1, 4, or 8 saponin moieties, preferably 1, 4, or 8 saponin moieties, and the oligonucleotide are covalently linked via a trifunctional linker, preferably wherein the GalNAc moiety, saponin or saponin moiety, and the oligonucleotide are each covalently linked to a separate arm of the trifunctional linker.

[0357] One embodiment is an oligonucleotide conjugate of the invention, comprising at least one saponin covalently linked to a ligand of the asialoglycoprotein receptor (ASGPR), wherein the ligand of the ASGPR comprises at least one N-acetylgalactosamine (GalNAc) moiety, preferably three or four GalNAc moieties, more preferably three GalNAc moieties, and more preferably the ligand of the ASGPR comprises or consists of (GalNAc)3Tris, and further covalently linked to an oligonucleotide, wherein the at least one saponin is a 12,13-dehydrooleanane-type monodesmosidic or bisdesmosidic pentacyclic triterpene saponin, preferably having an aldehyde functional group at position C23 of the saponin aglycone core structure, and wherein the oligonucleotide conjugate comprises 1 to 16 saponin moieties, preferably 1 to 8 saponin moieties, more preferably 1 saponin moiety, 4 saponin moieties, or 8 saponin moieties.

[0358] One embodiment is an oligonucleotide conjugate of the invention, wherein at least one GalNAc moiety, preferably three GalNAc moieties, at least one saponin, preferably 1 to 16 saponin moieties, more preferably 1 to 8 saponin moieties, e.g., 1, 4, or 8 saponin moieties, and the oligonucleotide are covalently linked via a trifunctional linker, preferably wherein each of the GalNAc moiety, saponin or saponin moiety, and oligonucleotide is covalently linked to a separate arm of the trifunctional linker.

[0359] In some embodiments, the oligonucleotide conjugate of the invention comprises one saponin moiety, four saponin moieties, or eight saponin moieties.

[0360] One embodiment is an oligonucleotide conjugate of the invention, wherein at least one saponin moiety is linked via a hydrazone or semicarbazone bond. At least one saponin is preferably covalently attached to the oligonucleotide conjugate via a semicarbazone bond. Such a hydrazone or semicarbazone bond (with a linker) can be formed, for example, through an aldehyde group at the C23 atom of the quillaia acid aglycone core structure of the saponin or the gypsogenin aglycone core structure of the saponin (see Table A1 for these types of saponins).

[0361] One embodiment of the present invention relates to an oligonucleotide conjugate according to the molecule (EE):

[0362] [ka]

[0363] This molecule (EE) is a covalent conjugation product, obtained by covalent conjugation of a trifunctional linker according to formula (XXI) with:

[0364] [ka]

[0365] Saponin derivatives according to the molecule (AA):

[0366] [ka]

[0367] [In the formula,

[0368] [ka]

[0369] represents a saponin moiety according to formula (SM):

[0370] [ka]

[0371] In the formula, R 1 and R 2 are independently selected from hydrogen, monosaccharides, linear oligosaccharides, and branched oligosaccharides, wherein the saponin moiety according to formula (SM) is based on a saponin containing an aldehyde group at position C23; and (2) GalNAc conjugate according to molecule (FF):

[0372] [ka]

[0373] [In the formula,

[0374] [ka]

[0375] represents a tri-GalNAc conjugate according to molecule (DD1) or according to molecule (DD2):

[0376] [ka]

[0377] [In the formula, y1, y2, and y3 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, and most preferably 2 and 3; preferably, y1, y2, and y3 are the same; and y4 is an integer selected from the following: 1 to 100, preferably 2 to 80, more preferably 3 to 70, even more preferably 4 to 60, even more preferably 4 to 50, even more preferably 4 to 40, even more preferably 4 to 30, even more preferably 4 to 20, even more preferably 4 to 6, most preferably 4 to 5, for example 4]

[0378] [ka]

[0379] 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, and most preferably 2 and 3; 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, and most preferably 8 to 12, for example 9. Preferably, it represents a tri-GalNAc complex according to molecule (DD3) or according to molecule (DD4):

[0380] [ka]

[0381] and (3) Oligonucleotides with a linker according to the molecule (GG):

[0382] [ka]

[0383] where the molecule (GG) represents the conjugation product of the conjugation reaction between the linker (E)-1-(4-((2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)hydrazinylidene)methyl)benzamido)-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-3,6,9,12-tetraoxapentadecan-15-amide and the oligonucleotide-linker molecule according to the molecule (HH):

[0384] [ka]

[0385] Such oligonucleotide conjugates according to the molecules of the present invention (EE) exhibit surprisingly improved potency when the endosomal escape-enhancing activity of the saponin moiety is taken into account. It is believed that this improved efficacy is related to improved decoupling (release) of the saponin from the conjugate due to cleavage of the semicarbazone functional group under the influence of a slightly acidic pH in the endosomes of cells that have taken up the conjugate, mediated by the binding of the conjugate to ASGPR. This results in improved endosomal escape of the oligonucleotide and improved (gene silencing) activity of the oligonucleotide in the cytosol and / or nucleus compared to application of a similar conjugate but without the covalently linked saponin moiety.

[0386] An example of an oligonucleotide conjugate of the invention comprising a single saponin moiety is the oligonucleotide presented in Figure 35D as molecule 13. The synthesis of such an oligonucleotide of the invention comprising a single saponin moiety is exemplified in the Examples section below by a detailed description of the synthesis of molecule 13 (Figure 35D) and presented in Figure 35. The saponin moiety is linked to a trifunctional linker via a linker, where the saponin moiety is linked to the linker via a hydrazone bond. A further example of such an oligonucleotide conjugate of the invention comprising a single saponin moiety is the oligonucleotide presented in Figure 38C as molecule 31. The synthesis of such an oligonucleotide conjugate of the invention comprising a single saponin moiety is exemplified in the Examples section below by a detailed description of the synthesis of molecule 31 (Figure 38C) and presented in Figure 38. The saponin moiety is linked to a trifunctional linker via a linker, where the saponin moiety is linked to the linker via a semicarbazone bond (see, e.g., molecule 28 in Figure 38A). See also saponin derivatives according to molecule (AA) above herein.

[0387] In one embodiment, the oligonucleotide conjugate of the invention comprises four saponin moieties. In one embodiment, the oligonucleotide conjugate of the invention comprises four saponin moieties covalently attached to a dendron, preferably a G2 dendron. An example of such a (G2) dendron is N,N'-((9S,19S)-14-(6-aminohexanoyl)-1-mercapto-9-(3-mercaptopropanamido)-3,10,18-trioxo-4,11,14,17-tetraazatricosane-19,23-diyl)bis(3-mercaptopropanamide). The synthesis of a dendron linked to four saponin moieties is presented, for example, in Figure 39B and in the Examples section, for the synthesis of intermediate 20 (molecule 39) from molecule 37 (a saponin linked to a maleimide, involving a semicarbazone bond between the saponin, here SO1861, and a linker bearing a maleimide group) and molecule 38, which is dendron N,N'-((9S,19S)-14-(6-aminohexanoyl)-1-mercapto-9-(3-mercaptopropanamido)-3,10,18-trioxo-4,11,14,17-tetraazatricosane-19,23-diyl)bis(3-mercaptopropanamido) formate. Certain embodiments of the present invention relate to oligonucleotide conjugates according to molecule (PP):

[0388] [ka]

[0389] This molecule (PP) is a covalent conjugation product, a saponin-GalNAc conjugate according to molecule (LL):

[0390] [ka]

[0391] is obtained by covalent conjugation of with an oligonucleotide equipped with a linker according to the molecule (GG):

[0392] [ka]

[0393] where the molecule (GG) represents the conjugation product of the conjugation reaction between the linker (E)-1-(4-((2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)hydrazinylidene)methyl)benzamido)-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-3,6,9,12-tetraoxapentadecan-15-amide and the oligonucleotide-linker molecule according to the molecule (HH):

[0394] [ka]

[0395] This molecule (LL) is a covalent conjugation product, a saponin derivative according to the molecule (JJ):

[0396] [ka]

[0397] is obtained by covalent conjugation of the compound with a trifunctional linker and GalNAc according to the molecule (MM): [ka]

[0398] This molecule (JJ) is the conjugation product of N,N'-((9S,19S)-14-(6-aminohexanoyl)-1-mercapto-9-(3-mercaptopropanamido)-3,10,18-trioxo-4,11,14,17-tetraazatricosane-19,23-diyl)bis(3-mercaptopropanamide) with: First, Saponin derivatives according to the molecule (KK):

[0399] [ka]

[0400] [In the formula,

[0401] [ka]

[0402] represents a saponin moiety according to formula (SM):

[0403] [ka]

[0404] In the formula, R 1 and R 2 are independently selected from hydrogen, monosaccharides, linear oligosaccharides, and branched oligosaccharides according to the present invention, wherein the saponin moiety according to formula (SM) is based on a saponin comprising an aldehyde group at position C23, e.g., listed in Table A1, according to any one of the saponins of the present invention, e.g., QS-21, SO1861, SO1832; And afterwards, 2,5-dioxopyrrolidin-1-yl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate, wherein the molecule (MM) is a trifunctional linker according to formula (XXI):

[0405] [ka]

[0406] and a complex of GalNAc conjugates according to the molecule (NN):

[0407] [ka]

[0408] During the ceremony,

[0409] [ka]

[0410] represents a tri-GalNAc conjugate according to molecule (DD1) or molecule (DD2) as detailed herein above, preferably molecule (DD3) or molecule (DD4) as detailed herein above, more preferably molecule (DD3) as detailed herein above.

[0411] An example of such an oligonucleotide conjugate of the invention comprising four saponin moieties is the oligonucleotide conjugate presented in Figure 36E as molecule 22. The synthesis of such an oligonucleotide conjugate of the invention comprising four saponin moieties is exemplified in the Examples section below by a detailed description of the synthesis of molecule 22 (Figure 36E) and presented in Figure 36. The saponin moieties are linked to the dendron via a linker, where each saponin moiety is linked to the linker via a hydrazone bond. A further example of such an oligonucleotide conjugate of the invention comprising four saponin moieties is the oligonucleotide conjugate presented in Figure 39F as molecule 42. The synthesis of such an oligonucleotide conjugate of the invention comprising four saponin moieties is exemplified in the Examples section below by a detailed description of the synthesis of molecule 42 (Figure 39F) and presented in Figure 39. The saponin moieties are linked to the dendron via a linker, where each saponin moiety is linked to the linker via a semicarbazone bond. See also the saponin derivative according to molecule (KK) above herein.

[0412] One embodiment is an oligonucleotide conjugate of the invention comprising eight saponin moieties. As noted above, the following oligonucleotide conjugates of the invention are preferred: at least one GalNAc moiety, preferably three GalNAc moieties, at least one saponin, preferably 1 to 16 saponin moieties, more preferably 1 to 8 saponin moieties, e.g., eight saponin moieties, and an oligonucleotide are covalently linked via a trifunctional linker, preferably wherein the GalNAc moiety, single saponin or more than one saponin moiety, and oligonucleotide are each covalently linked to a separate arm of the trifunctional linker.

[0413] One embodiment is an oligonucleotide conjugate of the invention comprising eight saponin moieties covalently attached 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[(2S)-2,6-bis(3-sulfanylpropanamido)hexanamido]hexanamido]ethyl}hexanamido)ethyl]carbamoyl}-5-[(2S)-2,6-bis(3-sulfanylpropanamido)hexanamido]pentyl]-2,6-bis(3-sulfanylpropanamido)hexanamide. The synthesis of a dendron linked to eight saponin moieties is presented, for example, in Figure 40A and in the Examples section, for the synthesis of intermediate 25 (molecule 44) from: molecule 37 (a maleimide-linked saponin involving a semicarbazone bond between the saponin, here SO1861, and a linker bearing a maleimide group); and molecule 43, which is the dendron (2S)-N-[(1S)-1-{[2-(6-amino-N-{2-[(2S)-2,6-bis[(2S)-2,6-bis(3-sulfanylpropanamido)hexanamido]hexanamido]ethyl}hexanamido)ethyl]carbamoyl}-5-[(2S)-2,6-bis(3-sulfanylpropanamido)hexanamido]pentyl]-2,6-bis(3-sulfanylpropanamido)hexanamido formate.

[0414] One embodiment of the present invention relates to an oligonucleotide conjugate according to the molecule (SS):

[0415] [ka]

[0416] This molecule (SS) is a covalent conjugation product, a saponin-GalNAc conjugate according to the molecule (RR):

[0417] [ka]

[0418] is obtained by covalent conjugation of with an oligonucleotide equipped with a linker according to the molecule (GG):

[0419] [ka]

[0420] where the molecule (GG) represents the conjugation product of the conjugation reaction between the linker (E)-1-(4-((2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)hydrazinylidene)methyl)benzamido)-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-3,6,9,12-tetraoxapentadecan-15-amide and the oligonucleotide-linker molecule according to the molecule (HH):

[0421] [ka]

[0422] This molecule (RR) is a covalent conjugation product, a saponin derivative conforming to the molecule (QQ):

[0423] [ka]

[0424] is obtained by covalent conjugation of the compound with a trifunctional linker and GalNAc according to the molecule (MM):

[0425] [ka]

[0426] This molecule (QQ) is the conjugation product of (2S)-N-[(1S)-1-{[2-(6-amino-N-{2-[(2S)-2,6-bis[(2S)-2,6-bis(3-sulfanylpropanamido)hexanamido]hexanamido]ethyl}hexanamido)ethyl]carbamoyl}-5-[(2S)-2,6-bis(3-sulfanylpropanamido)hexanamido]pentyl]-2,6-bis(3-sulfanylpropanamido)hexanamido formate with: First, (a) Saponin derivative according to the molecule (KK):

[0427] [ka]

[0428] [In the formula,

[0429] [ka]

[0430] represents a saponin moiety according to formula (SM):

[0431] [ka]

[0432] In the formula, R 1 and R 2are independently selected from hydrogen, monosaccharides, linear oligosaccharides, and branched oligosaccharides according to the present invention, wherein the saponin moiety according to formula (SM) is based on a saponin comprising an aldehyde group at position C23, e.g., listed in Table A1, according to any one of the saponins of the present invention, e.g., QS-21, SO1861, SO1832; And afterwards, (b) 2,5-dioxopyrrolidin-1-yl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate, wherein the molecule (MM) is a trifunctional linker according to formula (XXI):

[0433] [ka]

[0434] and a GalNAc conjugate (see above here) according to molecule (NN), where:

[0435] [ka]

[0436] represents a tri-GalNAc conjugate according to molecule (DD1) or molecule (DD2) as detailed herein above, preferably molecule (DD3) or molecule (DD4) as detailed herein above, more preferably molecule (DD3) as detailed herein above.

[0437] An example of such an oligonucleotide conjugate of the invention comprising eight saponin moieties is the oligonucleotide conjugate presented in Figure 37C as molecule 26. The synthesis of such an oligonucleotide conjugate of the invention comprising eight saponin moieties is exemplified in the Examples section below by a detailed description of the synthesis of molecule 26 (Figure 37C) and presented in Figure 37. The saponin moieties are linked to the dendron via a linker, where each saponin moiety is linked to the linker via a hydrazone bond. A further example of such an oligonucleotide conjugate of the invention comprising eight saponin moieties is the oligonucleotide conjugate presented in Figure 40D as molecule 47. The synthesis of such an oligonucleotide conjugate of the invention comprising eight saponin moieties is exemplified in the Examples section below by a detailed description of the synthesis of molecule 47 (Figure 40D) and presented in Figure 40. The saponin moieties are linked to the dendron via a linker, where each saponin moiety is linked to a separate linker via a semicarbazone bond. See also saponin derivatives according to molecule (KK) above herein.

[0438] Preferred are oligonucleotide conjugates of the invention comprising one saponin moiety or four saponin moieties, preferably four saponin moieties, covalently attached to a dendron, preferably a G2 dendron, such as, for example, N,N'-((9S,19S)-14-(6-aminohexanoyl)-1-mercapto-9-(3-mercaptopropanamide)-3,10,18-trioxo-4,11,14,17-tetraazatricosane-19,23-diyl)bis(3-mercaptopropanamide), or eight saponin moieties, Preferably, eight saponin moieties are covalently attached to a dendron, preferably a G3 dendron, such as, for example, (2S)-N-[(1S)-1-{[2-(6-amino-N-{2-[(2S)-2,6-bis[(2S)-2,6-bis(3-sulfanylpropanamido)hexanamido]hexanamido]ethyl}hexanamido)ethyl]carbamoyl}-5-[(2S)-2,6-bis(3-sulfanylpropanamido)hexanamido]pentyl]-2,6-bis(3-sulfanylpropanamido)hexanamide.

[0439] An embodiment is an oligonucleotide conjugate according to the present invention and according to, for example, any one of molecules (EE), (PP), and (SS), wherein the oligonucleotide conjugate is based on a saponin according to any one of the saponins listed herein above and in Table A1.

[0440] The following oligonucleotide conjugates according to the invention and according to any one of the molecules (EE), (PP) and (SS) are preferred: the oligonucleotide conjugate is based on a saponin selected from SO1861, SO1832 and QS-21, preferably SO1861 and SO1832, more preferably SO1861 or SO1832. When the saponin is SO1861, a saponin derivative according to the molecule (VII)a:

[0441] [ka]

[0442] is conjugated to an oligonucleotide and at least one GalNAc moiety to provide an oligonucleotide conjugate of the invention.

[0443] Also preferred are the following oligonucleotide conjugates according to the invention and according to, for example, any one of the molecules (EE), (PP), and (SS): wherein the semicarbazone functional group undergoes hydrolysis in vivo under acidic conditions present in the endosomes and / or lysosomes of mammalian cells, preferably human cells, preferably at a pH between 4.0 and 6.5, more preferably at a pH ≦ 5.5.

[0444] In one embodiment is an oligonucleotide conjugate of the invention, wherein the saponin included in the conjugate is any one or more of the following: a) A saponin selected from any one or more of List A below: Quillaja saponaria saponin mixtures or saponins isolated from Quillaja saponaria, such as Quil-A, QS-17-api, QS-17-xyl, QS-21, QS-21A, QS-21B, QS-7-xyl, - Gypsophila elegans saponin mixture, or saponins isolated from Gypsophila elegans; - Saponinum albumensis saponin mixture or saponins isolated from Saponinum albumensis, - Saponaria officinalis saponin mixture or saponins isolated from Saponaria officinalis, and Quillaja bark saponin mixtures or saponins isolated from quillaja bark, such as Quil-A, QS-17-api, QS-17-xyl, QS-21, QS-21A, QS-21B, QS-7-xyl, or b) A saponin containing a gypsogenin aglycone core structure selected from List B below: SA1641, gypsoside A, NP-017772, NP-017774, NP-017777, NP-017778, NP-018109, NP-017888, NP-017889, NP-018108, SO1658, and phytolaccagenin, or c) A saponin containing a quillaric acid aglycone core structure selected from List C below: AG1856, AG1, AG2, agrostemoside E, GE1741, gypsophila saponin 1 (Gyp1), NP-017674, NP-017810, NP-003881, NP-017676, NP-017677, NP-017705, NP-017706, NP-017773, NP-017775, SA1657, saponariosides B, SO1542, SO1584, SO1674, SO1700, SO1730, SO1772, 10SO1832, SO1861, SO1862, SO1904, QS-7, QS-7api, QS-17, QS-18, QS-21A-apio, QS-21A-xylo, QS-21B-apio, and QS-21B-xylo; or d) Saponins containing a 12,13-dehydrooleanane type aglycone core structure without an aldehyde group at C23 of the aglycone selected from List D below: Escin Ia, escinate, alpha-hederin, AMA-1, AMR, AS6.2, AS64R, assamsaponin F, dipsacoside B, esculentoside A, macranthoidine A, NP-005236, NP-012672, primulic 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 lists A, B or C, more preferably from lists B or C, even more preferably from list C.

[0445] The following oligonucleotide conjugates of the present invention are preferred: the saponin is any one or more of AG1856, GE1741, saponin isolated from Quillaja saponaria, Quil-A, QS-17, QS-21, QS-7, SA1641, saponin isolated from Saponaria officinalis, saponarioside B, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862, and SO1904; preferably, wherein the saponin is any one or more of QS-21, SO1832, SO1861, SA1641, and GE1741; more preferably, wherein the saponin is QS-21, SO1832, or SO1861; and most preferably SO1861.

[0446] Also preferred are the following oligonucleotide conjugates of the present invention: the saponin is a saponin isolated from Saponaria officinalis, preferably wherein the saponin is any one or more of saponariosides B, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862, and SO1904, more preferably wherein the saponin is SO15 42, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862, and SO1904, and even more preferably, wherein the saponin is any one or more of SO1832, SO1861, and SO1862, and even more preferably, wherein the saponin is SO1832 and SO1861, and most preferably SO1861.

[0447] An embodiment is an oligonucleotide conjugate of the invention, wherein the saponin is SO1861, and wherein the oligonucleotide conjugate comprises an SO1861 saponin derivative according to molecule (VII)a: [ka]

[0448] is provided by conjugation of the above with an oligonucleotide and at least one GalNAc moiety to provide an oligonucleotide conjugate.

[0449] One embodiment is an oligonucleotide complex of the invention, wherein the oligonucleotide contained in the complex is defined as a nucleic acid no longer than 150 nt, preferably wherein the oligonucleotide has a size of 5 to 150 nt, preferably 8 to 100 nt, and most preferably 10 to 50 nt.

[0450] One embodiment is an oligonucleotide conjugate of the invention, wherein the semicarbazone functional group undergoes in vivo hydrolysis under acidic conditions present in the endosomes and / or lysosomes of mammalian, preferably human, cells, preferably at a pH of 4.0 to 6.5, more preferably at a pH of 5.5 or less, resulting in the formation of an aldehyde group of the saponin or saponin moiety contained in the oligonucleotide conjugate.

[0451] One embodiment is an oligonucleotide conjugate of the invention, wherein the hydrazone functional group undergoes in vivo hydrolysis under acidic conditions present in the endosomes and / or lysosomes of mammalian, preferably human, cells, preferably at a pH of 4.0 to 6.5, more preferably at a pH of 5.5 or less, resulting in the formation of an aldehyde group of the saponin or saponin moiety contained in the oligonucleotide conjugate.

[0452] An embodiment is an oligonucleotide conjugate according to the present invention comprising an oligonucleotide and according to, for example, any one of molecules (EE), (PP), and (SS), wherein the oligonucleotide is any one of an AON, e.g., a BNA, a xenonucleic acid, or an siRNA.

[0453] An embodiment is an oligonucleotide complex according to the invention and according to, for example, any one of 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 shaped 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 oligonucleotides (BNA-AONs).

[0454] An embodiment is an oligonucleotide conjugate according to the invention and according to, for example, any one of molecules (EE), (PP), and (SS), comprising an oligonucleotide, wherein the oligonucleotide is selected from any one or more of the following: an anti-miRNA, a BNA-AON, or an siRNA, for example, a BNA-based siRNA, selected from a chemically modified siRNA, a metabolically stable siRNA, and a chemically modified metabolically stable siRNA.

[0455] An embodiment is an oligonucleotide conjugate according to the invention comprising an oligonucleotide and according to, for example, any one of molecules (EE), (PP), and (SS), wherein the oligonucleotide is capable of silencing, for example, any one of the genes apolipoprotein B (apoB), HSP27, transthyretin (TTR), proprotein convertase subtilisin / kexin type 9 (PCSK9), TMPRSS6, delta-aminolevulinic acid synthase 1 (ALAS1), antithrombin 3 (AT3), glycolate oxidase (GO), complement component C5 (CC5), hepatitis B virus (HBV) X gene, HBV S gene, alpha-1 antitrypsin (AAT), miR-122, hepatitis B virus HbsAg, LDHA, CEBPA, and lactate dehydrogenase (LDH) when present inside a mammalian cell, preferably a human cell, and / or is capable of targeting, for example, an aberrant miRNA when present inside a mammalian cell.

[0456] An embodiment is an oligonucleotide conjugate according to the invention comprising an oligonucleotide and according to, for example, any one of molecules (EE), (PP), and (SS), wherein the oligonucleotide is an oligonucleotide capable of silencing any one of the genes apolipoprotein B (apoB) and HSP27 when present, for example, inside a mammalian cell, preferably inside a human cell.

[0457] An embodiment is an oligonucleotide conjugate according to the invention comprising an oligonucleotide and according to, e.g., any one of molecules (EE), (PP), and (SS), wherein the oligonucleotide is capable of targeting, e.g., when present inside a mammalian cell, preferably inside a human cell, an mRNA involved in the expression of any one of proteins apoB, HSP27, TTR, PCSK9, TMPRSS6, ALAS1, AT3, GO, CC5, an expression product of the HBV X gene, an expression product of the HBV S gene, AAT, miR-122, Hepatitis B virus HBsAg, LDHA, CEBPA, and LDH, or is capable of antagonizing or restoring miRNA function, e.g., inhibiting the repression of oncogenic miRNA (onco-miR) or onco-miR expression, e.g., when present inside a mammalian cell, preferably inside a human cell.

[0458] An embodiment is an oligonucleotide conjugate according to the invention comprising an oligonucleotide and according to, for example, any one of molecules (EE), (PP), and (SS), wherein the oligonucleotide is an oligonucleotide capable of targeting, for example, when present inside a mammalian cell, preferably when present inside a human cell, mRNA involved in the expression of any one of the following proteins: HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AAT, miR-122, Hepatitis B virus HbsAg, LDHA, and CEBPA.

[0459] An embodiment is an oligonucleotide conjugate according to the invention comprising an oligonucleotide and according to, for example, any one of molecules (EE), (PP), and (SS), wherein the oligonucleotide is an oligonucleotide capable of targeting mRNA involved in the expression of any one of proteins apoB and HSP2 when present, for example, inside a mammalian cell, preferably inside a human cell.

[0460] An embodiment of the present invention relates to a second pharmaceutical composition, comprising an oligonucleotide conjugate of the present invention and according to any one of molecules (EE), (PP), and (SS), and optionally a pharmaceutically acceptable excipient and / or optionally a pharmaceutically acceptable diluent.

[0461] An embodiment of the present invention relates to a second pharmaceutical composition of the present invention or to an oligonucleotide conjugate of the present invention and according to any one of, for example, molecules (EE), (PP), and (SS), for use as a medicament.

[0462] An embodiment of the present invention relates to the second pharmaceutical composition of the present invention or to an oligonucleotide conjugate of the present invention and according to any one of molecules (EE), (PP), and (SS), for use in the treatment or prevention of a disease or health problem involving the expression product of any one or more of the genes HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AT3, GO, CC5, HBV X gene, HBV S gene, AAT, miR-122, Hepatitis B virus HBsAg, LDHA, CEBPA, and LDH; and / or for use in the treatment or prevention of a disease or health problem involving any one or more of the genes HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AT3, GO, CC5, HBV X gene, HBV S gene, AAT, miR-122, Hepatitis B virus HBsAg, LDHA, CEBPA, and LDH.

[0463] An embodiment of the present invention relates to the second pharmaceutical composition of the present invention or to an oligonucleotide conjugate of the present invention and according to any one of molecules (EE), (PP), and (SS), for use in the treatment or prevention of a disease or health problem involving the expression product of any one or more of the genes HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AAT, miR-122, Hepatitis B virus HbsAg, LDHA, and CEBPA, and / or for use in the treatment or prevention of a disease or health problem involving any one or more of the genes HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AAT, miR-122, Hepatitis B virus HbsAg, LDHA, and CEBPA.

[0464] An embodiment is a second pharmaceutical composition of the invention or an oligonucleotide conjugate according to the invention and for example any one of molecules (EE), (PP) and (SS), for the uses outlined herein above, wherein said use is for use in the treatment or prevention of a disease or health problem involving the expression product of any one or more of the genes HSP27 and apoB, preferably apoB, and / or in the treatment or prevention of a disease or health problem involving any one or more of the genes HSP27 and apoB, preferably apoB.

[0465] An embodiment is a second pharmaceutical composition of the invention or an oligonucleotide conjugate according to any one of the invention and e.g. molecules (EE), (PP), and (SS), for the uses outlined herein above, 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 amyloidosis, hereditary TTR amyloidosis (hATTR), complement-related disease, hepatitis B infection, hepatitis C infection, α1-antitrypsin deficiency, β-thalassemia, or an autoimmune disease.

[0466] An embodiment is a second pharmaceutical composition of the invention, or an oligonucleotide conjugate according to any one of the invention and e.g. molecules (EE), (PP), and (SS), for the uses outlined herein above, wherein said use is in the treatment or prevention of cancer, e.g. endometrial cancer, breast cancer, lung cancer, or hepatocellular carcinoma, and / or cardiovascular disease, e.g. hypercholesterolemia, preferably hypercholesterolemia.

[0467] An embodiment of the present invention relates to the second pharmaceutical composition of the present invention or to an oligonucleotide conjugate of the present invention and according to any one of the molecules (EE), (PP), and (SS), for use in lowering LDL cholesterol in a subject. Preferably, the subject is a human subject.

[0468] The saponin of the oligonucleotide conjugate of the present invention, for example, according to any one of the molecules (EE), (PP), and (SS), is preferably a saponin derived from a plant. The saponin contained in the oligonucleotide conjugate of the present invention is preferably a saponin isolated from a plant. For example, the saponin is isolated from the root of a plant. Examples of such plants from which saponin is derived (isolated) include Quillaja saponaria, Gypsophila paniculata L., Saponaria officinalis, for example, Saponaria officinalis L., and Gypsophila elegans, for example, Gypsophila elegans M. Beebe. Preferably, the oligonucleotide conjugate comprises a single type of saponin, preferably a single type of saponin derived from plant material such as a plant root, such as SO1861, SO1862, or SO1832 from Saponaria officinalis (e.g., Saponaria officinalis L., preferably Saponaria officinalis L.) (root), or QS-21, QS-7, or QS-17 from Quillaja saponaria (root). Suitable sources for isolating saponins according to the present invention, i.e., those exhibiting endosomal escape-enhancing activity, are Quillaja saponaria, Gypsophila paniculate L., Saponaria officinalis, and Gypsophila elegans, and Quillaja bark.

[0469] An aspect of the present invention relates to an in vitro or ex vivo method for transferring an oligonucleotide complex of any one of the above-outlined embodiments of the present invention from the outside of a cell to the inside of said cell, preferably followed by transferring the oligonucleotide contained in said oligonucleotide complex into the cytosol of said cell, comprising the steps of: a) providing cells expressing ASGPR on their surface, preferably selected from liver cells, virus-infected cells, and tumor cells, and providing an oligonucleotide complex of the invention for transfection into the provided cells; b) contacting the cells of step a) in vitro or ex vivo with the oligonucleotide complex of step a), thereby resulting in the transfer of the oligonucleotide complex from outside the cells into said cells, and preferably thereby resulting in the subsequent transfer of the oligonucleotides contained in the oligonucleotide complex into the cytosol of said cells.

[0470] While the present invention has been described in terms of several embodiments, it is contemplated that alternatives, modifications, permutations, and equivalents thereof will become apparent to those skilled in the art upon reading the specification and studying the drawings. The present invention is in no way limited to the illustrated embodiments. Changes may be made without departing from the scope defined by the appended claims.

[0471] Embodiments of the present invention 1. A saponin conjugate comprising at least one saponin covalently linked to a ligand of the asialoglycoprotein receptor (ASGPR), wherein the ligand of the ASGPR comprises at least one N-acetylgalactosamine (GalNAc) moiety, preferably three or four GalNAc moieties, and more preferably the ligand of the ASGPR comprises or consists of (GalNAc)3Tris, wherein the at least one saponin is selected from monodesmosidic triterpenoid saponins and bisdesmosidic triterpenoid saponins.

[0472] 2. The saponin complex of embodiment 1, wherein the saponin comprises an aglycone core structure selected from the group consisting of: 2 alpha-hydroxyoleanolic acid, 16 alpha-hydroxyoleanolic acid, Hederagenin (23-hydroxyoleanolic acid), 16alpha,23-dihydroxyoleanolic acid, gypsogenin, Quillaja acid, Protoessigenin-21(2-methylbut-2-enoate)-22-acetate, 23-oxo-valintgenol C-21,22-bis(2-methylbut-2-enoate), 23-oxo-valintgenol C-21(2-methylbut-2-enoate)-16,22-diacetate, digitogenin, 3,16,28-trihydroxyoleanan-12-ene, Dipsogenic acid, and their derivatives. Preferably, the saponin comprises an aglycone core structure selected from quillaic acid and gypsogenin or a derivative thereof, more preferably the aglycone core structure of the saponin is quillaic acid or a derivative thereof.

[0473] 3. The saponin complex of embodiment 1 or 2, wherein: Saponins comprise a sugar chain attached to an aglycone core structure selected from Group A below: 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-, and their derivatives, or Saponins comprise a sugar chain attached to an aglycone core structure selected from Group B below: Glc-, Gal-, Rha-(1→2)-[Xyl-(1→4)]-Rha-, Rha-(1→2)-[Ara-(1→3)-Xyl-(1→4)]-Rha-, Ara-, Xyl-, Xyl-(1→4)-Rha-(1→2)-[R1-(→4)]-Fuc-, where R1 is 4E-methoxycinnamic acid; Xyl-(1→4)-Rha-(1→2)-[R2-(→4)]-Fuc-, where R2 is 4Z-methoxycinnamic acid; Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-4-OAc-Fuc-, Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-3,4-di-OAc-Fuc-, Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R3-(→4)]-3-OAc-Fuc-, where 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→3)-(Ara- or Xyl-)(1→4)-(Rha- or Fuc-)(1→2)-[4-OAc-(Rha- or Fuc-)(1→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→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R4-(→4)]-Fuc-, where R4 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)-[R5-(→4)]-Fuc-, where 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→3)-Xyl-(1→4)-Rha-(1→2)-[3,4-di-OAc-Qui-(1→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→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R6-(→4)]-Fuc-, where R6 is 5-O-[5-O-Rha-(1→2)-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid; Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R7-(→4)]-Fuc-, where R7 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid; Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R8-(→4)]-Fuc-, where R8 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)-[R9-(→4)]-Fuc-, where R9 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)-[R10-(→4)]-Fuc-, where R10 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)-[R11-(→3)]-Fuc-, where 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-(→3)]-Fuc-, where R12 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid; Glc-(1→3)-[Glc-(1→6)]-Gal-, and their derivatives, or Saponins are bisdesmosidic triterpene glycosides that contain a first sugar chain selected from Group A attached to an aglycone core structure and a second sugar chain selected from Group B attached to the aglycone core structure.

[0474] 4. 4. The saponin complex of any one of embodiments 1-3, wherein the saponin is selected from the group consisting of: Quillaja bark saponin, dipsacoside B, saikosaponin A, saikosaponin D, macrantoidin A, esculentoside A, phytolaccagenin, essinate, AS6.2, NP-005236, AMA-1, AMR, alpha-hederin, NP-012672, NP-017777, NP-017778, NP-017774, NP-018110, NP-017772, NP-018109, NP-017888, NP-017889, NP-018108, SA1641, AEX55, NP-017674, NP-017810, AG1, NP-003881, NP-0176 76, NP-017677, NP-017706, NP-017705, NP-017773, NP-017775, SA1657, AG2, SO1861, GE1741, S O1542, SO1584, SO1658, SO1674, SO1832, SO1862, SO1904, QS-7, QS1861, QS-7api, QS1862, QS-1 7, 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, assamsaponin F, digitonin, primulic acid 1, and AS64R, their stereoisomers, derivatives, and combinations thereof. Preferably, the saponin is selected from the group consisting of QS-21, QS-21 derivatives, SO1861, SO1861 derivatives, SA1641, SA1641 derivatives, GE1741, GE1741 derivatives, and combinations thereof; more preferably, the saponin is selected from the group consisting of QS-21 derivatives, SO1861 derivatives, and combinations thereof; most preferably, the saponin is a SO1861 derivative.

[0475] 5. The saponin complex of embodiment 3 or 4, wherein the saponin is a saponin derivative, wherein: i. The saponin derivative contains an aglycone core structure that includes an aldehyde group that is derivatized; ii. The saponin derivative comprises a sugar chain, preferably a sugar chain selected from Group A as defined in embodiment 3, wherein the sugar chain comprises a carboxyl group that is derivatized; or iii. The saponin derivative comprises a sugar chain, preferably a sugar chain selected from Group B as defined in embodiment 3, wherein the sugar chain comprises a derivatized acetoxy (Me(CO)O-) group; or iv. Saponin derivatives include any combination of derivatizations i, ii, and iii, preferably any combination of two of derivatizations i, ii, and iii.

[0476] 6. The saponin complex of any one of embodiments 1-5, wherein the saponin is any one or more of the following: SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21A-api, QS-21A-xyl, QS-21B, QS-21B-api, QS-21B-xyl, QS-7-xyl, QS-7-api. , QS-17-api, QS-17-xyl, QS1861, QS1862, Quillajasaponin, Saponinum albumensis, QS-18, Quil-A, Gyp1, gypsoside A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, SO1904, their stereoisomers, their derivatives, and combinations thereof. Preferably, the saponin is selected from the group consisting of QS-21, QS-21 derivatives, SO1861, SO1861 derivatives, SA1641, SA1641 derivatives, GE1741, GE1741 derivatives, and combinations thereof. More preferably, the saponin is selected from the group consisting of QS-21 derivatives, SO1861 derivatives, and combinations thereof. Most preferably, the saponin is a SO1861 derivative.

[0477] 7. The saponin complex of any one of embodiments 1-6, wherein the saponin is a saponin derivative of quillaia acid saponin or gypsogenin saponin of embodiment 2, represented by molecule 1:

[0478] [ka]

[0479] During the ceremony, A1 represents hydrogen, a monosaccharide, or a linear or branched oligosaccharide, preferably A1 represents a saccharide chain selected from Group A as defined in embodiment 3, more preferably A1 represents a saccharide chain selected from Group A as defined in embodiment 3, and A1 comprises or consists of a glucuronic acid moiety; A2 represents hydrogen, a monosaccharide, or a linear or branched oligosaccharide, preferably A2 represents a sugar chain selected from Group B as defined in embodiment 3, more preferably A2 represents a sugar chain selected from Group B as defined in embodiment 3, and A2 comprises at least one acetoxy (Me(CO)O-) group, for example 1, 2, 3, or 4 acetoxy groups; wherein at least one of A1 and A2 is not hydrogen, and preferably both A1 and A2 are oligosaccharide chains; R is the hydrogen of gypsogenin or the hydroxyl of quillaic 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 Quillaia acid or gypsogenin 23 wherein the aldehyde group in ii. A1 represents a glycan selected from Group A as defined in embodiment 3, and when A1 comprises or consists of a glucuronic acid moiety, the carboxyl group of the glucuronic acid moiety of A1 is derivatized; and iii. When A2 represents a glycan selected from Group B as defined in embodiment 3, and A2 contains at least one acetoxy group, one or more, preferably all, of the acetoxy groups of one sugar moiety or of two or more sugar moieties of A2 are derivatized.

[0480] 8. The saponin complex of embodiment 7, wherein A1 represents a glycan selected from group A as defined in embodiment 3 and comprises or consists of a glucuronic acid moiety, wherein the carboxyl group of the glucuronic acid moiety of A1 is derivatized, and / or wherein A2 represents a glycan selected from group B as defined in embodiment 3 and A2 comprises at least one acetoxy group, wherein at least one acetoxy group of A2 is derivatized.

[0481] 9. The saponin complex of embodiment 7 or 8, wherein the saponin represented by molecule 1 is a bisdesmosidic triterpene saponin.

[0482] 10. The saponin conjugate of any one of embodiments 7-9, wherein the saponin derivative corresponds to the saponin represented by molecule 1, and wherein at least one, preferably one or two, more preferably one of the following derivatizations is present: i. Position C of Quillaia acid or gypsogenin 23 The aldehyde group in has been derivatized with: - reduction to alcohol, - conversion to a hydrazone bond by reaction with N-ε-maleimidocaproic hydrazide (EMCH), thereby providing saponin-Ald-EMCH, e.g., SO1861-Ald-EMCH or QS-21-Ald-EMCH, wherein the maleimide group of EMCH is optionally derivatized by formation of a thioether bond with mercaptoethanol; - conversion to a hydrazone bond by reaction with N-[β-maleimidopropionic acid] hydrazide (BMPH), where the maleimide group of BMPH is optionally derivatized by formation of a thioether bond with mercaptoethanol, or - conversion to a hydrazone bond by reaction with N-[κ-maleimidoundecanoic acid]hydrazide (KMUH), wherein the maleimide group of KMUH is optionally derivatized by formation of a thioether bond with mercaptoethanol; ii. A1 represents a sugar chain selected from Group A as defined in embodiment 3, and when A1 comprises or consists of a glucuronic acid moiety, the carboxyl group of the glucuronic acid moiety of A1 is derivatized 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 saponin-Glu-AMPD, e.g., QS-21-Glu-AMPD or SO1861-Glu-AMPD, or saponin-Glu-AEM, e.g., QS-21-Glu-AEM or SO1861-Glu-AEM; and iii. When A2 represents a glycan selected from Group B as defined in embodiment 3, and A2 contains at least one acetoxy group, one or more, preferably all, of the acetoxy groups of one or more sugar moieties of A2 are derivatized by conversion to a hydroxyl group (HO-) by deacetylation.

[0483] 11. The saponin conjugate of any one of embodiments 7 to 10, wherein A1 is Gal-(1→2)-[Xyl-(1→3)]-GlcA and / or A2 is Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc, and preferably the saponin represented by molecule 1 is 3-O-beta-D-galactopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)]-beta-D-glucuronopyranosyl. and more preferably the saponin is any one or more of SO1861, GE1741, SA1641, and QS-21, or derivatives thereof, most preferably SO1861 or a derivative thereof.

[0484] 12. The saponin complex of any one of embodiments 5-11, wherein the saponin is a saponin derivative, wherein: i. The saponin derivative contains an aglycone core structure containing an aldehyde group that is derivatized by: - reduction to alcohol, - conversion to a hydrazone bond by reaction with N-ε-maleimidocaproic hydrazide (EMCH), thereby providing saponin-Ald-EMCH, e.g., SO1861-Ald-EMCH or QS-21-Ald-EMCH, wherein the maleimide group of EMCH is optionally derivatized by formation of a thioether bond with mercaptoethanol; - conversion to a hydrazone bond by reaction with N-[β-maleimidopropionic acid] hydrazide (BMPH), where the maleimide group of BMPH is optionally derivatized by formation of a thioether bond with mercaptoethanol, or - conversion to a hydrazone bond by reaction with N-[κ-maleimidoundecanoic acid]hydrazide (KMUH), wherein the maleimide group of KMUH is optionally derivatized by formation of a thioether bond with mercaptoethanol; ii. the saponin derivative comprises a sugar chain, preferably a sugar chain selected from Group A as defined in embodiment 3, wherein the sugar chain comprises 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 2-amino-2-methyl-1,3-propanediol (AMPD) or N-(2-aminoethyl)maleimide (AEM), thereby providing saponin-Glu-AMPD, e.g., QS-21-Glu-AMPD or SO1861-Glu-AMPD, or saponin-Glu-AEM, e.g., QS-21-Glu-AEM or SO1861-Glu-AEM; or iii. The saponin derivative comprises a sugar chain, preferably a sugar chain selected from Group B as defined in embodiment 3, wherein the sugar chain comprises an acetoxy (Me(CO)O-) group that has been derivatized by conversion to a hydroxyl group (HO-) by deacetylation; or iv. Saponin derivatives include any combination of derivatizations i, ii, and iii, preferably any combination of two of derivatizations i, ii, and iii. Preferably, the saponin derivative comprises an aglycone core structure, wherein the aglycone core structure comprises an aldehyde group that has been derivatized by conversion to a hydrazone bond by reaction with EMCH, wherein the maleimide group of EMCH is optionally derivatized by formation of a thioether bond with mercaptoethanol.

[0485] 13. The saponin complex of embodiment 12, wherein the saponin is a saponin derivative, wherein: i. Saponin derivatives include an aglycone core structure containing an aldehyde group that has been derivatized by conversion to a hydrazone bond by reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), thereby providing saponin-Ald-EMCH, e.g., SO1861-Ald-EMCH or QS-21-Ald-EMCH; ii. the saponin derivative comprises a sugar chain, preferably a sugar chain selected from Group A as defined in embodiment 3, wherein the sugar chain comprises 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 saponin-Glu-AEM, e.g., QS-21-Glu-AEM or SO1861-Glu-AEM; or iii. Saponin derivatives include combinations of derivatizations i and ii.

[0486] 14. The saponin conjugate of embodiment 12, wherein the saponin derivative comprises an aglycone core structure, wherein the aglycone core structure comprises an aldehyde group, and wherein the saponin derivative comprises a saccharide chain, preferably selected from Group A defined in embodiment 3, wherein the saccharide chain comprises a carboxyl group, preferably the 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).

[0487] 15. The saponin complex of any one of embodiments 1-12, wherein the saponin is a saponin derivative represented by molecule 2:

[0488] [ka]

[0489] Or, wherein the saponin is a saponin derivative represented by molecule 3:

[0490] [ka]

[0491] 16. The saponin conjugate of any one of embodiments 1 to 15, wherein the at least one saponin and the ligand of the ASGPR are covalently linked directly or via at least one linker.

[0492] 17. The saponin conjugate of any one of embodiments 1-16, wherein the GalNAc moiety is linked to the saponin S, preferably via a saponin linker Ls, by a linker of formula (II): S are combined as shown in:

[0493] [ka]

[0494] 18. The saponin conjugate of any one of embodiments 1-16, wherein the GalNAc moieties are each separately covalently linked to a central bridging moiety B via an oxygen at position "1" of the GalNAc moiety, which completes the bridge between the GalNAc moieties and the saponin moiety, preferably via a saponin moiety linker L. S via the formula (III) S Effectively forming as shown in:

[0495] [ka]

[0496] wherein n is an integer greater than or equal to 2, preferably n is 3, and L S is the saponin moiety linker and S is the saponin moiety.

[0497] 19. The saponin conjugate of embodiment 18, wherein the GalNAc moiety is linked to the bridging moiety B by a GalNAc linker L. GAL via formula (IV) S are combined as shown in:

[0498] [ka]

[0499] wherein n is an integer greater than or equal to 2, preferably n is 3, and L S is the saponin moiety linker and S is the saponin moiety.

[0500] 20. The saponin conjugate of any one of embodiments 17-19, wherein the saponin moiety linker Ls connects the saponin to formula (II) S or to GalNAc of formula (III) S and (IV) S represents any chemical moiety suitable for covalently bonding to the bridging moiety B of

[0501] 21. The saponin conjugate of any one of embodiments 17-20, wherein the saponin moiety linker L S comprises at least a first precursor L covalently linked to a GalNAc or bridging moiety B S1 and a second precursor L covalently linked to the saponin moiety. S2 where L is the result of a coupling reaction between S1 is the saponin moiety linker L S is a precursor of L, which is covalently linked to GalNAc or to a bridging moiety B S2 is the saponin moiety linker L S It is a precursor of , which is covalently bound to a saponin moiety.

[0502] 22. The saponin conjugate of any one of embodiments 17-21, wherein the saponin moiety linker L S comprises at least a first precursor L covalently linked to a GalNAc or bridging moiety B S1 and a second precursor L covalently linked to the saponin moiety. S2 wherein the coupling reaction is selected from the group consisting of azide-alkyne cycloaddition, thiol-maleimide coupling, Staudinger reaction, nucleophilic ring-opening of a strained heterocyclic electrophile, non-aldol carbonyl reaction, and addition to a carbon-carbon double bond; preferably, wherein the coupling reaction is azide-alkyne cycloaddition, thiol-maleimide coupling, Staudinger reaction, nucleophilic ring-opening of a strained heterocyclic electrophile; more preferably, wherein the coupling reaction is azide-alkyne cycloaddition or thiol-maleimide coupling.

[0503] 23. The saponin conjugate of any one of embodiments 17-22, wherein the saponin moiety linker L S is a first precursor L covalently linked to a GalNAc or bridging moiety B S1 and a second precursor L covalently linked to the saponin moiety. S2 is the result of a coupling reaction between the first precursor L S1 contains an azide, and the second precursor LS2 comprises an alkyne, preferably a hydrazone resulting from hydrazide / aldehyde coupling to the aldehyde of the saponin moiety.

[0504] 24. The saponin complex of any one of embodiments 21-23, wherein precursor L S1 is the following azide of formula (XVII):

[0505] [ka]

[0506] 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, where precursor L S1 The structure of includes the following azide of formula (XVIII):

[0507] [ka]

[0508] In the formula, c represents an integer equal to or greater than 0, preferably c represents an integer in the range of 5 to 15, and more preferably c represents 9.

[0509] 25. The saponin complex of any one of embodiments 21-24, wherein precursor L S2 The structure includes the following hydrazone having the formula (XIX):

[0510] [ka]

[0511] In the formula, a represents an integer greater than or equal to 0, preferably a represents an integer in the range of 2 to 6, more preferably a represents 4, and wherein L S2a represents an alkyne-containing moiety.

[0512] 26. The saponin complex of embodiment 25, wherein L S2a contains less than 20 carbon atoms, preferably L S2a represents a moiety according to formula (XX):

[0513] [ka]

[0514] 27. The saponin conjugate of any one of embodiments 18-26, wherein the bridging moiety is a compound of formula (XV):

[0515] [ka]

[0516] wherein the oxygen atom of the compound of formula (XV) is linked to GalNAc or to a GalNAc linker L GAL and the nitrogen atom of the compound of formula (XV) is linked to a saponin moiety linker L S is combined with

[0517] 28. The saponin complex of any one of embodiments 19-27, wherein L GAL represents any chemical moiety suitable for covalently linking GalNAc to bridging moiety B.

[0518] 29. The saponin complex of any one of embodiments 19-28, wherein L GAL contains 2 to 25 carbon atoms, preferably 7 to 15 carbon atoms, more preferably 11 carbon atoms, wherein L GAL contains at least one, preferably two, amide moieties.

[0519] 30. The saponin complex of any one of embodiments 19-29, wherein L GAL is a compound according to formula (XVI): [ka]

[0520] 31. The saponin conjugate of any one of embodiments 17-30, wherein the ligand of the ASGPR is (GalNAc)3Tris, represented by the molecule (DD3) or the molecule (DD4):

[0521] [ka]

[0522] Alternatively, where the ligand of the ASGPR is monoGalNAc, represented by molecule II':

[0523] [ka]

[0524] 32. The saponin conjugate of any one of embodiments 1 to 31, wherein at least one saponin is covalently attached to molecule (DD3) or molecule (DD4) or molecule II' of embodiment 31 via a linker represented by molecule III':

[0525] [ka]

[0526] where the hydrazide moiety of molecule III' forms a covalent hydrazone bond with the aldehyde group on the saponin, and the dibenzocyclooctyne group forms a covalent bond with the azide group of molecule (DD3) or molecule (DD4) or molecule II'.

[0527] 33. The saponin conjugate of any one of embodiments 1-32, wherein at least one saponin is covalently attached to the ligand of the ASGPR via at least one cleavable linker.

[0528] 34. The saponin conjugate of embodiment 33, wherein the cleavable linker undergoes cleavage under acidic, reductive, enzymatic, and / or light-induced conditions, preferably the cleavable linker comprises a cleavable bond selected from: hydrazone and hydrazide bonds that undergo cleavage under acidic conditions, and / or bonds that are susceptible to proteolysis, for example proteolysis by cathepsin B, and / or bonds that are susceptible to cleavage under reductive conditions, such as disulfide bonds.

[0529] 35. The saponin conjugate of embodiment 33 or 34, wherein the cleavable linker undergoes cleavage in vivo under acidic conditions, e.g., present in the endosomes and / or lysosomes of mammalian cells, preferably human cells; preferably, the cleavable linker undergoes cleavage in vivo at a pH of 4.0 to 6.5, more preferably at a pH of ≦5.5.

[0530] 36. The saponin complex of any one of embodiments 1-35, wherein the complex comprises 1, 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, 128, or 1-100 saponin moieties, or any number therebetween, e.g., 7, 9, 12 saponin moieties.

[0531] 37. Pharmaceutical combinations including: - a first pharmaceutical composition comprising the saponin complex of any one of embodiments 1 to 36, and optionally a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent; and - a second pharmaceutical composition comprising a second complex of an effector molecule and a ligand of ASGPR (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 or consists of (GalNAc)3Tris) or a third complex of an effector molecule and a binding molecule comprising a binding site for a cell surface molecule, and optionally comprising a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent.

[0532] 38. A pharmaceutical composition comprising: - the saponin complex of any one of embodiments 1 to 36; - a second complex of an effector molecule and a ligand of ASGPR (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 or consists of (GalNAc)3Tris) or a third complex of an effector molecule and a binding molecule comprising a binding site for a cell surface molecule, and optionally a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent.

[0533] 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 the following: a small molecule, e.g., a drug molecule, a toxin, e.g., a protein toxin, an oligonucleotide, e.g., an AON, e.g., a BNA, a xenonucleic acid, or an siRNA, an enzyme, a peptide, a protein, or any combination thereof. Preferably, the effector molecule is a toxin, an enzyme, or an oligonucleotide.

[0534] 40. The pharmaceutical combination of embodiment 37 or 39 or the pharmaceutical composition of embodiment 38 or 39, wherein the ligand of ASGPR comprises or is (GalNAc)3Tris, and / or wherein the ligand of ASGPR and the effector molecule are conjugated via a covalent bond, preferably via at least one linker.

[0535] 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 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 oligonucleotides (BNA-AONs).

[0536] 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 the following: an anti-miRNA, a BNA-AON, or an ...

Claims

1. An oligonucleotide conjugate comprising at least one saponin covalently linked to a ligand of the asialoglycoprotein receptor (ASGPR) and further covalently linked to an oligonucleotide, said ligand of ASGPR being in accordance with the following molecule (DD1): (GalNAc): ３ comprising or consisting of a Tris moiety, 【Chemistry 1】 wherein y1, y2, and y3 are each independently an integer selected from 0 to 20, 1 to 15, 2 to 12, 2 to 10, 2 to 8, 2 and 3, y1, y2, and y3 are the same, and y4 is an integer selected from 1 to 100, 2 to 80, 3 to 70, 4 to 60, 4 to 50, 4 to 40, 4 to 30, 4 to 20, 4 to 6, 4 to 5, and 4; wherein the at least one saponin is a monodesmosidic or bisdesmosidic pentacyclic triterpene saponin of the 12,13-dehydrooleanane type, or has an aldehyde functional group at position C23 of the aglycone core structure of the saponin; the oligonucleotide conjugate comprises 1-16 saponin moieties, or 1-8 saponin moieties, or 1 saponin moiety, 4 saponin moieties, or 8 saponin moieties; Oligonucleotide conjugates.

2. The oligonucleotide complex of claim 1, wherein the (GalNAc) 3 Tris portion conforms to the following molecule: (DD3): 【Chemistry 2】 3. The oligonucleotide complex of claim 1, wherein the (GalNAc) 3 Tris moiety, the at least one saponin, or 1 to 16 saponin moieties, or 1 to 8 saponin moieties, e.g., 1, 4, or 8 saponin moieties, and the oligonucleotide are covalently linked via a trifunctional linker, and each of the (GalNAc) 3 Tris moiety, the saponin or saponin moieties, and the oligonucleotide is covalently linked to a separate arm of the trifunctional linker.

4. One saponin moiety or four saponin moieties or dendrons or G2 dendrons, such as, for example, N,N'-((9S,19S)-14-(6-aminohexanoyl)-1-mercapto-9-(3-mercaptopropanamido)-3,10,18-trioxo-4,11,14,17-tetraazatricosane-19,23-diyl)bis(3-mercaptopropanamide), or eight saponin moieties or dendrons or G3 dendrons, such as, for example, (2S 4. The oligonucleotide conjugate of claim 1, comprising eight saponin moieties covalently attached to the oligonucleotide of claim 1, the oligonucleotide of claim 2, and the oligonucleotide of claim 3, the oligonucleotide of claim 2, the oligonucleotide of claim 3 being substituted with 5-[(2S)-2,6-bis(3-sulfanylpropanamido)hexanamide] hexanamide, and the oligonucleotide of claim 2 being substituted with 5-[(2S)-2,6-bis(3-sulfanylpropanamido)hexanamide] pentyl]-2,6-bis(3-sulfanylpropanamido)hexanamide.

5. The saponin contained in the complex is any one or more of the following: a) a saponin selected from any one or more of List A below: Quillaja saponaria saponin mixtures or saponins isolated from Quillaja saponaria, such as Quil-A, QS-17-api, QS-17-xyl, QS-21, QS-21A, QS-21B, QS-7-xyl, - Gypsophila elegans saponin mixture or saponins isolated from Gypsophila elegans, - Saponinum album saponin mixture or saponin isolated from Saponinum album, - Saponaria officinalis saponin mixture or saponin isolated from Saponaria officinalis, and Quillaja bark saponin mixtures or saponins isolated from Quillaja bark, such as Quil-A, QS-17-api, QS-17-xyl, QS-21, QS-21A, QS-21B, QS-7-xyl, or b) A saponin containing a gypsogenin aglycone core structure selected from List B below: SA1641, gypsoside A, NP-017772, NP-017774, NP-017777, NP-017778, NP-018109, NP-017888, NP-017889, NP-018108, SO1658, and phytolaccagenin, or c) Saponins containing a quillaric acid aglycone core structure selected from List C below: AG1856, AG1, AG2, agrostemoside E, GE1741, gypsophila saponin 1 (Gyp1), NP-017674, NP-017810, NP-003881, NP-017676, NP-017677, NP-017705, NP-017706, NP-017773, NP-017775, SA1657, saponariosi B, SO1542, SO1584, SO1674, SO1700, SO1730, SO1772, 10SO1832, SO1861, SO1862, SO1904, QS-7, QS-7api, QS-17, QS-18, QS-21A-apio, QS-21A-xylo, QS-21B-apio, and QS-21B-xylo, or d) Saponins comprising a 12,13-dehydrooleanane type aglycone core structure without an aldehyde group at the C23 position of the aglycone selected from List D below: Escin Ia, escinate, alpha-hederin, AMA-1, AMR, AS6.2, AS64R, assamsaponin F, dipsacoside B, esculentoside A, macranthoidin A, NP-005236, NP-012672, primulic acid 1, saikosaponin A, saikosaponin D, tea seed saponin I, and tea seed saponin J, Alternatively, the saponin is one or more of a saponin selected from list A, B or C, or a saponin selected from list B or C, or a saponin selected from list C. The oligonucleotide complex according to any one of claims 1 to 4.

6. The saponins include AG1856, GE1741, saponins isolated from Quillaja saponaria, Quil-A, QS-17, QS-21, QS-7, SA1641, saponins isolated from Saponaria officinalis, saponarioside B, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1 or wherein the saponin is one or more of QS-21, SO1832, SO1861, SA1641, and GE1741; or wherein the saponin is QS-21, SO1832, or SO1861; or wherein the saponin is SO1861; The oligonucleotide complex according to any one of claims 1 to 5.

7. The saponin is a saponin isolated from Saponaria officinalis, or the saponin is any one or more of saponariosides B, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862, and SO1904, or the saponin is any one or more of SO1542, SO1584, SO 1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862, and SO1904; or the saponin is one or more of SO1832, SO1861, and SO1862; or the saponin is SO1832 and SO1861; or the saponin is SO1861; The oligonucleotide complex according to any one of claims 1 to 6.

8. The saponin is SO1861 and the oligonucleotide conjugate is a SO1861 saponin derivative according to molecule (VII)a 【Chemistry 3】 with an oligonucleotide and said (GalNAc) 3 Tris moiety to provide said oligonucleotide conjugate. The oligonucleotide complex according to any one of claims 1 to 7.

9. The oligonucleotide complex according to any one of claims 1 to 8, wherein the oligonucleotides comprised in the complex are defined as nucleic acids not longer than 150 nt, or the oligonucleotides have a size between 5 and 150 nt, or between 8 and 100 nt, or between 10 and 50 nt.

10. A second pharmaceutical composition comprising the oligonucleotide conjugate according to any one of claims 1 to 9 and / or a pharma- ceutically acceptable excipient and / or a pharma- ceutically acceptable diluent.

11. A second pharmaceutical composition according to claim 10 or an oligonucleotide conjugate according to any one of claims 1 to 10 for use as a medicament.

12. and / or for use in the treatment or prevention of a disease or health problem involving any one or more expression products of the genes HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AT3, GO, CC5, HBV X gene, HBV S gene, AAT, miR-122, Hepatitis B virus HbsAg, LDHA, CEBPA, and LDH. The second pharmaceutical composition of claim 10 or the oligonucleotide conjugate of any one of claims 1 to 9 for use in the treatment or prevention of a disease or health problem involving any one or more of PCSK9, TMPRSS6, ALAS1, AT3, GO, CC5, HBV X gene, HBV S gene, AAT, miR-122, Hepatitis B virus HbsAg, LDHA, CEBPA, and LDH.

13. The second pharmaceutical composition according to claim 10 or the oligonucleotide conjugate according to any one of claims 1 to 9 for use in the treatment or prevention of a disease or health problem involving any one or more expression products of the genes HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AAT, miR-122, Hepatitis B virus HbsAg, LDHA and CEBPA and / or for use in the treatment or prevention of a disease or health problem involving any one or more of the genes HSP27, apoB, TTR, PCSK9, TMPRSS6, ALAS1, AAT, miR-122, Hepatitis B virus HbsAg, LDHA and CEBPA.

14. The second pharmaceutical composition according to claim 10 or the oligonucleotide conjugate according to any one of claims 1 to 9 for use according to claim 12 or 13, wherein said use is in the treatment or prevention of a disease or health problem in which one or more of the genes HSP27 and apoB, or the expression product of apoB, is involved, and / or for use in the treatment or prevention of a disease or health problem in which one or more of the genes HSP27 and apoB, or the expression product of apoB is involved.

15. 15. The second pharmaceutical composition according to claim 10 or the oligonucleotide conjugate according to any one of claims 1 to 9 for use according to any one of claims 12 to 14, wherein the use is in the treatment or prevention of cancer, an infectious disease, a viral infection, hypercholesterolemia, a cardiovascular disease, primary hyperoxaluria, hemophilia A, hemophilia B, AAT-related liver disease, acute hepatic porphyria, TTR amyloidosis, hereditary TTR amyloidosis (hATTR), complement-related diseases, hepatitis B infection, hepatitis C infection, alpha 1-antitrypsin deficiency, beta-thalassemia, or an autoimmune disease.

16. The second pharmaceutical composition according to claim 10 or the oligonucleotide complex according to any one of claims 1 to 9 for use according to any one of claims 12 to 15, wherein said use is in the treatment or prevention of cancer, such as endometrial cancer, breast cancer, lung cancer or hepatocellular carcinoma, and / or cardiovascular diseases, such as hypercholesterolemia.

17. The second pharmaceutical composition of claim 10 or the oligonucleotide conjugate of any one of claims 1 to 9 for use in lowering LDL cholesterol in a subject.

18. 10. An in vitro or ex vivo method for transferring an oligonucleotide complex according to any one of claims 1 to 9 from the outside of a cell to the inside of said cell, or subsequently transferring said oligonucleotide contained in said oligonucleotide complex into the cytosol of said cell, comprising: a) providing a cell expressing ASGPR on its surface, said cell being selected from a liver cell, a virus-infected cell, and a tumor cell, and providing an oligonucleotide complex according to any one of claims 1 to 9 for transfection into said provided cell; b) contacting the cells of step a) in vitro or ex vivo with the oligonucleotide complex of step a), thereby resulting in the transfer of the oligonucleotide complex from outside the cell into the cell, thereby subsequently resulting in the transfer of the oligonucleotides contained in the oligonucleotide complex into the cytosol of the cell; The method includes:

19. The oligonucleotide complex according to any one of claims 1 to 9, wherein the saponin contained in the oligonucleotide complex is isolated from a plant.

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