Treatment for cardiovascular disease

Abstract

The present invention relates to the field of therapy. More in particular, the invention relates to therapeutic methods of treating and / or preventing and / or ameliorating cardiovascular disease (CVD). The therapeutic methods involve a therapeutic combination comprising an oligonucleotide-based medicament and a saponin component. The invention also relates to the therapeutic combination as such. More in particular, the invention relates to the therapeutic combination for use in the treatment and / or prevention and / or amelioration of CVD, wherein the oligonucleotide-based medicament is adapted to target and bind to a nucleic acid molecule present in a human cell. The therapeutic methods particularly involve treating or preventing or ameliorating the CVDs by modulating the expression of a gene implicated in the CVD, typically by targeting of (pre-)mRNA by the oligonucleotide-based medicament in the therapeutic combination. The invention also relates to a pharmaceutical formulation, and a kit of parts comprising the therapeutic combination comprising an oligonucleotide-based medicament and a saponin component is also part of the current invention. The invention also relates to the therapeutic combination comprising the oligonucleotide-based medicament and the saponin component.

Description

[0001] TREATMENT FOR CARDIOVASCULAR DISEASE

[0002] TECHNICAL FIELD

[0003] The present invention relates to the field of therapy. More in particular, the invention relates to therapeutic methods of treating and / or preventing and / or ameliorating cardiovascular disease (CVD). The therapeutic methods involve a therapeutic combination comprising an oligonucleotide-based medicament and a saponin component. The invention also relates to the therapeutic combination as such. More in particular, the invention relates to the therapeutic combination for use in the treatment and / or prevention and / or amelioration of CVD, wherein the oligonucleotide-based medicament is adapted to target and bind to a nucleic acid molecule present in a human cell. The therapeutic methods particularly involve treating or preventing or ameliorating the CVDs by modulating the expression of a gene implicated in the CVD, typically by targeting of (pre-)mRNA by the oligonucleotide-based medicament in the therapeutic combination. The invention also relates to a pharmaceutical formulation, and a kit of parts comprising the therapeutic combination comprising an oligonucleotide-based medicament and a saponin component is also part of the current invention. The invention also relates to the therapeutic combination comprising the oligonucleotide-based medicament and the saponin component.

[0004] BACKGROUND OF THE INVENTION

[0005] CVDs remain a leading cause of morbidity and mortality worldwide, encompassing a broad spectrum of conditions. The complexity of CVDs arises from its multifactorial etiology, which involves an interplay of genetic predisposition, environmental influences, lifestyle factors, and metabolic conditions. CVD is not confined to a single organ system but often involves multiple organs and pathways.

[0006] For example, a range of conditions that affect the kidney, share common risk factors, such as hypertension, diabetes (in particular diabetes mellitus type 2 (T2DM)), and (chronic) inflammation. The bidirectional relationship between these diseases is well established; cardiovascular conditions often contribute to kidney dysfunction, while impaired kidney function exacerbates cardiovascular risks. Chronic kidney disease (CKD) is recognized as a significant independent risk factor for cardiovascular events, including myocardial infarction, heart failure, and arrhythmia. Conversely, cardiovascular disorders can accelerate the progression of CKD through mechanisms such as hemodynamic stress and reduced renal perfusion. This intertwined pathology underscores the urgent need for therapeutic strategies that address the interconnected pathways of these conditions.

[0007] A variety of risk factors exists for the development of CVDs, wherein elevated levels of plasma low-density lipoprotein cholesterol (LDL-C), triglycerides (TG) and / or fibrinogen is amongst the well-recognized risk factors for CVDs. LDL-C, commonly referred to as “bad cholesterol,” plays a direct role in the pathogenesis of atherosclerosis. High levels of LDL-C contribute to the build-up of lipid-laden plaques within the arterial walls. Over time, these plaques harden, leading to reduced arterial elasticity and an increased likelihood of arterial blockages, which can precipitate events such as myocardial infarction or stroke. Similarly, the elevated TG levels are increasingly recognized as an independent risk factor for CVDs, especially in patients with concurrent LDL-C elevation. TG is the primary form of fat in the body, and serve as an energy source. However, the elevated lipid levels are frequently associated with various forms of lipid metabolism disorders, including hyperlipidemia, dyslipidemia, hypertriglyceridemia, and hypercholesterolemia. When triglyceride levels are elevated, these can exacerbate the deposition of cholesterol in arterial walls leading to hypertension, especially in patients who also have elevated LDL-C, compounding cardiovascular risk.

[0008] Several genetic disorders exacerbate these risks. Familial hypercholesterolemia (FH), or Type II hyperlipoproteinemia, is a common inherited disorder that results in extremely high LDL-C from birth due to impaired LDL clearance, which can lead to early-onset atherosclerosis and heart disease if untreated. Familial hypertriglyceridemia (an inherited form of hypertriglyceridemia) and familial chylomicronemia (Type I hyperlipoproteinemia) similarly involve excessive lipid levels in the bloodstream due to genetic mutations affecting triglyceride metabolism.

[0009] The interplay between these lipid disorders underscores the importance of targeting both LDL-C and TG levels in therapeutic approaches to treat CVDs. Addressing conditions like hyperlipidemia, hypertriglyceridemia, and hypercholesterolemia, particularly managing patients with genetic disorders such as familial chylomicronemia and familial hypercholesterolemia, may offer substantial preventative benefits for patients with CVD or at risk of CVD. Traditional therapies for lowering plasma cholesterol including LDL-C and subsequently treating CVDs include: statins, cholesterol absorption inhibitors, cholesteryl ester transfer protein (CETP) inhibitor, peroxisome proliferator activator receptor (PPAR) a / y modulators, microsomal triglyceride transfer protein and / or apolipoprotein B (MTP / Apo B) secretion inhibitors, squalene synthetase or squalene epoxidase or squalene cyclase or combined squalene epoxidase / squalene cyclase inhibitors, enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitors, gastric and / or pancreatic lipase inhibitors, bile acid sequestrants, anti-diabetic compounds such as GLP-1 analogues, GLP-1 / GIP dual agonists, GLP-1 / GIP / GCG triple agonists.

[0010] CVDs may co-occur with CVDs such as diabetes and dyslipidemia, systemic inflammatory states, renal dysfunction, and neurological complications. Lifestyle factors such as poor diet, physical inactivity, smoking, and chronic stress exacerbate the risk and progression of CVD. This multifaceted nature makes the diagnosis, prevention, and treatment of CVD challenging and highly individualized. For the same reasons, the treatment of CVD is inherently complex due to the variability in its manifestation among patients. Factors such as age, sex, genetic make-up, coexisting medical conditions, and individual lifestyle choices influence the course of the disease and its response to treatment. Consequently, therapeutic strategies that work effectively for one patient may be less efficacious or even contraindicated for another. This variability necessitates a personalized approach to therapy, often requiring iterative adjustments based on patient response. Current treatment paradigms for CVD typically involve a combination of pharmacological agents, including antihypertensives, anticoagulants, lipid-lowering drugs, and other targeted therapies. However, these treatments are not always sufficient to address the systemic and multifactorial nature of CVD. Moreover, persistent adherence to prescribed therapies and prolonged continuation of healthimproving lifestyle modifications remain a significant barrier, as many patients find it challenging to maintain long-term changes in diet, exercise, and other habits critical for cardiovascular health.

[0011] Given these challenges, there is an urgent need for innovative therapeutic strategies for the treatment of CVDs that considers their multifactorial and systemic nature while accommodating patient-to-patient variability.

[0012] Recently identified targets in the fight against CVDs include targeting various genes involved in the regulation of lipid metabolism, plasma lipid re-uptake, clearance of triglyceride-rich lipoproteins, lipoprotein lipase (LPL) enzyme activity-modulator and / or by inducing cardio-protective effect. The concept of interfering with proteins on a nucleic acid level to prevent or change the course of a disease has been studied since long. Over the classical gene therapies (TALEN and CRISPR-based technologies), more recently oligonucleotide-based therapies have also emerged to prominence for targeting genes. The existing oligonucleotide-based therapies related to the treatment of elevated levels of LDL-C, TG and / or fibrinogen includes: mipomersen and inclisiran.

[0013] Despite having huge potential, oligonucleotide therapeutics are known to suffer from an extremely inefficient cellular uptake, which prevents them from effectively reaching the cytoplasmic and / or the nuclear intracellular compartments where these therapeutics are supposed to act upon their targets, and leads to insufficient therapeutic efficacy. It is further known that less than 2% from a therapeutic dose of an oligonucleotide drug becomes correctly internalised, possibly due to an estimated 98% thereof being retained within the endosomal compartment and eventually degraded in the lysosomes (Gilleron et al., 2013). However, for the renal cells, this leads to oligonucleotide drug accumulation raising safety concerns (Echevarria & Goyenvalle, 2022; Crooke etal., 2021). Therefore, increasing the oligonucleotide drug dose further exacerbates the accumulation related toxicity. Hence, a frequent administration is most often adapted for the delivery of a therapeutically effective dose of the oligonucleotide drug. Apparently, which further leads to injection-site reactions such as pruritus, redness, or swelling.

[0014] Consequently, there exist a need for increasing of the efficacy, bioavailability, and long termeffects of oligonucleotide-based medicaments, to minimise the risks of nephrotoxic effects with improved dosing regimen, especially for the provision of new therapeutic modalities for treating / preventing / ameliorating CVDs.

[0015] SUMMARY OF THE INVENTION It is an objective of the present invention to provide an improved oligonucleotide-based treatment, in particular for reducing or avoiding the risk for adverse events such as an immune response and / or inflammatory response, and prevent or limit off-target activity of the oligonucleotide-based medicament, extending the duration of effect of an oligonucleotide therapeutic and / or extending the dosing interval of an oligonucleotide therapeutic and / or reducing of the dose and / or the dosing frequency of an oligonucleotide therapeutic. It is further an objective of the present disclosure to provide an efficacious and safe treatment of a cardiovascular disease. It is another objective to provide a solution to the problem of inefficacious delivery of oligonucleotide-based medicaments into the target cells and which solution also addresses the problems of off-target activity and / or undesired adverse effects and potentially hepatotoxic and / or nephrotoxic effects.

[0016] Based on this finding, provided herein are a therapeutic combination comprising an oligonucleotide-based medicament and saponin component, a pharmaceutical formulation thereof and pharmaceutical kits thereof and methods involving their administration, for use in the treatment and / or prevention and / or amelioration of a cardiovascular disease (CVD). The oligonucleotide-based medicament(s) as contained herein is / are adapted to target and bind to a nucleic acid molecule present in kidney cells and / or present in liver cells (and thereby to modulate gene expression in the kidney cells and / or liver cells), and a saponin component comprising a penta-cyclic triterpene saponin comprising an aglycone core of 12,13-dehydrooleanane type. The treatment and / or prevention and / or amelioration comprises administration of the therapeutic combination or the pharmaceutical formulation thereof or the pharmaceutical kits to a human subject in the need thereof, wherein the saponin component and the oligonucleotide-based medicament are delivered to the kidney cells and / or liver cells and wherein the human disease is optionally or preferably a disease affecting other organs in addition to affecting the kidney and / or to affecting the liver, advantageously wherein the human disease is a cardiovascular disease.

[0017] The presented herein therapeutic solutions recite on a series of unexpected findings in combination made during multiple in vitro and in vivo delivery experiments with low dosages of oligonucleotide-based medicament such as antisense oligonucleotide (ASO) effector and small or short interfering RNA (siRNA) together with endosomal escape enhancing (EEE) saponins.

[0018] First, in in vivo targeted hepatic delivery experiments, it was observed that in the presence of the EEE saponins, intravenously administered liver-targeted therapeutic oligonucleotides (also referred to as oligonucleotide-based medicaments) were not only effectively silencing the expression of the target nucleic acid in the liver cells, but also in the kidney cells, furthermore without showing any visible hallmarks of nephrotoxicity. This serendipitous and surprising observation indicated that the oligonucleotide therapeutic that was provided at a very low dose, was not only taken up by the kidney cells, but also that in the presence of the EEE saponin, it was also effectively released after its uptake into the kidney cell cytoplasm where it could act on its target mRNA instead of remaining unproductively trapped in the renal subcellular compartments. As it is demonstrated in the in vivo experiments performed in mice and in a non-human primate (NHP), as shown below, the same was observed for therapeutic combinations of non-targeted-oligonucleotide-saponin conjugates and in a co-delivery setting involving a targeted-oligonucleotide-based medicament and a separate targeted EEE saponin component. Additional in vitro tests based on cell-based bioassays further confirmed the striking finding that efficacious therapeutic effects of an oligonucleotide-based medicament can be established not only in the liver but in addition (or uniquely) in the cells of the kidney under influence of the administration of a saponin component together with the oligonucleotide-based medicament and / or after said administration.

[0019] Based on these findings, provided herein are therapeutic combinations, compositions, formulations, and methods involving their administration, for use in the treatment and / or prevention and / or amelioration of a (human) disease, which are comprising an oligonucleotide-based medicament adapted to target and bind to a nucleic acid molecule present in liver cells and / or present in kidney cells (and thereby to modulate gene expression in the liver cells and / or in the kidney cells), and a saponin component comprising a penta-cyclic triterpene saponin comprising an aglycone core of 12,13-dehydrooleanane type; wherein the treatment and / or prevention and / or amelioration comprises administration of the saponin component and the oligonucleotide-based medicament to a subject, preferably a human subject, in the need thereof, wherein the saponin component and the oligonucleotide-based medicament are delivered to the liver cells and / or to the kidney cells and wherein the human disease is preferably a disease affecting the kidney and / or affecting the liver and possibly affecting other organs in addition to affecting the kidney and / or the liver, advantageously wherein the human disease is a kidney disease and / or a liver disease.

[0020] Second, the present invention resides in the finding that an oligonucleotide-based medicament, (as well as other therapeutic effector molecules, also jointly with the oligonucleotide-based medicament referred to as ‘effector component’) that require cellular uptake to become effective, persist for very long periods of time in cellular endosomes and that such effector components can be released from these endosomes by the action of a saponin component, long after the oligonucleotide-based medicament was administered, in amounts sufficient to cause a therapeutically meaningful boost in the effect of said oligonucleotide-based medicament (without actually administering a further dose of it). The durability of this response has proven to be remarkably high. More in particular, as shown in the experimental section of this document (‘Examples1), this principle has for example been demonstrated in an in vivo experiment (refer, e.g., to Figure 2), by treating mice with tri-antennary A / -acetylgalactosamine (GN3)-siTTR (an siRNA with advanced enhanced stabilization chemistry (ESC) design) followed by the administration of GN3-SO1861 (a trimeric GalNAc - saponin conjugate, also referred to as a trivalent GalNAc - saponin conjugate) at different points in time. It has been shown in these experiments that GN3-siTTR depots are accessible with GN3-saponin to release the siRNA, with a maximum response (suppression of transthyretin (TTR) protein expression) higher than achieved with GN3-siTTR treatment alone, even when the interval between GN3-siTTR and GN3-saponin administration was as long as 28 days. The experiment showed that GN3-saponin administration did not cause endosomal disruption or tolerability issues. Multiple in vitro experiments have shown in addition that comparable effects can be attained with other type of oligonucleotide-based medicaments as well as other (non-oligonucleotide) effector components. Taken together, these data show that, surprisingly, saponins can increase the potency of a preloaded oligonucleotide-based medicament (also referred to as an ‘effector component’) in a timed and inducible manner ( / .e. a controllable manner, for increasing potency of the oligonucleotide-based medicament at a selected time and / or to a desired extent), after a (controlled and selected) resting period. The effects observed thus support the use of a saponin component to attain a meaningful extension of the duration of effect and / or a meaningful extension of the dosing interval and / or a meaningful reduction of the dosing frequency of an oligonucleotide-based medicament and / or a meaningful (delayed) boost in the effect of the oligonucleotide-based medicament, such as, in particular, an ASO comprising therapeutic or an siRNA comprising therapeutic. The experiments underlying the present invention also included oligonucleotide-based medicament with advanced ESC designs. Even with these oligonucleotides, which already possess significantly increased metabolic stability, substantial further extension of the duration of effect has been demonstrated. From the literature, it is known that pharmacokinetic and / or pharmacodynamic effects of oligonucleotide-based medicament observed in animals / animal models, can reliably be extrapolated / translated to other species, including humans. McDougal et al. (The Nonclinical Disposition and Pharmacokinetic / Pharmacodynamic Properties of N-Acetylgalactosamine-Conjugated Small Interfering RNA Are Highly Predictable and Build Confidence in Translation to Human; Drug Metab Dispos 50:781-797, June 2022), for instance report the results of studies demonstrating that the pharmacokinetics / pharmacodynamics (PK / PD) and absorption, distribution, metabolism, and excretion (ADME) properties of GalNAc-conjugated siRNAs are highly conserved across species. Based on their results McDougal et al. state that results obtained in animals can accurately be scaled to human, allowing to identify efficacious and safe clinical dosing regimens in the absence of human liver PK profiles.

[0021] According to the inventor’s best knowledge, the literature concerning the use of saponins for endosomal escape enhancement has never alluded to the administration regimens and / or effects that are the subject of the present invention. At present, most of the literature concerning saponins as endosomal escape enhancing moieties teaches or hints at the concurrent use of the saponin and the effector component. These prior art teachings do not in any way disclose or hint at the general concept underlying the present invention, according to which the saponin component is used / administered with the purpose of extending the duration of effect, extending the dosing interval, reducing the dosing frequency and / or creating a (delayed) boost in the effect of the oligonucleotide-based medicament. More in particular, in these examples the effector component and the saponin component are invariably administered together in the in vitro and in vivo models for assessing the stimulatory effect of the saponin on the activity and efficacy of the effector molecule.

[0022] To the extent that sequential treatment has been disclosed in the art, it involved priming with a saponin, followed by the administration of the effector component. For instance, Mitdank et al. (Suicide nanoplasmids coding for ribosome-inactivating proteins; European Journal of Pharmaceutical Sciences 170 (2022) 106107), describe an in vivo study (in mice) wherein AG1856 was administered (subcutaneously) 1 hour prior to the (intravenous) administration of ‘suicide nanoplexes’. Bachran et al. (The distribution of saponins in vivo affects their synergy with chimeric toxins against tumours expressing human epidermal growth factor receptors in mice; British Journal of Pharmacology (2010) 159 345–352), describe the results of an in vivo study (in tumour-bearing mice) relying on the sequential administration of Saponinum album followed by the administration of a chimeric toxin against the epidermal growth factor receptor, ErbB1. Bachran et al. report that there was high antitumour efficacy (66% inhibition of tumour growth) when the toxin was administered after 60 minutes, following pretreatment with the saponin, but no significant inhibition when it was administered already after 10 minutes following pre-treatment. Panjideh et al. (Improved Therapy of B-Cell Non-Hodgkin Lymphoma by Obinutuzumab-Dianthin Conjugates in Combination with the Endosomal Escape Enhancer SO1861; Toxins (2022) 14, 478, doi.org / 10.3390 / toxins14070478) describe the results of an in vivo study (in mice), where mice in the treatment group received the saponin SO1861 subcutaneously, followed by the administration (intraperitoneally) of antibody-drug conjugate (ADC) obinutuzumab-dianthin, one hour later.

[0023] The extension of the duration of effect, extension of the dosing interval, reduction in the dosing frequency and / or the delayed boost in the effect are particularly pronounced and advantageous in case wherein the effector component is an oligonucleotide-based medicament. However, as will be apparent to those skilled in the art, based on the present teachings, similar advantageous effects can be attained in case of other types of effector components (that require cellular uptake to become effective), such as toxins, enzymes, small molecule therapeutics, etc., and such embodiments are thus also encompassed by the present invention.

[0024] Hence, generally stated, the invention also concerns a therapeutic method of treatment or prevention or amelioration of a subject suffering from a disease or condition, preferably a CVD; said therapeutic method of treatment or prevention or amelioration comprising:

[0025] i) the administration, to said (human) subject, preferably the repeated administration, of an oligonucleotide-based medicament that is capable of modulating an intracellular process involved in the disease or condition or of an intracellular process that can aid in the ailment of said disease or condition, including the relief of a symptom of the disease or condition; and

[0026] ii) the administration, to said (human) subject, of a saponin component;

[0027] wherein the administration of the saponin component results in an extension of the effect of the effector component and / or in an extension of the dosing interval of the effector component and / or in a reduction of the dosing frequency of the effector component and / or in a (delayed) boost of the effect of the oligonucleotide-based medicament (effector component).

[0028] Third, the present invention resides in the finding that exposure of human cells in vitro and of animal organs and the blood in vivo to a saponin component is well tolerated and does not result in adverse events or side reactions commonly observed with oligonucleotide components or oligonucleotide-based medicaments. That is to say, the inventors demonstrate that the saponin component does not activate nuclear factor-kappa B (NF-KB) since the NF-KB pathway is not activated upon exposure to the saponin component. In addition, in the examples it is shown that the saponin component does not cause an undesired innate immune response (e.g. involving a Toll-like receptor mediated response) in vivo. Furthermore, exposure of human blood to the saponin component does not induce an increase in the level of any one of the cytokines interleukin-1β (IL-1β), interleukin-2 (IL-2), interferon-a2a (IFNa2a), IFNy, tissue necrosis factor-a (TNFa) and INF0. Moreover, in in vivo experiments (as outlined in the ‘Examples’ section), it is demonstrated that exposure of organs and the blood circulation to the saponin component is well tolerated and does not induce any sign of adverse events or undesired side effects. That is to say, administration of the saponin component intravenously (i.v.) to mice provided treatments that were well tolerated, and bodyweight and serum biomarkers of kidney function (creatinine) and liver function (ALT) were not affected by any of the applied treatments. Moreover, and even more relevant, administration of the saponin component to non-human primates (NHP) (subcutaneously (s.c.)), provided the observation that the doses were well tolerated without any clinical observations, including acceptable tolerability as assessed in an extensive clinical pathology panel assessing haematology, coagulation, and clinical chemistry, containing serum ALT and creatinine levels, amongst a battery of standard serum tests, as well as a gross organ morphology and liver and kidney histopathology. These findings were further substantiated with a large panel of in vitro cell-based bioassay experiments providing a series of examples showing that the saponin component or the combination of the saponin component and the oligonucleotide-based medicament has no to fairly low levels of cytotoxicity towards a panel of human test cells (see ‘Examples’ section).

[0029] These combined findings let to the surprising and highly relevant conclusion that the saponin component at the doses tested and when combined with a (relatively low dose of an) oligonucleotide-based medicament, can be administered in the therapeutic setting in a safe manner, with no to low apparent side effects or even absence of such side effects relating to e.g. inflammatory reactions, (innate) immunogenicity, etc.

[0030] Fourth, the present invention resides in the striking recognition that the here-above combined observations together with the combined observations as disclosed in the here-above listed previous international applications of Sapreme Technologies, overall and in joint combination made new therapeutic treatment methods available for conquering systemic diseases that affect single or multiple organs in the human body. The large group of diseases and health problems jointly referred to as CVDs, are more often than not diseases and health problems with a systemic nature. That is to say, hallmarks are that multiple organs are affected or involved in disease pathology, including for example the blood, the liver, the spleen, the pancreas, the kidneys, and in several occasions even the central nervous system (CNS) and / or the eyes. Involvement of health-threatening events in multiple organs, or even when apparent in a single organ, in patients suffering from one or more CVDs, may require a multifactorial therapeutic treatment approach involving the targeting of one or more different molecules (e.g. a gene, gene transcript, protein, peptide, hormone, cytokine, cellular surface receptor, etc., etc.) with one or a combination of therapeutic agents. Indeed, combination therapy, or adjunctive therapy, is often considered and applied when a human subject is treated for a multi-factorial CVD. It is now recognized for the first time that application of the saponin component in therapeutic methods involving the administration of an oligonucleotide-based medicament for treatment of such CVD can provide a beneficial treatment option for a multitude of CVDs. These CVDs may affect single or multiple organs, may involve the negative consequences of an activated inflammatory pathway such as the NF-KB pathway, and may progress in time, differ in severity and extent amongst different subjects, and above all are a diverse group of devastating diseases encompassing for example T2DM, metabolic disorder, such as accompanied by NASH (such as NASH), liver fibrosis and / or liver cirrhosis, (diabetic or chronic) kidney disease, including kidney fibrosis and kidney failure, metabolic syndrome e.g. involving a CVD, a liver- and / or lung disease related to AATD, (NASH and / or liver fibrosis related to) hereditary hemochromatosis and cholestasis (such as cholestasis accompanied with NASH and / or liver fibrosis and inflammation in the liver), etc., etc. Therefore, not only the therapeutic combination as provided for the first time now by the current inventors, provides in itself a new and helpful contribution to the arsenal of treatment options for such CVDs, as a mono-therapy. In addition, the therapeutic combinations as now provided also provide for the first time new treatment modalities involving adjunctive therapy, for which the therapeutic combination is combined with the administration of any one or more of the existing therapeutic agents applied for treating e.g. T2DM with kidney failure, NASH, cholestasis, liver cirrhosis, etc., which therapeutic agents range from small molecule therapeutic agents to antibodies to oligonucleotide-based medicaments, etc.

[0031] Hence, generally stated, the invention also concerns a therapeutic method of treatment or prevention or amelioration of a subject suffering from a CVD; said therapeutic method of treatment or prevention or amelioration comprising:

[0032] i) the administration, to said (human) subject, preferably the repeated administration, of an oligonucleotide-based medicament that is capable of modulating an intracellular process involved in the CVD or of an intracellular process that can aid in the ailment of said CVD, including the relief of a symptom of the CVD; and

[0033] ii) the administration, to said (human) subject, of a saponin component;

[0034] wherein the administration of the saponin component and the oligonucleotide-based medicament is preceded by, accompanied by or followed by administration of one or more further therapeutic agent(s) capable of modulating an intracellular process involved in the CVD or of an intracellular process that can aid in the ailment of said CVD, including the relief of a symptom of the CVD.

[0035] It is therefore one of the objectives of the disclosed further herein embodiments to provide a solution to the problem of inefficacious delivery of oligonucleotide-based medicaments into the kidney cells and / or into the liver cells e.g. of a human subject at risk for developing a CVD or suffering from a CVD.

[0036] Another one of the objectives of the disclosed herein embodiments is to provide a solution to the problem of low efficiency of target engagement by oligonucleotide-based medicaments, which appears to be the likely cause of the effective dose of the oligonucleotide-based medicaments being too low at the target site (e.g. kidney cell, liver cell, etc.) following systemic administration.

[0037] A further one of the objectives of the disclosed herein embodiments is to provide a solution to any one or more of the problems of insufficient delivery of the required quantity of oligonucleotide-based medicaments to the target organ(s) (e.g. kidney, liver), the abrogated or suboptimal therapeutic efficacy of the administered oligonucleotide-based medicaments following said delivery, as well as the off-target activity and / or undesired adverse effects and potentially nephrotoxic effects of such oligonucleotide-based medicaments. Last but not least, another one of the several objectives of the disclosed herein embodiments is to provide a solution to the problem of insufficient safety characteristics of the currently existing oligonucleotide-based medicaments, in particular those relating to adverse effects related to nephrotoxicity and / or hepatotoxicity, when the oligonucleotide-based medicaments are systemically administered to subjects, preferably human patients, in need thereof, in particular the toxic side-effects caused by the administration of excessive doses.

[0038] In one aspect, the present disclosure relates to a therapeutic combination comprising: a. an oligonucleotide-based medicament adapted to target and bind to a nucleic acid molecule present in the cytosol of a human cell and / or present in the nucleus of a human cell;

[0039] b. a saponin component comprising a penta-cyclic triterpene saponin, comprising an aglycone core of 12,13-dehydrooleanane type;

[0040] for use in treatment and / or prevention and / or amelioration of a cardiovascular disease;

[0041] wherein the treatment and / or prevention and / or amelioration comprises administration of the therapeutic combination to a human subject in the need thereof;

[0042] wherein the saponin component and the oligonucleotide-based medicament are delivered to the human cell wherein said human cell is a cell of the kidney and / or is a liver cell such as a hepatocyte.

[0043] In another aspect, the present disclosure relates to a therapeutic combination for treatment and / or prevention and / or amelioration of a cardiovascular diseases comprising:

[0044] a. an oligonucleotide-based medicament adapted to target and bind to a nucleic acid molecule present in the cytosol of a human cell and / or present in the nucleus of a human cell;

[0045] b. a saponin component comprising a penta-cyclic triterpene saponin, comprising an aglycone core of 12,13-dehydrooleanane type;

[0046] wherein the saponin component and the oligonucleotide-based medicament are delivered to the human cell wherein said human cell is a cell of the kidney and / or is a liver cell such as a hepatocyte.

[0047] In one aspect, the present disclosure relates to a therapeutic combination for treatment and / or prevention and / or amelioration of a cardiovascular disease comprising:

[0048] a. an oligonucleotide-based medicament adapted to target and bind to a nucleic acid molecule present in the cytosol of a human cell and / or present in the nucleus of a human cell; and

[0049] b. a saponin component comprising a penta-cyclic triterpene saponin comprising an aglycone core of 12,13-dehydrooleanane type;

[0050] wherein the oligonucleotide-based medicament and the saponin component are either:

[0051] co-formulated in a single pharmaceutical composition optionally comprising any one or more of a pharmaceutically acceptable excipient, diluent and carrier, for simultaneous administration; or formulated separately as at least two pharmaceutical formulations comprising a first pharmaceutical formulation comprising the saponin component and optionally comprising any one or more of a pharmaceutically acceptable excipient, diluent and carrier, and a second pharmaceutical formulation comprising the oligonucleotide-based medicament and optionally comprising any one or more of a pharmaceutically acceptable excipient, diluent and carrier;

[0052] wherein the saponin component and the oligonucleotide-based medicament are delivered to the human cell wherein said human cell is a cell of the kidney and / or is a liver cell such as a hepatocyte.

[0053] In one aspect, the present disclosure relates to a therapeutic combination comprising: a. an oligonucleotide-based medicament adapted to target and bind to a nucleic acid molecule present in the cytosol of a human cell and / or present in the nucleus of a human cell; b. a saponin component comprising a penta-cyclic triterpene saponin, comprising an aglycone core of 12,13-dehydrooleanane type;

[0054] for use in treatment and / or prevention and / or amelioration of a cardiovascular disease;

[0055] wherein the oligonucleotide-based medicament and the saponin component are either:

[0056] co-formulated in a single pharmaceutical composition optionally comprising any one or more of a pharmaceutically acceptable excipient, diluent and carrier, for simultaneous administration; or formulated separately as at least two pharmaceutical formulations comprising a first pharmaceutical formulation comprising the saponin component and optionally comprising any one or more of a pharmaceutically acceptable excipient, diluent and carrier, and a second pharmaceutical formulation comprising the oligonucleotide-based medicament and optionally comprising any one or more of a pharmaceutically acceptable excipient, diluent and carrier;

[0057] wherein the treatment and / or prevention and / or amelioration comprises administration of the therapeutic combination to a human subject in the need thereof;

[0058] wherein the saponin component and the oligonucleotide-based medicament are delivered to the human cell wherein said human cell is a cell of the kidney and / or is a liver cell such as a hepatocyte.

[0059] In one aspect, the second pharmaceutical formulation is administered first and subsequently the first pharmaceutical formulation is administered after an interval of at least 1 day, preferably after an interval of at least one week, more preferably after an interval of at least one month, most preferably after an interval of at least 3-6 months.

[0060] In yet one aspect, the administration of co-formulated single pharmaceutical composition and / or separately formulated two pharmaceutical formulations is further continued after an interval of at least 1 day, preferably after an interval of at least one week, more preferably after an interval of at least one month, most preferably after an interval of at least 3-6 months, with a boosting administration of the saponin component that is further referred to as a booster.

[0061] In another aspect, the present disclosure relates to a method of treatment and / or prevention and / or amelioration of a cardiovascular disease in a human subject that is any one or more of: caused by, treatable by, prevented by and ameliorated by an alteration in the expression of a gene and / or a modulation of the expression level of a gene; said method of treatment comprising the steps of: i) the administration, to said human subject, preferably the repeated administration, of the oligonucleotide-based medicament, that is capable of modulating the expression of said gene and / or that is capable of modulating the expression level of said gene; and

[0062] ii) the administration, to said human subject, of the saponin component;

[0063] wherein the saponin component is administered simultaneously with the oligonucleotide-based medicament and / or wherein the saponin component is administered some time, optionally after an interval of at least 1 day, preferably at least one week, more preferably at least one month, most preferably at least 3-6 months, after the oligonucleotide-based medicament has been administered, resulting in an extension of the duration of effect of the oligonucleotide-based medicament and / or in an extension of the dosing interval of the oligonucleotide-based medicament and / or in a reduction of the dosing frequency of the oligonucleotide-based medicament and / or in a (delayed) boost in the effect of the oligonucleotide-based medicament.

[0064] In yet another aspect, the present disclosure relates to use of the saponin component and the oligonucleotide-based medicament in the manufacture of a medicament for use in a method of treatment and / or prevention and / or amelioration of a cardiovascular disease in a human subject that is any one or more of: caused by, treatable by, prevented by and ameliorated by an alteration in the expression of a gene and / or a modulation of the expression level of a gene; said method of treatment comprising the steps of:

[0065] i) the administration, to said human subject, preferably the repeated administration, of the oligonucleotide-based medicament that is capable of modulating the expression of said gene and / or that is capable of modulating the expression level of said gene; and

[0066] ii) the administration, to said human subject, of the saponin component;

[0067] wherein the saponin component is administered simultaneously with the oligonucleotide-based medicament and / or wherein the saponin component is administered some time, optionally after an interval of at least 1 day, preferably at least one week, more preferably at least one month, most preferably at least 3-6 months, after the oligonucleotide-based medicament has been administered, resulting in an extension of the duration of effect of the oligonucleotide-based medicament and / or in an extension of the dosing interval of the oligonucleotide-based medicament and / or in a reduction of the dosing frequency of the oligonucleotide-based medicament and / or in a (delayed) boost in the effect of the oligonucleotide-based medicament.

[0068] In one aspect, the nucleic acid molecule targeted by the oligonucleotide-based medicament is a gene transcript present in kidney cells and / or in liver cells and is selected from: beta-1, 4-galactosyltransferase 1 (B4GALT1), proprotein convertase subtilisin / kexin type 9 (PCSK9), angiopoietin-like 3 (ANGPTL3), cytochrome P450 family 7 subfamily A member 1 (CYP7A1), arachidonate 12-lipoxygenase, 12S type (ALOX12) and serpin family H member 1 (SERPINA1), preferably selected from: B4GALT1, CYP7A1 and ALOX12.

[0069] In one aspect, the present disclosure relates to the treatment and / or prevention and / or amelioration of cardiovascular disease, which is related to (an) elevated plasma concentration(s) of low- density lipoprotein cholesterol (LDL-C) and / or triglycerides, optionally related to (an) expression or expression level of any one or more of the genes selected from: B4GALT1, PCSK9, ANGPTL3, CYP7A1, ALOX12 and SERPINA1, more preferably B4GALT1, CYP7A1 and ALOX12, and wherein the cardiovascular disease is optionally accompanied with inflammation.

[0070] In yet another aspect, the present disclosure relates to the treatment and / or prevention and / or amelioration of cardiovascular disease, wherein the cardiovascular disease is accompanied with inflammation and the treatment and / or prevention and / or amelioration (also) comprises treating, preventing or ameliorating inflammation in the human subject, optionally NF-KB pathway related inflammation, and / or inhibition of NIK.

[0071] In one aspect, the present disclosure relates to:

[0072] a first pharmaceutical kit comprising a package comprising:

[0073] a) one or more dosage units comprising the saponin component;

[0074] b) one or more dosage units comprising the oligonucleotide-based medicament; and

[0075] c) printed instructions to use the dosage units comprised in the kit in a therapeutic method of treatment of a cardiovascular disease related to a defect in (the expression of) a gene and / or that is treatable by modulating the expression and / or expression level of a gene, said method of treatment comprising: i) the administration, preferably the repeated administration, of the oligonucleotide-based medicament that is capable of regulating the expression of said gene and / or that is capable of modulating the expression level of said gene;

[0076] ii) the administration, to said subject, of the saponin component;

[0077] wherein the saponin component is administered simultaneously with the oligonucleotide-based medicament and / or wherein the saponin component is administered some time, optionally after an interval of at least 1 day, preferably at least one week, more preferably at least one month, most preferably at least 3-6 months, after the oligonucleotide-based medicament has been administered, resulting in an extension of the duration of effect of the oligonucleotide-based medicament and / or in an extension of the dosing interval of the oligonucleotide-based medicament and / or in a reduction of the dosing frequency of the oligonucleotide-based medicament and / or in a (delayed) boost in the effect of the oligonucleotide-based medicament; or

[0078] a second pharmaceutical kit comprising a package comprising:

[0079] a) one or more dosage units comprising the saponin component and comprising the oligonucleotide-based medicament; and

[0080] b) printed instructions to use the dosage units comprised in the kit in a method of treatment of a cardiovascular disease related to a defect in (the expression of) a gene and / or that is treatable by modulating the expression and / or expression level of a gene, said method of treatment comprising: - the administration, preferably the repeated administration, of the dosage units comprising the oligonucleotide-based medicament that is capable of regulating the expression of said gene and / or that is capable of modulating the expression level of said gene; or

[0081] a third pharmaceutical kit comprising a package comprising:

[0082] a) one or more first dosage units comprising the saponin component and comprising the oligonucleotide-based medicament;

[0083] b) one or more second dosage units comprising the saponin component; and

[0084] c) printed instructions to use the dosage units comprised in the kit in a method of treatment of a cardiovascular disease in a human subject related to a defect in (the expression of) a gene and / or that is treatable by modulating the expression and / or expression level of a gene, said method of treatment comprising:

[0085] i. the administration, preferably the repeated administration, to said human subject, of the first dosage units comprising the oligonucleotide-based medicament that is capable of regulating the expression of said gene and / or that is capable of modulating the expression level of said gene, and comprising the saponin component;

[0086] ii. the administration, to said human subject, of the second dosage unit comprising only the saponin component;

[0087] wherein the second dosage unit is administered some time, optionally after an interval of at least 1 day, preferably at least one week, more preferably at least one month, most preferably at least 3-6 months, after the first dosage unit comprising the oligonucleotide-based medicament has been administered, resulting in an extension of the duration of effect of the oligonucleotide-based medicament and / or in an extension of the dosing interval of the oligonucleotide-based medicament and / or in a reduction of the dosing frequency of the oligonucleotide-based medicament and / or in a (delayed) boost in the effect of the oligonucleotide-based medicament.

[0088] DEFINITIONS

[0089] The terms “cardiovascular disease” or “CVD”, as used herein refers to disease conditions associated with the heart and / or with the vascular system. CVD has its regular meaning as recognised in the field of biology, which encompass a broad spectrum of disorders involving the heart and / or vascular system, including but not limited to: atherosclerotic cardiovascular disease (ASCVD), selected from: coronary heart disease (CHD), such as myocardial infarction, angina and coronary artery stenosis; cerebrovascular disease, such as a transient ischemic attack and ischemic stroke; peripheral artery disease, such as claudication; and aortic atherosclerotic disease, such as abdominal aortic aneurysm and descending thoracic aneurysm; hyperlipidemia; dyslipidemia; hypertriglyceridemia; familial chylomicronemia (type I hyperlipoproteinemia); familial hypercholesterolemia (type II hyperlipoproteinemia), such as heterozygous familial hypercholesterolemia (heFH) or homozygous familial hypercholesterolemia (HoFH); hypercholesterolemia; hemorrhagic disease; thrombosis; congenital disorder of glycosylation type lid (CDG-lld); blood clotting defects; arterial plaque formation; hypertension, etc. The conditions are influenced by its multifactorial etiology, which involves an interplay of genetic predisposition, environmental influences, lifestyle factors, and metabolic conditions. CVD is not confined to a single organ system but often involves multiple organs and pathways, for example a range of conditions that affect the heart, the kidney and vascular system. Many of these etiologies involve key regulator pathways associated with specific genes. The interplay of these genes with cardiovascular disease highlights the complex molecular mechanisms underlying pathogenesis. Such genes are selected from, including but not limited to:

[0090] B4GALT1: is implicated in glycosylation processes that affect cell signaling and lipid metabolism. Variations in this gene may influence lipoprotein function, contributing to atherosclerosis and other lipid-related cardiovascular conditions.

[0091] PCSK9: plays a critical role in cholesterol homeostasis by regulating low-density lipoprotein receptor (LDLR) levels. Mutations or dysregulation in PCSK9 can lead to altered cholesterol clearance, significantly affecting the risk of coronary artery disease and myocardial infarction.

[0092] ANGPTL3: regulates lipid metabolism, particularly the inhibition of lipoprotein lipase (LPL), impacting triglyceride and cholesterol levels. Genetic mutations in ANGPTL3 have been associated with altered plasma lipid levels and varying susceptibility to atherosclerosis.

[0093] CYP7A1: is a key enzyme in bile acid synthesis, regulating cholesterol catabolism. Variants in this gene can influence cholesterol homeostasis and contribute to hypercholesterolemia, a major risk factor for cardiovascular diseases.

[0094] ALOX12: is involved in lipid oxidation and inflammation through the metabolism of arachidonic acid. Its role in oxidative stress and vascular inflammation links it to the development of atherosclerosis and thrombosis.

[0095] SERPINA1 encodes alpha-1 antitrypsin, a protein that inhibits proteolytic enzymes. Deficiency or mutations in SERPINA1 can result in heightened vascular inflammation and oxidative stress, exacerbating risks for conditions like aneurysms and coronary artery disease.

[0096] The terms “thrombosis”, as used herein has its regular scientific meaning and here refers to the formation of a blood clot (thrombus) within the vascular system, obstructing blood flow and contributing to the pathogenesis of various CVDs. Thrombosis plays a pivotal role in CVD by disrupting normal blood circulation, promoting ischemia, and triggering inflammatory responses. Its underlying mechanisms are influenced by genetic factors (e.g., prothrombotic mutations) and molecular pathways (e.g., platelet activation, coagulation cascade dysregulation), highlighting the importance of targeting thrombotic processes in the development of diagnostic and therapeutic solutions for cardiovascular health. In arterial thrombosis, clots form in arteries due to endothelial damage, platelet aggregation, and atherosclerosis, leading to conditions such as myocardial infarction, ischemic stroke, and peripheral artery disease. The terms “therapeutic combination” and “pharmaceutical combination”, as used herein, are to be construed as synonymous and as referring to a combined use of at least two components (herein being an oligonucleotide-based medicament and a saponin component that potentiates the effects of the oligonucleotide-based medicament) in treating of a disease and / or in preventing that a disease develops. In the present context, the two components of the therapeutic combination can be covalently conjugated together to form a single larger component (“1 -component”) and provided in a single therapeutic (pharmaceutical) composition, or can be kept as two separate components that can either be co-formulated in a single (2-component) therapeutic (pharmaceutical) composition, or can be provided independently in separate therapeutic (pharmaceutical) formulations e.g. at least two pharmaceutical formulations: a first pharmaceutical formulation comprising the saponin component and a second pharmaceutical formulation comprising the oligonucleotide-based medicament. Wherein, said at least two pharmaceutical formulations are optionally comprising any one or more of a pharmaceutically acceptable excipient, diluent and carrier. Also, said at least two pharmaceutical formulations are optionally in a form of a kit or a kit of parts containing two or more separate products packaged together in a single package or as a unit, wherein each product contains a separate pharmaceutical formulation of a different component of the presented herein therapeutic combination. As it will be understood by the skilled person, different modes of formulating the two compounds of the therapeutic combination have different advantages. For example, 1 -component (conjugate) and 2-component (co-formulation) therapeutic compositions have the advantage of ease of administration to the subject. On the other hand, two separate formulations allow more flexibility for fine-tuning the treatment by e.g. using different administration routes and having a choice between preforming a simultaneous or sequential administration.

[0097] The terms “kidney” and “kidneys” have their regular meaning as recognised in the field of biology that relate to one or more of usually two excretory filtration organs that belong to the urinary system and produce urine by filtering blood. The skilled person will be familiar with the anatomy, histology and the functioning of the kidney and, therefore, will know the substructures and different types of cells (as used herein, kidney cells) that build the kidney and molecular markers associated therewith (Agarwal et al., 2021). In brief, a typical mammalian kidney has a renal capsule, cortex, medulla, one or more renal calyces, and a renal pelvis (although the latter two may be absent in some species). The medulla usually contains one or more renal pyramids, forming papillae with their innermost parts. Generally, the urine produced by the cortex and the medulla drains from the papillae into the calyces, and then into the renal pelvis, from which it exits the kidney through the ureter. The cortex and the medulla contain the structural and functional units of the kidney known as the nephrons, which are composed of a renal corpuscle and a renal tubule. The blood is filtered inside of the corpuscles, which are primarily located in the cortex, while the medulla of a mammalian kidney contains the major sections of the tubules including loops of Henle, which are responsible for urine concentration. The renal corpuscle is made of a tuft of capillaries called the glomerulus that is covered by a Bowman's capsule. The renal tubule is connected to and extends from the capsule, and both are composed of epithelial cells and have a lumen. The tuft is structurally supported by the mesangium (the space between the blood vessels), composed of intraglomerular mesangial cells, which make up about 30–40% of the glomerular cell population (Ebefors et al., 2022). The blood is filtered across the capillary walls to form a filtrate that passes to the renal tubule. During the filtration, the blood passes through three layers that form the glomerular filtration barrier, which are: the endothelial cells of the capillary wall, its basement membrane, and between the podocyte foot processes of the lining of the capsule. The barrier filters molecules based on size and charge and is made from three types of filters present in the different layers: (i) the endothelial fenestrations in glomerular capillaries, (ii) the glomerular basement membrane, and (iii) the visceral epithelial cell podocyte slit diaphragm. The tubule has adjacent peritubular capillaries that run between the descending and ascending portions of the tubule. As the filtrate from the capsule flows down into the tubule, it is processed by the epithelial cells lining of the tubule: water is reabsorbed and substances are exchanged (some are added, others are removed); first with the interstitial fluid outside the tubules, and then into the plasma in the adjacent peritubular capillaries through the endothelial cells lining that capillary. This process regulates the volume of body fluid as well as levels of many body substances. At the end of the tubule, the remaining fluid —urine— exits: it is composed of water, metabolic waste, and toxins. Human kidney anatomy and function are part the common general knowledge as evidenced by academic textbooks and review articles (cf. e.g. Alallam et al., 2023).

[0098] The term “subject” as used herein refers to a human suffering from or at risk of a certain health-related disorder, such as a disease or other pathological condition. The terms “subject” and “patient” are used interchangeably herein.

[0099] The term “treatment” as used herein has its conventional meaning and refers to a medical intervention or management of a subject with the intention to cure, ameliorate, or stabilise a health-related disorder. The term “treatment” includes e.g. active treatment that is a type of an action directed specifically toward the improvement of a health-related disorder and also includes causal treatment that is a treatment directed towards a removal of the cause of the associated therewith health-related disorder. The terms “prevention” and “prophylaxis” as used herein are to be construed as referring to a medical intervention or management of a subject with the intention to maintain health or the normal bodily functions and / or prevent the otherwise anticipated development of a health-related disorder. The terms “amelioration” as used herein is to be construed as the process of improving or alleviating the severity, symptoms and / or progression of a disease or medical condition. It does not necessarily imply a cure, but implies to enhance the patient's quality of life, well-being and reduce the disease’s impact on daily functioning.

[0100] As used herein the term “administration” is to be construed as referring to the way of providing a substance, such as a compound, or a composition to a subject. Conversely, as used herein the term “delivery” is to be construed as referring to the way a compound reaches its destination site, e.g. specific zone, organ, its sub-compartment, cell or tissue type, for example the kidney, the kidney cortex, kidney medulla, a specific kidney cell type etc. Administration can relate to e.g. an intravenous, subcutaneous or other way of providing one or more compounds into the subject’s body, with an intended delivery to e.g. kidney cells as the destination site. Usually, the terms “administering” or “administration” will be construed as relating to the provision of a substance that is physiologically and / or pharmacologically useful (e.g. medicament) to treat or prevent a disease or a pathological condition in the subject. As used herein, the term “oligonucleotide-based medicament” is to be construed as a substance containing an oligomeric molecule that has a functionality of an oligonucleotide in the sense of being capable of sequence-dependent binding to a target nucleic acid (as used herein “targeting and binding”) that can be present within a cell, and through this binding, modulating the outcomes related to the target nucleic acid within the cell, for example by silencing the nucleic acid’s expression or by leading to different gene editing result (mRNA), whereby the successful modulation will be generally expected to have a positive therapeutic or prophylactic effect against a disease or development thereof. As a consequence, as used herein, an oligonucleotide-based medicament does not have to be a clinically-proven and / or Regulatory Authority-approved oligonucleotide-based medicament, although it certainly can be. However, it can also be an oligonucleotide-based medicament that is at a stage of pre-clinical trials, or is discontinued as a result of a failed clinical trial stage (e.g. phase III) but is credible to be able to produce a positive therapeutic or prophylactic effect but has failed e.g. due safety and / or toxicity concerns, often in relation to formulation and / or (possibly too high) dose. One of the advantages of the presented herein therapeutic combinations, formulations, and / or compositions containing the saponin component is the ability to use much lower doses of the credibly potentially-effective oligonucleotide-based medicaments to have them safely delivered into the cells, thus allowing to unleash their therapeutic potential at lower safety risks and / or likelihood of toxicity-related failures. As sometimes used herein, the oligonucleotide-based medicament will be referred to as an “effector component”, in which the therapeutic oligomeric nucleic acid and / or nucleic acid analogue molecule or moiety will be considered to function as an “effector molecule”, or “effector moiety”, respectively, for its ability to exert its desired therapeutic modulatory effect through binding to its target intracellular nucleic acid in the cytosol (cytoplasm) and / or in the nucleus of a cell, herein being a kidney cell. A typical example of an oligonucleotide-based medicament effector component suitable for the presented herein purposes can be a so-called antibody-oligonucleotide conjugate (AOC). The term “oligonucleotide-based medicament” will in general be clear to the skilled person who will be familiar with different possible chemistries of the oligomeric molecule contained therein and other known in the art modifications and addition thereto, examples of which can be found in Roberts etal., 2020.

[0101] As used herein, the terms “nucleic acid” and “polynucleotide” are synonymous to one another and are to be construed as encompassing any polymeric molecule made of units, wherein a unit comprises at least a nucleobase (or simply “base”) e.g. being a canonical nucleobase like adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U), or any known non-canonical, modified, or synthetic nucleobase like 5-methylcytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 7-methylguanine; 5,6-dihydrouracil etc., or a functional equivalent thereof, which renders said polymeric molecule capable of engaging in hydrogen bond-based nucleobase pairing (such as Watson–Crick base pairing) under appropriate hybridisation conditions with naturally-occurring nucleic acids such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which naturally-occurring nucleic acids are to be understood as being polymeric molecules made of units being nucleotides, whereby each nucleotide consists of a pentose sugar, a phosphate group and one of the nucleobases.

[0102] From the chemistry perspective, the term nucleic acid under the present definition can be construed as encompassing polymeric molecules that chemically are DNA or RNA, as well as polymeric molecules that are nucleic acid analogues, also known as xeno nucleic acids (XNA) or artificial nucleic acids, which are polymeric molecules wherein one or more (or all) of the units are modified nucleotides or are functional equivalents of nucleotides. Nucleic acid analogues are well known in the art and due to improved properties, such as specificity and / or affinity, higher binding strength to their target and / or increased stability in vivo, they are extensively used in research and medicine. Typical examples of nucleic acid analogues include but are not limited to locked nucleic acid (LNA) (that is also known as bridged nucleic acid (BNA)), phosphorodiamidate morpholino oligomer (PMO also known as Morpholino), peptide nucleic acid (PNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), hexitol nucleic acid (HNA), 2'-O, 4'-C-ethylene-bridged nucleic acid (ENA), 2’-deoxy-2’-fluoroarabinonucleic acid (FANA or FNA), 2’-deoxy-2’-fluororibonucleic acid (2’-F RNA or FRNA); altritol nucleic acids (ANA), cyclohexene nucleic acids (CeNA), nucleic acids containing modified linkages like phosphoryl guanidine (PN) backbone linkages etc. In line with the above, in some instance, the nucleic acid of the present disclosure may be chemically modified. For example, the nucleic acid may be modified on its backbone. Examples of modifications that can be performed on the backbone of a nucleic acid include, but are not limited to, phosphorothioate (PS), PN linkages, boranophosphate, phosphonoacatate (PACE), morpholine, peptide nucleic acid backbone modification (PNA), and amid-linked bases. The nucleic acid may also be modified on the sugar moiety and / or on the base moiety. Examples of modifications that can be performed on the sugar and / or the base moieties include, but are not limited to, locked nucleic acid (LNA), phosphoramidate (PN), 2'F-RNA, 2'-O methoxyethyl (2'MOE), 2'O-methyl (2'OMe), 2'-O-fluoro (2'-F) 5-bromouracil, 5-iodouracil, 5-methylcytosine, ethylene bridged nucleic acids (ENA), diaminopurine, 2-thiouracil, 4-thiouracil, pseudouracil, hypoxantine, 2-aminoadenine, 6-methyl or other alkyl derivates of adenine and guanine, 2-propyl and other derivative of adenine and guanine, 6-azo-uracil, 8-halo, 8-amino, 8-thiol, 8-hydroxyk and other 8-substituted adenines and guanines, constrained ethyl sugar moiety (cET), ribofuranosy I, 2'-0,4'-C-methylene and 2'-0,4'-C-ethylene bicyclic nucleotide analogues, acyclic nucleotides (UNA and PNA), and dihydrouridine modification. Other modifications that may be performed on nucleic acids are, but are not limited to, modifications that include deoxyribonucleotide bases incorporated in a ribonucleotide sequence. The incorporations may be limited to the overhang structure in the canonical siRNA architecture or may be distributed in the sequence, and non-limiting examples thereof are shown in Hu et al., 2020. Modifications to RNA molecules include, but are not limited to blunt-ended siRNA, 25-27mer siRNA, single strand siRNA, short hairpin siRNA, dumbbell siRNA, asymmetric siRNA, short interspaced siRNA, hybrid between siRNA and antisense oligonucleotides (ASO). Other analogue nucleic acids that may be contemplated include those with non-ribose backbones. In addition, mixtures of naturally occurring nucleic acids, analogues, and both may be made. Nucleic acids include but are not limited to DNA, RNA and hybrids where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, 5-methylcytidine, pseudouridine, etc. Modified 5' cap structures such as 3'-O-Me-m7G(5')ppp(5')G (anti-reverse cap analogue), may also be used for increased translation of mRNA. Nucleic acids include DNA in any form, RNA in any form, including triplex, duplex or single-stranded, antisense, siRNA, ribozymes, deoxyribozymes, polynucleotides, oligonucleotides, chimeras, and derivatives thereof.

[0103] In accordance with the cannon, length of a nucleic acid is expressed herein as the number of units from which a single strand of a nucleic acid is build. Because each unit corresponds to exactly one nucleobase capable of engaging in one base pairing event, the length is frequently expressed in so called "base pairs" or "bp" regardless of whether the nucleic acid in question is a single stranded (ss) or double stranded (ds) nucleic acid. In ss nucleic acids 1 bp usually corresponds to 1 nucleotide, abbreviated to 1 nt. For example, a ss nucleic acid made of 1000 nucleotides (or a ds nucleic acid made of two complementary strands each of which is made of 1000 nucleotides) is described as having a length of 1000 base pairs or 1000 bp, which length can also be expressed as 1000 nt or as 1 kilobase that is abbreviated to 1 kb. 2 kilobases or 2 kb are equal to the length of 2000 bp which equates 2000 nt in an ssRNA or ssDNA. To avoid confusion however, in view of the fact the nucleic acids as defined herein may comprise or consist of units not only chemically being nucleotides but also being functional equivalents thereof, the length of nucleic acids will preferentially be expressed herein in "bp" or "kb" rather than in the equally common in the art denotation "nt".

[0104] As used herein, an oligonucleotide (or simply “an oligo”) is to be construed as a relatively short nucleic acid, usually no longer than 500 bp, preferably no longer than 200 bp, i.e. in accordance with the above provided definition, being any polymeric molecule made of no more than 200 units, wherein each unit comprises a nucleobase or a functional equivalent thereof, which renders said oligonucleotide capable of engaging in nucleobase pairing under appropriate hybridisation conditions with DNA or RNA. Within the ambit of said definition, it will immediately be appreciated that the disclosed herein oligonucleotides can comprise or consist of units not only being from a purely chemical point of view nucleotides, but also units being synthetic equivalents thereof. In other words, from chemistry perspective, as used herein, the term oligonucleotide will be construed as possibly comprising or consisting of RNA, DNA, or one or more different or same nucleic acid analogues such as but not limited to LNA (BNA), PMO (Morpholino), PNA, GNA, TNA, HNA, FANA, FRNA, ANA, CeNA and / or the like.

[0105] The term “saponin" has its regular scientific meaning and refers to a chemical compound from a group of amphipathic glycosides that comprise one or more hydrophilic glycone moieties (usually arranged in chains containing at least one sugar group, but more frequently containing an often branched glycan chain of several sugar groups), which one or more glycone moieties are covalently bound to a lipophilic aglycone core of steroid or terpenoid structure that is termed sapogenin.

[0106] In the context of saponins, the terms “aglycone core”, “sapogenin”, and “aglycone core structure”, “aglycone glycoside core (structure)” are used interchangeably and in line with their scientifically accepted meaning in the field. Namely, these terms refer to the lipophilic part of a saponin, which part has a steroid or terpenoid structure and to which one or more glycone moieties are attached (these glycone moieties are sometimes also referred to as “glycone antennae” or “sugar antennae”).

[0107] The term “saccharide chain” or “carbohydrate chain” has its regular scientific meaning and here refers to any of a glycan, a carbohydrate antenna, a single saccharide moiety (monosaccharide) or a chain comprising multiple saccharide moieties (oligosaccharide, polysaccharide). The saccharide chain can consist of only saccharide moieties or may also comprise further moieties such as any one of 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), such as for example present in QS-21.

[0108] The term “Api / Xyl-“ or “Api- or Xyl-“ in the context of the name of a saccharide chain has its regular scientific meaning and here refers to the saccharide chain either comprising an apiose (Api) moiety, or comprising a xylose (Xyl) moiety.

[0109] As it will be apparent from the present description, a specific group of saponins with an aglycone core of terpenoid structure will form part of the presented herein pharmaceutical combinations, compositions, formulations and therapeutic methods. Because of this aglycone core structure, the saponins are classified as penta-cyclic triterpene saponins which, in particular, comprise an aglycone core of 12,13-dehydrooleanane type. The chemical structure of this aglycone core type is schematically shown in a saponin presented in Scheme of SAPONIN A (in Detailed Description). Examples of aglycone cores of 12,13-dehydrooleanane type include saponin aglycone cores of quillaic acid and of gypsogenin, which in naturally-occurring forms in addition also contain an aldehyde function at position C-23 of the aglycone core. For example, quillaic acid is the aglycone glycoside core structure for SO1861, SO1832, AG1856.

[0110] A saponin may be naturally occurring or non-naturally occurring, e.g. modified during isolation process, partial degradation, chemical modification, or can be partially or entirely synthetic.

[0111] Consequently, as used herein, the term “saponin” should be construed as referring to any glycoside compound (that is free or conjugated to another compound) as long as this glycoside compound comprises at least one hydrophilic glycone moiety that is covalently bound to a lipophilic aglycone core moiety of a steroid or terpenoid structure, regardless whether this glycoside compound is identical to a naturally-occurring saponin, or appears to largely correspond in structure to a naturally-occurring saponin but possesses at least one chemical group modification on either one of the glycone moiety or aglycone core moiety as compared to its corresponding naturally-occurring saponin, or is a glycoside compound that does not seem to correspond to any naturally-occurring saponin but by the above definition is a saponin, which could have been synthetically obtained through chemical and / or biotechnological synthesis routes and for this reason does not resemble any naturally-occurring saponin but still visibly comprises at least one hydrophilic glycone moiety that is covalently bound to a lipophilic aglycone core moiety of a steroid or terpenoid structure.

[0112] As already indicated above, as used herein, the term “saponin” shall be construed as encompassing: (i) non-conjugated (“free”) saponins, which are further referred to herein using a term “saponin molecule” in the context of the disclosed herein saponin components of the pharmaceutical compositions and therapeutic methods; and (ii) “unconjugated saponin moiety” further refers to a derivatized saponin molecule, which is not yet conjugated to at least one non-saponin moiety, (iii) conjugated saponins that are covalently conjugated to other compound types and, hence, form part of conjugates comprising at least one saponin as a “saponin moiety” of the conjugate, which saponin moiety is conjugated to an at least one non-saponin moiety such as a linker for further conjugation, or as an effector molecule like an oligonucleotide, or a targeting ligand recognised by a cell-surface receptor, for example an endocytic receptor etc. Hence, in the context of the disclosed herein saponin components of the therapeutic (pharmaceutical) combinations, compositions, formulations and therapeutic methods of the disclosure, such covalently conjugated saponins will be further referred to herein using a term “saponin moiety”, to discern them from the non-conjugated (“free”) saponins or “saponin molecules”.

[0113] As used herein, the term “saponin component" refers to a component of a pharmaceutical combination, composition, formulation or of a therapeutic method (to be construed as synonymous to the term “method of treatment”), which component comprises a saponin as defined above. Consequently, saponin component can be present in the disclosed herein pharmaceutical combinations, compositions, formulations, or therapeutic methods in an unconjugated form (as used herein, as an “saponin molecule” or “unconjugated saponin moiety”), or in a form that is covalently bound (conjugated) to at least one other chemical compound that is not a saponin, thus forming a part of a conjugate comprising the saponin (as used herein, as a “saponin moiety” of said conjugate) and the at least other chemical compound that is not a saponin (as used herein, as a “non-saponin moiety” of said conjugate). For example, as used herein the “saponin molecule” can correspond to a naturally-occurring saponin molecule found in or isolatable from natural sources, such as plant material. While the “unconjugated saponin moiety” can correspond to a non-naturally-occurring saponin molecule that has a chemical group modification as compared to the naturally-occurring saponin, which is a result of e.g. degradation and / or oxidation. In case such saponin molecule or unconjugated saponin moiety becomes covalently conjugated to another compound, for example a linker moiety that can be used for further conjugation steps, the saponin part of such formed conjugate will be referred to as a “saponin moiety”.

[0114] For comparison, in case a saponin component comprises a penta-cyclic triterpene saponin comprising an aglycone core of 12,13-dehydrooleanane type and an acid sensitive covalent bond with one or several atoms and which bond can be seen as simply replacing the aldehyde function at position C-23 of the aglycone core for the reason that said one or several atoms cannot be further classified functionally (e.g. said one or several atoms are not a linker with a chemical group for further conjugation reactions; nor a ligand for binding a receptor) or structurally (e.g. said one or several atoms are not an oligonucleotide, a peptide, an oligosaccharide, etc.), in such a case, such saponin component could be further referred to using the term “saponin molecule” rather than the term “saponin moiety” depending on the specific context, which will be clear to the skilled person. If however such saponin component comprises a penta-cyclic triterpene saponin comprising an aglycone core of 12,13-dehydrooleanane type and an acid sensitive covalent bond with another functionally or structurally distinct non-saponin moiety (e.g. a linker, a ligand, an oligonucleotide, etc.), in such a case, such saponin will be further referred to using the term “saponin moiety” rather than the term “saponin molecule”. The above distinction will be clear to the skilled person and requires not further elaboration.

[0115] The term “Saponinum album” has its normal meaning and here refers to a mixture of saponins produced by Merck KGaA (Darmstadt, Germany) containing saponins from Gypsophila paniculata and Gypsophila arostii, containing SA1657 and mainly SA1641. The term “Quillaja saponin” has its normal meaning and here refers to the saponin fraction of Quillaja saponaria and thus the source for all other QS saponins, mainly containing QS-18 and QS-21.

[0116] “QS-21” or “QS21” has its regular scientific meaning and here refers to a mixture of QS-21 A-apio (-63%), QS-21 A-xylo (-32%), QS-21 B-apio (-3.3%), and QS-21 B-xylo (-1.7%).

[0117] Similarly, “QS-21 A” has its regular scientific meaning and here refers to a mixture of QS-21 A-apio (-65%) and QS-21 A-xylo (-35%).

[0118] Similarly, “QS-21 B” has its regular scientific meaning and here refers to a mixture of QS-21 B-apio (-65%) and QS-21 B-xylo (-35%).

[0119] The term “Quil-A” refers to a commercially available semi-purified extract from Quillaja saponaria and contains variable quantities of more than 50 distinct saponins, many of which incorporate the triterpene-trisaccharide substructure Gal-(1→2)-[Xyl-(1→3)]-GlcA- at the C-3beta-OH group found in QS-7, QS-17, QS-18, and QS-21. The saponins found in Quil-A are listed in van Setten (1995), Table 2 [Dirk C. van Setten, Gerrit van de Werken, Gijsbert Zomer and Gideon F. A. Kersten, Glycosyl Compositions and Structural Characteristics of the Potential Immuno-adjuvant Active Saponins in the Quillaja saponaria Molina Extract Quil A, RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 9,660-666 (1995)]. Quil-A and also Quillaja saponin are fractions of saponins from Quillaja saponaria and both contain a large variety of different saponins with largely overlapping content. The two fractions differ in their specific composition as the two fractions are gained by different purification procedures.

[0120] The term “QS1861” and the term “QS1862” refer to QS-7 and QS-7 api. QS1861 has a molecular mass of 1861 Dalton, QS1862 has a molecular mass of 1862 Dalton. QS1862 is described in Fleck et al. (2019) in Table 1, row no. 28 [Juliane Deise Fleck, Andresa Heemann Betti, Francini Pereira da Silva, Eduardo Artur Troian, Cristina Olivaro, Fernando Ferreira and Simone Gasparin Verza, Saponins from Quillaja saponaria and Quillaja brasiliensis: Particular Chemical Characteristics and Biological Activities, Molecules 2019, 24, 171; doi:10.3390 / molecules24010171]. The described structure is the api-variant QS1862 of QS-7. The molecular mass is 1862 Dalton as this mass is the formal mass including proton at the glucuronic acid. At neutral pH, the molecule is deprotonated. When measuring in mass spectrometry in negative ion mode, the measured mass is 1861 Dalton.

[0121] The terms “SO1861” and “SO1862” refer to the same saponin of Saponaria officinalis, though in deprotonated form or api form, respectively. The molecular mass is 1862 Dalton as this mass is the formal mass including a proton at the glucuronic acid. At neutral pH, the molecule is deprotonated. When measuring the mass using mass spectrometry in negative ion mode, the measured mass is 1861 Dalton. Similarly, the terms “SO1903” and “SO1904” refer to the same saponin of Saponaria officinalis, as well as do “SO1831” and “SO1832”.

[0122] The term “conjugate” has its regular scientific meaning and herein refers to at least a first molecule (further termed “first moiety”) that is covalently bound to at least a second molecule (“second moiety”), therewith forming a covalently coupled assembly comprising or consisting of the first moiety and the second moiety. Typical conjugates are an ADC, an AOC, and SO1861 -EMCH (EMCH linked to the aldehyde function of the aglycone glycoside core structure of the saponin, according to formula (I) (see below)). As used herein, the term “conjugate” is thus to be construed as a combination of two or more different moieties, which before the covalent conjugation, as used herein (purely to discern between the conjugated and unconjugated state), were referred to as the two or more molecules. For example, different moieties forming a conjugate as disclosed herein may include one or more saponins or saponin moieties with one or more ligands that bind to an endocytic receptor present on a surface of the target cells (herein being kidney cells), preferably wherein the ligand is e.g. one or more GalNAc moieties or an antibody or a binding fragment thereof, such as an IgG, a monoclonal antibody (mAb), a single domain antibody such as a VHH domain or another nanobody type, a bivalent nanobody molecule comprising two single domain antibodies, a camelid VH, or a humanized VHH with humanised a human (IgG1-derived) Fc and / or humanized VHH-Fc antibody etc. In some instances, the disclosed herein conjugates may be made by covalently linking different moieties via one or more intermediary moieties such as linkers, for example for linking to a central or further linker. In a conjugate, not all of the two or more different moieties need to be directly covalently bound to each other. Different moieties in the conjugate may also be covalently bound by being both covalently bound to the same intermediary moiety such as a linker or each by being covalently bound to an intermediary moiety such as a further linker or a central linker wherein these two intermediary moieties such as two (different) linkers, are covalently bound to each other. According to this definition even more intermediary moieties such as linkers, may be present between the two different moieties in a conjugate, as long as there is a chain of covalently bound atoms in between.

[0123] As used herein, the term “ligand” is to be understood as any molecule that binds to or can be recognised by a another, usually bigger, molecule, most frequently - a receptor. A typical ligand will be proteinaceous but can also be or comprise a polysugar, i.e. contain or consist of one or more sugar moieties such as (1, 2, 3 or 4) GalNAc moieties.

[0124] The term “GalNAc” has its regular scientific meaning and refers to A / -acetylgalactosamine and / or to the IUPAC name thereof: 2-(acetylamino)-2-deoxy-D-galactose. Ligands containing GalNAc are well known in the art and are usually employed for liver cell-targeting purposes for the reason of binding to the ASGPR1 receptor expressed on the liver cells. Consequently, ligands based on GalNAc are usually considered as liver cell-specific ligands and are not considered as kidney cell-specific ligands (cf Bhingardeve et al., 2020; Chan et al., 2005) although, as demonstrated by the data presented herein below, these ligands based on GalNAc appear to undergo endocytic uptake at the kidney cells as well (i.e. can be considered as belonging to non-kidney cell specific ligand for kidney cells), possibly via multifunctional kidney endocytic receptors such as megalin and cubilin that are highly expressed at e.g. renal proximal tubules and that rescue a variety of ligands filtered from the blood including lipoproteins, vitamin-binding proteins, and various carriers, etc.

[0125] Proteinaceous ligands advantageously comprise or consist of an antibody or a fragment thereof; but they can also be or contain a protein, a peptide, a glycoprotein, or a fragment of any one thereof that, in the present context, is advantageously capable of being recognised by an endocytic receptor. As used herein, the term “an endocytic receptor” is to be understood as any one of cell surface molecules, likely receptors or transporters that are accessible to their specific ligands from the external side or surface of cell membrane (also known as plasmalemma) and capable of undergoing internalisation via endocytic pathway e.g., upon external stimulation, such as ligand binding to the receptor. In some embodiments, an endocytic receptor can be internalized by clathrin-mediated endocytosis but can also be internalized by a clathrin-independent pathway, such as, for example, phagocytosis, macropinocytosis, caveolae- and raft-mediated uptake or constitutive clathrin-independent endocytosis. In some embodiments, the endocytic receptor comprises an intracellular domain, a transmembrane domain, and / or (e.g., and) an extracellular domain, which may optionally further comprise a ligand-binding domain. In some embodiments, the endocytic receptor becomes internalized by the cell after ligand binding. In some embodiments, a ligand may be a specific-cell-targeting agent, for example a natural ligand (or a synthetic fragment thereof) or an antibody or a binding fragment thereof.

[0126] The term “proteinaceous” has its regular scientific meaning and refers to a molecule that is at least protein-like, meaning that the molecule possesses, to some degree, the physicochemical properties characteristic of a protein, is of protein, relating to protein, containing protein, pertaining to protein, consisting of protein, resembling protein, or being a protein. The term “proteinaceous” as used in for example ‘proteinaceous molecule’ refers to the presence of at least a part of the molecule that resembles or is a protein, wherein ‘protein’ is to be understood to include a chain of amino-acid residues at least two residues long, preferably at least 3, 4, 5, 6, 7, 8, 9, 10 residues long, thus including a peptide, a polypeptide and a protein and an assembly of proteins or protein domains. In the proteinaceous molecule, the at least two amino-acid residues are for example linked via (an) amide bond(s), such as (a) peptide bond(s). In the proteinaceous molecule, the amino-acid residues are natural amino-acid residues and / or artificial amino-acid residues such as modified natural amino-acid residues. In a preferred embodiment, a proteinaceous molecule is a molecule comprising at least two amino-acid residues, preferably between two and about 2.000 amino-acid residues. In one embodiment, a proteinaceous molecule is a molecule comprising from 2 to 20 (typical for a peptide) amino acids. In one embodiment, a proteinaceous molecule is a molecule comprising from 21 to 1.000 (typical for a polypeptide, a protein, a protein domain, such as an antibody, VHH, a Fab, an scFv, a ligand for a receptor such as EGF) amino acids. Preferably, the amino-acid residues are (typically) linked via (a) peptide bond(s). As disclosed herein, said amino-acid residues are or comprise (modified) (non-)natural amino acid residues.

[0127] As used herein, the term “antibody or a binding fragment thereof or a binding domain thereof’ refers to a polypeptide that includes at least one immunoglobulin variable domain or at least one antigenic determinant, e.g., paratope that specifically binds to an antigen. In some embodiments, an antibody is a full-length antibody. In some embodiments, an antibody is a chimeric antibody. In some embodiments, an antibody is a humanized antibody. However, in some embodiments, an antibody is a Fab fragment, a F(ab’) fragment, a F(ab')2 fragment, a Fv fragment or a scFv fragment. In some embodiments, an antibody is a nanobody derived from a camelid antibody or a nanobody derived from a shark antibody. In some embodiments, an antibody is a diabody. In some embodiments, an antibody comprises a framework having a human germline sequence, for example an antibody comprising camelid VHH domain(s) and human Fc (VHH-Fc (dimerized) antibody). In another embodiment, an antibody comprises a heavy chain constant domain selected from the group consisting of IgG, lgG1, lgG2, lgG2A, lgG2B, lgG2C, lgG3, lgG4, IgAI, lgA2, IgD, IgM, and IgE constant domains. In some embodiments, an antibody comprises a heavy (H) chain variable region (abbreviated herein as VH), and / or (e.g., and) a light (L) chain variable region (abbreviated herein as VL). In some embodiments, an antibody comprises a constant domain, e.g., an Fc region. An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences and their functional variations are known. With respect to the heavy chain, in some embodiments, the heavy chain of an antibody described herein can be an alpha (a), delta (D), epsilon (e), gamma (g) or mu (m) heavy chain. In some embodiments, the heavy chain of an antibody described herein can comprise a human alpha (a), delta (D), epsilon (e), gamma (g) or mu (m) heavy chain. In a particular embodiment, an antibody described herein comprises a human gamma 1 CH1, CH2, and / or (e.g., and) CH3 domain. In some embodiments, the amino acid sequence of the VH domain comprises the amino acid sequence of a human gamma (g) heavy chain constant region, such as any known in the art. Non-limiting examples of human constant region sequences have been described in the art, e.g., see U. S. Pat. No. 5,693,780 and Kabat E A etal., (1991) supra. In some embodiments, the VH domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the variable chain constant regions provided herein. In some embodiments, an antibody is modified, e.g., modified via glycosylation, phosphorylation, sumoylation, and / or (e.g., and) methylation. In some embodiments, an antibody is a glycosylated antibody, which is conjugated to one or more sugar or carbohydrate molecules. In some embodiments, the one or more sugar or carbohydrate molecule(s) is / are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and / or (e.g., and) phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecule(s) is / are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecule(s) is / are a branched oligosaccharide or a branched glycan. In some embodiments, the one or more sugar or carbohydrate molecule include(s) a mannose unit, a glucose unit, an N-acetylglucosamine unit, an A / -acetylgalactosamine unit, a galactose unit, a fucose unit, or a phospholipid unit. In some embodiments, an antibody is a construct that comprises a polypeptide comprising one or more antigen binding fragments of the disclosure linked to a linker polypeptide or an immunoglobulin constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Examples of linker polypeptides have been reported (see e.g., Holliger, P, etal. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., etal. (1994) Structure 2:1121-1123). Still further, an antibody may be part of a larger immuno-adhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immuno-adhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal poly-histidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058).

[0128] The term “single domain antibody”, or “sdAb”, in short, or ‘nanobody’, has its regular scientific meaning and here refers to an antibody fragment consisting of a single monomeric variable antibody domain, unless referred to as more than one monomeric variable antibody domain such as for example in the context of a bivalent sdAb, which comprises two of such monomeric variable antibody domains e.g. in tandem. A bivalent nanobody is a molecule comprising two single domain antibodies targeting epitopes on molecules present at the extracellular side of a cell, such as epitopes on the extracellular domain of a cell surface molecule that is present on the cell. Preferably the cell-surface molecule is a cell-surface receptor. A bivalent nanobody is also named a bivalent single domain antibody. Preferably the two different single domain antibodies are directly covalently bound or covalently bound through an intermediate molecule that is covalently bound to the two different single domain antibodies. Preferably the intermediate molecule of the bivalent nanobody has a molecular weight of less than 10,000 Dalton, more preferably less than 5000 Dalton, even more preferably less than 2000 Dalton, most preferably less than 1500 Dalton.

[0129] The term “antibody-oligonucleotide conjugate” or “AOC” has its regular scientific meaning and here refers to any conjugate of an antibody such as an IgG, a Fab, an scFv, an immunoglobulin, an immunoglobulin fragment, one or multiple VHdomains, one or multiple single-domain antibodies, one or multiple VHH, one or more camelid VH, etc., and any polynucleotide (oligonucleotide) molecule that can exert a therapeutic effect when contacted with cells of a subject such as a human patient, such as an oligonucleotide selected from a natural or synthetic string of nucleic acids encompassing DNA, modified DNA, RNA, mRNA, modified RNA, synthetic nucleic acids, presented as a single-stranded molecule or a double-stranded molecule, such as a BNA, an antisense oligonucleotide (ASO, AON), a short or small interfering RNA (siRNA; silencing RNA), an anti-sense DNA, anti-sense RNA, etc.

[0130] As used herein, the term “covalently linked” refers to a characteristic of two or more molecules being linked together via at least one covalent bond, i.e. directly, or via a chain of covalent bonds, i.e. via a linker comprising at least one or more atoms.

[0131] The term “moiety” as used herein will usually refer to a molecule that is bound, linked, conjugated to a further molecule, linker, assembly of molecules, etc., and therewith forming part of a larger molecule, conjugate, assembly of molecules. Typically, a moiety is a first molecule that is covalently bound to a second molecule (second moiety), involving one or more chemical groups initially present on the first and second molecules. For example, when a saponin molecule is covalently linked via at least one linker to one or more GalNAc molecules, both the saponin molecule is a saponin moiety in the formed saponin-GalNAc conjugate and the GalNAc molecule(s) is / are a moiety / moieties in said conjugate. For example, a nucleic acid such as an antisense oligonucleotide, that is conjugated to an endocytic receptor binding ligand such as an antibody or one or more GalNAc molecules, is a nucleic acid moiety in the nucleic acid - GalNAc conjugate or in the nucleic acid - antibody conjugate.

[0132] As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0133] The terms first, second, third and the like in the description and in the claims, are used for distinguishing between for example similar elements, compositions, constituents in a composition, or separate method steps, and not necessarily for describing a sequential or chronological order. The terms are interchangeable under appropriate circumstances and the disclosed herein embodiments can operate in other sequences than described or illustrated herein, unless specified otherwise.

[0134] The term “comprising”, used in the claims, should not be interpreted as being restricted to for example the elements or the method steps or the constituents of a compositions listed thereafter; it does not exclude other elements or method steps or constituents in a certain composition. It needs to be interpreted as specifying the presence of the stated features, integers, (method) steps or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps or components, or groups thereof. Thus, the scope of the expression “a method comprising steps A and B” should not be limited to a method consisting only of steps A and B, rather with respect to the present disclosure, the only enumerated steps of the method are A and B, and further the claim should be interpreted as including equivalents of those method steps. The scope of the expression “a method comprising steps A and B” encompasses a method consisting of steps A and B, unless the context clearly requires otherwise. Thus, the scope of the expression “a composition comprising components A and B” should not be limited to a composition consisting only of components A and B, rather with respect to the present disclosure, the only enumerated components of the composition are A and B, and further the claim should be interpreted as including equivalents of those components. The scope of the expression “a composition comprising components A and B” encompasses a composition consisting of components A and B, unless the context clearly requires otherwise.

[0135] In addition, reference to an element or a component by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element or component are present, unless the context clearly requires that there is one and only one of the elements or components. The indefinite article "a" or "an" thus usually means "at least one".

[0136] The use of terms in brackets in the text, with the exception of chemical and / or mathematical formulae, usually means that the term within brackets specifies a possible option or a possible meaning and should thus not be considered limiting.

[0137] The embodiments as described herein can operate in combination and cooperation, unless specified otherwise. Furthermore, the various embodiments, although referred to as “preferred” or “e.g.” or “for example” or “in particular” and the like are to be construed as exemplary manners in which the disclosed herein concepts may be implemented rather than as limiting.

[0138] For all Figures, “Figure” and “Fig.” refer to the same. BRIEF DESCRIPTION OF THE DRAWINGS

[0139] Figure 1: Structure of trivalent GalNAc-oligonucleotide, for example trivalent GalNAc-siRNA also referred to as GN3-siRNA, or in a specific example GN3-siTTR.

[0140] Figure 2A, 2B, 2C, 2D, 2E: Depot release by saponin components in vivo: efficacy and durability of effect (here, serum TTR protein reduction) of co-administration of saponin component (here, GN3-SC-SO1861) and an oligonucleotide, here GN3-siTTR. GN3-siTTR was always administered on day 0 and GN3-SC-SO1861 was administered at the timepoints indicated by the arrow; n = 6 mice in all groups except vehicle, where n = 3; shown is mean TTR serum level ± SD. (A) GN3-siTTR administered together with saponin component at day 0, (B) GN3-siTTR administered at day 0, saponin component administered at day 7 (arrow), (C) GN3-siTTR administered at day 0, saponin component administered at day 14 (arrow), (D) GN3-siTTR administered at day 0, saponin component administered at day 21 (arrow), (E) GN3-siTTR administered at day 0, saponin component administered at day 28 (arrow).

[0141] Figure 3A, 3B, 3C, 3D, 3E: Depot release of liver targeted oligonucleotides, here GN3-siTTR, by saponin components in vitro measured by reduction in Ttr RNA levels. (A) Incubation scheme of depot release from murine primary hepatocytes (MPH) with saponin components with 6 hr loading of GN3-siTTR, wash out and subsequent release by saponin components (or PBS for a control condition), (B) Ttr RNA quantification following 6 hr loading with GN3-siTTR (wherein the oligonucleotide was designed as an oligonucleotide with advanced enhanced stabilization chemistry (AdvESC) with DV18 chemistry) followed by different saponin components (or PBS) for 24 hrs (C) Ttr RNA quantification following 6 hr loading with GN3-siTTR (wherein the oligonucleotide was designed as an oligonucleotide with advanced enhanced stabilization chemistry (AdvESC) with DV18 chemistry) followed by different saponin components (or PBS) for 48 hrs, (D) Incubation scheme of depot release with saponin components with a 6 hr loading of GN3-siTTR, then wash and resting period in medium, followed by short saponin component pulse for 3 hrs (top scheme) or 6 hrs (bottom scheme), followed by wash and resting period before analysis, (E) Ttr RNA quantification following loading of GN3-siTTR, wash and resting period, and then saponin component pulse for 6 hrs for release, before analysis after an additional 24 hrs.

[0142] Figure 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H, 4I, 4J, 4K, 4L: Depot release in various cancer cell lines by saponin components in vitro: efficacy and durability enhancement of STAT3 ASO by saponin components measured by STAT3 RNA levels. (A) Incubation scheme of co-administration of STAT3 ASO with saponin components for 48 hrs (positive control), (B) Incubation scheme of depot release with saponin components, by first loading cells with STAT3 ASO followed by wash and release incubation with saponin components for 48 hrs, (C) Incubation scheme of loading with STAT3 ASO, followed by wash and a 6 hr resting period in medium, followed by saponin components for 48 hrs, (D) Incubation scheme of loading with STAT3 ASO, followed by wash and a 24 hr resting period in medium, followed by saponin components for 48 hrs, (E - H) Co-treatment in an EGFR-high expressing (EGFR++) epidermoid carcinoma cell line (A431): STAT3 RNA quantification following co-administration of STAT3 ASO and saponin components (or PBS): (E) for co-administration of STAT3 ASO and saponin component as described in Figure 4A, (F) for STAT3 ASO loading followed by saponin component as described in Figure 4B, (G) for STAT3 ASO loading, 6 hrs resting and after that saponin component as described in Figure 4C, (H) for STAT3 ASO loading, 24 hrs resting and after that saponin component as described in Figure 4D. (I - L): Co-treatment in a metastatic melanoma cell line (A2058), lacking the EGFR receptor (EGFR-), the target for cetuximab: STAT3 RNA quantification following the timed release by different saponin component treatments (or PBS): (I) for co-administration of STAT3 ASO and saponin component as described under (Figure 4A), (J) for STAT3 ASO loading followed by saponin component as described in Figure 4B, (K) for STAT3 ASO loading, 6 hrs resting and after that saponin component as described in Figure 4C, (L) for STAT3 ASO loading, 24 hrs resting and after that saponin component as described in Figure 4D.

[0143] Figure 5A, 5B: Visualized gradual depot release by different saponin components in A431 cells. (A) Incubation scheme for imaging gradual depot release with saponin components, by first loading cells with Cy5-labeled cetuximab (Cet-Cy5) for 48 hrs, followed by wash and resting phase in medium for 24 hrs, before release trigger with saponin components, or PBS as control, (B) Quantification of Cy5-release by measuring total integrated Cy5-fluorescence intensity under influence of different saponin components.

[0144] Figure 6A, 6B, 6C, 6D, 6E, 6F, 6G: Depot release of proteinaceous payload (toxin) in epidermoid carcinoma cells by saponin components: in vitro efficacy and durability enhancement of an immunotoxin anti-CD71 -saporin (aCD71-SPRN or protein toxin) under influence of saponin components of in A431 cells data. (A) Incubation scheme of co-administration of aCD71-SPRN with saponin components (positive control), (B) Incubation scheme of timed depot release with saponin components. After 24 hr loading with aCD71-SPRN, followed by wash, saponin components were added and cells were analyzed after 48 hrs, (C) Incubation scheme of timed depot release with saponin components after short resting phase: after 24 hr loading with aCD71-SPRN, followed by wash and a 6 hr resting period in medium, saponin components were added and cells were analyzed after 48 hrs, (D) Incubation scheme of depot release with saponin components after longer resting phase: after 24 hr loading with aCD71-SPRN, followed by wash and a 24 hr resting period in medium, saponin components were added and cells were analyzed after 48 hrs, (E) Relative cell viability for the different treatment conditions (Figure 6A-6D) for a concentration range of saponin component 1 (SO1861) at a fixed concentration of 5 pM aCD71-SPRN in A431 cells, (F) Relative cell viability for the different treatment conditions (Figure 6A-6D) for a concentration range of saponin component 2 (SO1861-AH-Maleimide-Block, also referred to as SO1861-AH-Block, a saponin molecule according to formula (V)) at a fixed concentration of 5 pM aCD71-SPRN in A431 cells, (G) Relative cell viability for the different treatment conditions (Figure 6A-6D) for a concentration range of saponin component 3 (cetuximab-AH-SO1861) at a fixed concentration of 5 pM aCD71-SPRN in A431 cells.

[0145] Figure 7A, 7B: Depot release of proteinaceous payload in a metastatic melanoma cells under influence of saponin components: in vitro efficacy and durability enhancement of of immunotoxin anti-CD71 -saporin (aCD71-SPRN) under influence of saponin components in A2058 cells data. (A) Relative cell viability for the different treatment conditions (Figure 6A-6D) for a concentration range of saponin component 1 (SO1861; see the formula of SO1861 in Figure 8) at a fixed concentration of 5 pM aCD71 -SPRN in A2058 cells, (B) Relative cell viability for the different treatment conditions (Figure 6A-6D) for a concentration range of saponin component 2 (SO1861-AH-Maleimide-Block, also referred to as SO1861-AH-Block, a saponin molecule according to formula (V) at a fixed concentration of 5 pM aCD71-SPRN in A2058 cells.

[0146] Figure 8: Synthesis and chemical structure of SO1861-AH-azide

[0147] Figure 9: Synthesis and chemical structure of SO1861-SC-azide

[0148] Figure 10: Synthesis and chemical structure of GN3-AH-SO1861

[0149] Figure 11: Synthesis and chemical structure of GN3-SC-SO1861

[0150] Figure 12A, Figure 12B, Figure 12C: Efficacy enhancement by saponin components at different co-doses (of either 0, 0.3, 1 or 3 mg / kg GN3-AH-SO1861, also referred to as GN3-saponin, or GN3-SPT) an oligonucleotide of 0.3 mg / kg GN3-siAT3 in the non-human primate (NHP). Shown are AT3 serum levels. (A) GN3-siAT3 administered alone, or together with saponin component (at 0.3 mg / kg) at day 0, (B) GN3-siAT3 administered alone at day 0, or together with saponin component (at 1 mg / kg) administered at day 0, (C) GN3-siAT3 administered alone at day 0, or together with saponin component administered (3 mg / kg) at day 0. In all cases, no marked AT3 serum protein reduction is observed when GN3-siAT3 is administered alone, but in combination with saponin component, efficacy is observed as AT3 protein reduction: here, already 0.3 mg / kg saponin component show marked efficacy increase that is not further increased by a higher saponin compound dose, indicating that already 0.3 mg / kg is at or above a maximum release efficacy dose.

[0151] Figure 13: Depot release by saponin components in vivo in NHP: efficacy and durability of effect with co-administration of saponin component (here, GN3-AH-SO1861, also referred to as GN3-saponin, or GN3-SPT) and an oligonucleotide, here GN3-siAT3. GN3-siAT3 was administered on day 0 in n = 2 NHPs; in one NHP, 0.1 mg / kg saponin component (in the form of GN3-saponin) was administered at day 28 (indicated by the arrow); shown are AT3 serum levels. Only when the saponin component was added, a marked decrease in AT3 serum levels was observed, and to levels considered clinically meaningful (between 15 - 35%, also referred to as the AT3 target range).

[0152] Figure 14. In vivo co-administration of ligand-saponin with ligand-ASO#02 dose-dependently enhances the ASO-effector potency in kidney cortex and medulla. Relative ApoB expression analysis in mouse cortex and medulla 72 hr (A) and 336 hr (B) after intravenous administration of vehicle (DPBS), ASO#02 alone, ligand-ASO#02 alone or co-administration of ligand-ASO#02 with ligand-saponin. Data are shown as mean ± SEM, n=2-3. Legend to be read from top to bottom, which corresponds to the bars from left to right (for both (A) cortex and (B) medulla).

[0153] Figure 15. Administration of ligand-saponin-ASO#02 enhances the effector potency compared to ligand-ASO#02 treatment (without saponin component) in kidney cortex and medulla. Relative ApoB expression analysis in mouse cortex and medulla 72 hr (A) and 336 hr (B) after intravenous administration of vehicle (DPBS), ligand-ASO#02 or ligand-saponin-ASO#02. Data are shown as mean ± SEM, n=2-3.

[0154] Figure 16. Delayed ligand-saponin administration to ligand-siRNA causes an efficacy enhancement in NHP kidneys. Relative SERPINC1 expression in NHP kidneys at day 45 after subcutaneous administration of (i) ligand-siRNA (at day 1) or (ii) ligand-siRNA (at day 1) followed by ligand-saponin (at day 28). Data are shown as mean normalized expression (relative to TBP and DDX3X) ± SEM, n=1.

[0155] Figure 17. Dose dependent efficacy enhancement of ligand-saponin co-administration to ligand-siRNA in NHP kidneys. Relative SERPINC1 expression analysis in NHP kidneys after 11-17 days of subcutaneous co-administration of ligand-saponin and ligand-siRNA, or 45 days of subcutaneous administration of ligand-siRNA alone. Data are shown as average mean normalized expression (relative to TBP and DDX3X) ± SD, n=1-2.

[0156] Figure 18. Administration of ligand-saponin-PMO enhances the effector potency compared to ligand-PMO treatment in kidney cortex and medulla. Average DMD exon 23 skipping in (A) mouse cortex and (B) medulla after intravenous administration of vehicle (DPBS), ligand-PMO or ligand-saponin-PMO. Data are shown as mean ± SEM, n=2-3.

[0157] Figure 19. Non-kidney cell-specific ligand (GalNAc) conjugation to ASO#01 (in ligand-ASO#01) does not improve the ASO efficacy in HEK293-FT kidney cells compared to ASO#01 alone. Relative HSP27 expression in HEK293-FT cells upon treatment with a titration of ASO#01 or ligand-ASO#01.

[0158] Figure 20. Both saponin-compound and ligand-saponin co-administration enhance (ligand-)ASO#01 efficacy in HEK293-FT cells. (A) Relative HSP27 expression in HEK293-FT cells upon treatment with a titration of saponin-compound or ligand-saponin co-dosed with 100 nM ASO#01. (B) Relative HSP27 expression in HEK293-FT cells upon treatment with a titration of saponin-compound or ligand-saponin co-dosed with 100 nM ligand-ASO#01.

[0159] Figure 21. Saponin co-administration enhances ASO#01 and ligand-ASO#01 efficacy in HEK293-FT cells. Relative HSP27 expression in HEK293-FT cells upon treatment with a titration of saponin co-dosed with 100 nM ASO#01 or ligand-ASO#01.

[0160] Figure 22. Non-targeted ASO#02-saponin shows a high potency in mouse liver in vivo. Relative ApoB 100 expression in mouse livers after intravenous administration of vehicle (DPBS), ASO#02 alone or ASO#02-saponin. Data are shown as mean ± SEM, n=3-6.

[0161] Figure 23. Saponin component does not activate NF-KB pathway. (A) Neither the saponin component ‘Cetux-DoL4 SPT’ (also referred to as ‘Cetuximab-SPT DoL4-l’), nor the control monoclonal anti-EGFR antibody cetuximab alone (‘Cetux’), induce activation of the NF-KB pathway in HEK293-FT cells incubated with the compounds for 5-6 hours. Cetux-DoL4 SPT is a covalent conjugate of cetuximab with 4 saponin moieties covalently bound to it as described in detail in international application W02020126627, page 98, line 1-3 and line 22-25, page 117, line 9-16, page 161, line 10-19, page 148, line 1-20, in conjunction with the figures 50, 60, 63 and 66A. “ug / ml” is dosage in microgram per millilitre. (B) The NF-KB pathway is activated in HEK293-FT cells in response to TNF-alpha (‘TNFa’), used as a positive control, whereas the negative control, cell-culture medium (‘Ct NT’) does not induce NF-KB pathway activation. (C) HEK293-FT cells transfected with the NF-KB-inducible luciferase reporter plasmid pGL4.32 are exposed to an increasing-concentration series of TNFa in the cell-culturing medium, resulting in activation of the NF-KB pathway. Read-out: luciferase luminescence following activated NF-KB-dependent induction of the luciferase reporter. (D) HEK293-FT cells are exposed to saponin component in the cell-culture medium, herein provided as a saponin moiety covalently conjugated with a linker via a hydrazone bond involving the C-23 aldehyde function of the saponin (‘SPT-AH (block)’), which does not result in activation of the NF-KB pathway; “uM” is dosage in micromoles. The synthesis of the saponin component is described in international application W02020126627, page 31, line 21-25, page 98, line 1-3, page 117, line 9-16, page 148, line 1-29, in conjunction with the figures 60, 61 B, 63 and 66A. (E) Neither the saponin component ‘SO1861’ (a saponin molecule), nor the saponin component ‘SPT-AH-Mal’ (conjugate of SO1861 with EMCH) induce activation of the NF-kB pathway in HEK293-FT cells incubated with the compounds for 5-6 hours. (F) The saponin components Cetuximab-SPT DoL4-l (a first batch), Cetuximab-SPT DoL4-ll (a second batch) and ‘GalNac-SPT DoL 1 (a conjugate comprising a GalNAc moiety and comprising a SO1861 moiety as described in international application WO2021261992, Figure 3 in conjunction with the Examples section relating to its synthesis), tested at a concentration of 10 microgram / ml dose, did not activate the NF-kB pathway, neither did Cetuximab (‘Cetuximab-hlgG1)’ and buffer control ‘Ct NT’, whereas the NF-KB pathway is activated in the HEK293-FT cells in response to a dose of 10 ng / ml TNFa (‘TNFa’). (G) None of the saponin components ‘OKT9-SPT DoL4’, an antibody binding to transferrin receptor (TfR) conjugated with SO1861, ‘SO1861’ (a saponin molecule) ‘SPT-AH-Mal’ (conjugate of SO1861 with EMCH), tested at a concentration of 10 microgram / ml dose, did activate the NF-kB pathway, neither did the anti-TfR antibody OKT9 and buffer control ‘Ct NT’, whereas the NF-KB pathway is activated in the HEK293-FT cells in response to a dose of 10 ng / ml TNFa (‘TNFa’).

[0162] Figure 24. Administration of different ligand-saponin-PMO conjugates enhances the effector potency compared to PMO and ligand-PMO treatment in kidney, heart and liver in a humanized, disease relevant mouse model. Average exon skipping after intravenous administration of vehicle (DPBS), PMO, ligand-PMO or ligand-saponin-PMO. Data are shown as mean ± SEM, n=4 per group. Shown is exon23 skip in heart, liver, kidney (cortex) and kidney (medulla) of CD-1 mice treated with Vehicle, aCD71- PMO or aCD71-SPT-PMO. Exon skip was determined by ddPCR at D14 after dosing.

[0163] Figure 25. Exon skip in hDMDdel52 / mdx mice, in heart, liver, kidney.

[0164] Figure 26. 3M12-lgG1 antibody binding to endogenous hTfR1 in HEK293FT cells.

[0165] Figure 27: (A) Relative MALAT1 mRNA expression in HEK293FT cells after treatment with a titration of hCD71-SPT (also shown as aCD71-SPT) alone or hCD71-SPT + 200 nM ASO-2, for 72 hr. (B) Relative cell viability of HEK293FT cells after treatment with a titration of hCD71-SPT alone or hCD71-SPT + 200 nM ASO-2, for 72 hr.

[0166] Figure 28: Relative MALAT1 mRNA expression in HEK293FT cells after treatment with a titration of ASO-2, for 72 hr. Figure 29: (A) Relative STAT3 mRNA expression in HEK293FT cells after treatment with a titration of hCD71-SPT (also shown as aCD71-SPT) alone or hCD71-SPT + 200 nM ASO-1, for 72 hr. (B) Relative cell viability of HEK293FT cells after treatment with a titration of hCD71-SPT alone or hCD71-SPT + 200 nM ASO-1, for 72 hr.

[0167] Figure 30: Relative STAT3 mRNA expression in HEK293FT cells after treatment with a titration of ASO-1, for 72 hr.

[0168] Figure 31: (A) Relative STAT3 mRNA splice switching in HEK293FT cells after treatment with a titration of hCD71-SPT (also shown as aCD71-SPT) alone or hCD71-SPT + 1 pM PMO-1, for 72 hr. (B) Relative cell viability of HEK293FT cells after treatment with a titration of hCD71-SPT alone or hCD71-SPT + 1 pM PMO-1, for 72 hr.

[0169] Figure 32: Relative STAT3 mRNA splice switching in HEK293FT cells after treatment with a titration of PMO-1, for 72 hr.

[0170] Figure 33: Tolerability of GN3-SPT in mice at exaggerated doses: histopathology. Mice received a single subcutaneous dose of vehicle [DPBS] or GN3-SPT. (A) Liver histopathology. (B) Kidney histopathology. See Table S17 for clinical observations; gross necroscopy and histopathology description.

[0171] DETAILED DESCRIPTION

[0172] Therapeutic use of oligonucleotides including small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs, AONs) is appreciated as holding great promise for the treatment of numerous diseases. Their ability to specifically recognise and bind in a sequence-specific manner to an intracellular nucleic acid and thereby modulate gene expression via a range of processes including RNA interference (RNAi), target degradation by RNase H-mediated cleavage, splicing modulation, ADAR-based RNA editing, non-coding RNA inhibition, gene activation, etc. (Roberts et al., 2020), makes the oligonucleotide-based medicines one of the most flexible and pragmatic therapeutic modalities for treating any disorders and conditions associated with a specific genetic target.

[0173] Till date, several oligonucleotide-based drugs, encompassing various oligonucleotide chemistries, have gained commercial approval from the U. S. Food and Drug Administration (FDA), including ASOs, siRNAs, aptamers, and mixed single / double-stranded DNA. The existing oligonucleotide-based therapies related to the treatment of elevated levels of plasma low-density lipoprotein cholesterol (LDL-C), triglycerides (TG) and / or fibrinogen includes: mipomersen and inclisiran. While oligonucleotide-based therapies under development would include: zodasiran (GalNAc conjugated ARO-ANG3 ds-siRNA), solbinsiran (GalNAc conjugated Dicer-substrate siRNA; LY3561774), vupanorsen (GalNAc conjugated ASO), CiVi007 (cepadacursen sodium), AZD-8233 (ION-449; IONIS-AZ4-2.5-LRx), ALNAAT-02 (GalNAc-siRNA conjugate), belcesiran (DCR-A1AT) -siRNA / GalXC-siRNA, fazirsiran (TAK-999 / ARO-AAT; RNA interference (RNAi), WVE-006. Despite having huge potential, oligonucleotide therapeutics are known to suffer from an extremely inefficient cellular uptake, which prevents them from effectively reaching the cytoplasmic and / or the nuclear intracellular compartments where they are supposed to act upon their generic targets and leading to insufficient therapeutic efficacy. For example, vupanorsen was discontinued because the magnitude of non-HDL-C and TG reduction was not sufficient to continue it further.

[0174] It is further known that less than 2% from a therapeutic dose of an oligonucleotide drug becomes correctly internalised, possibly due to an estimated 98% thereof being retained within the endosomal compartment and eventually degraded in the lysosomes (Gilleron et al., 2013). However, for the renal cells, this leads to oligonucleotide drug accumulation raising safety concerns (Echevarria & Goyenvalle, 2022; Crooke etal., 2021). Therefore, merely increasing the oligonucleotide drug dose would not solve the low efficacy issue, rather it would further exacerbate the accumulation related toxicity. For example, nusinersen, inotersen, golodirsen, viltolarsen, casimersen and mipomersen comes with the black-box warning for hepatotoxicity, while nephrotoxicity data obtained during their preclinical studies and clinical trials mentions renal tubular degeneration, glomerulonephritis, and increased urinary protein levels (Wu et al., 2022).

[0175] Hence, a frequent administration is most often adapted for the delivery of a therapeutically effective dose of the oligonucleotide drug. Apparently, which further leads to injection-site reactions such as pruritus, redness, or swelling. Additional toxicological properties of oligonucleotides have been comprehensively and extensively summarised (Blom etal., 2022; Andersson etal., 2019; Frazier 2015). For example: inclisiran, AZD-8233 and belcesiran also demonstrated various side-effects, including but not limited to neurocognitive events, new-onset diabetes, hemorrhagic stroke (Zhou et al., 2024), gastroenteritis, back pain, nasopharyngitis, death from cardiovascular causes, fatal or nonfatal stroke, diabetes mellitus, bronchitis, dyspnea, upper respiratory tract infections, arthralgia, upper respiratory tract infection (Blom et al., 2022).

[0176] Consequently, the present disclosure addresses the existing need of increasing the efficacy, improving the bioavailability and a long term-effect of oligonucleotide-based therapeutics. Solutions are now provided by the inventors, allowing to lower the doses and improve dosing regimen of such oligonucleotide-based therapeutics, to minimise the risks of associated side-effects.

[0177] The available strategies on improving the delivery and efficacy of oligonucleotide-based medicament, thereby enhancing target engagement, and development of an efficient intracellular delivery platform are based on a specific group of penta-cyclic triterpene saponins comprising an aglycone core of 12,13-dehydrooleanane type, believed to have endosomal escape-enhancing (EEE) properties. Saponins of this specific type were characterised and reported in e.g. W02020126620 as possessing an EEE activity towards various antibody-drug conjugates (ADCs) in several cancer cell lines. This group of saponins was further disclosed in W02020126626, W02020126627, WO2020126620, WO2020126627, WO2020126064, WO2020126604, WO2020126600, and W02020126609 as being capable of dramatically improving cancer treatment using oligonucleotide therapeutics, as demonstrated by enhanced by the saponin silencing of the HSP27 gene transcript, using HSP27-specific BNA-based oligonucleotide in different tumour models and cell lines. The EEE activity of such saponins was further shown in W02020126610, WO2021261992, WO2021261992, WO2022055351, WO2022265493 as potentiating the effects of antisense oligonucleotides in the liver cells following GalNAc-mediated targeting of these cells with different therapeutic combinations of the oligonucleotide-based effector and the saponin. Lastly, advantageous effects of such saponins were also shown in WO2023121444, WO2023121445, and WO2023121446 to potentiate exon-skipping effects of therapeutic oligos in differentiated muscle cells.

[0178] However, it was never before observed or demonstrated that these oligonucleotide-based medicaments could be delivered into human kidney and / or liver cells in vivo, and can be efficacious for use in the treatment of a cardiovascular disease.

[0179] As for the first time documented in the accompanying examples and outlined in the current specification, following systemic administration in vivo, a therapeutic combination of an oligonucleotide-based medicament and a penta-cyclic triterpene saponin comprising an aglycone core of 12,13-dehydrooleanane type, not only underwent a successful co-delivery into the kidney and / or into the liver cells without becoming fully eliminated by renal filtration or becoming unproductively trapped within kidney cells, but also it visibly resulted in sufficient oligonucleotide therapeutic bioavailability to induce its use in the treatment and / or prevention and / or amelioration of a cardiovascular disease, without resulting in apparent signs of oligonucleotide-associated known adverse-effects.

[0180] The presented examples and embodiments herein are in part an unexpected observation made during in vivo targeted hepatic delivery experiments with low dosages of antisense oligonucleotide (ASO) effectors together with the EEE saponins. Namely, it was observed that in the presence of the EEE saponins, intravenously administered liver-targeted therapeutic oligonucleotides were not only effectively silencing the expression of the target nucleic acid in the liver cells, but also in the kidney cells, and further used in the treatment, amelioration and / or prevention of a cardiovascular disease (e.g. bleeding disorder, thrombosis). This serendipitous and surprising observation indicated that the oligonucleotide therapeutic ( / .e. the oligonucleotide-based medicament) that was provided at a very low dose, was not only taken up by NHP kidney and / or liver cells, but also that in the presence of the EEE saponin, it was also effectively released after its uptake into the human cells’ cytoplasm where it could act on its target mRNA instead of remaining unproductively trapped in the organ compartments. As it is demonstrated in the in vivo experiments performed in mice and in a non-human primate (NHP), as shown below, the same was observed for therapeutic combinations of non-targeted-oligonucleotide-saponin conjugates and in a co-delivery setting involving a targeted-oligonucleotide-based medicament and a separate targeted EEE saponin component.

[0181] Disclosed herein are the therapeutic combination, pharmaceutical formulations thereof and the pharmaceutical kits thereof, as well as methods of treatment involving their administration, as well as use of a saponin component and an oligonucleotide-based medicament for the manufacture of a therapeutic combination as a medicament for use in a method treatment and / or prevention and / or amelioration of a cardiovascular diseases, which comprise an oligonucleotide-based medicament that acts on an intracellular nucleic acid target in the kidney and / or liver cells, and an endosomal-escape enhancing (EEE) saponin component that enables the effective release of said oligonucleotide-based medicament inside of the target cells, thus allowing its use at a much lower and safer dose than it would be required in the absence of the EEE saponin component. As a consequence, the presented herein combinations of oligonucleotide-based medicaments and saponins enable effective modulation of gene expression in the kidney and / or liver cells and, therefore, open new treatment options for cardiovascular diseases.

[0182] The innovative concepts as presented herein will be described in relation to particular aspects and embodiments of the disclosure, which should be regarded as descriptive and not as limiting the matter as described in the claims. The aspects and / or the embodiments as described herein can operate in combination and cooperation, unless specified otherwise. While the disclosed herein innovative concepts are described with reference to these aspects and / or embodiments, it is contemplated that alternatives, modifications, permutations and equivalents thereof will become apparent to one having ordinary skill in the art upon reading the specification in view of the drawings and / or graphs. The disclosed matter is not limited in any way to specific embodiments as illustrated herein, and changes to these embodiments should be construed as acceptable as long as they can be made without departing from the scope as defined by the appended claims.

[0183] A therapeutic combination

[0184] In one aspect, the present disclosure relates to a therapeutic combination (suitable) for treatment and / or prevention and / or amelioration of a cardiovascular disease comprising:

[0185] a. an oligonucleotide-based medicament adapted to target and bind to a nucleic acid molecule present in the cytosol of a human cell and / or present in the nucleus of a human cell; b. a saponin component comprising a penta-cyclic triterpene saponin, comprising an aglycone core of 12,13-dehydrooleanane type.

[0186] In one aspect, the present disclosure relates to a therapeutic combination for treatment and / or prevention and / or amelioration of a cardiovascular disease comprising:

[0187] a. an oligonucleotide-based medicament adapted to target and bind to a nucleic acid molecule present in the cytosol of a human cell and / or present in the nucleus of a human cell; b. a saponin component comprising a penta-cyclic triterpene saponin, comprising an aglycone core of 12,13-dehydrooleanane type;

[0188] wherein the saponin component and the oligonucleotide-based medicament are for delivery to the human cell wherein said human cell is a cell of the kidney and / or is a liver cell such as a hepatocyte.

[0189] In one aspect, the present disclosure relates to a therapeutic combination comprising: a. an oligonucleotide-based medicament adapted to target and bind to a nucleic acid molecule present in the cytosol of a human cell and / or present in the nucleus of a human cell; b. a saponin component comprising a penta-cyclic triterpene saponin, comprising an aglycone core of 12,13-dehydrooleanane type;

[0190] for use in treatment and / or prevention and / or amelioration of a cardiovascular disease; wherein the treatment and / or prevention and / or amelioration comprises administration of the therapeutic combination to a human subject in the need thereof;

[0191] wherein the saponin component and the oligonucleotide-based medicament are delivered to the human cell wherein said human cell is a cell of the kidney and / or is a liver cell such as a hepatocyte.

[0192] In another aspect, the present disclosure relates to a method of treatment and / or prevention and / or amelioration of a cardiovascular disease comprising administration of a therapeutic combination to a human subject in the need thereof, wherein the therapeutic combination comprises:

[0193] a. an oligonucleotide-based medicament adapted to target and bind to a nucleic acid molecule present in the cytosol of a human cell and / or present in the nucleus of a human cell; b. a saponin component comprising a penta-cyclic triterpene saponin, comprising an aglycone core of 12,13-dehydrooleanane type;

[0194] wherein the saponin component and the oligonucleotide-based medicament are delivered to the human cell wherein said human cell is a cell of the kidney and / or is a liver cell optionally a hepatocyte.

[0195] In yet another aspect, the present disclosure relates to use of a saponin component and an oligonucleotide-based medicament in the manufacture of a therapeutic combination as a medicament for use in a method of treatment and / or prevention and / or amelioration of a cardiovascular disease; wherein the therapeutic combination comprises:

[0196] a. an oligonucleotide-based medicament adapted to target and bind to a nucleic acid molecule present in the cytosol of a human cell and / or present in the nucleus of a human cell; b. a saponin component comprising a penta-cyclic triterpene saponin, comprising an aglycone core of 12,13-dehydrooleanane type;

[0197] wherein the saponin component and the oligonucleotide-based medicament are delivered to the human cell wherein said human cell is a cell of the kidney and / or is a liver cell, optionally a hepatocyte.

[0198] The references to the therapeutic combination in the above paragraphs are also to be interpreted as references to the pharmaceutical formulation thereof and the pharmaceutical kits thereof being suitable for treatment and / or prevention and / or amelioration of the cardiovascular diseases. The detailed embodiments of the pharmaceutical compositions thereof and the pharmaceutical kits thereof are described further in the description.

[0199] Further, the therapeutic combination as described herein is comprised of an oligonucleotide-based medicament and a saponin component.

[0200] In one embodiment, the therapeutic combination comprises the oligonucleotide-based medicament and the saponin component which are co-formulated in a single pharmaceutical composition.

[0201] In an additional embodiment, the single pharmaceutical composition is selected from any one or more of the following: 2-component saponin formulation, defined as comprising the saponin component comprising a saponin moiety and wherein the 2-component saponin formulation further comprises the oligonucleotide-based medicament that optionally comprises a second ligand recognised by a second endocytic receptor;

[0202] 2-component linker-saponin formulation, defined as comprising the saponin component comprising the saponin moiety and wherein the saponin moiety is covalently conjugated with the linker; wherein the 2-component linker-saponin formulation further comprises the oligonucleotide-based medicament that optionally comprises the second ligand recognised by the second endocytic receptor;

[0203] 2-component targeted-saponin formulation, defined as comprising the saponin component comprising the saponin moiety and wherein the saponin moiety is covalently conjugated with the first ligand, and preferably wherein a non-saponin moiety comprises the linker; and wherein the 2-component targeted-saponin formulation further comprises the oligonucleotide-based medicament that optionally comprises the second ligand;

[0204] 1-component formulation, defined as comprising a saponin-oligonucleotide conjugate, optionally wherein the saponin-oligonucleotide conjugate further comprises the first ligand.

[0205] In another embodiment, the single pharmaceutical composition is a 1-component formulation, defined as comprising the saponin-oligonucleotide conjugate, wherein optionally the saponinoligonucleotide conjugate is provided with a covalently bound targeting ligand, such as the first or second ligand. The single pharmaceutical composition further optionally encompasses a separate targeting ligand-oligonucleotide conjugate (here, a second oligonucleotide-based medicament) and / or a targeting ligand-saponin conjugate (saponin component).

[0206] In an alternative embodiment, the therapeutic combination comprises the oligonucleotide-based medicament and the saponin component which are formulated separately in at least two pharmaceutical formulations, wherein a first pharmaceutical formulation comprises the saponin component and a second pharmaceutical formulation comprises the oligonucleotide-based medicament.

[0207] In an additional embodiment, the therapeutic combination comprises a combination of the first pharmaceutical formulation with the second pharmaceutical formulation, preferably being a 2-component targeted-saponin combination defined as comprising the first pharmaceutical formulation, wherein the saponin component comprises the saponin moiety and wherein the saponin moiety is covalently conjugated with the first ligand, and preferably wherein the non-saponin moiety comprises the linker, and the second pharmaceutical formulation, wherein the oligonucleotide-based medicament possibly comprises the second ligand.

[0208] In another embodiment, (as part of the therapeutic combination) the oligonucleotide-based medicament is packed in a lipid nanoparticle (LNP).

[0209] In an optional embodiment, the co-formulated single pharmaceutical composition or the separately formulated first pharmaceutical formulation and second pharmaceutical formulation, further comprise(s) a pharmaceutically acceptable excipient, pharmaceutically acceptable carrier and / or pharmaceutically acceptable diluent. In a general embodiment, the treatment and / or prevention and / or amelioration comprises administering to a patient in need thereof an amount of the therapeutic combination which delivers a therapeutic effective amount of the oligonucleotide-based medicament to the human cell wherein optionally and preferably said human cell is a cell of the kidney and / or is a liver cell such as a hepatocyte.

[0210] In yet another embodiment, the therapeutic combination is administered via a route of administration selected from: oral, intravenous injection, intravenous infusion, subcutaneous (injection), intraperitoneal (injection), renal artery (injection), retrograde renal vein (injection), renal parenchyma (injection) and retrograde ureteral (injection), portal vein (injection), preferably wherein the route of administration is selected from intravenous administration and subcutaneous administration.

[0211] In one aspect, the present disclosure relates to the therapeutic combination (suitable) for treatment and / or prevention and / or amelioration or to the therapeutic combination for use in treatment and / or prevention and / or amelioration of a cardiovascular disease, comprising:

[0212] a. an oligonucleotide-based medicament adapted to target and bind to a nucleic acid molecule present in the cytosol of a human cell and / or present in the nucleus of a human cell; and b. a saponin component comprising a penta-cyclic triterpene saponin comprising an aglycone core of 12,13-dehydrooleanane type;

[0213] wherein the oligonucleotide-based medicament and the saponin component are either:

[0214] co-formulated in a single pharmaceutical composition optionally comprising any one or more of a pharmaceutically acceptable excipient, diluent and carrier, for simultaneous administration; or formulated separately as at least two pharmaceutical formulations comprising a first pharmaceutical formulation comprising the saponin component and optionally comprising any one or more of a pharmaceutically acceptable excipient, diluent and carrier, and a second pharmaceutical formulation comprising the oligonucleotide-based medicament and optionally comprising any one or more of a pharmaceutically acceptable excipient, diluent and carrier.

[0215] In yet another embodiment, the first pharmaceutical formulation and second pharmaceutical formulation can be administered either simultaneously or sequentially.

[0216] Pharmaceutical formulation

[0217] In one embodiment, the therapeutic combination is in the form of a pharmaceutical formulation. In an embodiment, the treatment and / or prevention and / or amelioration comprises administration of the therapeutic combination to a human subject in the need thereof in the form of a pharmaceutical formulation.

[0218] In one aspect, the present disclosure relates to a pharmaceutical formulation comprising: i. a therapeutic combination comprising:

[0219] i. an oligonucleotide-based medicament;

[0220] ii. a saponin component comprising a penta-cyclic triterpene saponin moiety, comprising an aglycone core of 12,13-dehydrooleanane type; and

[0221] ii. a pharmaceutically acceptable excipient, carrier and / or diluent; for use in a method of treatment, amelioration and / or prevention of a cardiovascular disease; wherein the method comprises administration of the pharmaceutical formulation to a human subject in the need thereof.

[0222] In another aspect, the present disclosure relates to a pharmaceutical formulation comprising: i. a therapeutic combination, wherein the oligonucleotide-based medicament and the saponin component are co-formulated in a single pharmaceutical composition; and

[0223] ii. a pharmaceutically acceptable excipient, carrier and / or diluent;

[0224] for use in a method of treatment, amelioration and / or prevention of a cardiovascular disease; wherein the method comprises administration of the pharmaceutical composition to a human subject in the need thereof;

[0225] wherein the saponin component comprises a penta-cyclic triterpene saponin moiety, comprising an aglycone core of 12,13-dehydrooleanane type.

[0226] In another aspect, the present disclosure relates to a pharmaceutical composition comprising: a therapeutic combination, wherein the oligonucleotide-based medicament and the saponin component are formulated separately in at least two pharmaceutical formulations, wherein:

[0227] (i) a first pharmaceutical formulation comprising the saponin component, and a pharmaceutically acceptable excipient, carrier and / or diluent; and

[0228] (ii) a second pharmaceutical formulations comprising the oligonucleotide-based medicament, and a pharmaceutically acceptable excipient, carrier and / or diluent;

[0229] for use in a method of treatment, amelioration and / or prevention of a cardiovascular disease; wherein the method comprises administration of the pharmaceutical composition to a human subject in the need thereof;

[0230] wherein the saponin component comprising a penta-cyclic triterpene saponin moiety, comprising an aglycone core of 12,13-dehydrooleanane type.

[0231] An embodiment is the pharmaceutical composition comprising the therapeutic combination in the from any one or more of: 2-component saponin formulation; 2-component linker-saponin formulation; 2-component targeted-saponin formulation; and / or 1 -component formulation.

[0232] In a general embodiment, the treatment and / or prevention and / or amelioration comprises administering the pharmaceutical formulation to a patient in need thereof which delivers a therapeutic effective amount of the oligonucleotide-based medicament to the human cell wherein said human cell is optionally and preferably a cell of the kidney and / or is a liver cell, optionally and preferably a hepatocyte.

[0233] In yet another embodiment, the pharmaceutical formulation is administered via a route of administration selected from: oral, intravenous injection, intravenous infusion, subcutaneous (injection), intraperitoneal (injection), renal artery (injection), retrograde renal vein (injection), renal parenchyma (injection) and retrograde ureteral (injection), portal vein (injection), preferably wherein the route of administration is selected from intravenous administration and subcutaneous administration.

[0234] Kit

[0235] The therapeutic combination and / or the pharmaceutical formulations thereof may be supplied in the form of a kit. In a general embodiment, the kit comprises the therapeutic combination and / or the pharmaceutical formulations and printed instructions on how to use said therapeutic combination and / or the pharmaceutical formulations.

[0236] In one aspect, the present disclosure relates to:

[0237] - a first pharmaceutical kit comprising a package comprising:

[0238] a) one or more dosage units comprising the saponin component;

[0239] b) one or more dosage units comprising the oligonucleotide-based medicament; and

[0240] c) printed instructions to use the dosage units comprised in the kit in a therapeutic method of treatment of a cardiovascular disease related to a defect in (the expression of) a gene and / or that is treatable by modulating the expression and / or expression level of a gene, said method of treatment comprising: i) the administration, preferably the repeated administration, of the oligonucleotide-based medicament that is capable of regulating the expression of said gene and / or that is capable of modulating the expression level of said gene,

[0241] ii) the administration, to said subject, of the saponin component;

[0242] wherein the saponin component is administered simultaneously with the oligonucleotide-based medicament and / or wherein the saponin component is administered some time, optionally after an interval of at least 1 day, preferably at least one week, more preferably at least one month, most preferably at least 3-6 months, after the oligonucleotide-based medicament has been administered, resulting in an extension of the duration of effect of the oligonucleotide-based medicament and / or in an extension of the dosing interval of the oligonucleotide-based medicament and / or in a reduction of the dosing frequency of the oligonucleotide-based medicament and / or in a (delayed) boost in the effect of the oligonucleotide-based medicament; or

[0243] a second pharmaceutical kit comprising a package comprising

[0244] a) one or more dosage units comprising the saponin component and comprising the oligonucleotide-based medicament; and

[0245] b) printed instructions to use the dosage units comprised in the kit in a method of treatment of a cardiovascular disease related to a defect in (the expression of) a gene and / or that is treatable by modulating the expression and / or expression level of a gene, said method of treatment comprising: - the administration, preferably the repeated administration, of the dosage units comprising the oligonucleotide-based medicament that is capable of regulating the expression of said gene and / or that is capable of modulating the expression level of said gene; or

[0246] a third pharmaceutical kit comprising a package comprising

[0247] a) one or more first dosage units comprising the saponin component and comprising the oligonucleotide-based medicament;

[0248] b) one or more second dosage units comprising the saponin component; and

[0249] c) printed instructions to use the dosage units comprised in the kit in a method of treatment of a cardiovascular disease in a human subject related to a defect in (the expression of) a gene and / or that is treatable by modulating the expression and / or expression level of a gene, said method of treatment comprising:

[0250] i. the administration, preferably the repeated administration, to said human subject, of the first dosage units comprising the oligonucleotide-based medicament that is capable of regulating the expression of said gene and / or that is capable of modulating the expression level of said gene, and comprising the saponin component,

[0251] ii. the administration, to said human subject, of the second dosage unit comprising only the saponin component;

[0252] wherein the second dosage unit is administered some time, optionally after an interval of at least 1 day, preferably at least one week, more preferably at least one month, most preferably at least 3-6 months, after the first dosage unit comprising the oligonucleotide-based medicament has been administered, resulting in an extension of the duration of effect of the oligonucleotide-based medicament and / or in an extension of the dosing interval of the oligonucleotide-based medicament and / or in a reduction of the dosing frequency of the oligonucleotide-based medicament and / or in a (delayed) boost in the effect of the oligonucleotide-based medicament.

[0253] In another embodiment, the pharmaceutical kits are (suitable) for treatment and / or prevention and / or amelioration of a cardiovascular disease, or the pharmaceutical kits are for use in treatment and / or prevention and / or amelioration of a cardiovascular disease. In an optional embodiment, the present disclosure provides a method for the treatment and / or prevention and / or amelioration of a cardiovascular disease using the pharmaceutical kits as described herein.

[0254] Cardiovascular disease

[0255] As described herein above, the therapeutic combination, the pharmaceutical formulations thereof and / or the pharmaceutical kits thereof is / are for use in treatment and / or prevention and / or amelioration of a cardiovascular disease. Wherein the cardiovascular disease(s) is / are according to the forgoing aspects and / or embodiments. As the skilled person would recognize, the forgoing description shall not be construed as limiting, rather can be combined with any of the above and / or forgoing aspects and / or embodiments, which would provide literal basis for each individual features, such as the therapeutic combination, the pharmaceutical formulations thereof and / or the pharmaceutical kits thereof.

[0256] In one embodiment, the cardiovascular disease is related to (an) elevated plasma concentration(s) of low-density lipoprotein cholesterol (LDL-C) and / or triglycerides, wherein the cardiovascular disease is optionally accompanied with inflammation.

[0257] In one embodiment, the cardiovascular disease (CVD) is selected from any one or more of: i. atherosclerotic cardiovascular disease (ASCVD), selected from: coronary heart disease (CHD), such as myocardial infarction, angina and coronary artery stenosis; cerebrovascular disease, such as a transient ischemic attack and ischemic stroke; peripheral artery disease, such as claudication; and aortic atherosclerotic disease, such as abdominal aortic aneurysm and descending thoracic aneurysm;

[0258] ii. hyperlipidemia;

[0259] iii. dyslipidemia;

[0260] iv. hypertriglyceridemia;

[0261] v. familial chylomicronemia (type I hyperlipoproteinemia);

[0262] vi. familial hypercholesterolemia (type II hyperlipoproteinemia);

[0263] vii. hypercholesterolemia;

[0264] viii. hemorrhagic disease;

[0265] ix. thrombosis;

[0266] x. congenital disorder of glycosylation type IId (CDG-IId);

[0267] xi. blood clotting defects;

[0268] xii. arterial plaque formation;

[0269] xiii. hypertension; and

[0270] xiv. elevated levels of fibrinogen.

[0271] In one embodiment, the cardiovascular disease is familial hypercholesterolemia (type II hyperlipoproteinemia), such as heterozygous familial hypercholesterolemia (heFH) or homozygous familial hypercholesterolemia (HoFH).

[0272] In one embodiment, the cardiovascular disease is a disease and / or a disorder that is any one or more of: caused by, treatable by, prevented by and ameliorated by an alteration in the expression and / or a modulation of the expression level of one or more gene(s).

[0273] In one embodiment, the cardiovascular disease is a disease and / or a disorder that is any one or more of: caused by, treatable by, prevented by and ameliorated by an alteration in the expression and / or a modulation of the expression level of any one or more of the genes selected from: beta-1, 4-galactosyltransferase 1 (B4GALT1), proprotein convertase subtilisin / kexin type 9 (PCSK9), angiopoietin-like 3 (ANGPTL3), cytochrome P450 family 7 subfamily A member 1 (CYP7A1), arachidonate 12-lipoxygenase, 12S type (ALOX12) and serpin family H member 1 (SERPINA1), preferably selected from: B4GALT1, CYP7A1 and ALOX12. In one embodiment, the cardiovascular disease is a disease and / or a disorder that is any one or more of: caused by, treatable by, prevented by and ameliorated by an alteration in the expression and / or a modulation of the expression level of any one or more of the genes selected from:

[0274] B4GALT1, wherein the cardiovascular disease is selected from any one or more of the disorders and diseases listed here above, preferably, the disorder or disease is hyperlipidemia, dyslipidemia, hypertriglyceridemia, congenital disorder of glycosylation type lid, blood clotting defects, arterial plaque formation, and / or elevated levels of fibrinogen;

[0275] PCSK9, wherein the cardiovascular disease is selected from any one or more of the disorders and diseases listed here above, preferably, the disorder or disease is atherosclerotic cardiovascular disease (ASCVD), selected from: coronary heart disease (CHD), such as myocardial infarction, angina and coronary artery stenosis, cerebrovascular disease, such as a transient ischemic attack and ischemic stroke, peripheral artery disease, such as claudication, and aortic atherosclerotic disease, such as abdominal aortic aneurysm and descending thoracic aneurysm; hyperlipidemia; dyslipidemia; hypertriglyceridemia; familial chylomicronemia (type I hyperlipoproteinemia); familial hypercholesterolemia (type II hyperlipoproteinemia); heterozygous familial hypercholesterolemia (heFH); homozygous familial hypercholesterolemia (HoFH), and / or hypercholesterolemia;

[0276] ANGPTL3, wherein the cardiovascular disease is selected from any one or more of the disorders and diseases listed here above, preferably, the disorder or disease is hyperlipidemia, dyslipidemia, hypertriglyceridemia, familial chylomicronemia (type I hyperlipoproteinemia), familial hypercholesterolemia (type II hyperlipoproteinemia), heterozygous familial hypercholesterolemia (heFH), homozygous familial hypercholesterolemia (HoFH) and / or hypercholesterolemia;

[0277] CYP7A1, wherein the cardiovascular disease is selected from any one or more of the disorders and diseases listed here above, preferably, the disorder or disease is selected from any one or more of: (i) atherosclerotic cardiovascular disease (ASCVD), selected from: coronary heart disease (CHD), such as myocardial infarction, angina and coronary artery stenosis; cerebrovascular disease, such as a transient ischemic attack and ischemic stroke; peripheral artery disease, such as claudication; and aortic atherosclerotic disease, such as abdominal aortic aneurysm and descending thoracic aneurysm; (ii) hyperlipidemia; (iii) dyslipidemia; (iv) hypertriglyceridemia; (v) familial chylomicronemia (type I hyperlipoproteinemia); (vi) familial hypercholesterolemia (type II hyperlipoproteinemia); (vii) hypercholesterolemia; (viii) hemorrhagic disease; (ix) thrombosis; (x) congenital disorder of glycosylation type lid (CDG- lld); (xi) blood clotting defects; (xii) arterial plaque formation; and (xiii) elevated levels of fibrinogen; and / or heterozygous familial hypercholesterolemia (heFH) or homozygous familial hypercholesterolemia (HoFH);

[0278] ALOX12, wherein the cardiovascular disease is selected from any one or more of the disorders and diseases listed here above, preferably, the disorder or disease is selected from any one or more of: (i) atherosclerotic cardiovascular disease (ASCVD), selected from: coronary heart disease (CHD), such as myocardial infarction, angina and coronary artery stenosis; cerebrovascular disease, such as a transient ischemic attack and ischemic stroke; peripheral artery disease, such as claudication; and aortic atherosclerotic disease, such as abdominal aortic aneurysm and descending thoracic aneurysm; (ii) hyperlipidemia; (iii) dyslipidemia; (iv) hypertriglyceridemia; (v) familial chylomicronemia (type I hyperlipoproteinemia); (vi) familial hypercholesterolemia (type II hyperlipoproteinemia); (vii) hypercholesterolemia; (viii) hemorrhagic disease; (ix) thrombosis; (x) congenital disorder of glycosylation type lid (CDG- lld); (xi) blood clotting defects; (xii) arterial plaque formation; and (xiii) elevated levels of fibrinogen; and / or heterozygous familial hypercholesterolemia (heFH) or homozygous familial hypercholesterolemia (HoFH);

[0279] SERPINA1, wherein the cardiovascular disease is selected from any one or more of the disorders and diseases listed here above, preferably, the disorder or disease is selected from any one or more of: (i) atherosclerotic cardiovascular disease (ASCVD), selected from: coronary heart disease (CHD), such as myocardial infarction, angina and coronary artery stenosis; cerebrovascular disease, such as a transient ischemic attack and ischemic stroke; peripheral artery disease, such as claudication; and aortic atherosclerotic disease, such as abdominal aortic aneurysm and descending thoracic aneurysm; (ii) hyperlipidemia; (iii) dyslipidemia; (iv) hypertriglyceridemia; (v) familial chylomicronemia (type I hyperlipoproteinemia); (vi) familial hypercholesterolemia (type II hyperlipoproteinemia); (vii) hypercholesterolemia; (viii) hemorrhagic disease; (ix) thrombosis; (x) congenital disorder of glycosylation type IId (CDG-IId); (xi) blood clotting defects; (xii) arterial plaque formation; and (xiii) elevated levels of fibrinogen; and / or heterozygous familial hypercholesterolemia (heFH) or homozygous familial hypercholesterolemia (HoFH), preferably, the disorder or disease is hemorrhagic disease, and / or thrombosis.

[0280] In one embodiment, the cardiovascular disease is a disease and / or a disorder that is any one or more of: caused by, treatable by, prevented by and / or ameliorated by an alteration in the expression and / or a modulation of the expression level of any one or more of the genes selected from: B4GALT1, CYP7A1 and ALOX12.

[0281] In a related general aspect, the cardiovascular disease is optionally accompanied with inflammation, wherein the treatment and / or prevention and / or amelioration of a cardiovascular disease is or comprises the treatment or amelioration of inflammation in the human subject.

[0282] In one embodiment, the inflammation accompanied by the cardiovascular disease is optionally nuclear factor-kappa B (NF-KB) pathway related inflammation, optionally activated NF-KB and / or NF-KB inducing kinase (NIK) signalling related inflammation.

[0283] In one embodiment, the treatment and / or prevention and / or amelioration of the cardiovascular disease does not activate or induce the NF-KB pathway, optionally does not activate NF-KB signalling and / or does not activate NIK, and / or does not worsen, cause or increase inflammation in the human subject. In another embodiment, the treatment and / or prevention and / or amelioration of the cardiovascular disease does not result in an innate immune response, optionally involving TLR-mediated innate immune response.

[0284] In yet another embodiment, the treatment and / or prevention and / or amelioration of the cardiovascular disease does increase the intracellular level and / or extracellular level, optionally the blood serum concentration, with less than 30%, less than 20%, less than 10%, less than 5%, less than 2%, less than 1% or less than 0.5%, of any one or more of: interleukin-1β (IL-1β), interleukin-2 (IL-2), interferon-α2a (IFNα2a), IFNγ, tissue necrosis factor-α (TNFα) and INFβ.

[0285] Preferred is the embodiment relating to the therapeutic combination for use or the pharmaceutical formulations thereof or the pharmaceutical kit thereof or the method thereof or the use thereof, wherein the administration of the saponin component and the oligonucleotide-based medicament comprises treating, preventing or ameliorating of inflammation in the human subject, optionally nuclear factor-kappa B (NF-κB) pathway related inflammation, and / or inhibition of the NF-κB pathway, optionally inhibition of NF-κB and / or inhibition of NF-κB inducing kinase (NIK), wherein preferably the cardiovascular disease relates to expression or expression level of any one or more of the genes B4GALT1, PCSK9, ANGPTL3, CYP7A1, ALOX12 and SERPINA1, more preferably B4GALT1, CYP7A1 and ALOX12.

[0286] In a preferred embodiment relating to the therapeutic combination for use or the pharmaceutical formulations thereof or the pharmaceutical kit thereof or the method thereof or the use thereof (or the use of the saponin component and the oligonucleotide-based medicament in the manufacture of a medicament), the treatment and / or prevention and / or amelioration of the cardiovascular disease comprises treating, preventing or ameliorating inflammation in the human subject, optionally NF-κB pathway related inflammation, and / or inhibition of the NF-κB pathway, optionally inhibition of NF-κB and / or inhibition of NIK, wherein preferably the cardiovascular disease relates to expression or expression level of any one or more of the genes B4GALT1, PCSK9, ANGPTL3, CYP7A1, ALOX12 and SERPINA1, more preferably B4GALT1, CYP7A1 and ALOX12.

[0287] Preferred is the embodiment relating to the therapeutic combination for use or the pharmaceutical formulations thereof or the pharmaceutical kit thereof or the method thereof or the use thereof, wherein the treatment and / or prevention and / or amelioration of the CVD does not activate or induce the NF-KB pathway, optionally does not activate NF-κB signalling and / or NIK, and / or does not worsen, cause or increase inflammation in the human subject, optionally NF-κB pathway related inflammation, and / or wherein the treatment and / or prevention and / or amelioration of the CVD does not result in an innate immune response, and / or wherein the treatment and / or prevention and / or amelioration of the CVD does increase the intracellular level and / or extracellular level, optionally the blood serum concentration, with less than 30%, less than 20%, less than 10%, less than 5%, less than 2%, less than 1% or less than 0.5%, of any one or more of: IL-1β, IL-2, IFNα2a, IFNγ, TNFα and INFβ, wherein preferably the CVD relates to expression or expression level of any one or more of the genes B4GALT1, PCSK9, ANGPTL3, CYP7A1, ALOX12 and SERPINA1, more preferably B4GALT1, CYP7A1 and ALOX12. Preferred is the embodiment relating to the therapeutic combination for use or the pharmaceutical formulations thereof or the pharmaceutical kit thereof or the method thereof or the use thereof, wherein the administration of the saponin component and the oligonucleotide-based medicament, does not activate or induce the NF-KB pathway, optionally does not activate NF-κB signalling and / or NIK, and / or does not worsen, cause or increase inflammation in the human subject, optionally NF-κB pathway related inflammation, and / or wherein the treatment and / or prevention and / or amelioration of the CVD does not result in an innate immune response, and / or wherein the treatment and / or prevention and / or amelioration of the CVD does increase the intracellular level and / or extracellular level, optionally the blood serum concentration, with less than 30%, less than 20%, less than 10%, less than 5%, less than 2%, less than 1% or less than 0.5%, of any one or more of: IL-1β, IL-2, IFNα2a, IFNγ, TNFα and INFβ, wherein preferably the cardiovascular disease relates to expression or expression level of any one or more of the genes B4GALT1, PCSK9, ANGPTL3, CYP7A1, ALOX12 and SERPINA1, more preferably B4GALT1, CYP7A1 and ALOX12.

[0288] In a general embodiment, the treatment and / or prevention and / or amelioration comprises administering to a patient in need thereof an amount of the therapeutic combination or the pharmaceutical formulation thereof which delivers a therapeutic effective amount of the oligonucleotide-based medicament to the human subject in need thereof, wherein the saponin component and the oligonucleotide-based medicament are delivered to the human cell wherein said human cell is a cell of the kidney and / or is a liver cell, preferably a hepatocyte.

[0289] In a general embodiment, the treatment and / or prevention and / or amelioration refers to a method of treatment and / or prevention and / or amelioration, wherein the method comprises administration of the therapeutic combination or the pharmaceutical formulation or administration of the saponin component and the oligonucleotide-based medicament to the human subject in need thereof, wherein the saponin component and the oligonucleotide-based medicament are delivered to the human cell wherein said human cell is a cell of the kidney and / or is a liver cell and optionally and preferably a hepatocyte.

[0290] Dosage Regimen / Frequencies of Administration

[0291] As described herein above, the therapeutic combination, the pharmaceutical formulations thereof and / or the pharmaceutical kits thereof is / are for use in treatment and / or prevention and / or amelioration of a CVD. Wherein the treatment and / or prevention and / or amelioration comprises an administration of the therapeutic combination according to the forgoing aspects and / or embodiments. As the skilled person would recognize, the forgoing description shall not be construed as limiting, rather can be combined with any of the above and / or forgoing aspects and / or embodiments, which would provide literal basis for each individual features, such as the therapeutic combination, the pharmaceutical formulations thereof and / or the pharmaceutical kits thereof. In one embodiment, the treatment and / or prevention and / or amelioration comprises an administration of the therapeutic combination comprising the oligonucleotide-based medicament and the saponin component being co-formulated in a single pharmaceutical composition.

[0292] In an alternative embodiment the treatment and / or prevention and / or amelioration comprises an administration of the therapeutic combination comprising the oligonucleotide-based medicament and the saponin component being formulated separately in at least two pharmaceutical formulations, wherein a first pharmaceutical formulation comprises the saponin component and a second pharmaceutical formulation comprises the oligonucleotide-based medicament.

[0293] In one embodiment, the administration comprises provision to the human subject of the oligonucleotide-based medicament and of the saponin component which are co-formulated in a single pharmaceutical composition for simultaneous administration.

[0294] In an alternative embodiment, the administration comprises provision to the human subject of the oligonucleotide-based medicament and of the saponin component which are formulated separately as at least two pharmaceutical formulations wherein a first pharmaceutical formulation comprises the saponin component and a second pharmaceutical formulation comprises the oligonucleotide-based medicament, that can be administered either simultaneously or sequentially.

[0295] In one embodiment, the administration comprises provision to the human subject of the oligonucleotide-based medicament and of the saponin component which are co-formulated in a single pharmaceutical composition for simultaneous administration, wherein the single pharmaceutical composition is selected from any one or more of the following:

[0296] 2-component saponin formulation, defined as comprising the saponin component comprising a saponin moiety and wherein the 2-component saponin formulation further comprises the oligonucleotide-based medicament that possibly comprises the second ligand recognised by the second endocytic receptor;

[0297] 2-component linker-saponin formulation, defined as comprising the saponin component comprising the saponin moiety and wherein the saponin moiety is covalently conjugated with the linker; wherein the 2-component linker-saponin formulation further comprises the oligonucleotide-based medicament that possibly comprises a second ligand recognised by a second endocytic receptor;

[0298] 2-component targeted-saponin formulation, defined as comprising the saponin component comprising the saponin moiety and wherein the saponin moiety is covalently conjugated with the first ligand, and preferably wherein the non-saponin moiety comprises the linker; and wherein the 2-component targeted-saponin formulation further comprises the oligonucleotide-based medicament that possibly comprises the second ligand;

[0299] 1 -component formulation, defined as comprising the saponin-oligonucleotide conjugate, possibly wherein the saponin-oligonucleotide conjugate further comprises the first ligand.

[0300] In a next embodiment, the administration comprises provision of the at least two pharmaceutical formulations comprising a combination of the first pharmaceutical formulation with the second pharmaceutical formulation selected from any one or more of the following: non-targeted combination defined as comprising:

[0301] the first pharmaceutical formulation, wherein the saponin component does not comprise a ligand and preferably comprises or consists of the unconjugated saponin moiety and / or the saponin molecule wherein the saponin moiety is covalently conjugated with the linker, and

[0302] the second pharmaceutical formulation, wherein the oligonucleotide-based medicament does not comprise a ligand; or

[0303] targeted-effector combination defined as comprising:

[0304] the first pharmaceutical formulation, wherein the saponin component does not comprise a ligand and preferably comprises or consists of the unconjugated saponin moiety and / or the saponin molecule, wherein the saponin moiety is covalently conjugated with the linker, and

[0305] the second pharmaceutical formulation, wherein the oligonucleotide-based medicament comprises the second ligand; or

[0306] 2-component targeted-saponin combination defined as comprising:

[0307] the first pharmaceutical formulation, wherein the saponin component comprises the saponin moiety, wherein the saponin moiety is covalently conjugated with the first ligand, and preferably wherein the non-saponin moiety comprises the linker, and

[0308] the second pharmaceutical formulation, wherein the oligonucleotide-based medicament possibly comprises the second ligand.

[0309] In a preferred embodiment, the first and the second pharmaceutical formulation can be administered either simultaneously or sequentially, preferably the second pharmaceutical formulation is administered first and subsequently the first pharmaceutical formulation is administered after an interval of at least 1 day, preferably after an interval of at least one week, more preferably after an interval of at least one month, most preferably after an interval of at least 3-6 months.

[0310] In yet another embodiment, the administration of co-formulated single pharmaceutical composition and / or separately formulated two pharmaceutical formulations is further continued after an interval of at least 1 day, preferably after an interval of at least one week, more preferably after an interval of at least one month, most preferably after an interval of at least 3-6 months, with a boosting administration of the saponin component that is further referred to as a booster, e.g. when the at least two pharmaceutical formulations are previously administered simultaneously. The inventors surprisingly established that that such booster performed with the saponin component (the boosting saponin component) results in an extension of the duration of effects of the oligonucleotide-based medicament and / or in an extension of the dosing interval and / or in a reduction of the dosing frequency of the oligonucleotide-based medicament and / or a (delayed) potentiation of its therapeutic effects.

[0311] Possibly, the booster comprises an unconjugated saponin moiety or a saponin molecule covalently conjugated with the non-saponin moiety being at least the linker or at least the first ligand or at least the linker and the first ligand. In certain embodiments, the boosting application can be performed at a site of the administration or at another administration site, and / or using the same mode of administration (e.g. IV or SC). In possible embodiments, the interval is at least 1 day after the administration, preferably at least 2 days, at least 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 6 months after the administration.

[0312] In certain embodiments, the booster can be applied directly into the kidney (e.g. into the parenchyma or renal artery etc.), and / or can be applied directly into the liver (e.g. by portal vein injection) or can be applied IV or SC. In a particular embodiment, the booster can be performed at a site of the administration, or if the administration involved application at multiple sites, the boosting application can be made in one of these sites.

[0313] In case of multiple sites of the administration or repeated administrations or in case of the administration encompassing multiple partial administrations, e.g. wherein the administration involves provision of two or more pharmaceutical formulations as separate and possibly timed doses, the site of the administration is to be construed as at least one of the sites of the administration.

[0314] Consequently, in one aspect, the present disclosure relates to a therapeutic combination comprising:

[0315] a. an oligonucleotide-based medicament adapted to target and bind to a nucleic acid molecule present in the cytosol of a human cell and / or present in the nucleus of a human cell; b. a saponin component comprising a penta-cyclic triterpene saponin, comprising an aglycone core of 12,13-dehydrooleanane type;

[0316] for use in treatment and / or prevention and / or amelioration of a CVD caused by, treatable by, prevented by and ameliorated by an alteration in the expression of a gene and / or a modulation of the expression level of a gene;

[0317] wherein the treatment and / or prevention and / or amelioration comprising the steps of:

[0318] i) the administration, to said human subject, preferably the repeated administration, of the oligonucleotide-based medicament, that is capable of modulating the expression of said gene and / or that is capable of modulating the expression level of said gene; and

[0319] ii) the administration, to said human subject, of the saponin component;

[0320] wherein the saponin component is administered simultaneously with the oligonucleotide-based medicament and / or wherein the saponin component is administered some time, optionally after an interval of at least 1 day, preferably at least one week, more preferably at least one month, most preferably at least 3-6 months, after the oligonucleotide-based medicament has been administered, resulting in an extension of the duration of effect of the oligonucleotide-based medicament and / or in an extension of the dosing interval of the oligonucleotide-based medicament and / or in a reduction of the dosing frequency of the oligonucleotide-based medicament and / or in a (delayed) boost in the effect of the oligonucleotide-based medicament.

[0321] In another aspect, the present disclosure relates to a method of treatment and / or prevention and / or amelioration of a CVD in a human subject that is any one or more of: caused by, treatable by, prevented by and ameliorated by an alteration in the expression of a gene and / or a modulation of the expression level of a gene; said method of treatment comprising the steps of:

[0322] i) the administration, to said human subject, preferably the repeated administration, of the oligonucleotide-based medicament, that is capable of modulating the expression of said gene and / or that is capable of modulating the expression level of said gene; and

[0323] ii) the administration, to said human subject, of the saponin component;

[0324] wherein the saponin component is administered simultaneously with the oligonucleotide-based medicament and / or wherein the saponin component is administered some time, optionally after an interval of at least 1 day, preferably at least one week, more preferably at least one month, most preferably at least 3-6 months, after the oligonucleotide-based medicament has been administered, resulting in an extension of the duration of effect of the oligonucleotide-based medicament and / or in an extension of the dosing interval of the oligonucleotide-based medicament and / or in a reduction of the dosing frequency of the oligonucleotide-based medicament and / or in a (delayed) boost in the effect of the oligonucleotide-based medicament.

[0325] In yet another aspect, the present disclosure relates to use of the saponin component and the oligonucleotide-based medicament in the manufacture of a medicament for use in a method of treatment and / or prevention and / or amelioration of a CVD in a human subject that is any one or more of: caused by, treatable by, prevented by and ameliorated by an alteration in the expression of a gene and / or a modulation of the expression level of a gene; said method of treatment comprising the steps of:

[0326] i) the administration, to said human subject, preferably the repeated administration, of the oligonucleotide-based medicament that is capable of modulating the expression of said gene and / or that is capable of modulating the expression level of said gene; and

[0327] ii) the administration, to said human subject, of the saponin component;

[0328] wherein the saponin component is administered simultaneously with the oligonucleotide-based medicament and / or wherein the saponin component is administered some time, optionally after an interval of at least 1 day, preferably at least one week, more preferably at least one month, most preferably at least 3-6 months, after the oligonucleotide-based medicament has been administered, resulting in an extension of the duration of effect of the oligonucleotide-based medicament and / or in an extension of the dosing interval of the oligonucleotide-based medicament and / or in a reduction of the dosing frequency of the oligonucleotide-based medicament and / or in a (delayed) boost in the effect of the oligonucleotide-based medicament.

[0329] The references to the therapeutic combination in the above paragraphs are also to be interpreted as references to the pharmaceutical formulation thereof and the pharmaceutical kits thereof being suitable for treatment and / or prevention and / or amelioration of the CVDs.

[0330] Oligonucleotide-based medicament In one embodiment, the oligonucleotide-based medicament is adapted to target and bind to a nucleic acid molecule present in the cytosol of a human cell and / or present in the nucleus of a human cell.

[0331] In another embodiment, the nucleic acid molecule present in the cytosol of a human cell and / or present in the nucleus of a human cell is a gene or a gene transcript present in kidney cells and / or in liver cells, wherein the gene or the gene transcript is selected from B4GALT1, PCSK9, ANGPTL3, CYP7A1, ALOX12 and SERPINA1, preferably selected from B4GALT1, CYP7A1 and ALOX12. The gene transcript is a transcript, preferably pre-mRNA or mRNA, more preferably, mRNA from any one or more of the genes selected from B4GALT1, PCSK9, ANGPTL3, CYP7A1, ALOX12 and SERPINA1, preferably selected from B4GALT1, CYP7A1 and ALOX12

[0332] In one embodiment, the oligonucleotide-based medicament comprises a deoxyribonucleic acid (DNA)-based therapeutic oligonucleotide and / or ribonucleic acid (RNA)-based therapeutic oligonucleotide and / or a nucleic acid analogue-based therapeutic oligonucleotide comprising one or more nucleotide or nucleoside analogues and / or backbone modifications, preferably selected from: DNA aptamer, antisense oligonucleotide (ASO, AON), DNA ASO, RNA ASO, small or short interfering RNA (siRNA), microRNA (miRNA), RNA miRNA inhibitor (anti-microRNA, anti-miRNA, anti-miR) and / or RNA miRNA inhibitor ASO, RNA aptamer, ribozyme, RNA decoy, short hairpin RNA (shRNA), anti-hairpin-shaped microRNA; or mixed DNA / RNA therapeutic, preferably comprising one or more of the following analogues or modifications: phosphoramidate morpholino oligomer (PMO, Morpholino), peptide nucleic acid (PNA), phosphorothioate-modified antisense oligonucleotide (PS-ASO), antisense oligonucleotides containing phosphoryl guanidine (PN) backbone linkages (PN-ASO; PGO); 2'-O-methyl (2-OMe) phosphorothioate RNA, 2'-O-methoxyethyl (2-O-MOE) RNA (2’-O-methoxyethyl-RNA (2-MOE, MOE)), locked nucleic acid (LNA, bridged nucleic acid, BNA; for example 2’-O,4’-aminoethylene bridged nucleic acid (BNA-NC), BNA-based siRNA, BNA-based antisense oligonucleotide (BNA-ASO), BNA-based anti-microRNA etc.), 2'-O, 4'-C-ethylene-bridged nucleic acid (ENA), 2’-deoxy-2’-fluoroarabino nucleic acid (FANA), 3’-fluoro hexitol nucleic acid (FHNA), glycol nucleic acid (GNA), threose nucleic acid (TNA).

[0333] In another embodiment, the oligonucleotide-based medicament comprises or consists of mixed DNA / RNA and / or synthetic nucleic acid therapeutic selected from: synthetic ASO, substantially DNA-based synthetic ASO, substantially RNA-based synthetic ASO preferably comprising 2'-MOE modification, substantially DNA-based synthetic aptamer, substantially RNA-based synthetic aptamer, synthetic gapmer, synthetic siRNA, synthetic miRNA, synthetic anti-miRNA and / or synthetic anti-miRNA ASO, preferably

[0334] wherein the backbone modification is selected from: 2'-O, 4'-C-ethylene-bridged (ENA) or 2-MOE nucleic acid modifications and / or phosphoryl guanidine (PN) backbone linkages (PN-ASO), more preferably being a mutation specific therapeutic, for example being a mutation specific ASO comprising one or more nucleotide analogues and / or backbone modifications, possibly designed to silence a gene implicated in the disorder and / or to induce exon skipping or induce oligonucleotide-directed RNA editing.

[0335] In yet another embodiment, the oligonucleotide-based medicament is an oligonucleotide therapeutic defined as a nucleic acid therapeutic that is not longer than 200 nt, preferably has a size of 5- 150 nt, more preferably 8 - 100 nt, most preferably 10 - 50 nt, preferably wherein the oligonucleotide therapeutic is capable of treating and / or preventing and / or ameliorating the cardiovascular disease by modulating the expression of a gene implicated in the cardiovascular disease, optionally in liver cells and / or in kidney cells.

[0336] In one embodiment, the oligonucleotide-based medicament comprises an siRNA or an ASO, preferably an ASO for reducing expression of a gene, wherein the ASO optionally comprises PMO, or an ASO for promoting exon skipping, wherein the ASO optionally comprises PMO, or an ASO capable of inducing RNA editing of a target pre-mRNA or of a target mRNA, optionally adenosine deaminase acting on RNA (ADAR)-mediated adenosine to inosine (A-to-l) RNA editing.

[0337] In a generic embodiment, the oligonucleotide-based medicament is selected from: mipomersen, inclisiran, CTX-310, lipisense, solbinsiran, zodasiran, ALNANG-3, JS-401, RBD-7022, RN-0191, SGB-3403, SRSD-101, SYH-2053, VERVE-101, VERVE-102, YOLT-101, VSA-003, YKYY-015, ADGN-xxx PCSK9, ANGsiR10, AX-1412, CiVi-008, CTX-330, EMD-302, Epigenetic Editor Targeting Human PCSK9, LNP-ETR, PBGENE-PCSK9, RBD-3045, RP-910, VERVE-201, Csi-103, miniaturized single-targeting RNAi triggers (mxRNA), ALN-ANG, ALN-PCS02, BLSM-201, cepadacursen, DCRPCSK-9, ISIS-394814, SPC-5001, STP-133G, STP-135G, vupanorsen, AZD-8233, BMS-844421 and TKM-HTG or pharmaceutically acceptable salt thereof.

[0338] In a specific embodiment, the oligonucleotide-based medicament is selected from: mipomersen, inclisiran, lipisense, solbinsiran, zodasiran, cepadacursen (CiVi-007), vupanorsen, olpasiran, SPC-5001, AZD-8233 (ION 449), RN-0191, SGB-3403, SRSD-101, SYH-2053, VSA-003, YKYY-015, ANGsiR10, AX-1412, CiVi-008, RP-910, ALN-PCS02, ISIS-394814, STP-133G, STP-135G and BMS-844421 or pharmaceutically acceptable salt thereof, preferably selected from mipomersen, inclisiran, lipisense, solbinsiran, zodasiran, SPC-5001, AZD-8233 (ION 449), RN-0191, SGB-3403, SRSD-101, SYH-2053, VSA-003, YKYY-015, ANGsiR10, AX-1412, CiVi-008 and RP-910 or pharmaceutically acceptable salt thereof.

[0339] In an embodiment, the oligonucleotide-based medicament or a pharmaceutically acceptable salt thereof is selected from oligonucleotide therapeutics that are associated with the treatment or prevention of diseases which may cause or contribute to the development of cardiovascular conditions, as listed in TABLE X.

[0340] TABLE X: Exemplary drug development programs comprising oligonucleotide therapeutics

[0341]

[0342]

[0343]

[0344]

[0345]

[0346] In an additional embodiment, the oligonucleotide-based medicament or pharmaceutically acceptable salt thereof is selected from the corresponding therapeutics in connection to a cardiovascular disease as listed Table I.

[0347] TABLE I: Exemplary drug development programs targeting a cardiovascular disease

[0348]

[0349]

[0350] preferably, selected from:

[0351]

[0352]

[0353] Preferred is the embodiment relating to the therapeutic combination of the invention, the therapeutic combination for use according to the invention, the first, second or third kit of the invention, the therapeutic method of the invention and / or the use (of the saponin component and the oligonucleotide-based medicament in the manufacture of a medicament) according to the invention, wherein the oligonucleotide-based medicament further comprises a second ligand recognised by a second endocytic receptor, possibly wherein the second endocytic receptor is / are present on the kidney and / or liver cells, wherein the second ligand is / are a proteinaceous ligand, for example a naturally existing peptide or protein ligand or a receptor-interacting part thereof, or is an antibody or a binding fragment thereof; and / or wherein the second ligand comprises one or more sugar moieties, preferably being or comprising at least one N-acetylgalactosamine (GalNAc) moiety or three GalNAc moieties.

[0354] In yet another embodiment, the oligonucleotide-based medicament binds to a nucleic acid molecule present in the cytosol of a human cells and / or present in the nucleus of a human cell, selected from liver cells, preferably hepatocytes, and / or kidney cells, preferably kidney cortex cells and / or kidney medulla cells. Based on the presented herein data with the presently disclosed therapeutic combination, it will be apparent to the skilled person that without limiting to a specific oligonucleotide-based medicament, there appear to have been unlocked an enormous potential for reducing the effective dose of such oligonucleotide-based medicament while increasing the intracellular efficacy of the oligonucleotide moiety comprised by the oligonucleotide-based medicament, which could improve the therapeutic window and eliminate the toxicity issues which could have led to the failure.

[0355] The skilled person will be aware of gene expression patterns underlying different CVDs and their location within the kidney and / or liver cells, if known, and will be aware that different targeting strategies have been developed based on different oligonucleotide chemistries and use of targeting ligands and / or carriers. Consequently, the skilled person will be sufficiently equipped to develop therapeutic combinations in accordance with the present disclosure, to target a nucleic acid that is expressed in a specific structure or type of kidney cells and / or liver (hepatocyte) cells.

[0356] Saponin component

[0357] The development of the presented herein advantageous combinations in accordance with all and any one of the aspects and embodiments as presented herein, was based on the surprising realisation that thanks to the inclusion of the endosomal-escape-enhancing saponin in the presented herein therapeutic combinations, an oligonucleotide-based medicament, such as an siRNA or an ASO, effectively enters into kidney cells and / or effectively enters into liver cells following systemic delivery, and binds to the target of the oligonucleotide moiety comprised by the oligonucleotide-based medicament.

[0358] The “saponin component" as disclosed herein comprises a penta-cyclic triterpene saponin comprising an aglycone core of 12,13-dehydrooleanane type at their structure (also referred to as sapogenin or aglycone), usually shown as a penta-cyclic C30 terpene skeleton. Examples of such known saponins are shown in Table II.

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366] a, b: Different names refer to different isolates of the same structure

[0367] c, d: Different names refer to different isolates of the same structure

[0368] 1) Jia et al., Major Triterpenoid Saponins from Saponaria officinalis, J. Nat. Prod. 1998, 61, 11, 1368-1373, Publication Date: September 19, 1998, https: / / doi.org / 10.1021 / np980167u

[0369] 2) The structure of Agrostemmoside E (also referred to as AG1856 or AG2.8) is given in Fig. 4 of J. Clochard et al, A new acetylated triterpene saponin from Agrostemma githago L. modulates gene delivery efficiently and shows a high cellular tolerance, International Journal of Pharmaceutics, Volume 589, 15 November 2020, 119822.

[0370] 3) Structures of S01700, SO1730, SO1772, SO1904 are given in Moniuszko-Szajwaj et al., Highly Polar Triterpenoid Saponins from the Roots of Saponaria officinalis L., Helv. Chim. Acta, V99, pp. 347 - 354, 2016 (doi.org / 10.1002 / hlca.201500224).

[0371] 4) See for example:

[0372] - thesis by Dr Stefan Böttger (2013): Untersuchungen zur synergistischen Zytotoxizitat zwischen Saponinen und Ribosomen inaktivierenden Proteinen Typ I; and

[0373] - Sama et al., Structure-Activity Relationship of Transfection-Modulating Saponins - A Pursuit for the Optimal Gene Trafficker, Planta Med. Volume 85, pp. 513-518, 2019 (doi:10.1055 / a-0863-4795); and - Fuchs et al., Glycosylated Triterpenoids as Endosomal Escape Enhancers in Targeted Tumor Therapies, Biomedicine, Volume 5, issue 14, 2017 (doi:10.3390 / biomedicines5020014).

[0374] 5) Sama et al., Sapofectosid - Ensuring non-toxic and effective DNA and RNA delivery, International Journal of Pharmaceutics, Volume 534, Issues 1-2, 20 December 2017, Pages 195-205 (dx.doi.org / 10.1016 / j.ijpharm.2017.10.016) & Moniuszko-Szajwaj et al., Highly Polar Triterpenoid Saponins from the Roots of Saponaria officinalis L., Helv. Chim. Acta, V99, pp. 347 - 354, 2016 (doi.org / 10.1002 / hlca.201500224).

[0375] 6) See for example: doi:10.1016 / s0040-4039(01)90658-6, Tetrahedron Letters No. 8, pp. 477-482, 1963 and and “Gipsoside.” National Center for Biotechnology Information. PubChem Compound Database, U. S. National Library of Medicine, 8 Aug. 2005, pubchem.ncbi.nlm.nih.gov / compound / Gipsoside.

[0376] 7) The structure of Sodium Aescinate is for example given in the National Library of Medicine PubChem Compound Database (“Sodium Aescinate.” National Center for Biotechnology Information. PubChem Compound Database, U. S. National Library of Medicine, 26 Mar. 2005, pubchem.ncbi.nlm.nih.gov / compound / Sodium- aescinate,)

[0377]

[0378] As it can be seen from Table II, naturally existing (unmodified) EEE saponins frequently comprise an aldehyde function at position C-23 of the saponin’s aglycone core structure. This notable feature can be seen in the exemplary chemical structure of such saponin that is schematically depicted below in the Scheme of SAPONIN A:

[0379]

[0380] (SAPONIN A) Without wishing to be bound by any theory, it was observed that the presence of said aldehyde function (sometimes referred as “aldehyde group”; which in the present context should be construed as synonymous) can be particularly beneficial for the capacity of the saponin to stimulate and / or potentiate the endosomal escape of effector molecules, such as oligonucleotide-based medicaments or at least the oligonucleotide moiety comprised by the oligonucleotide-based medicament.

[0381] Preferred is the embodiment relating to the therapeutic combination of the invention, the therapeutic combination for use according to the invention, the first, second or third kit of the invention, the therapeutic method of the invention and / or the use (of the saponin component and the oligonucleotide-based medicament in the manufacture of a medicament) according to the invention,, wherein the saponin component further comprises a first ligand recognised by a first endocytic receptor, possibly wherein the first endocytic receptor is / are present on the kidney and / or liver cells, wherein the first ligand is / are a proteinaceous ligand, for example a naturally existing peptide or protein ligand or a receptor-interacting part thereof, or is an antibody or a binding fragment thereof; and / or wherein the first ligand comprises one or more sugar moieties, preferably being or comprising at least one GalNAc moiety or three GalNAc moieties.

[0382] In one embodiment, the penta-cyclic triterpene saponin further comprises

[0383] an aldehyde function at position C-23 of the aglycone core, or

[0384] an acid-sensitive covalent bond configured to break under acidic conditions so as to create the aldehyde function at position C-23 of the aglycone core, preferably wherein the acid-sensitive covalent bond is selected from any one or more of: a hydrazone bond, a semicarbazone bond, an imine bond, an acetal bond including a 1,3-dioxolane bond, a ketal bond, an ester bond, and / or an oxime bond, and preferably is a hydrazone bond.

[0385] As it can be appreciated from Table II, most of the naturally-occurring known penta-cyclic triterpene saponins comprising the aglycone core of 12,13-dehydrooleanane type and which also comprise the aldehyde function at position C-23 in their native or unconjugated form, are saponins for which the aglycone core is either quillaic acid or gypsogenin.

[0386] In line with this, it was observed that the penta-cyclic triterpene saponin comprises the aglycone core selected from quillaic acid, gypsogenin, and an aldehyde-substituted derivative of either one of quillaic acid or gypsogenin defined as a quillaic acid-based aglycone core or gypsogenin-based aglycone core, respectively, wherein the aldehyde function at position C-23 is substituted by the acid sensitive covalent bond configured to break under acidic conditions so as to create the aldehyde function at position C-23 of the aglycone core, preferably wherein the penta-cyclic triterpene saponin is selected from:

[0387] AG1856, AG1, AG2, Agrostemmoside 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, Saponarioside B, SO1542, SO1584, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862, SO1903, SO1904, QS-7, QS-7 apio, QS-17, QS-18, QS-21 A-apio, QS-21 A- xylo, QS-21 B-apio and QS-21 B-xylo, or the aldehyde-substituted derivative of any one thereof, respectively, preferably AG1856, AG1, AG2, Agrostemmoside 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, Saponarioside B, SO1542, SO1584, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862, SO1903, SO1904, or the aldehyde-substituted derivative of any one thereof, respectively; or

[0388] wherein the penta-cyclic triterpene saponin is selected from:

[0389] SA1641, gypsoside A, NP-017772, NP-017774, NP-017777, NP-017778, NP-018109, NP-017888, NP-017889, NP-018108 and SO1658, or the aldehyde-substituted derivative of any one thereof, respectively.

[0390] Saponins can comprise one or more saccharide chains attached to the aglycone core structure. Preferred saponins according to the disclosure comprise a single chain ( / .e. are mono-desmosidic) or two chains ( / .e. are bis-desmosidic) attached to the aglycone core structure.

[0391] In line with this, in one embodiment, the penta-cyclic triterpene saponin is a triterpenoid saponin and / or a bisdesmosidic triterpene saponin belonging to the type of a 12,13-dehydrooleanane with a branched carbohydrate side chain at the C-3 position wherein said carbohydrate side chain comprises glucuronic acid function, preferably also with an aldehyde function in position C-23 and preferably further with a branched carbohydrate side chain at the C-28 position comprising deoxy carbohydrate.

[0392] In yet another embodiment, the penta-cyclic triterpene saponin is a triterpenoid saponin and / or a bisdesmosidic triterpene saponin belonging to the type of a 12,13-dehydrooleanane with an aldehyde function in position C-23 selected from the group consisting of saponins with a quillaic acid aglycon core and saponins with a gypsogenin aglycon core, and / or wherein the saponin comprises a branched carbohydrate side chain at the C-3 position wherein said carbohydrate side chain comprises glucuronic acid function, and wherein the saponin comprises a branched carbohydrate side chain at the C-28 position comprising deoxy carbohydrate.

[0393] In another embodiment, the penta-cyclic triterpene saponin is mono-desmosidic or bi-desmosidic, preferably comprising a first saccharide chain bound to a position C-3 of the aglycone core, more preferably wherein the first saccharide chain is selected from Group A listed in Table III:

[0394] Table III: GLYCANS

[0395]

[0396]

[0397] In another embodiment, the penta-cyclic triterpene saponin is mono-desmosidic or bi-desmosidic, preferably comprising a second saccharide chain bound to a position C-28 of the aglycone core, more preferably wherein the second saccharide chain bound to its aglycone core structure, selected from Group B listed in Table III:

[0398] Table III: GLYCANS

[0399]

[0400]

[0401] In a preferred embodiment, the first saccharide chain comprises a glucuronic acid group, preferably being a terminal glucuronic acid group, such as wherein the first saccharide chain comprises: Gal-(1 -»2)-[Xyl-(1 ->3)]-GlcA. In another embodiment, the penta-cyclic triterpene saponin is isolated from Saponaria officinalis, and is preferably any one or more of Saponarioside B, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862, SO1903 and SO1904 or the aldehydesubstituted derivative of any one thereof, respectively, more preferably any one or more of SO1832, SO1861 and SO1862 or the aldehyde-substituted derivative of any one thereof, respectively, even more preferably SO1832 or SO1861 or the aldehyde-substituted derivative of any one thereof, respectively, most preferably SO1861 or the aldehyde-substituted derivative of any one thereof, respectively.

[0402] Other possible optional and / or specific embodiments relating to different features of the saponin component are summarised in a section provided above the Examples.

[0403] In a specific embodiment, the saponin component as disclosed herein includes one or more unconjugated saponin moiety, and / or a saponin molecules or wherein the saponin component comprises a saponin moiety that is covalently conjugated with at least one non-saponin moiety (purely to discern them from their unconjugated free molecule counterparts).

[0404] In a possible embodiment, compatible with preceding embodiments, therapeutic combinations, compositions, formulations, and methods for the disclosed herein use are provided, wherein the saponin component comprises an unconjugated saponin molecule (defined as the penta-cyclic triterpene saponin that is not covalently conjugated to a non-saponin moiety, possibly wherein the saponin component consists of the unconjugated saponin molecule).

[0405] Saponin molecules that have been conjugated were experimentally shown to have certain advantages over unconjugated saponin molecules in e.g. showing improved efficacy and tolerability in vitro and in vivo. For example, compared to native SO1861, an engineered SO1861 that was ligated with a linker or a ligand (like GalNAc ligand) shows reduced haemolytic activity on red blood cells (RBCs), inducing haemolysis at a ~6-fold higher concentration (data not shown).

[0406] Hence, in a preferred embodiment, the saponin component comprises an unconjugated saponin moiety or a saponin molecule or wherein the saponin component comprises a saponin moiety that is covalently conjugated with at least one non-saponin moiety, preferably via an acid-sensitive covalent bond that breaks under acidic conditions, more preferably being an acid-sensitive covalent bond at the position C-23 of the aglycone core, even more preferably wherein the acid sensitive covalent bond at the position C-23 of the aglycone core is configured to break under acidic conditions so as to create the aldehyde function at position C-23 of the aglycone core thus resulting in a release of the penta-cyclic triterpene saponin comprising the aldehyde function at the position C-23 of the aglycone core from the non-saponin moiety, even more preferably wherein the acid-sensitive covalent bond is selected from any one or more of: a hydrazone bond, a semicarbazone bond, an imine bond, an acetal bond including a 1,3-dioxolane bond, a ketal bond, an ester bond, and / or an oxime bond, most preferably being a hydrazone bond.

[0407] In another embodiment, the saponin component comprises an unconjugated saponin moiety or a saponin molecule or the saponin component comprises a saponin moiety that is covalently conjugated with at least one non-saponin moiety, preferably via an acid-stable bond, more preferably the acid-stable bond being present at the glucuronic acid group on the first saccharide chain.

[0408] In a related embodiment, the non-saponin moiety comprises any one or more of:

[0409] a linker, preferably a click chemistry linker,

[0410] the first ligand,

[0411] the oligonucleotide-based medicament, and / or

[0412] a scaffold molecule,

[0413] preferably, wherein the saponin moiety is directly covalently conjugated with the linker;

[0414] more preferably wherein the linker is covalently conjugated to the saponin moiety via the acid sensitive covalent bond, more preferably at the position C-23 of the aglycone core, or via the acid-stable bond, preferably at the glucuronic acid group if said group is present;

[0415] even more preferably wherein the linker is further covalently conjugated to the first ligand and / or to the oligonucleotide-based medicament, possibly via the scaffold molecule;

[0416] for example wherein the scaffold molecule is a multi-functional linker scaffold molecule or a polymeric scaffold molecule possibly comprising a dendron, such as a poly-amidoamine (PAMAM) dendrimer, or a polysaccharide, or a peptide, or a poly-ethylene glycol, such as any of PEG3 – PEG30.

[0417] In another embodiment, the saponin moiety is covalently conjugated with the non-saponin moiety comprising the oligonucleotide-based medicament, which covalent conjugation results in a conjugate further termed a saponin-oligonucleotide conjugate, preferably wherein the saponinoligonucleotide conjugate further comprises the linker, more preferably wherein the linker is directly covalently conjugated to the saponin moiety possibly wherein the saponin-oligonucleotide conjugate further comprises the first ligand.

[0418] As used herein, the term scaffold molecule is to relate to a moiety of a conjugate which can serve as a scaffold for conjugating other moieties to a conjugate. In the present context, such scaffold molecule can be used for effectuating covalent linking between the saponin moiety, an effector moiety, and further possibly the first ligand. The linking to scaffold molecule can be effectuated either directly, or via the first, second, of any further linker.

[0419] Typical scaffold molecules as known in the art are based on an oligomeric or polymeric structure, frequently either being a dendron such as a poly-amidoamine (PAMAM) dendrimer, or a polysaccharide, or a peptide, or a poly-ethylene glycol such as any of PEG3 – PEG30. In preferred embodiments of the disclosure, any one of such scaffold molecules can be used. For example, it can advantageously be a polymeric or oligomeric structure being any one of PEG4 - PEG12 or any one of a G2 dendron, a G3 dendron, a G4 dendron and a G5 dendron, more preferably being a G2 dendron or a G3 dendron or a PEG3-PEG30. Dendrons appear particularly advantageous for the reason as they appear to be associated with advantageous properties from kidney-targeting perspective (Huang et al., 2021) but other scaffolds were also shown to potentially act as carriers for kidney -targeted delivery of medicaments (Alallam et al., 2023; Chade and Bidwell 2022; Trac et al., 2023), which was discussed above in the targeting context. In another example, compatible with the above ones, a multi-functional linker can be used as a scaffold (termed above “multi-functional linker scaffold molecule”). A multi-functional linker scaffold molecule can be made from a trifunctional linker, such as the one shown by Structure A in the example below, here represented in non-conjugated form:

[0420]

[0421] (Structure A)

[0422] In a possible embodiment, a conjugate can be comprising 1-4 of such trifunctional linkers for every molecule of the targeting ligand comprised by the conjugate, more preferably being 1-2 trifunctional linkers, most preferably being 1.2 - 1.8. trifunctional linkers on average.

[0423] In a conjugated form, the trifunctional linker in its conjugated form is represented by Structure B:

[0424]

[0425] wherein:

[0426] S is the at least one saponin moiety,

[0427] L1 is a linker bound to the saponin moiety;

[0428] NA is the oligonucleotide-based medicament,

[0429] L2 is a linker bound to the oligonucleotide-based medicament;

[0430] A is one or more molecules of the first ligand, preferably being an antibody or a binding fragment thereof or at least one GalNAc moiety, preferably 3 GalNAc moieties,

[0431] L3 is a linker bound to the first ligand,

[0432] wherein L1, L2 and L3 are the same or different.

[0433] In a particularly preferred embodiment, therapeutic combinations, compositions, formulations, and methods for the disclosed herein use are provided, wherein the saponin moiety is covalently conjugated with the non-saponin moiety comprising the oligonucleotide-based medicament, which covalent conjugation results in a conjugate further termed a saponin-oligonucleotide conjugate, preferably wherein the saponin-oligonucleotide conjugate further comprises the linker, more preferably wherein the linker is directly covalently conjugated to the saponin moiety possibly wherein the saponin-oligonucleotide conjugate further comprises the first ligand (and is further termed targeted a saponin-oligonucleotide conjugate).

[0434] In possible embodiments, the targeted saponin-oligonucleotide conjugate comprises 1 - 16 saponin moieties and 1 - 5 molecules of the nucleic acid (also termed effector moieties) per 1 ligand moiety, preferably wherein the targeted saponin-oligonucleotide conjugate comprises 2 - 8 saponin moieties per 1 ligand moiety; preferably 3 - 6 saponin moieties per 1 ligand moiety; more preferably 4 - 5 saponin moieties per 1 ligand moiety; most preferably wherein the targeted saponin-oligonucleotide conjugate comprises on average 4-4.5 saponin moieties per 1 molecule of the ligand.

[0435] In sum, as disclosed herein, the saponin component is:

[0436] a penta-cyclic triterpene saponin of the 12,13-dehydrooleanane type;

[0437] preferably comprising an aldehyde function at position C-23 of the aglycone core, or an acidsensitive covalent bond configured to break under acidic conditions so as to create the aldehyde function at position C-23 of the aglycone core, preferably wherein the acid-sensitive covalent bond is selected from any one or more of: a hydrazone bond, a semicarbazone bond, an imine bond, an acetal bond including a 1,3-dioxolane bond, a ketal bond, an ester bond, and / or an oxime bond, and preferably is a hydrazone bond;

[0438] mono-desmosidic or bi-desmosidic, preferably bi-desmosidic; and / or

[0439] comprising a first saccharide chain bound to its aglycone core structure, selected from Group A listed in Table III and / or comprising a second saccharide chain bound to its aglycone core structure, selected from Group B listed in Table III, and preferably a first saccharide chain and a second saccharide chain are comprised by the saponin molecule or saponin moiety:

[0440] and / or is: preferably comprising a first saccharide chain bound to position C-3 of its aglycone core structure, selected from Group A listed in Table III, wherein preferably said first saccharide chain of the saponin molecule comprises a glucuronic acid group or optionally said first saccharide chain of the saponin moiety comprises a glucuronic acid group; and / or

[0441] preferably comprising the first saccharide chain which comprises a terminal glucuronic acid residue and / or comprising the second saccharide chain which comprises at least four sugar residues in a branched configuration; and / or

[0442] preferably comprising the first saccharide chain Gal-(1→2)-[Xyl-(1→3)]-GlcA and / or a branched second saccharide chain of at least four sugar residues comprising a terminal fucose residue and / or a terminal rhamnose residue, preferably selected from Table III; and / or

[0443] preferably comprising a first saccharide chain at position C-3 of the saponin’s aglycone core structure and / or a second saccharide chain at position C-28 of the saponin’s aglycone core structure, preferably wherein the first saccharide chain is a carbohydrate substituent at the C-3beta- OH group of the saponin’s aglycone core structure and / or wherein the second saccharide chain is a carbohydrate substituent at the C-28-OH group of the saponin’s aglycone core structure; and / or optionally comprising at least one acetoxy (Me(CO)O-) group in the first saccharide chain and / or in the second saccharide chain, preferably in the second saccharide chain; and / or

[0444] comprising an aglycone core structure selected from:

[0445] quillaic acid;

[0446] gypsogenin;

[0447] 2alpha-hydroxy oleanolic acid;

[0448] 16alpha-hydroxy oleanolic acid;

[0449] hederagenin (23-hydroxy oleanolic acid);

[0450] 16alpha,23-dihydroxy oleanolic acid;

[0451] protoaescigenin-21(2-methylbut-2-enoate)-22-acetate;

[0452] 23-oxo-barringtogenol C-21,22-bis(2-methylbut-2-enoate);

[0453] 23-oxo-barringtogenol C-21 (2-methylbut-2-enoate)-16,22-diacetate;

[0454] 3, 16,28-trihydroxyoleanan-12-en;

[0455] gypsogenic acid; and

[0456] a derivative thereof; and / or

[0457] preferably comprising an aglycone core structure selected from quillaic acid, gypsogenin, and a derivative thereof; and / or

[0458] preferably comprising the aglycone core structure quillaic acid; and / or

[0459] selected from any one or more of the saponins listed in Table II (above);

[0460] and / or is:

[0461] a) selected from any one or more of list A: - Quillaja saponaria saponin mixture, or a saponin isolated from Quillaja saponaria, for example Quil-A, QS-17-api, QS-17-xyl, QS-21, QS-21A, QS-21 B, QS-7-xyl;

[0462] - Saponinum album saponin mixture, or a saponin isolated from Saponinum album',

[0463] - Saponaria officinalis saponin mixture, or a saponin isolated from Saponaria officinalis; and - Quillaja bark saponin mixture, or a saponin isolated from Quillaja bark, for example Quil-A, QS-17-api, QS-17-xyl, QS-21, QS-21 A, QS-21 B, QS-7-xyl; or

[0464] b) comprising a gypsogenin aglycone core structure and is selected from list B:

[0465] SA1641, gypsoside A, NP-017772, NP-017774, NP-017777, NP-017778, NP-018109, NP- 017888, NP-017889, NP-018108 and SO1658; or

[0466] c) comprising a quillaic acid aglycone core structure and is selected from list C:

[0467] AG1856, AG1, AG2, Agrostemmoside 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, Saponarioside B, SO1542, SO1584, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862, SO1904, QS-7, QS-7 api, QS-17, QS-18, QS-21 A-apio, QS-21 A-xylo, QS-21 B-apio and QS-21 B-xylo;

[0468] d) comprising a 12, 13-dehydrooleanane type aglycone core structure without an aldehyde group at the C-23 position of the aglycone and is selected from list D:

[0469] Aescin la, aescinate, alpha-Hederin, AMA-1, AMR, AS6.2, AS64R, Assamsaponin F, dipsacoside B, esculentoside A, macranthoidin A, NP-005236, NP-012672, Primula acid 1, saikosaponin A, saikosaponin D, Teaseed saponin I and Teaseedsaponin J, preferably, any one or more selected from list A, B or C, more preferably, selected from list B or C, even more preferably selected from list C; and / or

[0470] any one or more of 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 having a formula according to formula ‘SO1832'

[0471]

[0472] SO1832

[0473] , SO1861 having a formula according to formula ‘SO1861'

[0474]

[0475] , SO1862 and SO1904, preferably any one or more of QS-21, SO1832, SO1861, SA1641, AG1856 and GE1741, more preferably AG1856, SO1832 or SO1861, most preferably SO1861 or SO1832; and / or a saponin isolated from Saponaria officinalis, preferably any one or more of Saponarioside B, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862 and SO1904, more preferably any one or more of SO1832, SO1861 and SO1862, even more preferably SO1832 or SO1861, most preferably SO1861; and / or

[0476] a saponin molecule, wherein the carboxyl group of the glucuronic acid unit in the first saccharide chain bound to C-3 of the aglycone core structure of the saponin molecule is transformed into an amide bond through reaction with 2-amino-2-methyl-1,3-propanediol (AMPD) as shown for SO1861 in formula (3):

[0477]

[0478] or a saponin molecule having a formula according to one of the following formulas (9)-(12):

[0479]

[0480]

[0481] In certain preferred embodiments, the saponin comprises a glucuronic acid group in the carbohydrate substituent at the C-3beta-OH group, and preferably the saponin is selected from the group consisting of (refer to Table II for the structural details): NP-017777, NP-017778, NP-017774, NP-018110, NP-017772, NP-018109, NP-017888, NP-017889, NP-018108, SA1641, AE X55, SO1658, gypsoside A, Gypsophila saponin 1 (Gyp1), NP-017674, NP-017810, AG1, NP-003881, NP-017676, NP-017677, NP-017706, NP-017705, NP-017773, NP-017775, SA1657, AG2, GE1741, SO1542, SO1584, SO1674, SO1700, Saponarioside B, SO1730, SO1772, SO1832 (protonated SO1831; also referred to as Saponarioside A), SO1861 (deprotonated SO1862), SO1862 (protonated SO1861; also referred to as Sapofectosid), SO1904, QS-7 (also referred to as QS1861), QS-7 api (also referred to as QS1862), QS-17, QS-18, QS-21 A-apio, QS-21 A-xylo, QS-21 B-apio, QS-21 B-xylo, QS-21, Agrostemmoside E (also referred to as AG1856 or AG2.8), NP-005236, NP-012672, beta-Aescin (described: Aescin la), Aescinate, Teaseed saponin I, Teaseedsaponin J, Assamsaponin F, Primula acid 1.

[0482] In certain preferred embodiments, the saponin does not comprise an aldehyde function linked to the C-4 atom of the aglycon core structure, and preferably the saponin is selected from the group consisting of (refer to Table II for the structural details): NP-005236, AMA-1, AMR, alpha-Hederin, NP- 012672, beta-Aescin (described: Aescin la), Aescinate, dipsacoside B, esculentoside A, Teaseed saponin I, Teaseedsaponin J, Assamsaponin F, Primula acid 1, AS64R, Macranthoidin A, saikosaponin A, saikosaponin D, AS6.2.

[0483] In certain preferred embodiments, the saponin comprises a glucuronic acid group in the carbohydrate substituent at the C-3beta-OH group and the saponin does not comprise an aldehyde function linked to the C-4 atom of the aglycon core structure, and preferably the saponin is selected from the group consisting of (refer to Table II for the structural details): NP-005236, NP-012672, beta-Aescin (described: Aescin la, Aescinate, dipsacoside B, esculentoside A, Teaseed saponin I, Teaseedsaponin J, Assamsaponin F, Primula acid 1, Macranthoidin A, saikosaponin A, saikosaponin D.

[0484] In some particular embodiments, possibly compatible with preceding ones, pharmaceutical combinations for the disclosed herein use can be provided, wherein one, two or three, preferably one or two, more preferably one, of:

[0485] an aldehyde function in the aglycone core structure of the at least one saponin has been derivatised when present,

[0486] a carboxyl group of a glucuronic acid moiety in a first saccharide chain of the at least one saponin has been derivatised when present in the at least one saponin, and

[0487] at least one acetoxy (Me(CO)O-) group in a second saccharide chain of the at least one saponin has been derivatised if present.

[0488] In more particular embodiments, pharmaceutical combinations for the disclosed herein use can be provided wherein the at least one saponin comprises:

[0489] i. an aglycone core structure comprising an aldehyde function which has been derivatised by:

[0490] - reduction to an alcohol;

[0491] - transformation into a hydrazone bond through reaction with N-ε-maleimidocaproic acid hydrazide (EMCH) wherein the maleimide group of the EMCH is optionally derivatised by formation of a thioether bond with mercaptoethanol;

[0492] - transformation into a hydrazone bond through reaction with N-[ß-maleimidopropionic acid] hydrazide (BMPH) wherein the maleimide group of the BMPH is optionally derivatised by formation of a thioether bond with mercaptoethanol; or

[0493] - transformation into a hydrazone bond through reaction with N-[κ-maleimidoundecanoic acid] hydrazide (KMUH) wherein the maleimide group of the KMUH is optionally derivatised by formation of a thioether bond with mercaptoethanol; or

[0494] ii. a first saccharide chain comprising a carboxyl group, preferably a carboxyl group of a glucuronic acid moiety, which has been derivatised by transformation into an amide bond through reaction with 2-amino-2-methyl-1,3-propanediol (AMPD) or N-(2-aminoethyl)maleimide (AEM); or Hi. a second saccharide chain comprising an acetoxy group (Me(CO)O-) which has been derivatised by transformation into a hydroxyl group (HO-) by deacetylation; or iv. any combination of two or three derivatisations i., ii. and / or iii., preferably any combination of two derivatisations of i., ii. and iii.

[0495] In a specific embodiment, pharmaceutical combinations for the disclosed herein use is provided wherein the aldehyde function in position C-23 of the aglycone core structure of the at least one saponin is covalently bound to linker EMCH, which EMCH is covalently bound via a thio-ether bond to a sulfhydryl group in the oligomeric molecule or in the polymeric molecule of the covalent saponin conjugate, such as a sulfhydryl group of a cysteine.

[0496] Binding of the EMCH linker to the aldehyde group of the aglycone of the saponin results in formation of a hydrazone bond. Such a hydrazone bond is a typical example of a cleavable bond under the acidic conditions inside endosomes and lysosomes.

[0497] When the saponin component comprises the saponin moiety, the saponin moiety is any one of the here-above defined saponin molecules with covalently bound thereto:

[0498] a linker, such as a linker suitable for covalently binding the saponin molecule to a further molecule, wherein the linker comprises or is for example:

[0499] a. a polyethylene glycol (PEG) with a length of any number between 2 and 60 (PEG2, PEG3, PEG4, PEG5, PEG6, PEG7-PEG10, PEG11-PEG25, PEG25-PEG50, etc.); b. a peptide;

[0500] c. a linear or branched or cyclic alkyl, a linear or branched or cyclic alkenyl, a linear or branched or cyclic alkynyl;

[0501] d. a polymeric structure or an oligomeric structure, for example:

[0502] wherein the polymeric or oligomeric structure is selected from:

[0503] i. poly- or oligo(amines), such as polyethylenimine and poly(amidoamine), ii. polyethylene glycols,

[0504] iii. poly- or oligo(esters), such as poly(lactids),

[0505] iv. poly (lactams),

[0506] v. polylactide-co-glycolide copolymers,

[0507] vi. poly- or oligosaccharides, such as cyclodextrin and polydextrose, vii. poly- or oligo(amino acids), such as proteins, peptides and polylysine, and viii. DNA oligomers or polymers, RNA polymers, stabilized RNA polymers and PNA (peptide nucleic acid) polymers, and / or

[0508] ix. dendron of type G2, G3, G4 or G5;

[0509] a linker, such as a linker as hereabove defined, with a further molecule covalently bound to the linker wherein said further molecule is any one or more of:

[0510] a. a further linker, such as a linker as hereabove defined; and / or

[0511] b. an effector moiety, wherein the effector moiety is an oligonucleotide-based medicament and / or

[0512] c. a ligand for binding to an endocytic cell-receptor,

[0513] wherein the ligand is a proteinaceous ligand or a non-proteinaceous ligand or a combination thereof, preferably wherein the ligand is a proteinaceous ligand, and for examples is: a. a protein ligand capable of binding to a(n) endocytic cell-surface receptor, which binding results in internalization of the protein ligand;

[0514] b. an antibody, wherein the antibody is defined as an immunoglobulin (Ig) or a functional binding fragment or binding domain thereof.

[0515] The saponin component is suitable for passive or active transfer from outside a cell to inside said cell. Moreover, the saponin is suitable for transfer from outside a cell into said cell, being the transfer in the endosomes of said cell. The saponin component is suitable for entry into a cell upon binding of a ligand for binding to an endocytic cell-receptor, bound to the saponin moiety comprised by the saponin component, to said endocytic cell receptor, via endocytosis. Upon binding of the ligand, endocytosis occurs and the saponin component is delivered in the endosomes of the cell bearing the cell receptor.

[0516] Notable examples of such cell-surface receptors are megalin and cubilin, that are multifunctional endocytic receptors expressed in the kidney. Other suitable endocytosing cell-surface receptors are CD71 (transferrin receptor) or CD63 (tetraspanin).

[0517] When the proteinaceous ligand comprised by the saponin component (and suitable for binding to an endocytic cell-surface receptor) is an antibody, the antibody is for example selected from IgG, IgM, IgE, IgA, or IgD, or any antigen-binding fragment thereof, preferably is selected from a monoclonal antibody, polyclonal antibody, human antibody, humanized antibody, chimeric antibody, resurfaced antibody, anti-idiotypic antibody, mouse antibody, rat antibody, rat / mouse hybrid antibody, llama antibody, llama heavy-chain only antibody, heavy-chain only antibody, a molecule comprising or consisting of a VHH domain, a VH domain (for example camelid VH), a humanized VHH with a human (IgG1-derived) Fc and / or humanized VHH-Fc antibody a Fab, an scFv, an Fv, monomeric Vhh-Fc type antibody (e.g. wherein Vhh is from camelid origin, preferably alpaca; the Fc is from human origin and is preferably engineered to prevent Fc-gamma receptor binding), a single domain antibody (sdAb), an F(ab)2, Fcab fragment. A monoclonal antibody and a Fab and a Vhh-Fc type antibody and a single sdAb or a string of covalently linked sdAb’s is preferred.

[0518] The linker covalently bound to the saponin molecule, forming the saponin component comprising the saponin moiety and the linker (and in some embodiments a ligand covalently bound to the linker), is in preferred embodiments covalently bound to the saponin via a bond that is cleavable under conditions present in the endosome of mammalian cells, for example human cells. Such cleavable bond is for example subject to cleavage under acidic, reductive, enzymatic and / or light-induced conditions; preferably wherein the cleavable bond is selected from:

[0519] • a bond subject to cleavage under acidic conditions such as a hydrazone bond, a semicarbazone bond, an imine bond, an acetal bond including a 1,3-dioxolane bond, a ketal bond, an ester bond, and / or an oxime bond,

[0520] • a bond susceptible to proteolysis, for example amide or peptide bond, preferably subject to proteolysis by Cathepsin B;

[0521] • a red / ox-cleavable bond such as a disulfide bond, or a thiol-exchange reaction-susceptible bond such as a thio-ether bond preferably being an acid-sensitive bond subject to cleavage in vivo under acidic conditions present in endosomes and / or lysosomes of human cells, preferably at pH 4.0 - 6.5, and more preferably at pH ≤ 5.5; more preferably being an acid-sensitive bond selected from any one or more of: a hydrazone bond, a semicarbazone bond, an imine bond, an acetal bond including a 1,3-dioxolane bond, a ketal bond, an ester bond, and / or an oxime bond, even more preferably selected from a hydrazone bond and a semicarbazone bond; most preferably being a hydrazone bond.

[0522] In a further embodiment, the saponin component comprises a glucuronic acid function with a carboxylic acid functional group in a carbohydrate substituent at the C-3beta-OH group of the saponin, wherein the carboxylic acid functional group is transformed into an active ester.

[0523] In a next embodiment, the saponin component comprises a glucuronic acid function with a carboxylic acid functional group in a carbohydrate substituent at the C-3beta-OH group of the saponin, wherein the carboxylic acid functional group is transformed into an active ester upon binding of a linker to said carboxylic acid functional group.

[0524] In a next embodiment, the saponin component comprises a bisdesmosidic triterpene saponin belonging to the type of a 12,13-dehydrooleanane with an aldehyde function in position C-23 and comprising a glucuronic acid function with a carboxylic acid functional group in a carbohydrate substituent at the C-3beta-OH group of the saponin, and comprising a N-(2-aminoethyl)maleimide (AEM) moiety, wherein said AEM moiety is covalently coupled to said saponin via said carboxylic acid functional group, preferably wherein said AEM moiety is covalently coupled to said saponin via an amide bond formed with the glucuronic acid functional group.

[0525] In an embodiment, a ligand as hereabove defined is covalently bound to said linker which linker is bound to the saponin moiety. An example of such a saponin moiety comprising an active ester is the moiety resulting from activation of the carboxylic group of the saponin molecule selected for providing the saponin moiety, via 1-[Bis(dimethylamino)methylene]-1 H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU). An example is the saponin with the structure with formula (IV), referred to as SO1861 -HATU, in the list shown here below, suitable for e.g. binding to the amino group of the lysyl side chain of a Lys residue in a proteinaceous ligand. A further example of such a saponin moiety comprising an active ester is the moiety resulting from activation of the carboxylic group of the saponin molecule selected for providing the saponin moiety, via 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) and subsequently with N-(2-Aminoethyl)maleimide trifluoroacetate salt (AEM), therewith providing a maleimide group for forming a covalent bond with a thiol presented by the ligand, e.g. by a Cys residue in a proteinaceous ligand. An example is the saponin with the structure with formula (III), referred to as SO1861-S-Mal, in the list shown here below.

[0526] In embodiments, the linker that is bound to the saponin molecule in the saponin component further comprises an oligomeric or polymeric structure either being a dendron such as a polyamidoamine (PAMAM) dendrimer, or a polysaccharide, or a peptide, or a poly-ethylene glycol such as any of PEG3 - PEG30; preferably the polymeric or oligomeric structure being any one of PEG4 - PEG12 or any one of a G2 dendron, a G3 dendron, a G4 dendron and a G5 dendron, more preferably being a G2 dendron or a G3 dendron or a PEG3-PEG30.

[0527] For example, the saponin component comprises a saponin moiety comprising a covalently bound linker and is a molecule according to any one of formula (I) - (V):

[0528]

[0529]

[0530] and / or for example the saponin component comprises a saponin, wherein the carboxyl group of the glucuronic acid unit in the first saccharide chain bound to C-3 of the aglycone core structure of the saponin is transformed into an amide bond through reaction with N-(2-aminoethyl)maleimide (AEM) as shown for SO1861 in formula (18):

[0531]

[0532] or a saponin having a formula according to one of the following formulas (14)-(16) and (19)-(21):

[0533]

[0534]

[0535]

[0536] In a preferred embodiment, the saponin component is the molecule according to formula (I) herein above or is S01861 or is a conjugate of S01861 and the first ligand.

[0537] In a preferred embodiment, the saponin component comprises a ligand capable of binding to an endocytic cell-surface receptor.

[0538] In a preferred embodiment, the oligonucleotide-based medicament comprises a ligand capable of binding to an endocytic cell-surface receptor.

[0539] In a preferred embodiment, both the saponin component and / or the oligonucleotide-based medicament comprise(s) a ligand capable of binding to an endocytic cell-surface receptor.

[0540] In a preferred embodiment, the oligonucleotide-based medicament comprises a covalently bound thereto any one or more:

[0541] a linker selected from the any one or more linkers hereabove defined for the saponin moiety; a linker, such as a linker as hereabove defined, with a further molecule covalently bound to the linker wherein said further molecule is defined as hereabove defined for the saponin moiety, and is any one or more of:

[0542] a. a further linker, such as a linker as hereabove defined;

[0543] b. a ligand for binding to an endocytic cell-receptor, wherein the ligand is a proteinaceous ligand or a non-proteinaceous ligand or a combination thereof,

[0544] wherein the proteinaceous ligand is for example:

[0545] a. a protein ligand capable of binding to a cell-surface receptor, which binding results in internalization of the protein ligand;

[0546] an antibody, as defined hereabove for the saponin moiety

[0547] In a preferred embodiment, the saponin component comprises both a ligand capable of binding to an endocytic cell-surface receptor as herein above defined and the oligonucleotide-based medicament as here above defined.

[0548] A preferred embodiment is the therapeutic combination of, or therapeutic composition comprising any one of the saponin component as here above defined and any one of the oligonucleotide-based medicament as here above defined.

[0549] A preferred embodiment is the therapeutic combination of, or therapeutic composition comprising any one of the saponin component as here above defined and any one of the oligonucleotide-based medicament as here above defined.

[0550] A preferred embodiment is the therapeutic combination of, or therapeutic composition comprising any one of the saponin component as here above defined wherein the saponin component comprises a ligand as here above defined and any one of the oligonucleotide-based medicament as here above defined wherein the oligonucleotide-based medicament comprises a ligand as here above defined, for targeting an endocytic cell-surface molecule present on the same cell as the endocytic cellsurface molecule targeted by the ligand comprised by the saponin component.

[0551] A preferred embodiment is the saponin component consisting of a saponin molecule.

[0552] A preferred embodiment is a therapeutic combination of, or therapeutic composition comprising a saponin molecule and an oligonucleotide-based medicament.

[0553] A preferred embodiment is a therapeutic composition comprising or consisting of a saponin component comprising an oligonucleotide-based medicament and comprising a ligand as here above defined.

[0554] Covalently Conjugates

[0555] Preferred is the embodiment relating to the therapeutic combination of the invention, the therapeutic combination for use according to the invention, the first, second or third kit of the invention, the therapeutic method of the invention and / or the use (of the saponin component and the oligonucleotide-based medicament in the manufacture of a medicament) according to the invention,, wherein, in particular when targeting is desired, the disclosed herein therapeutic combinations, compositions, formulations, and methods, further comprise a ligand or a carrier, preferably a kidney cell specific ligand and / or a liver cell specific ligand, optionally a hepatocyte specific ligand, or alternatively a scaffold acting as a (nano)carrier such as PAMAM dendrimers or PEG-based nanoparticles which appear to be preferentially captured by kidney cells depending on their size and charge (cf. Huang et al., 2021; Alallam et al., 2023; Chade and Bidwell 2022; Trac et al., 2023). The skilled person will be aware that the scaffolds’ / carries’ size and / or charges will have an influence on kidney cellular uptake, circulation half-life, and kidney targeting. The influence of the physicochemical properties (size, shape, charge, material types) of various carriers on their renal clearance and accumulation is summarised in Alallam etal. 2023).

[0556] The skilled person will in general know and take into consideration when designing particular components of the presented herein therapeutic combinations, especially when designing multimolecule conjugates and considering targeting strategy for the kidney, that components of an average size of 100 nm have a longer half-life period than components of smaller sizes, which can be removed either by phagocytosis in the liver and spleen, or by renal excretion if they are smaller than 10 nm. The skilled person will also be aware that nanoparticles with sizes of approximately 75 ± 25 nm targeted the renal mesangium, whereas larger nanoparticles (>100 nm) cannot pass the glomerular filter.

[0557] In view of this knowledge, when considering a kidney targeting strategy for the presented herein combinations and components thereof, the skilled person will be aware to and will control the size of any ligand- and / or scaffold element that may be further comprised in the particular components of the presented herein therapeutic combinations.

[0558] Use of ligands is advantageous and, in many embodiments, it will be preferred over use of scaffolds, although either of these approaches or both could be combined in specific embodiments of the presented herein therapeutic combinations. In a preferred embodiment, the disclosed herein therapeutic combinations, compositions, formulations, and methods, are provided, wherein the saponin component further comprises a first ligand recognised by a first endocytic receptor, and / or wherein the oligonucleotide-based medicament further comprises a second ligand recognised by a second endocytic receptor. In embodiments, the first ligand and second ligand can be the same ligand. In embodiments, the first endocytic receptor and second endocytic receptor can be the same endocytic receptor. When the first ligand and the second ligand are different, the first endocytic receptor and the second endocytic receptor are present on the surface of the same cell that is targeted in the liver and / or targeted in the kidney. As already explained, in certain embodiments, the second endocytic receptor possibly is the same as the first endocytic receptor. In further possible embodiments, the second ligand is the same as the first ligand. In other embodiments, the second endocytic receptor differs from the first endocytic receptor with the proviso that the two different endocytic receptors are both present on the same kidney cell or liver cells.

[0559] In one embodiment, the saponin component further comprises a saponin moiety, being the penta-cyclic triterpene saponin, and further comprises a non-saponin moiety, being a first ligand recognised by a first endocytic receptor, and / or wherein the oligonucleotide-based medicament further comprises a second ligand recognised by a second endocytic receptor, possibly wherein the second endocytic receptor is the same as the first endocytic receptor, further possibly wherein the second ligand is the same as the first ligand, alternatively wherein the second endocytic receptor differs from the first endocytic receptor with the proviso that the two different endocytic receptors are both present on the same cells, for example the same liver cell or the same kidney cell; wherein the first ligand and / or the second ligand is / are a proteinaceous ligand, for example a naturally existing peptide or protein ligand or a receptor-interacting part thereof, or is an antibody or a binding fragment thereof; and / or wherein the first ligand and / or the second ligand comprises one or more sugar moieties, preferably being or comprising at least one GalNAc moiety or three GalNAc moieties.

[0560] In a possible embodiment in accordance with any of the preceding ones, the first ligand and / or the second ligand is / are a proteinaceous ligand, for example a naturally existing peptide or artificial peptide or protein ligand or a receptor-interacting part thereof.

[0561] In one embodiment, the first ligand and / or the second ligand is selected from an antibody or a binding fragment thereof binding to and / or a natural ligand or a fragment thereof recognised by a cellsurface receptor, preferably an endocytic cell-surface receptor, more preferably CD71 or CD63.

[0562] In one embodiment, the first ligand and / or the second ligand is selected from an antibody or a binding fragment thereof binding to and / or a natural ligand or a fragment thereof recognised by parathyroid hormone receptor 1 R (PTH1R).

[0563] In one embodiment, the first ligand and / or the second ligand is selected from:

[0564] antibody or a binding fragment thereof binding to any one of a liver-cell specific receptor and / or a hepatocyte specific receptor and / or is asialoglycoprotein receptor (ASGPR), and / or is a kidney-cell specific receptor;

[0565] natural ligand or a fragment thereof recognised by any one of a liver-cell specific receptor and / or a hepatocyte specific receptor and / or is asialoglycoprotein receptor (ASGPR), and / or is a kidney-cell specific receptor;

[0566] and / or wherein the first ligand and / or the second ligand is selected from:

[0567] megalin ligand such as EGF;

[0568] cubilin ligand such as FGF;

[0569] megalin and cubilin binder such as albumin;

[0570] transferrin (Tf) or a fragment thereof recognised by CD71;

[0571] parathyroid hormone receptor 1 R (PTH1R) ligand such as PTH-related protein (PTHrP); non-specific kidney ligand preferably comprising one or more GalNAc moieties; asialoglycoprotein receptor (ASGPR) ligand such as one or more galactose, galactosamines, GalNAc, asialoorosomucoid, asialofetuin, asialoceruloplasmin and / or asialotransferrin.

[0572] In view of the fact that the transferrin (Tf) receptor (CD71, TfR1) appears to undergo expression at the proximal tubules (Smith et al., 2019), in certain embodiments, the first ligand and / or the second ligand can be CD71 ligand(s). In other preferred embodiments, in view of the abundant expression at the proximal tubules of two multiligand receptors that are known as megalin (LRP2) and cubilin (or rather: CUBAM, consisting of three cubilin proteins bound to a single transmembrane chaperone amnionless or AMN; Larsen et al., 2018), the first ligand and / or the second can be ligands for megalin and / or cubilin ligands. Another example of cell-surface receptor is CD63 (also referred as cluster differentiation 63 or tetraspanin). Another cell surface receptor is parathyroid hormone receptor 1 R (PTH1R). PTH1R, was upregulated in chronic albuminuria in rats, with detectable expression on glomerular mesangial and endothelial cells, and abundant localization on proximal and distal tubular epithelial cells.

[0573] In an embodiment that is compatible with any one of preceding embodiments, therapeutic combinations, compositions, formulations, and methods are provided for the disclosed herein use, wherein the first endocytic receptor and / or the second endocytic receptor is / are a kidney-cell specific receptor and / or is / are selected from megalin (LRP2), cubilin, cluster differentiation 63 (CD63 or tetraspanin), parathyroid hormone receptor 1 R (PTH1R) and CD71 (transferrin receptor), preferably being selected from megalin and / or CD71.

[0574] Targeting megalin and cubilin is advantageous as they both are relatively kidney-specific endocytic receptors. Megalin is an LDL receptor that is expressed in epithelial cells of the kidney but also in the intestine, and several other tissues (Nielsen et al., 2016). On the apical plasma membrane of proximal tubule cells, it can bind with cubilin and mediate the endocytosis of many types of low molecular weight molecules that escape the glomerular filtration barrier (Christensen and Birn, 2002; Lin etal., 2013; Oroojalian et al., 2017; Xu et al., 2020).

[0575] The cytoplasmic tails of megalin and AMN contain NPXY and NPXF motifs, respectively, that engage the clathrin adaptor protein Dab2 to enable endocytic uptake of the receptors via clathrin coated pits that form at the base of proximal tubule microvilli. After uncoating, endocytic vesicles fuse with apical early endosomes, where luminal acidification triggers ligand dissociation from their receptors. Ligands are retained within fluid-rich maturing endosomal compartments and ultimately delivered to lysosomes for degradation, while megalin and cubilin (CUBAM) are recycled to the apical membrane in tubular structures (Rbaibi etal., 2023).

[0576] As megalin and cubilin mediate the recovery of different types of low molecular weight proteins like albumin, peptides, and other primarily proteinaceous molecules from the glomerular filtrate at the proximal tubules (Eshbach and Weisz, 2017, Christensen et al., 2012), in certain embodiments, such low molecular weight moieties could be included in one or more, preferably both, components of the presented herein pharmaceutical combinations for proximal tubule targeting-purposes. Examples of such ligands are listed in Table IV below (and e.g. in Table 1 of Eshbach and Weisz, 2017).

[0577] Table IV. Ligand table for megalin and cubilin (adapted from Nielsen et al., 2016)

[0578]

[0579] Transcobalamin-vitamin B12 Intrinsic factor vitamin B12

[0580] Vitamin D-binding protein Vitamin D-binding protein

[0581]

[0582]

[0583]

[0584] The skilled person will also be aware of other cellular targets and ligands and / or carriers like nanoparticles that allow targeting to specific kidney cell types as known in the art (Alallam etal., 2023).

[0585] Examples include but are not limited to use of targeting VCAM-1 receptor to target TNFalpha-activated podocytes (Alallam et al., 2023), and use of anti-E-selectin-specific antibodies to target glomerular endothelial cells, for use in the treatment of e.g. glomerulonephritis (Asgeirsdottir et al., 2008). Furthermore, the skilled person will know that to target glomerular mesangial cells inside of the glomerulus, the drug present in the circulation should be larger than 6 nm to prevent filtering by the urinary tract, but smaller than 70-90 nm to be captured in the glomerulus and pass through the endothelial fenestration (Ahn, L, etal., 2023). Consequently, it was attempted to target mesangial cells with naked siRNAs through administration via the renal artery (Ahn, I., et al., 2023). Alternative targeting approach for glomerular cells is by using a modified non-inhibitory plasminogen activator inhibitor 1R (PAI-1 R) as a ligand, although its cellular entry mechanism is not fully clear (Liu etal., 2022).

[0586] In a further embodiment that is compatible with any one of preceding embodiments, therapeutic combinations, compositions, formulations, and methods for the disclosed herein use are provided, wherein the first ligand and / or the second ligand is / are selected from:

[0587] antibody or a binding fragment thereof binding to any one of the above-listed receptors; natural ligand or a fragment thereof recognised by any one of the above-listed receptors; and / or wherein the first ligand and / or the second ligand is selected from:

[0588] megalin ligand, such as a megalin-specific antibody or a fragment thereof (anti-LPR2; preferably of a smaller format such as Fab, VHH, etc.) or any one or more of the megalin ligands listed in Table IV, e.g. EGF;

[0589] cubilin ligand, such as such as a cubilin-specific antibody or a fragment thereof (preferably of a smaller format such as Fab, VHH, etc.) or any one or more of the cubilin ligands listed in Table IV, e.g. FGF;

[0590] megalin and cubilin ligand such as any one or more of the megalin and cubilin ligands listed in Table IV, e.g. albumin;

[0591] transferrin (Tf) or a fragment thereof as recognised by CD71 (Tf also being a cubilin ligand) non-specific kidney ligand preferably comprising one or more GalNAc moieties, more preferably three GalNAc moieties.

[0592] VCAM 1 receptor ligand such as an antibody or a fragment thereof (preferably of a smaller format such as Fab, VHH, etc.)

[0593] E-selectin-specific ligand such as an antibody or a fragment thereof (preferably of a smaller format such as Fab, VHH, etc.)

[0594] In a next embodiment, therapeutic combinations, compositions, formulations, and methods for the disclosed herein use are provided, wherein the first ligand and / or the second ligand is / are selected from:

[0595] megalin ligand, such as a megalin-specific antibody or a fragment thereof (anti-LRP2; preferably of a smaller format such as Fab, VHH, etc.) or any one or more of the megalin ligands listed in Table IV, e.g. EGF;

[0596] cubilin ligand, such as a cubilin-specific antibody or a fragment thereof (preferably of a smaller format such as Fab, VHH, etc.) or any one or more of the cubilin ligands listed in Table IV, e.g. FGF;

[0597] megalin and cubilin ligand, such as any one or more of the megalin and cubilin ligands listed in Table IV, e.g. albumin;

[0598] transferrin (Tf) or a fragment thereof as recognised by CD71 (Tf also being a cubilin ligand). Artificial peptide ligands for binding e.g. megalin are known (e.g. Durinova et al., 2024). One example is the (KKEEE)3K peptide ([Lys-Lys-Glu-Glu-Glu]3-Lys; SEQ ID NO: 54) which was shown to achieve targeted kidney delivery through megalin-mediated endocytosis of proximal tubule cells (Wang et al., 2018). Different variations of (KKEEE)3K peptides were shown to possess targeting performance for the kidney (Janzer etal., 2016; Wischnjow etal., 2016; Huang et al., 2020), and could also form part of certain specific embodiments of the present disclosure.

[0599] In one embodiment, the first endocytic receptor and / or the second endocytic receptor is a livercell specific receptor and / or a hepatocyte specific receptor and / or is asialoglycoprotein receptor (ASGPR), and / or is a kidney-cell specific receptor and / or is selected from megalin (low density lipoprotein receptor-related protein 2; LRP2 receptor), cubilin, parathyroid hormone receptor 1 R (PTH1 R), cluster of differentiation 63 (CD63 or tetraspanin) and cluster of differentiation 71 (CD71 or transferrin receptor (TfR)).

[0600] In one embodiment, the first ligand and / or the second ligand is a proteinaceous ligand, preferably a peptide or protein ligand or a receptor-interacting part thereof.

[0601] In a preferred embodiment, the first ligand and / or the second ligand is / are an antibody or a binding fragment thereof, such as a F(ab')2 fragment, Fab' fragment, Fab fragment, scFv, dsFv, scFv-Fc, reduced IgG (rlgG), minibody, diabody, triabody, tetrabody, Fc fusion protein, nanobody, variable V domain, a single-domain antibody (sdAb), preferably a VHH, for example camelid VH, or a humanized VHH with a human (IgG 1 -derived) Fc and / or humanized VHH-Fc antibody, and / or a humanized VHH-Fc dimer antibody.

[0602] In an optional embodiment, the first ligand and / or the second ligand comprise(s) one or more sugar moieties, preferably being or comprising GalNAc such as 1, 2, 3 or 4 GalNAc moieties, preferably 3 GalNAc moieties, which is a kidney cell non-specific ligand that targets asialoglycoprotein receptor (ASGPR) but appears to enter in the kidney cells, as shown in the Examples section.

[0603] In another preferred embodiment, the ligand is an antibody or a binding fragment thereof, such as a F(ab')2 fragment, Fab' fragment, Fab fragment, scFv, dsFv, scFv-Fc, reduced IgG (rlgG), minibody, diabody, triabody, tetrabody, Fc fusion protein, nanobody, variable V domain, a single-domain antibody (sdAb), preferably a VHH, for example camelid VH, or a humanized VHH with a human (IgG1-derived) Fc and / or humanized VHH-Fc antibody, and preferably wherein the ligand is a humanized VHH-Fc dimer antibody.

[0604] In a more specific embodiment of the saponin component is based on SO1861 saponin and the first ligand and / or the second ligand is selected from: albumin or epidermal growth factor (EGF) or an antibody targeting CD71 or at least one GalNAc moiety, preferably three GalNAc moieties or an antibody or a binding fragment thereof binding to ASGPR.

[0605] The further general aspect is based on another also unexpected finding that a non-targeted ASO-saponin conjugate also showed a high potency in mouse liver in vivo (cf Figure 22) despite not being conjugated to a GalNAc-comprising ligand. This data shows that conjugates for non-targeted delivery like non-targeted saponin-oligonucleotide conjugates that do not possess a specific ligand for a cell-surface endocytosing receptor present on hepatocytes, can still be considered for delivery and / or treatment of the liver as long as a saponin component is delivered or co-delivered with the oligonucleotide-based medicament to the liver cells.

[0606] The presented herein below data shows that a co-treatment with a 2-component linkerformulation comprising a targeted saponin-component (exemplified by SO1861 conjugated with a GalNAc-comprising ligand moiety) strongly improved the potency of a targeted oligonucleotide-medicament (exemplified by an ASO also conjugated with a GalNAc-comprising ligand moiety such as GN3 (reference is made to the Examples section)), by at least two to three orders of magnitude. The improvement was also observed in a 1 -component formulation setting comprising a saponinoligonucleotide conjugate and / or a targeted saponin-oligonucleotide conjugate (1 -component system with the ASO covalently conjugated with SO1861 or with the ASO covalently conjugated with SO1861 also further conjugated to a ligand, such as a GalNAc-comprising ligand GN3) as well as in coadministration setting of the 2-component system, with all systems having at least a 10 to 100-fold safety margin. Together, the data showed below demonstrated that a co-administration is a safe treatment option which results in a 10-fold potency improvement as compared to oligonucleotide-based medicament when provided without the saponin component.

[0607] Combination Therapy

[0608] As would be acknowledged by the skilled person, the therapeutic combinations, pharmaceutical compositions thereof, pharmaceutical formulations thereof or kits thereof or the therapeutic method of the invention and / or the use (of the saponin component and the oligonucleotide-based medicament in the manufacture of a medicament) according to the invention may be conjointly administered with other therapeutic agents, such as other agents suitable for the treatment of cardiovascular disease and other complications around the same. Preferably, conjointly administering one or more additional therapeutic agents with a compound of the invention provides a synergistic effect. In certain embodiments, conjointly administering one or more additional therapeutic agents provides an additive effect.

[0609] In one embodiment, the treatment and / or prevention and / or amelioration of cardiovascular disease comprises administration of the therapeutic combination preceded by, concomitant with or followed by administration to the human subject of any one or more of the therapeutic agents selected from: (i) HMG-CoA reductase inhibitors (statins) such as atorvastatin, cilastatin, fluvastatin, lovastatin, pitavastatin pravastatin, rosuvastatin, simvastatin, cerivastatin; (ii) cholesteryl ester transfer protein (CETP) inhibitor such as torcetrapib, dalcetrapib, evacetrapib, anacetrapib, obicetrapib; (iii) peroxisome proliferator activator receptor (PPAR) modulators; (iv) microsomal triglyceride transfer protein and or apolipoprotein B (MTP / Apo B) secretion inhibitors such as implitapide; (iv) squalene synthetase or squalene epoxidase or squalene cyclase or combined squalene epoxidase / squalene cyclase inhibitors; (v) cholesterol absorption inhibitor such as ezetimibe; (vi) enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitors such as Avasimibe, CS-505, Eflucimibe; (vii) lipase inhibitors such as gastric and / or pancreatic lipase inhibitors lipstatin, tetrahydrolipstatin (orlistat), valilactone, esterastin, ebelactone A, and ebelactone B; (viii) bile acid sequestrants such as colestipol, colesevelam, cholestyramine, clofibrate, gemfibrozil, fenofibrate; (ix) anti-diabetic compounds such as biguanides such as metformin, insulin secretagogues such as sulfonylureas and glinides, glitazones, non-glitazone PPARy agonists, PPARp agonists, SGLT2 inhibitor such as dapagliflozin, empagliflozin, canagliflozin and ertugliflozin, DPP- IV inhibitors such as sitagliptin, saxagliptin, linagliptin, and alogliptin, glucagon-like peptide-1 (GLP-1) analogues, GLP-1 / gastric inhibitory peptide (GIP) dual agonists, GLP-1 / GIP / glucagon (GCG) triple agonists such as liraglutide, semaglutide, tirzepatide, mazedutide, retatrutide; (x) glycogen phosphorylase inhibitor; (xi) sorbitol dehydrogenase inhibitor; (xii) glucosidase inhibitor such as amylase inhibitor - tendamistat, acarbose, adiposine, voglibose, miglitol, emiglitate, camiglibose, tendamistate, trestatin, pradimicin-Q and salbostatin; (xiii) calcium channel blocker such as amlodipine, clevidipine, felodipine, isradipine, nicardipine, nifedipine, nimodipine, nisoldipine; (xiv) Angiotensin Converting Enzyme Inhibitors (ACE-lnhibitors) such as alacepril, benazepril, captopril, ceronapril, delapril, enalapril, fosinopril, imadapril, lisinopril, moveltopril, perindopril, quinapril, ramipril, spirapril, temocapril trandolapril; (xv) Angiotensin-ll receptor antagonists (A-ll antagonists); (xvi) Beta-adrenergic receptor blockers (beta-blockers); (xvii) Alpha-adrenergic receptor blockers (alpha-blockers); (xviii) coronary and / or peripheral vasodilators; (xix) benzothiadiazine and / or sulfonamide diuretic; (xx) an inhibitor of nuclear factor-kappa B (NF-KB) or NF-KB pathway, optionally any one or more selected from: NF-KB inhibitors ectinascidin 743, chromomycin A3, bortezomib, emetine, a tyrosine kinase inhibitor, optionally lestaurtinib (CEP-701), sorafenib tosylate (BAY-43-9006) or sunitinib malate (SU-11248), digitoxin, ouabain, MG 132, TPCA-1, Bay 11-7085, Piceatannol, RAGE antagonist peptide, Caffeic acid, phenethyl ester, Triptolide, ACHP, IKK 16, SUN C8079, Honokiol, Zoledronic Acid, Bay 11-7821, Cardamonin, Withaferin A, Celastrol, BMS 345541, Sulfasalazine, SP 100030, FPS ZM1, Arctigenin, IMD 0354, Luteolin, PF 184, Amlexanox, ML 120B dihydrochloride, IP7e, GSK 319347A, C-DIM 12, C25-140, IDR 1002 and / or therapeutic agents targeting the NF-KB pathway selected from alogliptin, AS62868, docosahexanoic acid, Etanercept, forsythoside B, Minocycline, pioglitazone, simulfilam, telmisartan, TPCA-1, VX-745 and Bay 11-7082, SN50, TPCA-1 and a Non-Steroidal Antiinflammatory Drug (NSAID) optionally indomethacin or ibuprofen;

[0610] an inhibitor of NF-KB inducing kinase (NIK) optionally any one or more selected from: B022, XT2, staurosporine, ZINC-1601221, SIM1, 6Z1T (Protein Data Bank code), 6Z1Q (Protein Data Bank code); menatine;

[0611] an anti-hypertensive agents optionally a drug selected from the class: thiazide diuretics such as metolazone, loop diuretics such as furosemide, potassium-sparing diuretic such as triamterene, aldosterone receptor blocker such as spironolactone, beta blocker such as nadolol, penbutolol and metoprolol, angiotensin converting enzyme inhibitor such as benazepril and ramipril, angiotensin II antagonist such as irbesartan and valsartan, calcium channel blocker such as verapamil, diltiazem, amlodipine and nisoldipine, alpha-1 blocker such as prazosin, alpha-2 agonist such as clonidine and reserpine, and direct vasodilator such as hydralazine and minoxidil;

[0612] and / or any one or more of the B4GALT1, PCSK9, ANGPTL3, CYP7A1 and ALOX12 inhibitor(s); preferably liraglutide, semaglutide, tirzepatide, mazedutide, retatrutide and / or dapagliflozin; and wherein preferably the cardiovascular disease relates to expression or expression level of any one or more of the gene B4GALT1, PCSK9, ANGPTL3, CYP7A1, ALOX12 and SERPINA1, more preferably of any one or more of the genes B4GALT1, CYP7A1 and ALOX12.

[0613] In an embodiment, the saponin component is selected and / or adapted and / or verified so as to not induce NF-KB pathway activation in kidney cells (e.g. by being provided at an in vitro and / or in vivo verified concentration and / or format including, for example, in vitro and / or in vivo verified combination of one or more verified saponin moiety with a linker and alternatively also a ligand).

[0614] The saponin component preferably does not comprise a QS saponin, such as QS-21. The saponin component preferably comprises a saponin with a quillaic acid aglycon or with a gypsogenin aglycon with the proviso that the saponin component does not comprise or consist of a QS saponin such as QS-21. Alternatively, preferred is the saponin component which comprises a saponin without an acyl chain in the glycan / oligosaccharide chain linked to C-28 of the aglycon, preferably quillaic acid or gypsogenin, such as the acyl chain comprised by the glycan linked to C-28 in QS-21, for example the acyl chain comprised by QS-21 apio, i.e. 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid (see Table II for more examples of QS saponins with such glycan comprising an acyl chain). Preferred is a saponin component that does not activate the NF-KB pathway, such as a saponin component comprising a saponin isolated from Saponaria officinalis, and is preferably any one or more of Saponarioside B, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862, SQ1903 and SQ1904, or the aldehyde-substituted derivative of any one thereof more preferably any one or more of SO1832, SO1861 and SO1862 or the aldehyde-substituted derivative of any one thereof, even more preferably SO1832 or SO1861, or the aldehyde-substituted derivative of any one thereof, most preferably SO1861, or the aldehyde-substituted derivative thereof, or, preferably, any one of AG1856, AG1, AG2, Agrostemmoside 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, Saponarioside B, SO1542, SO1584, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862, SO1903, SO1904, or the aldehyde-substituted derivative of any one thereof.

[0615] In another advantageous embodiment of the presented herein therapeutic combinations, compositions, formulations, and / or methods, the saponin component is selected and / or adapted and / or verified so as to it does not induce increase in one or more inflammatory marker(s) level at least in human whole blood, wherein the one or more inflammatory marker(s) level is preferably selected from IL-1β, IL-2, IFNγ, IFNβ, IFNα2a, and TNFα, preferably following the duration of at least 1 hr, preferably 2 hrs, more preferably 3 hrs, most preferably 4 hrs post the whole blood exposure to a pre-determined and / or clinically effective dose or concentration of the therapeutic combination, composition, or formulation, comprising the saponin component ( / .e. dose or concentration capable of producing the desired biological response).

[0616] In a further advantageous embodiment, the saponin component is selected and / or adapted and / or verified so as it has no to low propensity, preferably no propensity, for inducing anti-drug antibody formation (ADA) (as measured by any one of assays that are standard in the art) following intravenous injection of a clinically effective dose or concentration of the therapeutic combination, composition, or formulation, comprising the saponin component, at least 1 day following the injection, preferably at least 3 days following the injection, more preferably at least 1 week following the injection, even more preferably at least 2 weeks following the injection, most preferably more than 3 or 4 weeks following the injection, such as 29 days or more.

[0617] EXAMPLES

[0618] The following examples serve to illustrate the broad applicability of systemic or local co-ad ministration of different saponin components with a variety of oligonucleotides. Together these examples show that co-dosing of saponin components with oligonucleotides (simultaneously, with saponin component and oligonucleotide administered together, or consecutively, with first administering the oligonucleotide either or not together with the saponin component, and thereafter administering the saponin component (again; boosting effect)) markedly improves the efficacy of the oligonucleotides:

[0619] (1) in relevant target tissues, such as (but not limited to) the vasculature, the kidney, the liver, including examples for treatment of sporadic and inherited (familiar) genetic diseases, as well as non-inherited diseases that profit from RNA modulation and various types of cancers originating from or spreading to the liver or to the kidney;

[0620] (2) by targeting (the expression or expression level of) disease relevant genes for the preferred tissues, including but not limited to APOC3, LPA, PCSK9, HBV, DGAT2, GCGR, TAZ, PNPLA3, TM6SF2, HBsAg, LDHA, HSD17B13, PTGS2, TGFB1, AGT, circScdl, PGC-1, mlNDY, Fas death receptor, Drp1, HGF, BID, ANGPTL3, SERPINH1, SLC10A1, CYP7A1, ALOX12, ApoB, HFE, B4GALT1, ANGPTL3, and SERPINA1, in particular SLC10A1, CYP7A1, ABCB4, HFE, PNPLA3, ALOX12 and SERPINA1, or SLC10A1, CYP7A1, HFE and AL0X12, or SERPINA1, or B4GALT1, PCSK9, ANGPTL3, CYP7A1, ALOX12 and SERPINA1;

[0621] (3) by using a variety of oligonucleotide modalities to target these genes or to target RNA transcripts of these genes (pre-mRNA, mRNA) (such as PMO, or phosphorothioated (PS) 2’-M0E ASOs, or PS 2’-locked nucleic acid (LNA) ASOs, or siRNAs with different stabilizations), enabling different mechanisms of action such as

[0622] (a) exon skip to induce frame shift to

[0623] (i) lead to a premature termination codon and nonsense mediated mRNA decay (RNA degradation), or

[0624] (ii) lead to an altered viable transcript and subsequently a different / functional protein (isoform), or

[0625] (b) splice-site blocking to induce alternative splicing / aberrant transcripts leading to RNA degradation, or by

[0626] (c) stimulating RNA cleavage (degradation) through the recruitment of ribonuclease (RNase) H to cleave the RNA strand of a DNA-RNA duplex, or by

[0627] (d) post-transcriptionally halting, or silencing, gene expression of a target mRNA by siRNA, or by

[0628] (e) RNA editing (pre-RNA or mRNA editing) for example antisense RNA-guided (endogenous) adenosine deaminase acting on RNA (ADAR)-based programmable A-to-l editing, for example leading to correction of for example a disease-causing point mutation at the gene level and / or at the protein level, or for example leading to (transiently) inhibiting or silencing protein expression;

[0629] (4) by use of specific, pentacyclic 12,13-dehydrooleanane-type saponin components, but not steroid(-like) saponins / molecules,

[0630] (5) by use of specific, pentacyclic 12,13-dehydrooleanane-type saponin components that are either coadministered free / unconjugated (e.g., SO1861, or SO1861-AH-Block, or SO1861-SC-Mal or AG1856 or GE1741 or SO1832) or as covalently conjugated components (either to the oligonucleotide, e.g. as GN3-SO1861), i.e. either with or without a cell-receptor targeting ligand,

[0631] (6) for different routes of administration including but not limited to intravenously and subcutaneously administration.

[0632] The herein presented data shows that regardless of whether directly conjugated, ligand-conjugated or unconjugated, the pentacyclic 12,13-dehydrooleanane-type saponin component increases the potency of the oligonucleotide therapeutic provided to a tissue or cells of kidney or liver origin without inducing / substantially increasing (cyto)toxicity associated with the treatment.

[0633] The data also suggests that a direct conjugation of an oligonucleotide therapeutic and the pentacyclic 12,13-dehydrooleanane-type saponin component appears to be beneficial in reaching certain liver or kidney regions, in particular including those that are far from the injection site or less exposed to the blood circulation.

[0634] It further shows that such covalent conjugates ensure the desired synchronization of the cellular delivery of an oligonucleotide therapeutic and the saponin component, which results in improved therapeutic efficacy as compared to ASO or siRNA alone but also to ASO or siRNA co-administered with saponin. The data also show that administering a dose of saponin component days to weeks to months after an oligonucleotide-based medicament has been administered, results in a so-called “boosting” effect, determined as an enhanced effect of the oligonucleotide and / or a prolonged effect of the oligonucleotide. Furthermore, the data show that the saponin component does not inflict an inflammatory response mediated by the NF-KB pathway.

[0635] Furthermore, the data show that the saponin component does not trigger an innate immune response, neither does activate an inflammatory response in human blood.

[0636] The data further suggests that ligand-targeted conjugates of an oligonucleotide therapeutic and the pentacyclic 12,13-dehydrooleanane-type saponin (“1 -component conjugates”) are particularly advantageous for performing an endosomally enriched and synchronized delivery of the saponin component and the therapeutic payload into the same cellular compartment. Moreover, the delivery of the therapeutic payload such as an oligonucleotide-based medicament in the aimed cellular compartment (cytosol, nucleus) can be controlled when timing and extent of dose and efficacy is concerned, under influence of the timing of the administration of the saponin component and the timing of administration and the dosing of the oligonucleotide-based medicament. The examples show that by applying the invention it is now made possible to induce controlled release of the oligonucleotide-based medicament. For example, by first administering to a patient a dose of the oligonucleotide-based medicament, either or not together with the saponin component, and thereafter (days, weeks, months thereafter) administer (again) a saponin component. Therewith inducing a therapeutic effect of the oligonucleotide-based medicament for the first time, or therewith boosting the therapeutic effect.

[0637] Furthermore, the data also show that the saponin component does not induce an inflammatory response in kidney cells, as shown by an apparent lack of the NF-KB pathway activation in HEK293-FT cells in vitro. Furthermore, because of the low-immunogenicity of the saponin component, the presented herein pharmaceutical combinations, compositions and formulations appear to be suitable for parenteral administration as various forms of the saponin component do not appear to trigger innate immune or other inflammatory responses in circulating human whole blood obtained from donors, as verified by comparing levels of markers including IL-1β, IL-2, IFNγ, IFNβ, IFNα2a and TNFα following the donor circulating blood exposure to even relatively high concentrations of the saponin component versus control conditions, even after the duration of 4 hrs. These findings are further corroborated by the results of in vivo experiments, in which mice injected intravenously with an antibody-saponin conjugate did not appear to show signs of apparent immunogenicity or suffer from induction of innate immune responses against such conjugates and, importantly, even 29 days post injection, the determination of anti-drug antibody (ADA) levels showed that such saponin components have no to very low propensity for inducing ADA formation in vivo. Abbreviations

[0638] Ab Antibody

[0639] AH Acylhydrazone bond

[0640] AEM A / -(2-Aminoethyl)maleimide trifluoroacetate salt

[0641] AMPD 2-Amino-2-methyl-1,3-propanediol

[0642] BOP (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate

[0643] Cet Cetuximab

[0644] Cy5 Cyanine5 dye

[0645] DIPEA N,N-diisopropylethylamine

[0646] DMF N,N-dimethylformamide

[0647] DTT Dithiothreitol

[0648] EDCI.HCl 3-((Ethylimino)methyleneamino)-N,N-dimethylpropan-1-aminium chloride EMCH.TFA N-(ε-maleimidocaproic acid) hydrazide, trifluoroacetic acid salt

[0649] GalNAc N-Acetylgalactosamine

[0650] GN3 trimeric GalNAc also referred to as trivalent GalNAc

[0651] HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate

[0652] min minutes

[0653] NEM N-Ethylmaleimide

[0654] NMM 4-Methylmorpholine

[0655] r.t. retention time

[0656] SC Semicarbazone bond

[0657] SH Thiol

[0658] siTTR siRNA targeting murine transthyretin

[0659] SPT SO1861 saponin

[0660] TCEP tris(2-carboxyethyl)phosphine hydrochloride

[0661] Temp temperature

[0662] TFA trifluoroacetic acid

[0663] TTR Transthyretin Materials

[0664]

[0665]

[0666] Analytical methods

[0667] LC-MS method 1

[0668] Apparatus: Waters ICIass; Bin. Pump: UPIBSM, SM: UPISMFTN with SO; UPCMA, PDA: UPPDATC, 210-320 nm, SQD: ACQ-SQD2 ESI, mass ranges depending on the molecular weight of the product: neg or neg / pos within in a range of 1500-2400 or 2000-3000; ELSD: gas pressure 40 psi, drift tube temp: 50°C; column: Acquity C18, 50x2.1 mm, 1.7 pm Temp: 60°C, Flow: 0.6 mL / min, lin. Gradient depending on the polarity of the product:

[0669] At0= 2% A, t5.0min= 50% A, t6.0min= 98% A

[0670] ®to=2% A, tsOmin=98% A, teOmin=98% A

[0671] Posttime: 1.0 min, Eluent A: acetonitrile, Eluent B: 10 mM ammonium bicarbonate in water (pH=9.5).

[0672] LC-MS method 2 Apparatus: Waters ICIass; Bin. Pump: UPIBSM, SM: UPISMFTN with SO; UPCMA, PDA: UPPDATC, 210-320 nm, SQD: ACQ-SQD2 ESI, mass ranges depending on the molecular weight of the product: pos / neg 100-800 or neg 2000-3000; ELSD: gas pressure 40 psi, drift tube temp: 50 °C; column: Waters XSelect™ CSH C18, 50x2.1 mm, 2.5 pm, Temp: 25°C, Flow: 0.5 mL / min, Gradient: t0min= 5% A, t2.0min= 98% A, t2.7min= 98% A, Posttime: 0.3 min, Eluent A: acetonitrile, Eluent B: 10 mM ammonium bicarbonate in water (pH=9.5).

[0673] LC-MS method 3

[0674] Apparatus: Waters ICIass; Bin. Pump: UPIBSM, SM: UPISMFTN with SO; UPCMA, PDA: UPPDATC, 210-320 nm, SQD: ACQ-SQD2 ESI, mass ranges depending on the molecular weight of the product pos / neg 105-800, 500-1200 or 1500-2500; ELSD: gas pressure 40 psi, drift tube temp: 50°C; column: Waters XSelect™ CSH C18, 50x2.1 mm, 2.5pm, Temp: 40°C, Flow: 0.5 mL / min, Gradient: t0min= 5% A, t2.0min= 98% A, t2.7min= 98% A, Posttime: 0.3 min, Eluent A: 0.1% formic acid in acetonitrile, Eluent B: 0.1% formic acid in water.

[0675] LC-MS method 4

[0676] Apparatus: Waters ICIass; Bin. Pump: UPIBSM, SM: UPISMFTN with SO; UPCMA, PDA: UPPDATC, 210-320 nm, SQD: ACQ-SQD2 ESI, mass ranges depending on the molecular weight of the product: pos / neg 100-800 or neg 2000-3000; ELSD: gas pressure 40 psi, drift tube temp: 50°C column: Waters Acquity Shield RP18, 50x2.1 mm, 1.7 pm, Temp: 25°C, Flow: 0.5 mL / min, Gradient: t0min= 5% A, t2.0min= 98% A, t2.7min= 98% A, Posttime: 0.3 min, Eluent A: acetonitrile, Eluent B: 10 mM ammonium bicarbonate in water (pH=9.5).

[0677] LC-MS method 5

[0678] Apparatus: Waters ICIass; Bin. Pump: UPIBSM, SM: UPISMFTN with SO; UPCMA, PDA: UPPDATC, 210-320 nm, SQD: ACQ-SQD2 ESI, mass ranges depending on the molecular weight of the product: neg / pos within in a range of 1500-2700; ELSD: gas pressure 40 psi, drift tube temp: 50°C; column: Acquity Premier Peptide BEH C18, 50x2.1 mm, 1.7pm Temp: 25°C, Flow: 0.45 mL / min, Gradient depending on the polarity of the product:

[0679] At0= 2% B, t4.0min= 50% B, t5.0min= 98% B, t6.0min= 98% B

[0680] Bt0= 5% B, t6.0min= 98% B, t6.0min= 98% B

[0681] , Posttime: 1.0 min, Eluent A: 10 mM ammonium bicarbonate in water (pH=9.5), Eluent B: acetonitrile.

[0682] Preparative methods

[0683] Preparative MP-LC method 1

[0684] Instrument type: Reveleris™ prep MPLC; column: Waters XSelect™ CSH C18 (145x25 mm, 10 pm); Flow: 40 mL / min; Column temp: room temperature; Eluent A: 10 mM ammoniumbicarbonate in water pH = 9.0); Eluent B: 99% acetonitrile + 1% 10 mM ammoniumbicarbonate in water; Gradient:

[0685] At0min= 5% B, t1min= 5% B, t2min= 10% B, t17min= 50% B, t18min= 100% B, t23min= 100% B

[0686] At0min= 5% B, t1min= 5% B, t2min= 20% B, t17min= 60% B, t18min= 100% B, t23min= 100% B ; Detection UV: 210, 235, 254 nm and ELSD.

[0687] Preparative MP-LC method 2

[0688] Instrument type: Reveleris™ prep MPLC; Column: Phenomenex LUNA C18(3) (150x25 mm, 10 pm); Flow: 40 mL / min; Column temp: room temperature; Eluent A: 0.1% (v / v) Formic acid in water, Eluent B: 0.1% (v / v) Formic acid in acetonitrile; Gradient:

[0689] At0min= 5% B, t1min= 5% B, t2min= 20% B, t17min= 60% B, t18min= 100% B, t23min= 100% B

[0690] Bt0min= 2% B, t1min= 2% B, t2min= 2% B, t17min= 30% B, t18min= 100% B, t23min= 100% B

[0691] Ct0min= 5% B, t1min= 5% B, t2min= 10% B, t17min= 50% B, t18min= 100% B, t23min= 100% B

[0692] Dt0min= 5% B, t1min= 5% B, t2min= 5% B, t17min= 40% B, t18min= 100% B, t23min= 100% B

[0693] ; Detection UV: 210, 235, 254 nm and ELSD.

[0694] Preparative LC-MS method 3

[0695] MS instrument type: Agilent Technologies G6130B Quadrupole; HPLC instrument type: Agilent Technologies 1290 preparative LC; Column: Waters XSelect™ CSH (C18, 150x19 mm, 10 pm); Flow: 25 ml / min; Column temp: room temperature; Eluent A: 100% acetonitrile; Eluent B: 10 mM ammonium bicarbonate in water pH = 9.0; Gradient:

[0696] At0= 20% A, t2.5min= 20% A, t11min= 60% A, t13min= 100% A, t17min= 100% A

[0697] ®to=5% A, t2.5min=5% A, tl 1min=40% A, tl3min=100% A, tl7min=100% A

[0698] ; Detection: DAD (210 nm); Detection: MSD (ESI pos / neg) mass range: 100 - 800; Fraction collection based on DAD.

[0699] Preparative LC-MS method 4

[0700] MS instrument type: Agilent Technologies G6130B Quadrupole; HPLC instrument type: Agilent Technologies 1290 preparative LC; Column: Waters XBridge Protein (C4, 150x19 mm, 10 pm); Flow: 25 ml / min; Column temp: room temperature; Eluent A: 100% acetonitrile; Eluent B: 10 mM ammonium bicarbonate in water pH=9.0; Gradient:

[0701] At0= 2% A, t2.5min= 2% A, t11min= 30% A, t13min= 100% A, t17min= 100% A

[0702] ®to=10% A, t2.5min=10% A, tllmin=50% A, tl3min=100% A, tl7min=100% A

[0703] Ct0= 5% A, t2.5min= 5% A, t11min= 40% A, t13min= 100% A, t17min= 100% A

[0704] ; Detection: DAD (210 nm); Detection: MSD (ESI pos / neg) mass range: 100 - 800; Fraction collection based on DAD

[0705] Flash chromatography

[0706] Grace Reveleris X2® C-815 Flash; Solvent delivery system: 3-piston pump with auto-priming, 4 independent channels with up to 4 solvents in a single run, auto-switches lines when solvent depletes; maximum pump flow rate 250 mL / min; maximum pressure 50bar (725psi); Detection: UV 200-400nm, combination of up to 4 UV signals and scan of entire UV range, ELSD; Column sizes: 4-330g on instrument, luer type, 750g up to 3000g with optional holder.

[0707] UV-vis spectrophotometry

[0708] Antibody concentrations, and Sulfo-Cy5 concentrations and incorporations were determined using a Thermo Nanodrop 2000 spectrometer. Antibody concentrations in the conjugates were determined by BCA assay. BCA assays were conducted using a Thermo SkanIT plate reader.

[0709] Sulfo-Cy5; mass e646 = 355.7 M-1 cm-1, Rz (280:646) = 0.04.

[0710] Ellmans (TNB) e 412 = 14,150 M-1 cm-1

[0711] Cetuximab £ 280 = 1.4 (mg / ml)-1 cm-1

[0712] Cetuximab-SO1861; mass e280 = 1.4 (mg / ml)-1 cm-1

[0713] Thin Laver Chromatography

[0714] ~8 x 8 cm TLC cards were cut and Sulfo-Cy5 (1:100) and STB28 / 1-1 to 5 and 2-1 to 5 (1:1) spotted (0.5 pl) 6mm apart and allowed to dry. The TLC was run with methanol as mobile phase and inspected visually and under short / long-wave UV. For ‘quantitative’ measurement of residual free Sulfo-Cy5, Sulfo-Cy5 standards (10,000, 1,000, 100 and 0 ng / ml) were ran by TLC and analysed by Fluorescence spectrometry alongside the residual free Sulfo-Cy5 in conjugate samples.

[0715] Fluorescence Spectrometry

[0716] TLC plates were analysed using a Perkin Elmer LS55 Fluorescence Spectrometer with plate reader attachment, in TLC reader mode (Aex = 646 nm; Aem = 720 nm; slit widths = 10nm). Quantities of residual free Sulfo-Cy5 were estimated with respect to Sulfo-Cy5 standards, discounting differences in spot sizes / shapes.

[0717] SEC

[0718] Native antibody and conjugates were analysed by SEC using an Akta purifier 100 system and Biosep SEC-s3000 column eluting with DPBSJPA (85:15). % purity was determined by integration of the antibody peak with respect to trace aggregate peaks.

[0719] SDS-PAGE and Western Blotting

[0720] Native antibody and conjugates were analysed under heat denaturing non-reducing and reducing conditions by SDS-PAGE against a protein ladder using a 4-12% bis-tris gel and MOPS as running buffer (200V, 40 minutes). Samples were prepared to 0.5 mg / ml, comprising LDS sample buffer and MOPS running buffer as diluent. For reducing samples, DTT was added to a final concentration of 50mM. Samples were heat treated for 2 minutes at 90-95 °C and 5 pg (10 pl) added to each well. Protein ladder (10 pl) was loaded without pre-treatment. Empty lines were filled with 1 x LDS sample buffer (10 pl). After the gel was run, it was washed thrice with DI water (100 ml) with shaking (15 minutes, 200 rpm). Coomassie staining was performed by shaker-incubating the gel with PAGEBIue protein stain (30 ml) (60 minutes, 200 rpm). Excess staining solution was removed, rinsed twice with DI water (100 ml) and destained with DI water (100 ml) (60 minutes, 200 rpm). The resulting gel was imaged and processed using ImageJ.

[0721] For Western Blotting, washed gel (not Coomassie stained) was transferred to nitrocellulose membrane using the X-Cell blot module with the following setup (BP-BP-FP-Gel-NC-FP-BP-FP-Gel-NC-FP-BP-BP) and conditions (30V, 0.17 Amps, 60 minutes) and freshly prepared transfer buffer. BP - blotting pad; FP - Filter pad; NC - Nitrocellulose membrane. After, the NC were washed thrice with PBS-T (100 ml), non-specific sites blocked with blocking buffer (30 ml) with shaking (10 minutes, 200 rpm) then active sites labelled with a combination of Goat anti-Human Kappa - HRP (1:2000) and Goat anti-Human IgG - HRP (1:2000) (30 ml) diluted in blocking buffer with shaking (60 minutes, 200 rpm). After, the NC were washed with PBS-T (100 ml) and complexed antibody detected with CN / DAB substrate (25 ml) freshly prepared using stable peroxide substrate buffer. Colour development was observed visually and the resulting NC photographed.

[0722] SO1861-AH-Maleimide

[0723] SO1861-AH-Maleimide (also referred to as SO1861-AH-Mal or SO1861-EMCH) was produced as previously described in WO 2021 / 259507A1 (page 72, Example 3, referred to as “SO1861-EMCH synthesis”). To SO1861 (121 mg, 0.065 mmol) and EMCH. TFA (110 mg, 0.325 mmol) was added methanol (extra dry, 3.00 mL) and TFA (0.020 mL, 0.260 mmol). The reaction mixture stirred at room temperature. After 1.5 hours the reaction mixture was subjected to preparative MP-LC.1 Fractions corresponding to the product were immediately pooled together, frozen and lyophilized overnight to give the title compound (120 mg, 90%) as a white fluffy solid. Purity based on LC-MS 96%.

[0724] LRMS (m / z): 2069 [M-1]1-LC-MS r.t. (min): 1.084

[0725] SO1861-AH-Maleimide-Block (saponin molecule according to formula (V), also referred to as SO1861-AH-Block, SO1861-Ald-AH-mercaptoethanol, saponin component, saponin-compound and SO1861-Ald-EMCH-mercaptoethanol)

[0726] To SO1861-AH-Maleimide (0.1 mg, 48 nmol) 200 pL mercaptoethanol (18 mg, 230 pmol) was added and the solution was shaken for 1 h at 800 rpm and room temperature on a ThermoMixer C (Eppendorf). After shaking for 1 h, the solution was diluted with methanol and dialyzed extensively for 4 h against methanol using regenerated cellulose membrane tubes (Spectra / Por 7) with a MWCO of 1 kDa. After dialysis the SO1861-Ald-EMCH-mercaptoethanol was provided (saponin molecule according to formula (V)), an aliquot was taken out and analyzed via MALDI-TOF-MS.

[0727] (RP mode): m / z 2193 Da ([M+K]+, SO1861-AH-Block), m / z 2185 Da ([M+K]+, SO1861-AH-Block), m / z 2170 Da ([M+Na]+, SO1861-AH-Block).

[0728] SO1861 -AH-azide

[0729] To SO1861 (60 mg, 0.032 mmol) and 1-azido-3,6,9,12-tetraoxapentadecane-15-hydrazide (39.3 mg, 0.129 mmol) was added methanol (extra dry, 1.00 mL) and TFA (9.86 pl, 0.129 mmol) and the reaction mixture was shaken for 1 min and left standing at room temperature. After 2 hours the reaction mixture was subjected to preparative MP-LC.1Fractions corresponding to the product were immediately pooled together, frozen and lyophilized overnight to give the title compound (58.4 mg, 84%) as a white fluffy solid. Purity based on LC-MS 100%.

[0730] LRMS (m / z): 2150 [M-1]1-

[0731] LC-MS r.t. (min): 1.103B

[0732] See Figure 8 for the formula of SO1861-AH-azide (referred to as ‘SOI86I-AH-N3’ in Fig. 8).

[0733] SO1861-SC-azide synthesis

[0734] Intermediate 1:

[0735] tert-butyl 2-(4-(6-azidohexanoyl)piperazine-1-carbonyl)hydrazine-1 -carboxylate

[0736] 6-azidohexanoic acid (603 mg, 3.84 mmol), tert-butyl 2-(piperazine-1-carbonyl)hydrazine-1 -carboxylate (781 mg, 3.20 mmol), EDCLHCI (735 mg, 3.84 mmol) and Oxyma Pure (591 mg, 4.16 mmol) were dissolved in a mixture of dichloromethane (25 mL) and DIPEA (835 pL, 4.80 mmol) and the reaction mixture was stirred at room temperature. After 2 hours the reaction mixture was evaporated in vacuo and the residue was dissolved in ethyl acetate (50 mL). The resulting solution was washed with 0.5 N potassium bisulphate solution (50 mL), saturated sodium bicarbonate solution (2 x 50 mL) and brine (50 mL), dried over Na2SO4, filtered and evaporated in vacuo. The residue was purified by flash chromatography (DCM - 10% methanol in DCM (v / v) gradient 100:0 rising to 40:60) to give the title compound (864 mg, 70%) as a white solid. Purity based on LC-MS 96%.

[0737] LRMS (m / z): 284 / 328 / 406 [M-99 / M-55 / M+23]1+

[0738] LC-MS r.t. (min): 1.132

[0739] Intermediate 2:

[0740] 4-(6-azidohexanoyl)piperazine-1 -carbohydrazide 2,2,2-trifluoroacetate

[0741] te / Y-butyl 2-(4-(6-azidohexanoyl)piperazine-1-carbonyl)hydrazine-1 -carboxylate (50.0 mg, 130 pmol) was dissolved in a mixture of dichloromethane (1.00 mL) and TFA (1.00 mL) and the reaction mixture was stirred at room temperature. After 1 hour the reaction mixture was evaporated in vacuo and coevaporated with dichloromethane (3 x 5 mL) to give the crude title product as a white solid.

[0742] LRMS (m / z): 284 / 307 [M+1 / M+23]1+

[0743] SO1861-SC-azide

[0744] To SO1861 (60 mg, 0.032 mmol) and 4-(6-azidohexanoyl)piperazine-1 -carbohydrazide 2,2,2-trifluoroacetate (51.2 mg, 0.129 mmol) was added methanol (extra dry, 1.5 mL) and the reaction mixture was shaken for 1 min and left standing at room temperature. After 3 hours the reaction mixture was subjected to preparative MP-LC.1AFractions corresponding to the product were immediately pooled together, frozen and lyophilized overnight to give the title compound (55.6 mg, 81%) as a white solid. Purity based on LC-MS 96%.

[0745] LRMS (m / z): 2127 [M-1]1 LC-MS r.t. (min): 3.395A

[0746] See Figure 9 for the formula of SO1861-SC-azide (referred to as ‘SOI86I-SC-N3’ in Fig. 9).

[0747] Trivalent GalNAc (or GN3)-azide synthesis

[0748] Intermediate 1:

[0749] tert-butyl 1 -azido-17,17-bis((3-(tert-butoxy)-3-oxopropoxy)methyl)-15-oxo-3,6,9,12,19-pentaoxa-16-azadocosan-22-oate

[0750] Intermediate 1 was produced as previously described in WO2022 / 055351 (page 136, line 3 to page 139, line 1, Figure 8, Example 1 C).

[0751] To di-tert-butyl 3,3'-((2-amino-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionate (1.27 g, 2.51 mmol) was added a solution of 3-Azido(peg4)propionic acid N-hydroxysuccinimide ester (977 mg, 2.51 mmol) in DMF (10 mL). Next, DIPEA (657 pL, 3.77 mmol) was added and the reaction mixture was stirred overnight at room temperature. The reaction mixture was evaporated in vacuo and the residue was dissolved in ethyl acetate (100 mL). The resulting solution was washed with 0.5 N potassium bisulphate solution (2 x 100 mL) and brine (100 mL), dried over Na2SO4, filtered and evaporated in vacuo. The residue was purified by flash chromatography (DCM -10% methanol in DCM (v / v) gradient 100:0 rising to 0:100) to give the title compound (1.27 g, 65%) as a colorless oil. Purity based on LC-MS 100% (ELSD).

[0752] LRMS (m / z): 780 [M+1]1+

[0753] LC-MS r.t. (min): 2.102

[0754] Intermediate 2:

[0755] 1 -azido-17,17-bis((2-carboxyethoxy)methyl)-15-oxo-3,6,9,12,19-pentaoxa-16-azadocosan-22-oic acid

[0756] Intermediate 2 was produced as previously described in WO2022 / 055351 (page 136, line 3 to page 139, line 1, Figure 8, Example 1 C).

[0757] To a solution of tert-butyl 1 -azido-17,17-bis((3-(tert-butoxy)-3-oxopropoxy)methyl)-15-oxo-3,6,9,12,19-pentaoxa-16-azadocosan-22-oate (1.27 g, 1.63 mmol) in DCM (5.0 mL) was added TFA (5.0 mL, 65 mmol). The reaction mixture was stirred at room temperature. After 1.5 hours the reaction mixture was evaporated in vacuo, co-evaporated with toluene (3 x 10 mL) and DCM (3 x 10 mL) to give the crude title product as a colorless oil.

[0758] LRMS (m / z): 611 [M+1]1+

[0759] Intermediate 3:

[0760] di-tert-butyl (10-(1-azido-3,6,9,12-tetraoxapentadecan-15-amido)-10-(13,13-dimethyl-5,11-dioxo-2,12-dioxa-6,10-diazatetradecyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecane-1,19-diyl)dicarbamate Intermediate 3 was produced as previously described in WO2022 / 055351 (page 136, line 3 to page 139, line 1, Figure 8, Example 1 C). 1-azido-17,17-bis((2-carboxyethoxy)methyl)-15-oxo-3,6,9,12,19-pentaoxa-16-azadocosan-22-oic acid (997 mg, 1.63 mmol), Oxyma Pure (1.04 g, 7.35 mmol) and EDCI. HCI (1.17 g, 6.12 mmol) were dissolved in DMF (10.0 mL). Next, DIPEA (1.99 mL, 11.4 mmol) was added, followed directly by the addition of a solution of N-BOC-1,3-propanediamine (1.07 g, 6.12 mmol) in DMF (10.0 mL). The reaction mixture was stirred overnight at room temperature. The reaction mixture was evaporated in vacuo and the residue was dissolved in ethyl acetate (100 mL). The resulting solution was washed with 0.5 N potassium bisulphate solution (100 mL), saturated sodium bicarbonate solution (2 x 100 mL) and brine (100 mL), dried over Na2SO4, filtered and evaporated in vacuo. The residue was purified by flash chromatography (DCM - 10% methanol in DCM (v / v) gradient 0:100 rising to 100:0, staying at 100:0 until the product eluted) to give the title compound (1.16 g, 66%) as a yellowish viscous oil. LC-MS 99% (ELSD).

[0761] LRMS (m / z): 1080 [M+1]1+

[0762] LC-MS r.t. (min): 1.513

[0763] Intermediate 4:

[0764] 3,3'-((2-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-2-(1-azido-3,6,9,12-tetraoxapentadecan-15-amido)pro pane-1,3-diyl)bis(oxy))bis(N-(3-aminopropyl)pro panamide) tris(2,2,2-trifluoroacetate) synthesis

[0765] Intermediate 4 was produced as previously described in WO2022 / 055351 (page 136, line 3 to page 139, line 1, Figure 8, Example 1 C).

[0766] To a solution of di-tert-butyl (10-(1-azido-3,6,9,12-tetraoxapentadecan-15-amido)-10-(13,13-dimethyl-5,11-dioxo-2,12-dioxa-6,10-diazatetradecyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecane-1,19-diyl)dicarbamate (1.16 g, 1.08 mmol) in DCM (10 mL) was added TFA (10 mL, 131 mmol). The reaction mixture was stirred at room temperature. After 2 hours the reaction mixture was evaporated in vacuo, co-evaporated with toluene (3 x 10 mL) and DCM (3 x 10 mL) to give the crude title product as a yellowish viscous oil.

[0767] LRMS (m / z): 260 [M+3]3+, 390 [M+2]2+, 780 [M+1 ]1+,

[0768] Intermediate 5:

[0769] (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2,5-dioxopyrrolidin-1-yl)oxy)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate

[0770] Intermediate 5 was produced as previously described in WO2022 / 055351 (page 136, line 3 to page 139, line 1, Figure 8, Example 1 C).

[0771] 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid (obtain according J. Am. Chem Soc., 2014, 136, 16958-16961, 3.00 g, 6.70 mmol) and A / -Hydroxysuccinimide (926 mg, 8.05 mmol) were dissolved in DCM (50 mL). Next, EDCI. HCI (1.54 g, 8.05 mmol) and 4-(Dimethylamino)pyridine (82 mg, 0.67 mmol) were added and the reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with DCM and the resulting solution was washed with 0.5 N potassium bisulphate solution (150 mL), saturated sodium bicarbonate solution (150 mL) and brine (150 mL), dried over Na2SO4, filtered and evaporated in vacuo to give the title compound (3.60 g, 99%) as a white foam. Purity based on LC-MS 99% (ELSD).

[0772] LRMS (m / z): 545 [M+1]1+

[0773] LC-MS r.t. (min): 1.073

[0774] Intermediate 6:

[0775] [(3R,6R)-3,4-bis(acetyloxy)-6-{4-[(3-{3-[2-(1-azido-3,6,9,12-tetraoxapentadecan-15-amido)-3-(2-{[3-(5-{[(2R,5R)-4,5-bis(acetyloxy)-6-[(acetyloxy)methyl]-3-acetamidooxan-2-yl]oxy}pentanamido)propyl]carbamoyl}ethoxy)-2-[(2-{[3-(5-{[(2R,5R)-4,5-bis(acetyloxy)-6-[(acetyloxy)methyl]-3-acetamidooxan-2-yl]oxy}pentanamido)propyl]carbamoyl}ethoxy)methyl]propoxy]propanamido}propyl)carbamoyl]butoxy}-5-acetamidooxan-2-yl]methyl acetate

[0776] Intermediate 6 was produced as previously described in WO2022 / 055351 (page 136, line 3 to page 139, line 1, Figure 8, Example 1 C).

[0777] 3,3'-((2-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-2-(1-azido-3,6,9,12-tetraoxapentadecan-15-amido)propane-1,3-diyl)bis(oxy))bis(N-(3-aminopropyl)propanamide) tris(2,2,2-trifluoroacetate) (1.21 g, 1.08 mmol) was dissolved in a mixture of DMF (10 mL) and DIPEA (1.69 mL, 9.70 mmol). Next, (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2,5-dioxopyrrolidin-1-yl)oxy)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (2.20 g, 4.04 mmol) was added and the reaction mixture was stirred over the weekend at room temperature. Next, the reaction mixture was evaporated in vacuo and the residue was purified by flash chromatography (DCM - 30% methanol in DCM (v / v) gradient 0:100 rising to 100:0) to give the title compound (1.84 g, 83%) as a yellowish foam. LC-MS 95% (ELSD).

[0778] LRMS (m / z): 2068 [M+1]1+

[0779] LC-MS r.t. (min): 1.183

[0780] Intermediate 7:

[0781] Trivalent GalNAc-azide

[0782] Trivalent GalNAc-azide was produced as previously described in WO2022 / 055351 (page 136, line 3 to page 139, line 1, Figure 8, Example 1 C).

[0783] [(3R,6R)-3,4-bis(acetyloxy)-6-{4-[(3-{3-[2-(1-azido-3,6,9,12-tetraoxapentadecan-15-amido)-3-(2-{[3-(5-{[(2R,5R)-4,5-bis(acetyloxy)-6-[(acetyloxy)methyl]-3-acetamidooxan-2-yl]oxy}pentanamido)propyl]carbamoyl}ethoxy)-2-[(2-{[3-(5-{[(2R,5R)-4,5-bis(acetyloxy)-6-[(acetyloxy)methyl]-3-acetamidooxan-2-yl]oxy}pentanamido)propyl]carbamoyl}ethoxy)methyl]propoxy]propanamido}propyl)carbamoyl]butoxy}-5-acetamidooxan-2-yl]methyl acetate (300 mg, 0.145 mmol) was dissolved in a mixture of triethylamine (2.00 mL, 14.4 mmol), methanol (2.00 mL) and water (2.00 mL) and the reaction mixture was stirred at room temperature. After 2 hours the reaction mixture was evaporated in vacuo. The residue was purified by preparative MP-LC.2BFractions corresponding to the product were immediately pooled together, frozen and lyophilized overnight to give the title compound (164 mg, 67%) as a white solid. Purity based on LC-MS 97%.

[0784] LRMS (m / z): 1688 [M-1]1-

[0785] LC-MS r.t. (min): 1.991A

[0786] Trivalent GalNAc-amine formate

[0787] Trivalent GalNAc-amine formate was produced as previously described in WO2022 / 055351 (page 143 - 144, Example 1D).

[0788] Trivalent GalNAc-azide (36.5 mg, 21.6 pmol) was dissolved in a solution of potassium carbonate (5.97 mg, 43.2 pmol) in water (1.00 mL) and acetonitrile (1.00 mL). Next, a 1.0 M trimethylphosphine solution in THF (216 pL, 216 pmol) was added and the resulting mixture was shaken for 1 min and left standing at room temperature. After 45 min the reaction mixture was evaporated in vacuo and the residue was dissolved in water / acetonitrile (9:1, v / v, 1 mL). The resulting solution was directly subjected to preparative MP-LC.2BFractions corresponding to the product were immediately pooled together, frozen and lyophilized overnight to give the title compound (36.1 mg, 98%) as a white solid. Purity based on LC-MS 100%.

[0789] LRMS (m / z): 1662 [M-1]1-

[0790] LC-MS r.t. (min): 1.621A

[0791] Intermediate 14: Trivalent GalNAc (or GN3)-DBCO

[0792] Trivalent GalNAc-DBCO was produced as previously described in WO2022 / 055351 (page 143 - 144, Example 1D).

[0793] Trivalent GalNAc-amine formate (17.4 mg, 10.2 pmol) and DBCO-NHS (6.14 mg, 15.3 pmol) were dissolved in a solution of NMM (2.24 pL, 20.3 pmol) in DMF (0.50 mL). The reaction mixture was shaken for 1 min and left standing at room temperature. After 2 hours the reaction mixture was evaporated in vacuo and the residue was dissolved in water / acetonitrile (8:2, v / v, 1 mL). The resulting solution was directly subjected to preparative MP-LC.2CFractions corresponding to the product were immediately pooled together, frozen and lyophilized overnight to give the title compound (14.2 mg, 72%) as a white solid. Purity based on LC-MS 96%.

[0794] LRMS (m / z): 1950 [M-1]1-

[0795] LC-MS r.t. (min): 1.861B

[0796] GN3-SC-SO1861

[0797] To SO1861-SC-N3 (18.0 mg, 8.45 pmol) and GN3-DBCO (16.5 mg, 8.45 pmol) was added a mixture of acetonitrile (250 pL) and 20 mM ammonium bicarbonate (750 pL). The reaction mixture was shaken for about 1 min and left standing at room temperature. After 1 hour the reaction mixture was subjected to preparative MP-LC.1AFractions corresponding to the product were immediately pooled together, frozen and lyophilized overnight to give the title compound (29.2 mg, 85%) as a white solid. Purity based on LC-MS 99%.

[0798] LRMS (m / z): 2038 [M-2H]2-

[0799] LC-MS r.t. (min): 2.195B

[0800] See Figure 11 for the formula of GN3-SC-SO1861.

[0801] N3-AH-SO1861

[0802] To SO1861-AH-N3 (30.0 mg, 13.9 pmol) and GN3-DBCO (27.2 mg, 13.9 pmol) was added a mixture of acetonitrile (250 pL) and 20 mM ammonium bicarbonate (750 pL). The reaction mixture was shaken for about 1 min and left standing at room temperature. After 1 hour the reaction mixture was subjected to preparative MP-LC.1AFractions corresponding to the product were pooled together, frozen and lyophilized overnight to give the title compound (50.0 mg, 87%) as a white solid. Purity based on LC-MS 99%.

[0803] LRMS (m / z): 2049 [M-2H]2-LC-MS r.t. (min): 2.155B

[0804] See Figure 10 for the formula of GN3-AH-SO1861.

[0805] GN3-siTTR

[0806] Trivalent GalNAc-siRNA targeting murine transthyretin (also referred to as the nucleic acid component GN3-siTTR [SEQ ID No: 1 for the sense strand, and SEQ ID No. 15 for the antisense strand]) with advanced enhanced stability chemistry backbone was custom-produced by BioSpring Gesellschaft fur Biotechnologie mbH, Germany, according to methods known in the art (Figure 1). GalNAc monomers were conjugated via their phosphate groups in a linear fashion to generate a trimeric (trivalent) GalNAc. The phosphate group of the third GalNAc links to the 3’ end of the oligonucleotide sequence resulting in the following conjugate with sense strand [SEQ ID No: 1]: 5’-6*6*7685451315875756566000; and antisense strand [SEQ ID No: 15]: 5’-5*1 *6564643668627275855*5*5; with 0 = GalNac, 1 = 2’-Fluoro-U, 2 = 2’-Fluoro-A, 3 = 2’-Fluoro-C, 4 = 2’-Fluoro-G, 5 = 2’OMe-rU, 6 = 2’OMe-rA, 7 = 2’OMe-rC, 8 = 2’OMe-rG, * = Thioate. Such a linear trimeric (trivalent) GalNAc could also be conjugated to saponin components to generate for example GN3-SC-SO1861 or GN3-AH-SO1861 by methods known in the art.

[0807] Cetuximab-AH-SO1861

[0808] To cetuximab (1087 mg, 4.800 mg / ml, 7.2 x 10-3 mmol, in TBS, 2.5 mM EDTA, pH 7.5) was added an aliquot of freshly prepared TCEP solution (1 mg / ml, 2.72 mole equivalents, 2.0 x 10-2 mmol, 5.65 mg), the mixture swirled by hand to mix then incubated for 210 minutes at 20 °C with roller-mixing. After incubation (priorto addition of SO1861-AH-Maleimide), a 2 mg (0.417 ml) aliquot of Ab-SH was removed and purified by gel filtration using zeba spin desalting column into TBS pH 7.5. This aliquot was characterized by UV-vis analysis and Ellman’s assay (3.693 mg / ml, thiol to Ab ratio = 4.0). To the bulk Ab-SH was added an aliquot of freshly prepared SO1861-AH-Maleimide solution (2 mg / ml, 5.2 mole equivalents, 3.8 x 10-2 mmol, 38.9 ml), the mixtures vortexed briefly then incubated for 120 minutes at 20 °C. Besides the conjugation reaction, two aliquots of desalted Ab-SH (0.5 mg, 0.135 ml, 3.33 x 10-6 mmol) were reacted with NEM (8.00 equivalents, 2.66 x 10-5 mmol, 3.3 pg, 13.3 pl of a 0.25 mg / ml solution) or TBS pH 7.5 buffer (13.3 pl) for 120 minutes at 20 °C, as positive and negative controls, respectively. After incubation (prior to addition of NEM), a ca. 2 mg (0.450 ml) aliquot of Ab - SO1861 mixture was removed and purified by gel filtration using zeba spin desalting column into TBS pH 7.5. This aliquot was characterized by UV-vis (3.271 mg / ml) and alongside positive and negative controls were characterized by Ellman’s assay to obtain SO1861 incorporation. To the bulk Ab - SO1861 mixture was added an aliquot of freshly prepared NEM solution (2.5 mg / ml, 5 mole equivalents, 3.6 x 10-2 mmol, 4.54 mg) and the mixture stored at 2-8 °C overnight. The conjugate was purified by 10 x 40 cm Sephadex G50M column eluting with DPBS pH 7.5 to give purified cetuximab - SO1861 conjugate. The aliquot was filtered to 0.2μm and dispensed. The result was a cetuximab - SO1861 conjugate. Yield = 1056 mg, 97%, SO1861 to Ab ratio = 3.9.

[0809] Cetuximab-Cyanine5 (Cet-Cy5)

[0810] To cetuximab (5 mg, 3.30 x 10-5 mmol, 4.982 mg / ml, 1 mL in DPBS) was added an aliquot of freshly prepared Sulfo-Cy5-NHS solution (100 mg / ml, 320 mole equivalents) followed by an aliquot of DPBS pH 7.5 buffer (to normalize reaction volumes), the mixture vortexed briefly then incubated for 120 minutes at 20 °C with roller-mixing. After, to reaction mixture was added an aliquot of freshly prepared glycine solution (100 mg / ml, 5 mole equivalents with respect to Sulfo-Cy5) to quench the reaction. The conjugate was purified by gel filtration using PD10 G25 columns eluting into DPBS pH 7.5. The resulting conjugate was characterized by UV-vis spectrophotometry and BCA assay to ascertain antibody concentrations and Sulfo-Cy5 incorporations. TLC using methanol as mobile phase was conducted to show that residual free Sulfo-Cy5 was present. The conjugate was further purified by Dialysis using float-a-lyser G2 dialysis devices against DPBST pH 7.5 (dialysate changes were made after 12, 21, 84 and 108 h). The conjugate was then analyzed by BCA assay to ascertain antibody concentration and filtered to 0.2 pm under laminar flow (UV-vis spectrophotometry was used to ascertain losses due to filtration were negligible). Employing careful handling techniques and handling under laminar flow where possible, the conjugate was concentrated via diafiltration using vivaspin T4 centrifuge filter tubes (2,000 g, 20 minutes, 5 °C). Sample was removed for characterization and the bulk product characterized by UV-vis spectrophotometry and BCA assay to ascertain antibody concentrations and Sulfo-Cy5 incorporations. Yield: 29%, Sulfo-Cy5 incorporation: 51.3, Purity: 98.1%.

[0811] GN3-siAT3

[0812] Trivalent GalNAc-siRNA targeting human SERPINC1 (and being cross-reactive with cercopithecine SERPINC1 of the non-human primate Macaca fascicularis), also referred to as the nucleic acid component GN3-siAT3 (SEQ ID No: 16 for the sense strand, and SEQ ID No. 17 for the antisense strand) with enhanced stability chemistry backbone was custom-made and manufactured by Biotage, United Kingdom (using the antisense-strand produced by BioSpring, Gesellschaft fur Biotechnologie mbH, Germany, according to methods known in the art (Figure 1)). GalNAc monomers were conjugated via their phosphate groups in a linear fashion to generate a trivalent GalNAc. The phosphate group of the third GalNAc links to the 3’ end of the oligonucleotide sequence resulting in the following conjugate with sense strand [SEQ ID No: 16]: 5’-6*G*8U5A7A775U8U5C8U7A5333; and antisense strand [SEQ ID No: 17]: 5’- U*8*G5A6U5A5UGG8G8U5A7C*A*G; with 3 = GalNAc C3; 5 = 2’-Fluoro-A; 6 = 2’-Fluoro-G; 7 = 2’-Fluoro-C; 8 =2’-Fluoro-U; Upper case letters = 2’-O-methyl; * = phosphorothioate linkage. RNA isolation and gene expression analysis

[0813] A sample of -50-100 mg was cut from frozen liver tissue. Each sample was cut into smaller fragments, transferred into a 2.0 ml (RNase free) safe-lock tube, and 1 ml TRIzol™ Reagent (Fisher Scientific) and a 5 mm stainless steel bead (Qiagen) were added. The tube was placed in a TissueLyser II (Qiagen) for 5 minutes at 30 Hz, to completely disrupt the tissue. Then, 0.2 ml chloroform was added, and the tube was shaken vigorously for 15 seconds. Samples were incubated for 2-3 minutes at room temperature, following centrifugation at 12.000 x g for 15 minutes at 4°C. Next, 400 pl of the upper phase was taken and transferred to a new 1.5 ml collection tube. To this, 400 pl (RNase free) isopropanol was added and the solution was mixed gently and then placed overni...

Claims

CLAIMS1. A therapeutic combination comprising:a. an oligonucleotide-based medicament adapted to target and bind to a nucleic acid molecule present in the cytosol of a human cell and / or present in the nucleus of a human cell; b. a saponin component comprising a penta-cyclic triterpene saponin, comprising an aglycone core of 12,13-dehydrooleanane type;for use in treatment and / or prevention and / or amelioration of a cardiovascular disease; wherein the treatment and / or prevention and / or amelioration comprises administration of the therapeutic combination to a human subject in the need thereof;wherein the saponin component and the oligonucleotide-based medicament are delivered to the human cell wherein said human cell is a cell of the kidney and / or is a liver cell, optionally a hepatocyte.

2. The therapeutic combination for use according to claim 1, wherein the cardiovascular disease is related to (an) elevated plasma concentration(s) of low-density lipoprotein cholesterol (LDL-C) and / or triglycerides, wherein the cardiovascular disease is optionally accompanied with inflammation.

3. The therapeutic combination for use according to any one of the claims 1 or 2, wherein the cardiovascular disease is selected from any one or more of:(i) atherosclerotic cardiovascular disease (ASCVD), selected from: coronary heart disease (CHD), such as myocardial infarction, angina and coronary artery stenosis; cerebrovascular disease, such as a transient ischemic attack and ischemic stroke; peripheral artery disease, such as claudication; and aortic atherosclerotic disease, such as abdominal aortic aneurysm and descending thoracic aneurysm;(ii) hyperlipidemia;(iii) dyslipidemia;(iv) hypertriglyceridemia;(v) familial chylomicronemia (type I hyperlipoproteinemia);(vi) familial hypercholesterolemia (type II hyperlipoproteinemia);(vii) hypercholesterolemia;(viii) hemorrhagic disease;(ix) thrombosis;(x) congenital disorder of glycosylation type lid (CDG-lld);(xi) blood clotting defects;(xii) arterial plaque formation;(xiii) hypertension; and(xiv) elevated levels of fibrinogen,and wherein the cardiovascular disease is optionally accompanied with inflammation, and / or wherein the cardiovascular disease is a human disease affecting the kidney and / or the liver.

4. The therapeutic combination for use according to claim 3, wherein familial hypercholesterolemia (type II hyperlipoproteinemia) is heterozygous familial hypercholesterolemia (heFH) or homozygous familial hypercholesterolemia (HoFH), and is optionally accompanied with inflammation.

5. The therapeutic combination for use according to any one of the preceding claims, wherein the oligonucleotide-based medicament and the saponin component are co-formulated in a single pharmaceutical composition.

6. The therapeutic combination for use according to any one of the claims 1-4, wherein the oligonucleotide-based medicament and the saponin component are formulated separately in at least two pharmaceutical formulations, wherein a first pharmaceutical formulation comprises the saponin component and a second pharmaceutical formulation comprises the oligonucleotide-based medicament.

7. The therapeutic combination for use according to any one of the preceding claims, wherein the oligonucleotide-based medicament is packed in a lipid nanoparticle (LNP).

8. The therapeutic combination for use according to any one of the claims 5-7, wherein the coformulated single pharmaceutical composition or the separately formulated first pharmaceutical formulation and second pharmaceutical formulation, further comprises a pharmaceutically acceptable excipient, carrier and / or diluent.

9. The therapeutic combination for use according to any one of the preceding claims, wherein the treatment and / or prevention and / or amelioration comprises administering to a patient in need thereof an amount of the therapeutic combination which delivers a therapeutic effective amount of the oligonucleotide-based medicament to the human cell wherein said human cell is a cell of the kidney and / or is a liver cell, optionally a hepatocyte.

10. The therapeutic combination for use according to any one of the preceding claims, wherein the therapeutic combination is administered via a route of administration selected from: oral, intravenous injection, intravenous infusion, subcutaneous, intraperitoneal, renal artery, retrograde renal vein, renal parenchyma and retrograde ureteral, portal vein injection, preferably wherein the route of administration is selected from intravenous administration and subcutaneous administration.

11. The therapeutic combination for use according to any one of the preceding claims, wherein the saponin component comprises a saponin moiety, being the penta-cyclic triterpene saponin, andfurther comprises a non-saponin moiety, being a first ligand recognised by a first endocytic receptor, and / or wherein the oligonucleotide-based medicament further comprises a second ligand recognised by a second endocytic receptor, possibly wherein the second endocytic receptor is the same as the first endocytic receptor, further possibly wherein the second ligand is the same as the first ligand, alternatively wherein the second endocytic receptor differs from the first endocytic receptor with the proviso that the two different endocytic receptors are both present on the same cells, for example the same liver cell or the same kidney cell; wherein the first ligand and / or the second ligand is / are a proteinaceous ligand, for example a naturally existing peptide or protein ligand or a receptor-interacting part thereof, or is an antibody or a binding fragment thereof; and / or wherein the first ligand and / or the second ligand comprises one or more sugar moieties, preferably being or comprising at least one N-acetylgalactosamine (GalNAc) moiety or three GalNAc moieties.

12. The therapeutic combination for use according to any one of the preceding claims, wherein the oligonucleotide-based medicament binds to a nucleic acid molecule present in the cytosol of a human cell and / or present in the nucleus of a human cell, selected from liver cells, preferably hepatocytes, and / or kidney cells, preferably kidney cortex cells and / or kidney medulla cells.

13. The therapeutic combination for use according to any one of the preceding claims, wherein the penta-cyclic triterpene saponin further comprises:an aldehyde function at position C-23 of the aglycone core; oran acid-sensitive covalent bond configured to break under acidic conditions so as to create the aldehyde function at position C-23 of the aglycone core, preferably wherein the acid-sensitive covalent bond is selected from any one or more of: a hydrazone bond, a semicarbazone bond, an imine bond, an acetal bond including a 1,3-dioxolane bond, a ketal bond, an ester bond, and / or an oxime bond, and preferably is a hydrazone bond.

14. The therapeutic combination for use according to any one of the preceding claims, wherein the penta-cyclic triterpene saponin is a triterpenoid saponin and / or a bisdesmosidic triterpene saponin belonging to the type of a 12,13-dehydrooleanane with a branched carbohydrate side chain at the C-3 position wherein said carbohydrate side chain comprises glucuronic acid function, preferably also with an aldehyde function in position C-23 and preferably further with a branched carbohydrate side chain at the C-28 position comprising deoxy carbohydrate.

15. The therapeutic combination for use according to any one of the preceding claims, wherein the penta-cyclic triterpene saponin is a triterpenoid saponin and / or a bisdesmosidic triterpene saponin belonging to the type of a 12,13-dehydrooleanane with an aldehyde function in position C-23 selected from the group consisting of saponins with a quillaic acid aglycon core and saponins with a gypsogenin aglycon core, and / or wherein the saponin comprises a branched carbohydrate side chain at the C-3 position wherein said carbohydrate side chain comprises glucuronic acid function,and wherein the saponin comprises a branched carbohydrate side chain at the C-28 position comprising deoxy carbohydrate.

16. The therapeutic combination for use according to any one of the preceding claims, wherein the penta-cyclic triterpene saponin is mono-desmosidic or bi-desmosidic, preferably comprising a first saccharide chain bound to a position C-3 of the aglycone core, more preferably wherein the first saccharide chain is selected from Group A listed in Table III, even more preferably wherein the first saccharide chain comprises a glucuronic acid group, preferably a terminal glucuronic acid group, most preferably wherein the first saccharide chain comprises or is: Gal-(1→2)-[Xyl-(1→3)]-GlcA.

17. The therapeutic combination for use according to any one of the preceding claims, wherein the penta-cyclic triterpene saponin comprises the aglycone core selected from quillaic acid, gypsogenin, and an aldehyde-substituted derivative of either one of quillaic acid or gypsogenin defined as a quillaic acid-based aglycone core or gypsogenin-based aglycone core, respectively, wherein the aldehyde function at position C-23 is substituted by the acid sensitive covalent bond configured to break under acidic conditions so as to create the aldehyde function at position C-23 of the aglycone core, preferably wherein the penta-cyclic triterpene saponin is selected from:AG1856, AG1, AG2, Agrostemmoside 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, Saponarioside B, SO1542, SO1584, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862, SO1903, SO1904, QS-7, QS-7 apio, QS-17, QS-18, QS-21 A-apio, QS-21 A- xylo, QS-21 B-apio and QS-21 B-xylo, or the aldehyde-substituted derivative of any one thereof, respectively,preferably AG1856, AG1, AG2, Agrostemmoside 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, Saponarioside B, SO1542, SO1584, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862, SQ1903, SQ1904, or the aldehyde-substituted derivative of any one thereof, respectively; orwherein the penta-cyclic triterpene saponin is selected from:SA1641, gypsoside A, NP-017772, NP-017774, NP-017777, NP-017778, NP-018109, NP-017888, NP-017889, NP-018108 and SO1658, or the aldehyde-substituted derivative of any one thereof, respectively.

18. The therapeutic combination for use according to any one of the preceding claims, wherein the penta-cyclic triterpene saponin is isolated from Saponaria officinalis, and is preferably any one or more of Saponarioside B, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862, SQ1903 and SQ1904 or the aldehyde-substituted derivative of any one thereof, respectively, more preferably any one or more of SO1832, SO1861 and SO1862 or the aldehyde-substituted derivative of any one thereof, respectively, even more preferably SO1832 orSO1861 or the aldehyde-substituted derivative of any one thereof, respectively, most preferably SO1861 or the aldehyde-substituted derivative of any one thereof, respectively.

19. The therapeutic combination for use according to any one of the preceding claims, wherein the saponin component comprises an unconjugated saponin moiety or a saponin molecule or wherein the saponin component comprises a saponin moiety that is covalently conjugated with at least one non-saponin moiety, preferably via an acid-sensitive covalent bond that breaks under acidic conditions,more preferably being an acid-sensitive covalent bond at the position C-23 of the aglycone core, even more preferably wherein the acid sensitive covalent bond at the position C-23 of the aglycone core is configured to break under acidic conditions so as to create the aldehyde function at position C-23 of the aglycone core thus resulting in a release of the penta-cyclic triterpene saponin comprising the aldehyde function at the position C-23 of the aglycone core from the non-saponin moiety, even more preferably wherein the acid-sensitive covalent bond is selected from any one or more of: a hydrazone bond, a semicarbazone bond, an imine bond, an acetal bond including a 1,3- dioxolane bond, a ketal bond, an ester bond, and / or an oxime bond, most preferably being a hydrazone bond.

20. The therapeutic combination for use according to any one of the preceding claims, wherein the saponin component comprises an unconjugated saponin moiety or a saponin molecule or wherein the saponin component comprises a saponin moiety that is covalently conjugated with at least one non-saponin moiety, preferably via an acid-stable bond, more preferably the acid-stable bond being present at the glucuronic acid group on the first saccharide chain.

21. The therapeutic combination for use according to any one of the claims 19 or 20, wherein the non- saponin moiety comprises any one or more of:a linker;the first ligand of claim 11;the oligonucleotide-based medicament; and / ora scaffold molecule;preferably, wherein the saponin moiety is directly covalently conjugated with the linker; more preferably wherein the linker is covalently conjugated to the saponin moiety via the acid sensitive covalent bond, more preferably at the position C-23 of the aglycone core, or via the acidstable bond, preferably at the glucuronic acid group if said group is present;even more preferably wherein the linker is further covalently conjugated to the first ligand and / or to the oligonucleotide-based medicament, possibly via the scaffold molecule;for example wherein the scaffold molecule is a multi-functional linker scaffold molecule or a polymeric scaffold molecule possibly comprising a dendron, such as a poly-amidoamine (PAMAM) dendrimer, or a polysaccharide, or a peptide, or a poly-ethylene glycol, such as any of PEG3 – PEG30.

22. The therapeutic combination for use according to any one of the preceding claims 11 -21, wherein the saponin moiety is covalently conjugated with the non-saponin moiety comprising the oligonucleotide-based medicament, which covalent conjugation results in a conjugate further termed a saponin-oligonucleotide conjugate, preferably wherein the saponin-oligonucleotide conjugate further comprises the linker, more preferably wherein the linker is directly covalently conjugated to the saponin moiety possibly wherein the saponin-oligonucleotide conjugate further comprises the first ligand of claim 11.

23. The therapeutic combination for use according to any one of the preceding claims, wherein the administration comprises provision to the human subject of the oligonucleotide-based medicament and of the saponin component which are co-formulated in a single pharmaceutical composition for simultaneous administration, and / or which are formulated separately as at least two pharmaceutical formulations wherein a first pharmaceutical formulation comprises the saponin component and a second pharmaceutical formulation comprises the oligonucleotide-based medicament, that can be administered either simultaneously or sequentially.

24. The therapeutic combination for use according to claim 23, wherein the first and the second pharmaceutical formulation can be administered either simultaneously or sequentially, preferably the second pharmaceutical formulation is administered first and subsequently the first pharmaceutical formulation is administered after an interval of at least 1 day, preferably after an interval of at least one week, more preferably after an interval of at least one month, most preferably after an interval of at least 3-6 months.

25. The therapeutic combination for use according to any one of the claims 23 or 24, wherein the administration of co-formulated single pharmaceutical composition and / or separately formulated two pharmaceutical formulations is further continued after an interval of at least 1 day, preferably after an interval of at least one week, more preferably after an interval of at least one month, most preferably after an interval of at least 3-6 months, with a boosting administration of the saponin component that is further referred to as a booster.

26. The therapeutic combination for use according to any one of the claims 23-25, wherein the single pharmaceutical composition is selected from any one or more of the following:2-component saponin formulation defined as comprising the saponin component comprising a saponin moiety of any one of the claims 13-18; wherein the 2-component saponin formulation further comprises the oligonucleotide-based medicament that possibly comprises the second ligand recognised by the second endocytic receptor of claim 11;2-component linker-saponin formulation defined as comprising the saponin component comprising the saponin moiety of any one of the claims 13-18, wherein the saponin moiety is covalently conjugated with the linker; wherein the 2-component linker-saponin formulationfurther comprises the oligonucleotide-based medicament that possibly comprises a second ligand recognised by a second endocytic receptor of claim 11;2-component targeted-saponin formulation defined as comprising the saponin component comprising the saponin moiety of any one of the claims 13-18, wherein the saponin moiety is covalently conjugated with the first ligand of the claim 11, and preferably wherein the nonsaponin moiety comprises the linker; and wherein the 2-component targeted-saponin formulation further comprises the oligonucleotide-based medicament that possibly comprises the second ligand of claim 11;1 -component formulation defined as comprising the saponin-oligonucleotide conjugate of claim 22, possibly wherein the saponin-oligonucleotide conjugate further comprises the first ligand of the claim 11.

27. The therapeutic combination for use according to any one of the claims 23-26, wherein the administration comprises provision of at least two pharmaceutical formulations wherein a combination of the first pharmaceutical formulation with the second pharmaceutical formulation, preferably being a 2-component targeted-saponin combination defined as comprising the first pharmaceutical formulation, wherein the saponin component comprises the saponin moiety of any one of the claims 13-18, wherein the saponin moiety is covalently conjugated with the first ligand, and preferably wherein the non-saponin moiety comprises the linker, and the second pharmaceutical formulation, wherein the oligonucleotide-based medicament possibly comprises the second ligand of the claims 11.

28. The therapeutic combination for use according to any one of the preceding claims, wherein the oligonucleotide-based medicament comprises a deoxyribonucleic acid (DNA)-based therapeutic oligonucleotide and / or ribonucleic acid (RNA)-based therapeutic oligonucleotide and / or a nucleic acid analogue-based therapeutic oligonucleotide comprising one or more nucleotide or nucleoside analogues and / or backbone modifications, preferably selected from: DNA aptamer, antisense oligonucleotide (ASO, AON), DNA ASO, RNA ASO, small or short interfering RNA (siRNA), microRNA (miRNA), RNA miRNA inhibitor (anti-microRNA, anti-miRNA, anti-miR) and / or RNA miRNA inhibitor ASO, RNA aptamer, ribozyme, RNA decoy, short hairpin RNA (shRNA), anti- hairpin-shaped microRNA; or mixed DNA / RNA therapeutic, preferably comprising one or more of the following analogues or modifications: phosphoramidate morpholino oligomer (PMO, Morpholino), peptide nucleic acid (PNA), phosphorothioate-modified antisense oligonucleotide (PS- ASO), antisense oligonucleotides containing phosphoryl guanidine (PN) backbone linkages (PN- ASO; PGO); 2'-O-methyl (2 -OMe) phosphorothioate RNA, 2-O-methoxyethyl (2-O-MOE) RNA (2 - O-methoxyethyl-RNA (2 -MOE, MOE)), locked nucleic acid (LNA, bridged nucleic acid, BNA; for example 2’-O,4’-aminoethylene bridged nucleic acid (BNA-NC), BNA-based siRNA, BNA-based antisense oligonucleotide (BNA-ASO), BNA-based anti-microRNA etc.), 2 -0, 4-C-ethylene-bridged nucleic acid (ENA), 2’-deoxy-2’-fluoroarabino nucleic acid (FANA), 3’-fluoro hexitol nucleic acid (FHNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), more preferably wherein theoligonucleotide-based medicament comprises or consists of mixed DNA / RNA and / or synthetic nucleic acid therapeutic selected from: synthetic ASO, substantially DNA-based synthetic ASO, substantially RNA-based synthetic ASO preferably comprising 2'-MOE modification, substantially DNA-based synthetic aptamer, substantially RNA-based synthetic aptamer, synthetic gapmer, synthetic siRNA, synthetic miRNA, synthetic anti-miRNA and / or synthetic anti-miRNA ASO, preferablywherein the backbone modification is selected from: 2'-O, 4'-C-ethylene-bridged (ENA) or 2'- MOE nucleic acid modifications and / or phosphoryl guanidine (PN) backbone linkages (PN-ASO), more preferably being a mutation specific therapeutic, for example being a mutation specific ASO comprising one or more nucleotide analogues and / or backbone modifications, possibly designed to silence a gene implicated in the disorder and / or to induce exon skipping or induce oligonucleotide- directed RNA editing; andwherein the oligonucleotide-based medicament is an oligonucleotide therapeutic defined as a nucleic acid therapeutic that is not longer than 200 nt, preferably has a size of 5 - 150 nt, more preferably 8 - 100 nt, most preferably 10 - 50 nt, preferably wherein the oligonucleotide therapeutic is capable of treating and / or preventing and / or ameliorating the cardiovascular disease by modulating the expression of a gene implicated in the cardiovascular disease, optionally in liver cells and / or in kidney cells.

29. The therapeutic combination for use according to any one of the preceding claims, wherein the oligonucleotide-based medicament comprises an siRNA or an ASO, preferably an ASO for reducing expression of a gene, wherein the ASO optionally comprises PMO, or an ASO for promoting exon skipping, wherein the ASO optionally comprises PMO, or an ASO capable of inducing RNA editing of a target pre-mRNA or of a target mRNA, optionally adenosine deaminase acting on RNA (ADAR)- mediated adenosine to inosine (A-to-l) RNA editing.

30. The therapeutic combination for use according to any one of the preceding claims, wherein the nucleic acid molecule targeted by the oligonucleotide-based medicament is a gene transcript present in kidney cells and / or in liver cells and is selected from: beta-1,4-galactosyltransferase 1 (B4GALT1), proprotein convertase subtilisin / kexin type 9 (PCSK9), angiopoietin-like 3 (ANGPTL3), cytochrome P450 family 7 subfamily A member 1 (CYP7A1), arachidonate 12-lipoxygenase, 12S type (ALOX 12) and serpin family H member 1 (SERPINA1).

31. The therapeutic combination for use according to any one of the preceding claims, wherein the nucleic acid molecule targeted by the oligonucleotide-based medicament is a gene transcript present in kidney cells and / or in liver cells and is selected from: B4GALT1, CYP7A1 and ALOX12.

32. The therapeutic combination for use according to any one of the preceding claims, wherein the oligonucleotide-based medicament and the cardiovascular disease are selected from the following combinations:or pharmaceutically acceptable salt thereof;preferably,or pharmaceutically acceptable salt thereof.

33. The therapeutic combination for use according to any one of the preceding claims, wherein the cardiovascular disease is a disease and / or a disorder that is any one or more of: caused by, treatable by, prevented by and ameliorated by an alteration in the expression and / or a modulation of the expression level of any one or more of the genes: B4GALT1, PCSK9, ANGPTL3, CYP7A1, ALOX12 and SERPINA1, optionally any one or more of the genes:B4GALT1, wherein the cardiovascular disease is selected from any one or more of the disorders and diseases listed in the claim 3, preferably, the disorder or disease is hyperlipidemia, dyslipidemia, hypertriglyceridemia, congenital disorder of glycosylation type lid, blood clotting defects, arterial plaque formation, and / or elevated levels of fibrinogen;PCSK9, wherein the cardiovascular disease is selected from any one or more of the disorders and diseases listed in any one or more of the claims 3 and 4, preferably, the disorder or disease is atherosclerotic cardiovascular disease (ASCVD), selected from: coronary heart disease (CHD), such as myocardial infarction, angina and coronary artery stenosis, cerebrovascular disease, such as a transient ischemic attack and ischemic stroke, peripheral artery disease, such as claudication, and aortic atherosclerotic disease, such as abdominal aortic aneurysm and descending thoracic aneurysm; hyperlipidemia; dyslipidemia; hypertriglyceridemia; familial chylomicronemia (type I hyperlipoproteinemia); familial hypercholesterolemia (type II hyperlipoproteinemia); heterozygous familial hypercholesterolemia (heFH); homozygous familial hypercholesterolemia (HoFH), and / or hypercholesterolemia;ANGPTL3, wherein the cardiovascular disease is selected from any one or more of the disorders and diseases listed in any one or more of the claims 3 and 4, preferably, the disorder or disease is hyperlipidemia, dyslipidemia, hypertriglyceridemia, familial chylomicronemia (type I hyperlipoproteinemia), familial hypercholesterolemia (type II hyperlipoproteinemia), heterozygous familial hypercholesterolemia (heFH), homozygous familial hypercholesterolemia (HoFH) and / or hypercholesterolemia;CYP7A1, wherein the cardiovascular disease is selected from any one or more of the disorders and diseases listed in any one or more of the claims 3 and 4;ALOX12, wherein the cardiovascular disease is selected from any one or more of the disorders and diseases listed in the claim 3, preferably, the disorder or disease is thrombosis;SERPINA1, wherein the cardiovascular disease is selected from any one or more of the disorders and diseases listed in the claim 3, preferably, the disorder or disease is hemorrhagic disease, and / or thrombosis.

34. The therapeutic combination for use according to any one of the preceding claims, wherein the first endocytic receptor and / or the second endocytic receptor is a liver-cell specific receptor and / or a hepatocyte specific receptor and / or is asialoglycoprotein receptor (ASGPR), and / or is a kidney-cell specific receptor and / or is selected from megalin (low density lipoprotein receptor-related protein 2; LRP2 receptor), cubilin, parathyroid hormone receptor 1 R (PTH1R), cluster of differentiation 63 (CD63 or tetraspanin) and cluster of differentiation 71 (CD71 or transferrin receptor (TfR)).

35. The therapeutic combination for use according to any one of the preceding claims, wherein the first ligand and / or the second ligand is a proteinaceous ligand, preferably a peptide or protein ligand or a receptor-interacting part thereof, more preferably wherein the ligand is an antibody or a binding fragment thereof, such as a F(ab')2 fragment, Fab' fragment, Fab fragment, scFv, dsFv, scFv-Fc, reduced IgG (rlgG), minibody, diabody, triabody, tetrabody, Fc fusion protein, nanobody, variable V domain, a single-domain antibody (sdAb), preferably a VHH, for example camelid VH, or a humanized VHH with a human (IgG1-derived) Fc and / or humanized VHH-Fc antibody, and / or a humanized VHH-Fc dimer antibody.

36. The therapeutic combination for use according to any one of the preceding claims, wherein the saponin component is based on SO1861 saponin and the first ligand and / or the second ligand is selected from: albumin or epidermal growth factor (EGF) or an antibody targeting CD71 or at least one GalNAc moiety, preferably three GalNAc moieties or an antibody or a binding fragment thereof binding to ASGPR.

37. A therapeutic combination for treatment and / or prevention and / or amelioration of a cardiovascular disease comprising:an oligonucleotide-based medicament adapted to target and bind to a nucleic acid molecule present in the cytosol of a human cell and / or present in the nucleus of a human cell according to any one or more of the claims 1, 12, 21, 22 and 28-36;anda saponin component comprising a penta-cyclic triterpene saponin comprising an aglycone core of 12,13-dehydrooleanane type according to any one or more of the claims 1, 11, 13-22 and 34-36; wherein the oligonucleotide-based medicament and the saponin component are either:co-formulated in a single pharmaceutical composition optionally comprising any one or more of a pharmaceutically acceptable excipient, diluent and carrier, for simultaneous administration; orformulated separately as at least two pharmaceutical formulations comprising a first pharmaceutical formulation comprising the saponin component and optionally comprising any one or more of a pharmaceutically acceptable excipient, diluent and carrier, and a second pharmaceutical formulation comprising the oligonucleotide-based medicament and optionally comprising any one or more of a pharmaceutically acceptable excipient, diluent and carrier.

38. A first pharmaceutical kit comprising a package comprisinga) one or more dosage units comprising the saponin component of any one or more of the claims 1, 11, 13-22 and 34-36;b) one or more dosage units comprising the oligonucleotide-based medicament of any one or more of the claims 1, 12, 21, 22 and 28-36; andc) printed instructions to use the dosage units comprised in the kit in a therapeutic method of treatment of a cardiovascular disease related to a defect in (the expression of) a gene and / or that is treatable by modulating the expression and / or expression level of a gene, said method of treatment comprising:i) the administration, preferably the repeated administration, of the oligonucleotide-based medicament that is capable of regulating the expression of said gene and / or that is capable of modulating the expression level of said gene,ii) the administration, to said subject, of the saponin component;wherein the saponin component is administered simultaneously with the oligonucleotide-based medicament and / or wherein the saponin component is administered some time, optionally after an interval of at least 1 day, preferably at least one week, more preferably at least one month, most preferably at least 3-6 months, after the oligonucleotide-based medicament has been administered, resulting in an extension of the duration of effect of the oligonucleotide-based medicament and / or in an extension of the dosing interval of the oligonucleotide-based medicament and / or in a reduction of the dosing frequency of the oligonucleotide-based medicament and / or in a (delayed) boost in the effect of the oligonucleotide-based medicament; ora second pharmaceutical kit comprising a package comprisinga) one or more dosage units comprising the saponin component of any one or more of the claims 1, 11, 13-22 and 34-36 and comprising the oligonucleotide-based medicament of any one or more of the claims 1, 12, 21, 22 and 28-36; andb) printed instructions to use the dosage units comprised in the kit in a method of treatment of a cardiovascular disease related to a defect in (the expression of) a gene and / or that is treatable by modulating the expression and / or expression level of a gene, said method of treatment comprising: - the administration, preferably the repeated administration, of the dosage units comprising the oligonucleotide-based medicament that is capable of regulating the expression of said gene and / or that is capable of modulating the expression level of said gene; ora third pharmaceutical kit comprising a package comprisinga) one or more first dosage units comprising the saponin component of any one or more of the claims 1, 11, 13-22 and 34-36 and comprising the oligonucleotide-based medicament of any one or more of the claims 1, 12, 21, 22 and 28-36;b) one or more second dosage units comprising the saponin component of any one or more of the claims 1, 11, 13-22 and 34-36; andc) printed instructions to use the dosage units comprised in the kit in a method of treatment of a cardiovascular disease in a human subject related to a defect in (the expression of) a gene and / or that is treatable by modulating the expression and / or expression level of a gene, said method of treatment comprising:i. the administration, preferably the repeated administration, to said human subject, of the first dosage units comprising the oligonucleotide-based medicament that is capable of regulating the expression of said gene and / or that is capable of modulating the expression level of said gene, and comprising the saponin component,ii. the administration, to said human subject, of the second dosage unit comprising only the saponin component;wherein the second dosage unit is administered some time, optionally after an interval of at least 1 day, preferably at least one week, more preferably at least one month, most preferably at least 3-6 months, after the first dosage unit comprising the oligonucleotide-based medicament has been administered, resulting in an extension of the duration of effect of the oligonucleotide-based medicament and / or in an extension of the dosing interval of the oligonucleotide-based medicament and / or in a reduction of the dosing frequency of the oligonucleotide-based medicament and / or in a (delayed) boost in the effect of the oligonucleotide-based medicament.

39. A method of treatment and / or prevention and / or amelioration of a cardiovascular disease in a human subject that is any one or more of: caused by, treatable by, prevented by and ameliorated by an alteration in the expression of a gene and / or a modulation of the expression level of a gene; said method of treatment comprising the steps of:i) the administration, to said human subject, preferably the repeated administration, of the oligonucleotide-based medicament of any one or more of the claims 1, 12, 21, 22 and 28-36, that is capable of modulating the expression of said gene and / or that is capable of modulating the expression level of said gene; andii) the administration, to said human subject, of the saponin component of any one or more of the claims 1, 11, 13-22 and 34-36;wherein the saponin component is administered simultaneously with the oligonucleotide-based medicament and / or wherein the saponin component is administered some time, optionally after an interval of at least 1 day, preferably at least one week, more preferably at least one month, most preferably at least 3-6 months, after the oligonucleotide-based medicament has been administered, resulting in an extension of the duration of effect of the oligonucleotide-based medicament and / or in an extension of the dosing interval of the oligonucleotide-based medicament and / or in a reduction of the dosing frequency of the oligonucleotide-based medicament and / or in a (delayed) boost in the effect of the oligonucleotide-based medicament.

40. Use of the saponin component of any one or more of the claims 1, 11, 13-22 and 34-36 and the oligonucleotide-based medicament of any one or more of the claims 1, 12, 21, 22 and 28-36 in the manufacture of a medicament for use in a method of treatment and / or prevention and / oramelioration of a cardiovascular disease in a human subject that is any one or more of: caused by, treatable by, prevented by and ameliorated by an alteration in the expression of a gene and / or a modulation of the expression level of a gene; said method of treatment comprising the steps of: i) the administration, to said human subject, preferably the repeated administration, of the oligonucleotide-based medicament that is capable of modulating the expression of said gene and / or that is capable of modulating the expression level of said gene; andii) the administration, to said human subject, of the saponin component;wherein the saponin component is administered simultaneously with the oligonucleotide-based medicament and / or wherein the saponin component is administered some time, optionally after an interval of at least 1 day, preferably at least one week, more preferably at least one month, most preferably at least 3-6 months, after the oligonucleotide-based medicament has been administered, resulting in an extension of the duration of effect of the oligonucleotide-based medicament and / or in an extension of the dosing interval of the oligonucleotide-based medicament and / or in a reduction of the dosing frequency of the oligonucleotide-based medicament and / or in a (delayed) boost in the effect of the oligonucleotide-based medicament.

41. The therapeutic combination for use according to any one of the claims 1-36 or according to claim 37 or the kit according to claim 38 or the method according to claim 39 or the use according to claim 40, wherein the administration of the saponin component of any one or more of the claims 1, 11, 13-22 and 34-36 and the oligonucleotide-based medicament of any one or more of the claims 1, 12, 21, 22 and 28-36 is preceded by, concomitant with or followed by administration to the human subject of any one or more of the therapeutic agents selected from: (i) HMG-CoA reductase inhibitors (statins) such as atorvastatin, cilastatin, fluvastatin, lovastatin, pitavastatin pravastatin, rosuvastatin, simvastatin, cerivastatin; (ii) cholesteryl ester transfer protein (CETP) inhibitor such as torcetrapib, dalcetrapib, evacetrapib, anacetrapib, obicetrapib; (iii) peroxisome proliferator activator receptor (PPAR) modulators; (iv) microsomal triglyceride transfer protein and or apolipoprotein B (MTP / Apo B) secretion inhibitors such as implitapide; (iv) squalene synthetase or squalene epoxidase or squalene cyclase or combined squalene epoxidase / squalene cyclase inhibitors; (v) cholesterol absorption inhibitor such as ezetimibe; (vi) enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitors such as Avasimibe, CS-505, Eflucimibe; (vii) lipase inhibitors such as gastric and / or pancreatic lipase inhibitors lipstatin, tetrahydrolipstatin (orlistat), valilactone, esterastin, ebelactone A, and ebelactone B; (viii) bile acid sequestrants such as colestipol, colesevelam, cholestyramine, clofibrate, gemfibrozil, fenofibrate; (ix) anti-diabetic compounds such as biguanides such as metformin, insulin secretagogues such as sulfonylureas and glinides, glitazones, non-glitazone PPARy agonists, PPARp agonists, SGLT2 inhibitor such as dapagliflozin, empagliflozin, canagliflozin and ertugliflozin, DPP- IV inhibitors such as sitagliptin, saxagliptin, linagliptin, and alogliptin, glucagon-like peptide-1 (GLP-1) analogues, GLP-1 / gastric inhibitory peptide (GIP) dual agonists, GLP-1 / GIP / glucagon (GCG) triple agonists such as liraglutide, semaglutide, tirzepatide, mazedutide, retatrutide; (x) glycogen phosphorylase inhibitor; (xi) sorbitol dehydrogenase inhibitor; (xii) glucosidase inhibitor such as amylase inhibitor - tendamistat,acarbose, adiposine, voglibose, miglitol, emiglitate, camiglibose, tendamistate, trestatin, pradimicin- Q and salbostatin; (xiii) calcium channel blocker such as amlodipine, clevidipine, felodipine, isradipine, nicardipine, nifedipine, nimodipine, nisoldipine; (xiv) Angiotensin Converting Enzyme Inhibitors (ACE-lnhibitors) such as alacepril, benazepril, captopril, ceronapril, delapril, enalapril, fosinopril, imadapril, lisinopril, moveltopril, perindopril, quinapril, ramipril, spirapril, temocapril trandolapril; (xv) Angiotensin-ll receptor antagonists (A-ll antagonists); (xvi) Beta-adrenergic receptor blockers (beta-blockers); (xvii) Alpha-adrenergic receptor blockers (alpha-blockers); (xviii) coronary and / or peripheral vasodilators; (xix) benzothiadiazine and / or sulfonamide diuretic; (xx) an inhibitor of nuclear factor-kappa B (NF-KB) or NF-KB pathway, optionally any one or more selected from: NF-KB inhibitors ectinascidin 743, chromomycin A3, bortezomib, emetine, a tyrosine kinase inhibitor, optionally lestaurtinib (CEP-701), sorafenib tosylate (BAY-43-9006) or sunitinib malate (SU-11248), digitoxin, ouabain, MG 132, TPCA-1, Bay 11-7085, Piceatannol, RAGE antagonist peptide, Caffeic acid, phenethyl ester, Triptolide, ACHP, IKK 16, SUN C8079, Honokiol, Zoledronic Acid, Bay 11-7821, Cardamonin, Withaferin A, Celastrol, BMS 345541, Sulfasalazine, SP 100030, FPS ZM1, Arctigenin, IMD 0354, Luteolin, PF 184, Amlexanox, ML 120B dihydrochloride, IP7e, GSK 319347A, C-DIM 12, C25-140, IDR 1002 and / or therapeutic agents targeting the NF-KB pathway selected from alogliptin, AS62868, docosahexanoic acid, Etanercept, forsythoside B, Minocycline, pioglitazone, simulfilam, telmisartan, TPCA-1, VX-745 and Bay 11-7082, SN50, TPCA-1 and a Non- Steroidal Anti-inflammatory Drug (NSAID) optionally indomethacin or ibuprofen;an inhibitor of NF-KB inducing kinase (NIK) optionally any one or more selected from: B022, XT2, staurosporine, ZINC-1601221, SIM1, 6Z1T (Protein Data Bank code), 6Z1Q (Protein Data Bank code); menatine;an anti-hypertensive agents optionally a drug selected from the class: thiazide diuretics such as metolazone, loop diuretics such as furosemide, potassium-sparing diuretic such as triamterene, aldosterone receptor blocker such as spironolactone, beta blocker such as nadolol, penbutolol and metoprolol, angiotensin converting enzyme inhibitor such as benazepril and ramipril, angiotensin II antagonist such as irbesartan and valsartan, calcium channel blocker such as verapamil, diltiazem, amlodipine and nisoldipine, alpha-1 blocker such as prazosin, alpha-2 agonist such as clonidine and reserpine, and direct vasodilator such as hydralazine and minoxidil;and / or any one or more of the B4GALT1, PCSK9, ANGPTL3, CYP7A1 and ALOX12 inhibitor(s); preferably liraglutide, semaglutide, tirzepatide, mazedutide, retatrutide and / or dapagliflozin; and wherein preferably the cardiovascular disease relates to expression or expression level of any one or more of the gene B4GALT1, PCSK9, ANGPTL3, CYP7A1, ALOX12 and SERPINA1, more preferably of any one or more of the genes B4GALT1, CYP7A1 and ALOX12.

42. The therapeutic combination for use according to any one of the claims 1-36 or 41, according to claim 37 or 41, the kit according to claim 38 or 41, the method according to claim 39 or 41, the use according to claim 40 or 41, wherein the administration of the saponin component of any one or more of the claims 1, 11, 13-22 and 34-36 and the oligonucleotide-based medicament of any one or more of the claims 1, 12, 21, 22 and 28-36 comprises the treatment or prevention or ameliorationof inflammation in the human subject, optionally nuclear factor-kappa B (NF-κB) pathway related inflammation, and / or inhibition of the NF-κB pathway, optionally inhibition of NF-κB and / or inhibition of NF-κB inducing kinase (NIK), wherein preferably the cardiovascular disease relates to expression or expression level of any one or more of the genes B4GALT1, PCSK9, ANGPTL3, CYP7A1, ALOX12 and SERPINA1, more preferably B4GALT1, CYP7A1 and ALOX12.

43. The therapeutic combination for use according to any one of the claims 1-36 or 41-42, according to claim 37 or 41-42, the kit according to claim 38 or 41-42, the method according to claim 39 or 41- 42, the use according to claim 40 or 41-42, wherein the treatment and / or prevention and / or amelioration of the cardiovascular disease comprises treating, preventing or ameliorating inflammation in the human subject, optionally NF-κB pathway related inflammation, and / or inhibition of the NF-κB pathway, optionally inhibition of NF-κB and / or inhibition of NIK, wherein preferably the cardiovascular disease relates to expression or expression level of any one or more of the genes B4GALT1, PCSK9, ANGPTL3, CYP7A1, ALOX12 and SERPINA1, more preferably B4GALT1, CYP7A1 and ALOX12.

44. The therapeutic combination for use according to any one of the claims 1-36 or 41-43, according to claim 37 or 41-43, the kit according to claim 38 or 41-43, the method according to claim 39 or 41- 43, the use according to claim 40 or 41-43, wherein the treatment and / or prevention and / or amelioration of the cardiovascular disease does not activate or induce the NF-KB pathway, optionally does not activate NF-KB signalling and / or NIK, and / or does not worsen, cause or increase inflammation in the human subject, optionally NF-KB pathway related inflammation, and / or wherein the treatment and / or prevention and / or amelioration of the cardiovascular disease does not result in an innate immune response, and / or wherein the treatment and / or prevention and / or amelioration of the cardiovascular disease does increase the intracellular level and / or extracellular level, optionally the blood serum concentration, with less than 30%, less than 20%, less than 10%, less than 5%, less than 2%, less than 1% or less than 0.5%, of any one or more of: interleukin-1β (IL-1β), interleukin-2 (IL-2), interferon-α2a (IFNα2a), IFNγ, tissue necrosis factor-α (TNFα) and INFβ, wherein preferably the cardiovascular disease relates to expression or expression level of any one or more of the genes B4GALT1, PCSK9, ANGPTL3, CYP7A1, ALOX12 and SERPINA1, more preferably B4GALT1, CYP7A1 and ALOX12.

45. The therapeutic combination for use according to any one of the claims 1-36 or 41-44, according to claim 37 or 41-44, the kit according to claim 38 or 41-44, the method according to claim 39 or 41- 44, the use according to claim 40 or 41-44, wherein the administration of the saponin component of any one or more of the claims 1, 11, 13-22 and 34-36 and the oligonucleotide-based medicament of any one or more of the claims 1, 12, 21, 22 and 28-36, does not activate or induce the NF-KB pathway, optionally does not activate NF-KB signalling and / or NIK, and / or does not worsen, cause or increase inflammation in the human subject, optionally NF-KB pathway related inflammation, and / or wherein the treatment and / or prevention and / or amelioration of the cardiovascular diseasedoes not result in an innate immune response, and / or wherein the treatment and / or prevention and / or amelioration of the cardiovascular disease does increase the intracellular level and / or extracellular level, optionally the blood serum concentration, with less than 30%, less than 20%, less than 10%, less than 5%, less than 2%, less than 1% or less than 0.5%, of any one or more of: interleukin-1β (IL-1β), interleukin-2 (IL-2), interferon-α2a (IFNα2a), IFNγ, tissue necrosis factor-α (TNFα) and INFβ, wherein preferably the cardiovascular disease relates to expression or expression level of any one or more of the genes B4GALT1, PCSK9, ANGPTL3, CYP7A1, ALOX12 and SERPINA1, more preferably B4GALT1, CYP7A1 and ALOX12.

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