Compositions Comprising Therapeutic Nucleic Acids and Targeted Saponins for the Treatment of Muscle Wasting Disorders

Abstract

The present invention relates to the field of treatment and prevention of muscle wasting disorders, in particular those involving genetic factors that can be targeted by delivery of therapeutic nucleic acids to muscle cells. In keeping with the latter aspect, pharmaceutical compositions and advantageous components thereof are disclosed that substantially facilitate effective delivery and release of therapeutic nucleic acids to the correct intracellular compartments of muscle cells, such as the cytosol and / or nucleus, where the therapeutic nucleic acid can reach and act on its genetic target. As disclosed herein, this substantial enhancement of delivery and release is achieved by providing a pharmaceutical composition comprising a therapeutic nucleic acid with an endosomal escape-promoting saponin that is specifically targeted to muscle cells by covalent conjugation with a ligand of an endocytic receptor present on the muscle cell. As demonstrated for the first time herein, this type of saponin surprisingly retains its endosomal escape-promoting properties in fully differentiated muscle cells.

Description

[Technical field]

[0001] The present invention relates to the field of treatment and prevention of muscle wasting disorders, in particular those involving genetic factors that can be targeted by delivery of therapeutic nucleic acids to muscle cells. In keeping with the latter aspect, pharmaceutical compositions and advantageous components thereof are disclosed that substantially facilitate effective delivery and release of therapeutic nucleic acids to the correct intracellular compartments of muscle cells, such as the cytosol and / or nucleus, where the therapeutic nucleic acid can reach and act on its genetic target. As disclosed herein, this substantial enhancement of delivery and release is achieved by providing in the pharmaceutical composition comprising the therapeutic nucleic acid an endosomal escape-promoting saponin that is specifically targeted to muscle cells by covalent conjugation with a ligand of an endocytic receptor present on the muscle cell. As demonstrated for the first time herein, this type of saponin surprisingly retains its endosomal escape-promoting properties in fully differentiated muscle cells. [Background technology]

[0002] Muscle wasting disorders are a major cause of human disease worldwide and may be caused by underlying genetic pathologies, as seen in various muscular dystrophies or congenital myopathies, among others [Cardamone, 2008], or may be age-related, such as the age-related muscle loss known as sarcopenia, or may result from traumatic muscle injury.

[0003] The cardinal feature of both hereditary and non-hereditary muscle wasting disorders is the weakening or loss of striated muscle tissue, a tissue that is especially responsible for whole body oxygenation, metabolic balance, and locomotion. Striated muscle tissue is composed of two types of striated muscle cells: skeletal muscle cells and cardiac muscle cells [Shadrin, 2016]. Skeletal muscle accounts for 30-40% of the total human body weight and is able to regenerate in response to muscle tears that occur during exercise or daily activities by activating, proliferating, and fusing upon injury to repair damaged or newly formed muscle fibers [Dumont, 2016]. In contrast, myocardium does not have a cardiomyogenic stem cell pool and has little to no regenerative capacity, leading to the formation of fibrous scars upon injury and ultimately to impaired pump function [Uygur, 2016].

[0004] Both of these cell types are terminally differentiated and highly specialized structurally and functionally, features that usually correlate with increased difficulty in targeting payloads to the internal compartments of such cells. In particular, muscle cells are surrounded by a unique type of cell membrane called the sarcolemma, which has excitability quite similar to that of neurons. Furthermore, these cells have a resilience, characterized in particular by contractility, extensibility and elasticity, which are key features required for their main function in muscle tissue, namely the generation of tension, which generates the force that causes the muscle cells to contract, thereby generating voluntary or involuntary movements of various body parts.

[0005] In light of this, it is also very well known that, although it is widely accepted that certain therapeutic payloads can produce beneficial effects when delivered to muscle cells, targeting these cells has proven to be a notoriously challenging task, as acknowledged, for example, in WO2021142227. This is especially true for cardiomyocytes, where any on-target and off-target activity of a drug poses significant safety hazards [Slordalm 2006]. Even for therapeutics that are specifically targeted to muscle, systemic delivery to the heart is known to have very limited efficacy, and even direct intramuscular injection into the heart has been shown to result in limited cardiac exposure [Ebner, 2015].

[0006] In addition to the above limitations, very few treatment options and strategies are available for patients with acquired, progressive or genetic muscle wasting disorders. In fact, for the majority of them, no drugs are available and palliative treatments are often the only available solution to alleviate the suffering of such patients.

[0007] In detail, to date, a surprisingly wide range of muscle cell-related genetic disorders (sometimes collectively referred to as hereditary myopathies) has been described; while the majority are catalogued as “rare diseases” due to their relatively low prevalence, taken together in their various forms, these disorders represent relatively common health problems that affect the quality of life of millions of patients worldwide, often causing debilitating complications that lead to death [Gonzalez-Jamett, 2017].

[0008] One common classification of muscle cell-related genetic disorders is based on the location of mutant protein products that arise from muscle cells. That is, congenital myopathies are considered to be caused by genetic defects of the contractile apparatus within the muscle cell and are defined by characteristic static histochemical or ultrastructural changes on muscle biopsies. In contrast, muscular dystrophies are described as diseases of the muscle membrane or its supporting proteins and are generally characterized by pathological evidence of ongoing muscle degeneration and regeneration [Cardamone, 2008].

[0009] Briefly, the contractile apparatus contains myofibrils, composed of actin and myosin, which form myofilaments that slide past each other to generate tension that changes the shape of the muscle cell. The function of the contractile apparatus relies heavily on the interaction of the reinforced muscle cell cytoskeleton with highly specialized structures in and around the sarcolemma, which, unlike most cell membranes in the human body, is heavily coated with a polysaccharide material called glycocalyx that contacts the basement membrane surrounding the muscle cell. This basement membrane contains numerous collagen fibrils and specialized extracellular matrix proteins, such as laminin. The matrix proteins provide a scaffold to which the muscle fibers can attach. The actin skeleton inside the muscle cell is connected to the basement membrane and the exterior of the cell through transmembrane proteins in the sarcolemma. Many such anchored muscle cells make up the muscle tissue, and the muscle cells can generate tension in a synchronized and controlled manner to generate large forces.

[0010] This structural and functional complexity of muscle cells, including its intracellular contractile apparatus, the protein web that underpins and explains the specialized functions and organization of the sarcolemma, and its outer multicomponent scaffolding, is the product of a large myocyte-specific proteome. A substantial part of this proteome is made up of large structural proteins translated from purely myocyte-specific transcripts that arise from often very large, multi-exonic genes that are prone to extensive alternative splicing events [Savarese, 2020]. Indeed, various mutations scattered along some of the largest genes in the human genome, including DMD, TTN, NEB, and RYR1, among others, are recognized as the underlying causes of some of the best-characterized myocyte-related genetic disorders.

[0011] Arguably the best-studied genetic muscle cell-related disorder is Duchenne muscular dystrophy (DMD), which is caused by mutations in the DMD gene that encodes the dystrophin protein, preventing the production of the muscle dystrophin isoform (Dp427m). DMD is a particularly severe disease, characterized by progressive weakness and replacement of skeletal muscle with fibrous, bony or fatty tissue, which ultimately leads to death, usually due to myocardial or respiratory failure. DMD is recessive and X-linked. Thus, most patients are male. On average, first symptoms occur around 2-3 years of age, patients become wheelchair bound around 10-12 years of age, and even with optimal care, death occurs between 20 and 40 years of age. The difference in the spectrum can be explained by the fact that DMD is not caused by precisely defined site-specific mutations or a single hotspot mutation in the DMD gene. Conversely, DMD, like many other muscle cell-related genetic disorders caused by a variety of different mutations in large, multi-exon genes, can be considered a spectrum of disorders with phenotypic severity depending on the extent to which the reading frame of the transcript is affected. DMD cases usually harbor frameshift or nonsense mutations that cause premature truncation leading to nonfunctional, unstable dystrophin. In contrast, a milder form of dystrophinopathy called Becker muscular dystrophy (BMD) is caused by in-frame mutations in the DMD gene, i.e. mutations that maintain the reading frame and lead to the production of a mutant dystrophin protein that is merely internally truncated.

[0012] Based on the observation that most out-of-frame mutations result in severe DMD, whereas the vast majority of in-frame mutations result in milder BMD, a variety of different antisense oligonucleotide (ASO)-based therapeutics have been tested and developed for DMD, with the goal of restoring the reading frame of the dystrophin transcript, thereby resulting in the production of an at least partially functional protein. These ASOs are short (20–30 nucleotides), often chemically modified nucleic acids or nucleic acid analogs that bind specifically to the target exon during pre-mRNA splicing, thereby resulting in the so-called skipping of the defective exon, i.e., preventing its inclusion in the mRNA. Exon-skipping ASO approaches are mutation-specific, since they require skipping a variety of different exons depending on the mutation location. However, skipping of certain exons is applicable to a larger patient population, including skipping of exon 51 (14%), exon 45 (8%), exon 53 (8%) and exon 44 (6%) [Bladen, 2013]. To date, four different morpholino ASOs designed to skip exon 51 (eteplirsen), exon 53 (golodirsen and viltolarsen) or exon 45 (casimersen) have shown some evidence of inducing dystrophin restoration in small cohorts of patients and have been granted FDA approval despite certain systemic side effects. Another exon 51 skipping ASO (drisapersen) with 2'-O-methyl phosphorothioate modifications was evaluated in a placebo-controlled trial but was ultimately not approved by the FDA due to, for example, the occurrence of injection site reactions, proteinuria and thrombocytopenia in some patients [Goemans, 2018]. For other compositions inducing exon skipping, reference can be made to WO2018129384.

[0013] Other nucleic acid-based approaches in DMD have attempted to deliver microdystrophin cDNA in high-dose vectors. Clinical trials are ongoing and some have already reported successful microdystrophin expression, although not without the observation of severe adverse effects in some patients, including transient renal failure, likely due to innate immune responses [Mendell, 2020]. Alternatively, efforts are underway to deliver cDNA for genes encoding proteins that can improve muscle mass, such as follistatin [Mendell, 2020], or proteins that target disease mechanisms, such as SERCA2a [Wasala, 2020]f). Further considerations involve the use of microRNAs, miRNA mimics, anti-miRs, and antagomirs, either alone or co-administered with other nucleic acid-based therapies, to stimulate growth and / or regeneration of awakened muscle cells [Aranega, 2021]. For example, WO2018080658 discloses miR-128-1 as an LNA-based ASO therapeutic for the treatment of DMD. Yet another alternative approach based on CRISPR / Cas9 technology with guide RNAs designed to restore the reading frame, for example by exon deletion or by disabling splice sites, has been proposed and proof-of-concept has been attempted in DMD cell lines and animal models [Chemello, 2020; Nelson 2017]. However, all genome editing efforts are still in the preclinical stage, and their systematic application in humans requires overcoming multiple challenges, including optimal delivery of genome editing components.

[0014] Thousands of different mutations of DMD have been found in patients with DMD or BMD [Blanden, 2015]. A similar situation exists for many other inherited muscle cell-related disorders for which gene mutation targets have been identified, including other muscular dystrophies, including, but not limited to, facioscapulohumeral muscular dystrophy (disease gene: DUX4 / double homeobox 4), myotonic dystrophy (DMPK), Emery-Dreifuss muscular dystrophy (disease genes: EMD / emerin and LMNA / lamin A / C), limb-girdle muscular dystrophy 1 (disease genes: MYOT / myotilin, LMNA / lamin A / C, etc.), congenital muscular dystrophies (disease genes: LAMA2 / either merosin or lamin α2 chain / COL6A gene encoding collagen 6A); or familial dilated cardiomyopathy (disease genes: Examples of myopathies include congenital myopathies including nemaline myopathies (disease genes: NEB / nebulin, ACTA / skeletal alpha actin, TPM3 / alpha tropomyosin-3, TPM2 / beta tropomyosin-2, TNNT1 / troponin T1, LMOD3 / leiomodin-3, MYPN / myopalladin, etc.) or congenital fiber type inequality myopathies (disease genes: TPM3 / alpha tropomyosin-3, CTA / skeletal alpha actin, RYR1 / ryanodine receptor channel), as well as any syndrome involving a mutation in the TTN gene (titin), for example. Thus, due to the variability in mutations even in defined gene targets underlying many different muscle wasting disorders, the use of therapeutic nucleic acids, such ASOs or antagomirs [Cerro-Herreros, 2020], appears to be a sensible and practical approach for the development of novel therapies for various muscle wasting disorders.

[0015] However, as already explained above, despite the great advantages, it is difficult to efficiently deliver such nucleic acid-based therapeutics to the appropriate compartments inside muscle cells, for example, into the muscle cell cytosol for antisense therapy or from there into the nucleus for direct gene editing, even when combined with various delivery systems. This low efficiency of in vivo muscle cell transfection inevitably results in the concentration of the nucleic acid-based therapeutic at its target site being too low to achieve effective and sustained outcomes. This therefore necessitates increased administration doses, which in turn cause off-target effects. The most common such side effects include activation of the complement cascade, inhibition of the coagulation cascade, and stimulation of the immune system via Toll-like receptors. Naturally, these effects are highly undesirable and carry the risk of inducing side effects that threaten the patient's health or even life, including organ failure. The occurrence of such and similar adverse events has led to the clinical failure of many nucleic acid-based therapeutics in trials, such as the DMD exon-skipping ASO drisapersen.

[0016] Therefore, it is highly desirable to provide new nucleic acid-based drug formulations for the treatment of muscle cell wasting disorders, which in particular would exhibit efficient nucleic acid delivery rates to muscle cells in vivo.Therefore, there is a need for formulations in which the therapeutic nucleic acid effect, among other things, (1) is highly specific to its gene target implicated in or causing muscle cell wasting disorders, (2) is sufficiently safe, (3) is effective, (4) is specifically directed to muscle cells with little to no off-target activity on other cells, (5) exhibits a sufficiently timely mode of action (e.g., the administered drug must reach the targeted site in a human patient within a certain time frame and remain at the targeted site for a certain time frame), and / or (6) has a sufficiently long-lasting therapeutic activity in the patient's body.

[0017] For a comprehensive review of different drug delivery approaches to striated muscle cells, see DC Ebner et al., 2015, Curr Pharm Des, 21(10):1327-36. doi:10.2174 / 1381612820666140929095755, which mentions peptides for muscle targeting, microbubbles, nanoparticles, virus-based, transporter-based and antibody-based targeting techniques, and also highlights the fact that cellular uptake remains a major problem in muscle cells in general. With regard to transporter-based and antibody-based targeting techniques, delivery of nucleic acids by targeting endocytosis (or internalization) receptors on the muscle cell surface through ligand mediation is shown, for example, in WO2018129384 or WO2020028857. Summary of the Invention [Problem to be solved by the invention]

[0018] However, to the best of the inventors' knowledge and experience, none of the known muscle-specific delivery techniques achieves all or at least a substantial portion of the beneficial features (1)-(6) outlined above. Thus, despite years of intensive research and progress in several areas of the field individually, there remains an urgent need to improve the efficiency of delivery of nucleic acid-based therapeutics to muscle cells in patients suffering from muscle wasting disorders. [Means for solving the problem]

[0019] To address this need, the present inventors have developed and herein described novel pharmaceutical compositions comprising therapeutic nucleic acids in combination with muscle cell-targeting triterpenoid saponins of the 12,13-dehydrooleanane type, which have been characterized and reported, for example, in WO2020126620, as possessing endosomal escape-promoting activity against various antibody-drug conjugates (ADCs) in several cancer cell types.

[0020] In the context of tumour cells, such saponins are further disclosed in WO2020126627, WO2020126064, WO2020126604, WO2020126600 and WO2020126609, which describe silencing of the HSP27 gene in tumour models by combining a first conjugate of a monoclonal antibody directed against a tumour cell marker with a saponin and a second conjugate of a monoclonal antibody directed against a tumour cell marker with a BNA to silence HSP27.

[0021] However, terminally differentiated muscle cells, specifically cardiomyocytes, are significantly different from genetically unstable, constantly dividing tumor cells in terms of metabolism as well as cell membrane organization and endocytic activity. Moreover, due to disturbed cell signaling pathways and unregulated proliferative activity, tumors are known to be supplied by a permeable and leaky angiogenesis [Hanahan and Weinberg, 2011], which is very different from the healthy tight junction-rich blood vessels that supply muscle tissue.

[0022] Despite these differences, the inventors observed that combining muscle endocytosis receptor ligand-conjugated 12,13-dehydrooleanane-type triterpenoid saponins with exon skipping therapeutic ASOs had an unexpectedly robust effect on exon skipping efficiency in human and mouse DMD transcripts. Without wishing to be bound by any theory, the inventors hypothesize that these findings indicate not only that this specific group of endosomal escape-promoting saponins is capable of stimulating efficient exit of therapeutic nucleic acids from muscle cell endosomes into the appropriate muscle cell internal compartments (in a phenomenon termed endosomal escape, which is highly desirable for therapeutics but poorly understood), but also that, unexpectedly, when formulated with ASOs and targeted by muscle cell endocytosis receptor-specific ligands, these saponins are successful in promoting exon skipping events, even at very low doses of ASO. Some preliminary data even suggest that such muscle cell targeted saponins, when co-administered intravenously, can even undergo in vivo endothelial cell transcytosis from the blood to the external environment of muscle cells, and surprisingly, this does not appear to result in any loss of payload-containing cargo via endosomal escape to the internal compartment of the endothelial cell.

[0023] To the best of our knowledge, the only example of combining saponins with nucleic acids for the treatment of muscle cell wasting disorders was attempted by Wang et al. [Wang, 2018, Molecular Therapy: Nucleic Acids; Wang, 2018-Drug Design, Development and Therapy]. However, in these reports, the authors focused on the membrane-perforating transfection activity of non-targeted nucleic acid complexes non-covalently bound to mainly steroidal saponins, such as the known in vitro transfection agent digitonin. However, none of the saponins as studied by Wang et al. were endosomal escape-promoting saponins of the 12,13-dehydrooleanane type, and none of these complexes specifically targeted muscle cells via endocytic receptor ligands. Thus, the concentrations of both saponins as well as nucleic acids as used by Wang et al. to induce exon skipping activity in their DMD model system significantly exceeded the concentrations of the respective components in the novel compositions as presented in the remainder of this specification.

[0024] In summary, to address the shortcomings of the prior art, we present herein novel pharmaceutical compositions for use in the treatment or prevention of muscle wasting disorders, in particular muscle cell-associated genetic disorders, as well as novel muscle-specific endocytic receptor-targeted conjugates of 12,13-dehydrooleanane-type endosomal escape-promoting saponins for the delivery of therapeutic nucleic acids to muscle cells, which the inventors have confirmed to have a unique ability to efficiently deliver therapeutic nucleic acids to striated muscle cells in vitro, presumably by facilitating endosomal escape specifically in target muscle cells. The findings presented herein pave the way for the development of novel, less therapeutically burdensome and therefore safer, therapeutic methods for patients suffering from muscle wasting disorders.

[0025] These and other advantages are further presented in the following sections. The innovative concepts presented herein are described with reference to detailed embodiments, which should be considered as illustrative and not limiting beyond what is set forth in the claims. These embodiments as described herein can function together in combination unless otherwise specified.

[0026] One objective of some embodiments of the present disclosure is to provide a solution to the problem of non-specificity faced when administering nucleic acid-based therapeutics to human patients suffering from muscle wasting disorders and in need of such therapeutics.

[0027] A further objective of some of the present embodiments is to provide a solution to the problem of inadequate safety profiles of current nucleic acid-based drugs when administered to human patients in need thereof, particularly in excessive doses that induce side effects.

[0028] Yet a further object of some of the embodiments of the present invention is to provide a solution to the problem that current nucleic acid based therapies are less effective than desired when administered to a human patient in need thereof due to their insufficient ability to reach and / or enter diseased muscle cells with little to no off-target activity against non-diseased cells.

[0029] At least one of the above objects relates to a pharmaceutical composition for use in the treatment or prevention of a muscle wasting disorder, in particular a muscle cell-associated genetic disorder such as a congenital myopathy or a muscular dystrophy, including in particular Duchenne muscular dystrophy, comprising a therapeutic nucleic acid, preferably an oligonucleotide such as an antisense oligonucleotide specific for a mutation in a muscle cell-specific transcript, and A first covalently linked conjugate comprising a saponin and a first ligand of an endocytic receptor on a muscle cell. wherein the saponin is an endosomal escape promoting triterpenoid 12,13-dehydrooleanane type saponin.

[0030] In a further aspect, at least one of the above objects is a therapeutic combination for the treatment or prevention of a muscle cell-associated genetic disorder, comprising: (a) antisense oligonucleotides specific for mutations in muscle cell-specific transcripts; (b) a third conjugate comprising a saponin covalently linked to a fourth ligand of an endocytic receptor on a muscle cell, the saponin being a triterpenoid 12,13-dehydrooleanane-type saponin. This is achieved by providing a therapeutic combination comprising:

[0031] In further aspects, there are provided further embodiments of the compositions for therapeutic or prophylactic use disclosed herein, and / or of the therapeutic combinations and / or muscle-targeted covalent conjugates of saponins according to the present disclosure, which further address one or more of the above mentioned objects.

[0032] In particularly advantageous aspects, various different embodiments of the present disclosure are provided that include advantageous muscle-targeted conjugates of various endosomal escape-promoting saponins, advantageous ligands or combinations thereof for targeting endocytic receptors on muscle cells, a variety of different therapeutic nucleic acids, such as, for example, antisense oligonucleotides configured to induce skipping of a defective exon of a wasting muscle cell disorder associated gene transcript, and advantageous covalent linkers linking at least the saponin together with the ligand, presumably also configured to be cleavable under conditions present in human endosomes.

[0033] These and other aspects of the present disclosure are presented in detail in subsequent sections.

[0034] definition The term "saponin" has its usual established meaning and is used herein to refer to a group of amphiphilic glycosides that contain one or more hydrophilic sugar chains combined with a lipophilic aglycone core, referred to as sapogenin. Saponins can be naturally occurring or synthetic (i.e., not naturally occurring). The term "saponin" includes naturally occurring saponins, functional derivatives of naturally occurring saponins, and saponins synthesized de novo through chemical and / or biotechnological synthetic routes. The saponins of the conjugates herein have a triterpene backbone, also referred to as sapogenin or aglycone, which is a pentacyclic C30 terpene backbone. Within the conjugates of the present invention, the saponin is not considered an effector molecule, nor is it considered an effector moiety of the conjugates of the present invention. Thus, in conjugates that include a saponin and an effector moiety, the effector moiety is a molecule that is distinct from the conjugated saponin. In the context of the conjugates of the invention, the term saponin refers to a saponin which, when present in the endosomes and / or lysosomes of a mammalian cell, such as a human cell, exerts an endosomal / lysosomal escape promoting activity towards an effector moiety comprised in the conjugate of the invention and present together with the saponin in said endosome / lysosome.

[0035] As used herein, the term "saponin derivatives" (also known as "modified saponins") shall be understood to refer to compounds corresponding to naturally occurring saponins (preferably having endosomal / lysosomal escape promoting activity towards therapeutic molecules, such as nucleic acids, when present together in endosomes or lysosomes of mammalian cells) that have been derivatized by one or more chemical modifications, such as oxidation of functional groups, reduction of functional groups and / or formation of covalent bonds with another molecule (also referred to as "conjugation" or "covalent conjugation"). Preferred modifications include derivatization of the aldehyde groups of the aglycone core, derivatization of the carboxyl groups of the sugar chains or derivatization of the acetoxy groups of the sugar chains. Typically, saponin derivatives have no natural counterpart, i.e., saponin derivatives are not naturally produced, for example, by plants or trees. The term "saponin derivative" also includes derivatives obtained by derivatizing naturally occurring saponins, as well as derivatives synthesized de novo through chemical and / or biotechnological synthetic routes that result in compounds corresponding to naturally occurring saponins derivatized by one or more chemical modifications. In the context of the present invention, a saponin derivative should be understood as a functional derivative of a saponin. In the context of a saponin derivative, "functionality" is understood as the ability or activity of the saponin or saponin derivative to promote endosomal escape of an effector molecule that contacts a cell together with the saponin or saponin derivative.

[0036] The term "aglycone core structure" is understood to refer to the aglycone core of the saponin, minus the carbohydrate antennae or sugar chains (glycans) attached thereto. For example, quilacic acid is the aglycone core structure of SO1861, QS-7 and QS21. Typically, the glycans of the saponin are monosaccharides or oligosaccharides, such as linear or branched glycans.

[0037] The term "glycan" has its usual scientific meaning and is used herein to refer to either a glycan, a carbohydrate antenna, a single sugar moiety (monosaccharide) or a chain containing multiple sugar moieties (oligosaccharide, polysaccharide). A glycan can consist of only sugar moieties or can also contain 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, as present in QS-21.

[0038] In connection with the names of glycans, the terms "Api / Xyl-" or "Api- or Xyl-" have their ordinary scientific meaning and refer herein to glycans that either contain an apiose (Api) moiety or a xylose (Xyl) moiety.

[0039] As used herein, the terms "nucleic acid" and "polynucleotide" are synonymous with each other and should be taken to encompass any polymeric molecule made of nucleobases (or simply "bases", e.g., standard nucleobases such as adenine (A), cytosine (C), guanine (G), thymine (T) or uracil (U), or any known non-standard, modified or synthetic nucleobase such as 5-methylcytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 7-methylguanine; 5,6-dihydrouracil, etc.) or functional equivalents thereof that confer upon said polymeric molecule the ability to associate by hydrogen bond-based nucleobase pairing (such as Watson-Crick base pairing) under suitable hybridization conditions with naturally occurring nucleic acids such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) (which naturally occurring nucleic acids should be understood to be polymeric molecules made of units that are nucleotides).

[0040] Thus, from a chemical point of view, the term nucleic acid as defined herein can be interpreted to include polymeric molecules that are chemically DNA or RNA, as well as polymeric molecules that are nucleic acid analogs, also known as xenonucleic acids (XNAs) or artificial nucleic acids, in which one or more (or all) units are modified nucleotides or functional equivalents of nucleotides. Nucleic acid analogs are well known in the art and are widely used in research and medicine due to various properties such as improved specificity and / or affinity, higher binding strength to their target and / or increased stability in vivo. Representative examples of nucleic acid analogs include, but are not limited to, locked nucleic acid (LNA) (also known as bridged nucleic acid (BNA)), phosphorodiamidate morpholino oligomers (PMO, also known as morpholino), peptide nucleic acid (PNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), hexitol nucleic acid (HNA), 2'-deoxy-2'-fluoroarabinonucleic acid (FANA or FNA), 2'-deoxy-2'-fluororibonucleic acid (2'-F RNA or FRNA); altritol nucleic acid (ANA), cyclohexene nucleic acid (CeNA), and the like.

[0041] According to the rules, the length of a nucleic acid is expressed herein as the number of units that make up a single strand of nucleic acid. Since each unit corresponds to exactly one nucleic acid base capable of associating in one base pairing event, the length is often expressed in so-called "base pairs" or "bp", regardless of whether the nucleic acid in question is a single-stranded (ss) or double-stranded (ds) nucleic acid. In naturally occurring nucleic acids, 1 bp corresponds to 1 nucleotide, abbreviated as 1 nt. For example, a single-stranded nucleic acid made of 1000 nucleotides (or a double-stranded nucleic acid made of two complementary strands, each complementary strand made of 1000 nucleotides) is described as having a length of 1000 base pairs or 1000 bp, which can also be expressed as 1 kilobase, abbreviated as 1000nt or 1 kb. 2 kilobases or 2 kb is equivalent to a length of 2000 base pairs, which is considered to be the same as a single-stranded RNA or DNA of 2000 nucleotides. However, to avoid confusion, and taking into account the fact that nucleic acids as defined herein may comprise or consist of units which are not only chemically nucleotides but also their functional equivalents, the length of nucleic acids will herein preferentially be expressed in "bp" or "kb" rather than the designation "nt" which is also commonly found in the art.

[0042] In advantageous embodiments as disclosed herein, the nucleic acid does not exceed 1 kb, preferably does not exceed 500 bp, most preferably does not exceed 250 bp.

[0043] In a particularly advantageous embodiment, the nucleic acid is an oligonucleotide (or simply oligo), defined as a nucleic acid not exceeding 150 bp, i.e. an oligonucleotide that is any polymeric molecule made of 150 units or less, where each unit contains a nucleobase or its functional equivalent that confers the oligonucleotide the ability to associate with DNA or RNA under suitable hybridization conditions by hydrogen bond-based nucleobase pairing, according to the definition provided above.Within the scope of said definition, it will be immediately recognized that the oligonucleotides disclosed herein may contain or consist of units that are not only nucleotides, but also their synthetic equivalents.In other words, from a chemical point of view, the term oligonucleotide as used herein will be interpreted as including or consisting of RNA, DNA or nucleic acid analogs, including but not limited to LNA (BNA), PMO (morpholino), PNA, GNA, TNA, HNA, FANA, FRNA, ANA, CeNA, etc.

[0044] As used herein, the term "endocytic receptor on a muscle cell" should be understood to refer to a surface molecule, which may be a receptor or transporter, whose specific ligand is accessible from the outside or surface of the muscle cell sarcolemma and has the ability to undergo internalization via an endocytic pathway upon an external stimulus, such as, for example, a ligand binding to the receptor. In some embodiments, the endocytic receptor on a muscle cell is internalized by clathrin-mediated endocytosis, but may also be internalized by clathrin-independent pathways, such as, for example, phagocytosis, macropinocytosis, caveolae- and raft-mediated uptake, or constitutive clathrin-independent endocytosis. In some embodiments, the endocytic receptor on a muscle cell comprises an intracellular domain, a transmembrane domain, and / or an extracellular domain, which may further include (for example, and) optionally a ligand-binding domain. In some embodiments, the endocytic receptor on a muscle cell becomes internalized by the muscle cell after ligand binding. In some embodiments, the ligand may be a muscle targeting agent or a muscle targeting antibody. In some embodiments, the internalization cell surface receptor is the transferrin receptor (CD71) or CD63 (also known as LAMP-3), which belongs to the tetraspanin family, for example.

[0045] The term "antibody-oligonucleotide conjugate" or "AOC" has its ordinary scientific meaning and is used herein to refer to an IgG, Fab, scFv, immunoglobulin, immunoglobulin fragment, one or more V H Domains, Single Domain Antibodies, V HH , Camelidae V Hand any conjugate of an antibody, such as an antibody or antibody fragment thereof, as well as any oligonucleotide selected from BNAs, antisense oligonucleotides (ASOs, AONs), short or small interfering RNA (siRNA; silencing RNA), antisense DNA, antisense RNA, etc., natural or synthetic stretches of nucleic acids including DNA, modified DNA, RNA, mRNA, modified RNA, synthetic nucleic acids, in the form of single or double stranded molecules.

[0046] As used herein, the term "antibody or binding fragment thereof" refers to a polypeptide that comprises at least one immunoglobulin variable domain or at least one antigenic determinant, e.g., a paratope, that specifically binds to an antigen. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. However, in some embodiments, the antibody is a Fab fragment, a F(ab') fragment, a F(ab')2 fragment, a Fv fragment, or a scFv fragment. In some embodiments, the antibody is a nanobody derived from a camelid antibody or a nanobody derived from a shark antibody. In some embodiments, the antibody is a diabody. In some embodiments, the antibody comprises a framework having human germline sequences. In another embodiment, the antibody comprises a heavy chain constant domain selected from the group consisting of IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgAl, IgA2, 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 and light chain constant domain amino acid sequences and functional variants thereof 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 particular embodiments, the antibody described herein comprises a human gamma 1 CHI, 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 are described in the art, see, for example, U.S. Pat. No. 5,693,780 and Kabat EA et al, (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, the antibody is modified, e.g., by glycosylation, phosphorylation, sumoylation and / or (e.g., and) methylation. In some embodiments, the antibody is a glycosylated antibody that is conjugated to one or more sugar or carbohydrate molecules. In some embodiments, one or more sugar or carbohydrate molecules are conjugated to the antibody by N-glycosylation, O-glycosylation, C-glycosylation, GPIylation (attachment of a GPI anchor) and / or (e.g., and) phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides or glycans. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, the one or more sugar or carbohydrate molecules comprise a mannose unit, a glucose unit, an N-acetylglucosamine unit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit or a phospholipid unit. In some embodiments, the antibody is a construct comprising a polypeptide comprising one or more antigen-binding fragments of the present disclosure linked to a linker polypeptide or an immunoglobulin constant domain. The linker polypeptide comprises two or more amino acid residues joined together by a peptide bond and is used to link one or more antigen-binding moieties. Examples of linker polypeptides have been reported (see, e.g., Holliger, P, et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123). Still further, an antibody may be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides.Examples of such immune adhesion molecules include the use of streptavidin core regions to generate tetrameric scFv molecules (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101) and the use of cysteine ​​residues, marker peptides and C-terminal polyhistidine tags to generate bivalent biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31:1047-1058).

[0047] The term "single domain antibody" or abbreviated "sdAb" or "nanobody" has its usual scientific meaning and refers herein to an antibody fragment consisting of a single monomeric variable antibody domain, unless it refers to two or more monomeric variable antibody domains, e.g. in the context of a bivalent sdAb, which comprises two of such monomeric variable antibody domains in tandem. A bivalent nanobody is a molecule comprising two single domain antibodies that target an epitope on a molecule present on the extracellular side of a cell, such as an epitope on the extracellular domain of a cell surface molecule present on a cell. Preferably, the cell surface molecule is a cell surface receptor. A bivalent nanobody is also named bivalent single domain antibody. Preferably, these two different single domain antibodies are covalently linked directly or through an intermediate molecule that covalently links the two different single domain antibodies. Preferably, the intermediate molecule of a bivalent Nanobody has a molecular weight of less than 10,000 Daltons, more preferably less than 5000 Daltons, even more preferably less than 2000 Daltons, and most preferably less than 1500 Daltons.

[0048] As used herein, the term "covalently linked" refers to the characteristic that two or more molecules are joined together by at least one covalent bond, i.e., directly linked, or through a chain of covalent bonds, i.e., through a linker that contains at least one or more atoms.

[0049] As used herein, the term "conjugate" should be construed as a combination of two or more different molecules that are covalently linked and covalently attached. For example, the different molecules forming the conjugate as disclosed herein may include one or more saponins or saponin molecules together with one or more ligands that bind to endocytic receptors present on the surface of muscle cells, preferably antibodies or binding fragments thereof, such as IgG, monoclonal antibodies (mAbs), VHH domains or another nanobody type, bivalent nanobody molecules including two single domain antibodies, etc. In some aspects, the conjugates disclosed herein may be made by covalently linking the different molecules through one or more intermediate molecules, such as linkers, for example, via linkage to a central linker or further linkers. In a conjugate, it is not necessary that all two or more, such as three, different molecules are directly covalently attached to each other. The different molecules in a conjugate may also be covalently linked by both being covalently linked to the same intermediate molecule, such as a linker, or by each being covalently linked to an intermediate molecule, such as a further or central linker, and the two intermediate molecules, such as two (different) linkers, being covalently linked to each other. According to this definition, there may be more intermediate molecules, such as linkers, between the two different molecules in a conjugate, as long as there is a chain of covalently linked atoms between them.

[0050] As used herein, the term "administering" or "administration" means providing a conjugate to a subject in a manner that is physiologically (e.g., to treat a condition in a subject) and / or pharmacologically useful.

[0051] As used herein, the term "approximately" or "about" when applied to one or more target values ​​refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that is within 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction from the stated reference value (greater or less), unless otherwise stated or otherwise clear from the context (except where such number would exceed 100% of possible values).

[0052] The terms first, second, third, etc. in this specification and claims are used, for example, to distinguish between similar elements, compositions, ingredients in a composition, or separate method steps, and are not necessarily used to describe a sequential or chronological order. These terms are interchangeable under appropriate circumstances, and those embodiments of the invention may function in other orders than those described or illustrated herein, unless otherwise specified.

[0053] The embodiments as described herein can function together in combination unless otherwise specified. Furthermore, references to various embodiments as "preferably" or "eg" or "for example" or "in particular" should not be construed as limitations but as exemplary ways in which the concepts disclosed in the present invention may be practiced.

[0054] The term "comprising" as used in the claims should not be construed as being limited to, for example, the subsequently recited elements or method steps or components of a composition; the term does not exclude other elements or method steps or components in a particular composition. The term should be construed as specifying the presence of the specified features, integers, (method) steps or components, but without excluding 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 of only steps A and B, but rather, for the purposes of the present invention, the only recited steps of the method are A and B, and the claims should be construed to include equivalents of those method steps. Thus, the scope of the expression "a composition comprising components A and B" should not be limited to a composition consisting of only components A and B, but rather, for the purposes of the present invention, the only recited components of the composition are A and B, and the claims should be construed to include equivalents of those components.

[0055] In addition, when an element or component is referred to with the indefinite article "a" or "an", this does not exclude the possibility that more than one element or component is present, unless the context clearly requires that there is only one of that element or component. Thus, the indefinite article "a" or "an" normally means "at least one".

[0056] The term "Saponinum album" has its ordinary scientific meaning and, as used herein, refers to a mixture of saponins manufactured by Merck KGaA (Darmstadt, Germany) containing saponins from Gypsophila paniculata and Gypsophila arostii, including SA1657 and primarily SA1641.

[0057] The term "Quillajasaponin" has its ordinary scientific meaning and is used herein to refer to the saponin fraction of Quillaja saponaria, and therefore the source of all other QS saponins, containing primarily QS-18 and QS-21.

[0058] "QS-21" or "QS21" has its ordinary scientific meaning and, as used herein, refers to a mixture of QS-21 A-apio (about 63%), QS-21 A-xylo (about 32%), QS-21 B-apio (about 3.3%) and QS-21 B-xylo (about 1.7%).

[0059] Similarly, "QS-21A" has its ordinary scientific meaning and is used herein to refer to a mixture of QS-21 A-apio (about 65%) and QS-21 A-xylo (about 35%).

[0060] Similarly, "QS-21B" has its ordinary scientific meaning and is used herein to refer to a mixture of QS-21 B-apio (about 65%) and QS-21 B-xylo (about 35%).

[0061] The term "Quil-A" refers to a commercially available semi-purified extract from Quillaja saponaria that contains varying amounts of over 50 different saponins, many of which incorporate the triterpene-trisaccharide substructure Gal-(1→2)-[Xyl-(1→3)]-GlcA- found in QS-7, QS-17, QS-18, and QS-21 at the C-3 β-OH group. The saponins found in Quil-A are listed in Table 2 of van Setten (1995) [Dirk C. van Setten, Gerrit van de Werken, Gijsbert Zomer and Gideon FAKersten, Glycosyl Compositions and Structural Characteristics of the Potential Immuno-adjuvant Active Saponins in Quillaja Saponaria Molina Extract Quil A, RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL.9, 660-666 (1995)]. Quil-A and also Quillaja saponins are fractions of saponins from Quillaja saponaria, both of which contain a wide variety of different saponins with largely overlapping contents. These two fractions are obtained by different purification procedures, so the two fractions differ in terms of their specific composition.

[0062] The terms "QS1861" and "QS1862" refer to QS-7 and QS-7 api. QS1861 has a molecular weight of 1861 Daltons and QS1862 has a molecular weight of 1862 Daltons. QS1862 is described in line 28 of Table 1 of Fleck et al. (2019) [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 structure described is the api variant of QS-7, QS1862. The molecular weight is 1862 Daltons, as this is the formal mass including the proton on the glucuronic acid. At neutral pH, the molecule is deprotonated. When measured by mass spectrometry in negative ion mode, the measured mass is 1861 Daltons.

[0063] The terms "SO1861" and "SO1862" refer to the same saponin from Saponaria officinalis, but in the deprotonated or api form, respectively. The molecular weight is 1862 Daltons, as this mass is the formal mass including the proton on the glucuronic acid. At neutral pH, the molecule is deprotonated. When the mass was measured using mass spectrometry in negative ion mode, the measured mass is 1861 Daltons. [Brief description of the drawings]

[0064] [Figure 1]Exon skipping using (A) DMD-ASO without (left panel) or with (right panel) co-administration of SO1861-EMCH and (B) DMD-PMO without (left panel) or with (right panel) co-administration of SO1861-EMCH in differentiated human myotubes from a non-DMD (healthy) donor (KM155). [Figure 2A] Synthesis of hCD71-PEG4-SPDP precursor [Figure 2B] Synthesis of hCD71-PEG4-SPDP precursor to generate hCD71-DMD-ASO [Figure 2C] Synthesis of hCD71-PEG4-SPDP precursor to create hCD71-DMD-PMO [Figure 2D] Synthesis of mCD71-SMCC [Figure 2E] Synthesis of mCD71-M23D [Diagram 3] Exon skipping using (A) hCD71-DMD-ASO (DAR2.1) without (left panel) or with (right panel) co-administration of SO1861-EMCH and (B) hCD71-DMD-PMO (DAR3.2) without (left panel) or with (right panel) co-administration of SO1861-EMCH in differentiated human myotubes from a non-DMD (healthy) donor (KM155); see also Figure 8. [Figure 4] Exon skipping using (A) hCD71-DMD-ASO without (left panel) or with (right panel) co-administration of SO1861-EMCH and (B) hCD71-DMD-PMO without (left panel) or with (right panel) co-administration of SO1861-EMCH in differentiated human myotubes from a DMD-affected donor (DM8036); note, in (A, right panel), the first sample at 0.013 nM (asterisk) indicates an empty lane. [Diagram 5] Exon skipping using mCD71-M23D PMO without (left panel) or with (right panel) co-administration of SO1861-SC-Mal in differentiated mouse C2C12 myotubes. [Figure 6A]Synthesis of mCD71-SO1861 using either SO1861-EMCH or SO1861-SC-maleimide resulting in an intact mAb conjugated to SO1861 via the interchain cysteines, held together by a variety of forces as known in the art; Note: the spacing between the heavy chains has been expanded in these figures for clarity of schematic representation. [Figure 6B] Generation of an mCD71-SH intermediate was used to obtain the synthesis of mCD71-SO1861, in which the intact mAb is conjugated to SO1861 via the interchain cysteines and held together by a variety of forces as known in the art; Note: For clarity of schematic representation, the spacing between the heavy chains has been expanded in these figures. [Figure 6C] Synthesis of mCD71-SO1861 was used to obtain an intact mAb conjugated to SO1861 via the interchain cysteines, held together by a variety of forces as known in the art. Synthesis of mCD71-SO1861; Note: For clarity of schematic representation, the spacing between the heavy chains has been expanded in these figures. [Figure 6D] IGF-1 ligand was conjugated to SO1861-hydrazone-NHS to generate IGF-1-SO1861 [Figure 7] Exon skipping using (A) mCD71-SO1861 (combined with SO1861-EMCH) + M23D (left panel) and mCD71-SO1861 (combined with SO1861-SC-Mal) + M23D (right panel), and (B) IGF-1-SO1861 + M23D (left panel) and control (right panel) in differentiated mouse C2C12 myotubes. [Figure 8] Exon skipping assessment using (A) hCD71-DMD-ASO (DAR2.2) without (left panel) or with (right panel) co-administration of SO1861-SC-Mal and (B) hCD71-DMD-PMO (DAR3.1) without (left panel) or with (right panel) co-administration of SO1861-SC-Mal in differentiated human myotubes from a non-DMD (healthy) donor (KM155). [Figure 9] Exon skipping assessment using (A) hCD71-DMD-ASO (DAR2.2) without (left panel) or with (right panel) co-administration of SO1861-SC-Mal and (B) hCD71-DMD-PMO (DAR3.1) without (left panel) or with (right panel) co-administration of SO1861-SC-Mal in differentiated human myotubes from a DMD-affected donor (DM8036). [Figure 10A] Exon skipping analysis of mice from single dose mCD71-M23D (group 2) and vehicle control (group 1) in gastrocnemius muscle on days 4, 14, and 28 after treatment [Figure 10B] Exon skipping analysis of mice from single dose mCD71-M23D (group 2) and vehicle control (group 1) in the diaphragm on days 4, 14, and 28 after treatment [Figure 10C] Exon skipping analysis of mice from single dose mCD71-M23D (Group 2) and vehicle control (Group 1) in hearts at days 4, 14, and 28 post-treatment [Figure 11] (A) Serum creatinine and (B) serum ALT analysis at days 4, 14, and 28 post-treatment from a single-dose study with mCD71-M23D (Group 2) and vehicle control (Group 1). [Figure 12A] Synthesis of DBCO-(M23D)2 via a synthetic scheme involving the synthesis of intermediate 3 (via intermediates 1 and 2). [Figure 12B] Synthesis of DBCO-(M23D)2 via a synthetic scheme involving the synthesis of intermediate 4. [Figure 12C] Synthesis of DBCO-(M23D)2 via a synthetic scheme involving coupling of intermediate 4 with M23D-SH (reduced form) to achieve synthesis of intermediate 5. [Figure 12D] Synthesis of DBCO-(M23D)2 via a synthetic scheme involving the coupling of intermediates 3 and 5 to give the desired final product DBCO-(M23D)2, a branched scaffold carrying two M23D PMO oligonucleotide payloads. [Figure 13A]Schematic representation of the conjugation procedure for mAb-M23D, such as mCD71-M23D and mCD63-M23D. Preparation of trimmed and azide-modified mAb glycans. [Figure 13B] Schematic representation of the conjugation procedure of mAb-M23D, including mCD71-M23D and mCD63-M23D. Conjugation between trimmed and azide-modified mAb glycans and DBCO-(M23D)2 via strain-promoted azide-alkyne click reaction to give mAb-(M23D)4. Note: For clarity of the schematic representation, mAb-M23D is denoted by DAR4. [Figure 13C] Schematic representation of the conjugation procedure for mAb-M23D, including mCD71-M23D and mCD63-M23D. Legend explaining the symbolically represented glycan residues. [Figure 13D] Schematic representation of the conjugation procedure for mAb-M23D, including mCD71-M23D and mCD63-M23D. Legend explaining the symbolically represented molecules. [Figure 14] Exon 23 skipping analysis of (A) mCD71-M23D without (left panel) or with (right panel) co-administration of SO1861-SC-Mal, and (B) mCD63-M23D without (left panel) or with (right panel) co-administration of SO1861-SC-Mal in differentiated mouse C2C12 myotubes. [Figure 15] Schematic representation of the conjugation procedure of mAb-SO1861, such as mCD63-SC-SO1861. Conjugation between mAb and SO1861 on the reduced interchain disulfide bond results in mAb-(SO1861)4. Note: For clarity of the schematic representation, mAb-SO1861 is shown with DAR4. [Figure 16A] Exon 23 skipping analysis of mCD63-SC-SO1861+M23D in differentiated mouse C2C12 myotubes. [Figure 16B] Exon 23 skipping analysis of mCD63-SC-SO1861+mCD71-M23D in differentiated mouse C2C12 myotubes. [Figure 16C]Exon skipping in differentiated mouse C2C12 myotubes using M23D, mCD63-SC-SO1861 or mCD71-M23D alone as a control. [Figure 17A] Schematic representation of the conjugation procedure for hAb-DMD-oligos, including hCD71-5'-SS-DMD-ASO, hCD71-5'-SS-DMD-PMO (1), hCD71-3'-SS-DMD-PMO (1-5) and hCD63-5'-SS-DMD-ASO, involving hAb functionalization with PEG4-SPDP at activated lysine (Lys) residues. Note: For clarity of the schematic representation, hAb-DMD-oligos are denoted with DAR4. [Figure 17B] Schematic representation of the conjugation procedure for hAb-DMD-oligos, including hCD71-5'-SS-DMD-ASO, hCD71-5'-SS-DMD-PMO (1), hCD71-3'-SS-DMD-PMO (1-5) and hCD63-5'-SS-DMD-ASO, involving activation of protected DMD-oligos. Note: For clarity of the schematic representation, hAb-DMD-oligos are indicated with DAR4. [Figure 17C] Schematic representation of the conjugation procedure of hAb-DMD-oligos, including hCD71-5'-SS-DMD-ASO, hCD71-5'-SS-DMD-PMO(1), hCD71-3'-SS-DMD-PMO(1-5) and hCD63-5'-SS-DMD-ASO, involving disulfide bond formation between activated DMD-oligo-SH and hAb-PEG4-SPDP. Note: For clarity of the schematic representation, hAb-DMD-oligo is indicated with DAR4. [Figure 17D] Schematic representation of the conjugation procedure of hAb-DMD-oligos, including hCD71-5'-SS-DMD-ASO, hCD71-5'-SS-DMD-PMO (1), hCD71-3'-SS-DMD-PMO (1-5) and hCD63-5'-SS-DMD-ASO, involving the figure legend. Note: For clarity of the schematic representation, hAb-DMD-oligos are indicated with DAR4. [Figure 18]Exon 51 skipping analysis of (A) hCD71-5'-SS-DMD-ASO (DAR2.1) without (left panel) or with (right panel) co-administration of SO1861-SC-Mal, and (B) hCD71-5'-SS-DMD-PMO(1) (DAR2.2) without (left panel) or with (right panel) co-administration of SO1861-SC-Mal in differentiated human myotubes from a non-DMD (healthy) donor (KM155). [Figure 19A] Exon 51 skipping analysis of hCD71-3'-SS-DMD-PMO(1)(DAR2.1) in differentiated human myotubes from a non-DMD (healthy) donor (KM155) without (left panel) or with (right panel) co-administration of SO1861-SC-Mal. [Figure 19B] Exon 51 skipping analysis of hCD71-3'-SS-DMD-PMO(2)(DAR3.0) without (left panel) or with (right panel) co-administration of SO1861-SC-Mal in differentiated human myotubes from a non-DMD (healthy) donor (KM155). [Figure 19C] Exon 51 skipping analysis of hCD71-3'-SS-DMD-PMO(3)(DAR2.6) without (left panel) or with (right panel) co-administration of SO1861-SC-Mal in differentiated human myotubes from a non-DMD (healthy) donor (KM155). [Figure 20] Exon 53 skipping analysis of (A) hCD71-3'-SS-DMD-PMO(4) (DAR2.3) without (left panel) or with (right panel) co-administration of SO1861-SC-Mal, and (B) hCD71-3'-SS-DMD-PMO(5) (DAR2.1) without (left panel) or with (right panel) co-administration of SO1861-SC-Mal in differentiated human myotubes from a non-DMD (healthy) donor (KM155). [Figure 21]Schematic representation of the conjugation procedure of hAb-SO1861, including hCD71-SC-SO1861 and hCD63-SC-SO1861. Conjugation between hAb and SO1861 on the interchain disulfide bond results in hAb-(SO1861)4. Note: For clarity of the schematic representation, hAb-SO1861 is shown with DAR4. [Figure 22] Exon 51 skipping analysis of (A) hCD71-5'-SS-DMD-ASO (DAR2.1) with co-administration of hCD63-SC-SO1861 (DAR4.8) and (B) hCD63-5'-SS-DMD-ASO (DAR2.3) with co-administration of hCD71-SC-SO1861 (DAR4.0) in differentiated human myotubes from a non-DMD (healthy) donor (KM155). [Figure 23] Exon 51 skipping analysis of hCD63-SC-SO1861 (DAR4.8) with co-administration of hCD71-5'-SS-DMD-ASO (DAR2.1) in (A) differentiated human myotubes from a non-DMD (healthy) donor (KM155) and (B) differentiated human myotubes from a DMD-affected donor (KM1328). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] Disclosed herein are improved biologically active compounds and pharmaceutical compositions comprising a therapeutic nucleic acid and a covalently linked conjugate of a muscle cell surface endocytic receptor targeting ligand and an endocytic escape-promoting saponin. The conjugates disclosed herein have particular advantages in that they exhibit highly desirable properties such as enhanced and efficient delivery of therapeutic nucleic acids, such as antisense oligonucleotides, to differentiated muscle cells, particularly striated muscle cells, including cardiomyocytes.

[0066] The innovative concepts presented herein are described based on detailed embodiments or aspects of the disclosure, which should be considered as illustrative and not limiting beyond what is described in the claims. The detailed aspects as described herein can work together in combination unless otherwise specified. Although the present invention has been described with reference to several embodiments, alternatives, improvements, modifications and equivalents will become apparent to those skilled in the art upon reading the specification and studying the drawings and graphs. The present invention is not limited in any way to the illustrated embodiments. Changes can be made without departing from the scope defined by the appended claims.

[0067] One of the objectives of some embodiments of the present disclosure is to provide a solution to the problem of non-specificity faced when administering nucleic acid-based therapeutics to human patients suffering from muscle wasting disorders and in need of such therapeutics. Another of the objectives of some embodiments is to provide a solution to the problem of insufficient safety profile of current nucleic acid-based drugs when administered to human patients in need thereof, particularly when administered in excessive doses that induce side effects. Yet another of the objectives of some embodiments of the present invention is to provide a solution to the problem that current nucleic acid-based therapies are less effective than desired when administered to human patients in need thereof due to insufficient ability to reach and / or enter diseased muscle cells with little to no off-target activity against non-diseased cells.

[0068] Without wishing to be bound by any theory, the novel pharmaceutical compositions disclosed herein were contemplated based on the observation that a specific group of triterpenoid 12,13-dehydrooleanane-type saponins appear to exhibit potent endosomal escape-promoting properties for nucleic acid-based therapeutics targeted to muscle cells via endocytosis receptor-mediated endocytosis.

[0069] The endocytic pathway is complex and poorly understood. It is currently hypothesized that it involves stable compartments linked by vesicular trafficking. Compartments are complex, multifunctional membrane organelles that are specialized for a specific set of essential functions in the cell. Vesicles are considered to be transient organelles with simple composition and are defined as membrane-bound containers that form de novo by budding from pre-existing compartments. In contrast to compartments, vesicles can undergo maturation, a series of physiologically irreversible biochemical changes. Early and late endosomes represent stable compartments in the endocytic pathway, while primary endocytic vesicles, phagosomes, multivesicular bodies (also called endosomal carrier vesicles), secretory granules and even lysosomes represent vesicles.

[0070] Endocytic vesicles originate in the plasma membrane, most notably from clathrin-coated pits, and initially fuse with early endosomes, the major sorting compartment with a pH of about 6.5. The majority of internalized cargo and membranes are recycled to the plasma membrane through recycling vesicles (recycling pathway). Components to be degraded are transported to acidic late endosomes (pH less than 6) via multivesicular bodies. Lysosomes are vesicles that can store mature lysosomal enzymes and deliver them to late endosomal compartments when needed. The resulting organelles are called hybrid organelles or endolysosomes. Lysosomes bud off hybrid organelles in a process called lysosomal remodeling. Late endosomes, lysosomes, and hybrid organelles are highly dynamic organelles, and the distinction between them is often difficult. Degradation of endocytosed molecules occurs inside endolysosomes.

[0071] Endosomal escape is the active or passive release of material from the lumen of any type of compartment or vesicle from the endocytic pathway, preferably the clathrin-mediated endocytosis or recycling pathway, into the cytoplasm. Thus, endosomal escape includes, but is not limited to, release from endosomes, endolysosomes or lysosomes, including intermediate and hybrid organelles. After entering the cytosol, the material may travel to other cellular units, such as the nucleus.

[0072] As will be demonstrated by the data presented herein, inclusion of triterpenoid 12,13-dehydrooleanane-type saponins in the therapeutic compositions and conjugates of the present disclosure not only appeared to stimulate efficient exit of therapeutic nucleic acids from muscle cell endosomes to the appropriate internal muscle cell compartment, but also, unexpectedly, preliminary data appears to indicate that it does not impede endothelial cell transcytosis in vivo from the blood to the external environment of the muscle cell, thus suggesting its suitability for intravenous delivery to muscle mass.

[0073] In light of these encouraging observations and findings, in a first general aspect the present invention provides a pharmaceutical composition for use in the treatment or prevention of a muscle wasting disorder, comprising: Nucleic acids, and A first covalently linked conjugate comprising a saponin and a first ligand of an endocytic receptor on a muscle cell. wherein the saponin is a triterpenoid 12,13-dehydrooleanane-type saponin.

[0074] As used herein, the term "covalently linked first conjugate", in reference to a covalently linked conjugate comprising a saponin and a ligand for an endocytic receptor on a muscle cell, should be taken to refer to a conjugate in which the saponin and the ligand are covalently linked together.

[0075] As used herein, the context will understand that the nucleic acid forming part of the composition disclosed herein for therapeutic purposes is selected to have a therapeutic activity for treating or preventing the selected muscle wasting disorder. In other words, the nucleic acid as contained in the composition as disclosed herein will be a therapeutic nucleic acid for one disorder, while for another disorder it may not provide any benefit. A person skilled in the art who aims to perform a specific treatment of a selected disorder will know how to perform the selection of a promising therapeutic nucleic acid and will be able to decide which therapeutic nucleic acid to include in a new composition as disclosed herein to perform an improved treatment, based either on his own knowledge of the mutations that cause such disorders or on the results of a genetic mutation screening of a given patient.

[0076] In a preferred embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein, wherein the muscle wasting disorder is a muscle cell associated genetic disorder which is preferably a congenital myopathy or muscular dystrophy (preferably, the congenital myopathy is selected from nemaline myopathy or congenital fiber type disproportion myopathy and / or the muscular dystrophy is selected from dystrophinopathy, facioscapulohumeral muscular dystrophy, myotonic dystrophy, Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy type 1B, congenital muscular dystrophy); or familial dilated cardiomyopathy; most preferably, the muscle wasting disorder is a muscle cell associated genetic disorder which is a dystrophinopathy, preferably Duchenne muscular dystrophy.

[0077] In particularly advantageous embodiments, compositions are provided for therapeutic or prophylactic use as disclosed herein, wherein the treatment or prevention of a muscle wasting disorder preferably involves antisense therapy involving exon skipping.

[0078] Depending on the application, in various embodiments, the pharmaceutical compositions disclosed herein may comprise the nucleic acid as part of a second conjugate, whereby the nucleic acid is covalently linked to a second ligand, or may alternatively comprise the nucleic acid in an unconjugated form, or at least in a non-targeted, i.e., non-covalently liganded, form.

[0079] Since no nucleic acid is conjugated to the first conjugate comprising a saponin and a first ligand, the size of the nucleic acid must be considered when co-administered with a muscle cell-targeted first conjugate comprising a saponin to achieve efficient endosomal escape-promoted intracellular delivery.

[0080] In light of the above, in a particularly advantageous embodiment, a composition for therapeutic or prophylactic use as disclosed herein is disclosed, wherein the nucleic acid is an oligonucleotide, defined as a nucleic acid not exceeding 150 nt, preferably wherein the oligonucleotide is between 5 and 150 nt in size, preferably between 8 and 100 nt, and most preferably between 10 and 50 nt.

[0081] In a related advantageous embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein, wherein the oligonucleotide is an antisense oligonucleotide (ASO), preferably a mutation-specific antisense oligonucleotide, most preferably an antisense oligonucleotide specific for a mutation in a muscle cell-specific transcript.

[0082] Indeed, the development of the advantageous compositions presented herein was based on the surprising realization that the inclusion of an endosomal escape-promoting saponin in the conjugates of the invention allows for the delivery of nucleic acid-based therapeutics, such as ASOs, into muscle cells with improved efficiency, thereby facilitating the treatment and / or prevention of muscle wasting disorders.

[0083] In view of this, in an advantageous embodiment, the nucleic acid is a therapeutic ASO adapted to target a mutated transcript of a gene affected by a particular muscle cell-associated genetic disorder. For a list of such potentially targetable gene targets and their associated muscle cell-associated genetic disorders, see, for example, Cardamone M, et al., 2008.

[0084] In a detailed embodiment, such a gene target is a mutated human dystrophin transcript whose expression is responsible for dystrophinopathies such as DMD, for which proof-of-concept experiments demonstrating the potential of the compositions disclosed herein are presented in the following sections. However, other mutations in known genes can also be targeted by antisense therapy, such as, but not limited to, those in DUX4 / double homeobox 4, which underlies facioscapulohumeral muscular dystrophy, or DMPK, which underlies myotonic dystrophy type 1, or EMD / emerin and LMNA / lamin A / C, which underlies Emery-Dreifuss muscular dystrophy, or MYOT / myotilin, LMNA / lamin A / C, which underlies limb-girdle muscular dystrophy 1. Further examples, either the LAMA2 / merosin or COL6A genes encoding laminin alpha 2 chain / collagen 6A, which become mutated in congenital muscular dystrophies, or LMNA / lamin A / C in familial dilated cardiomyopathy. Further mutations that may be targeted by the nucleic acids present in the conjugates and compositions disclosed herein can be found in genes such as NEB / nebulin, ACTA / skeletal alpha actin, TPM3 / alpha tropomyosin-3, TPM2 / beta tropomyosin-2, TNNT1 / troponin T1, LMOD3 / leiomodin-3, MYPN / myopalladin etc. (nemaline myopathy) or TPM3 / alpha tropomyosin-3, CTA / skeletal alpha actin, RYR1 / ryanodine receptor channel (congenital fiber type inequality myopathy) or advantageously the TTN gene (titin).

[0085] In accordance with the above, in another advantageous aspect, provided herein is a therapeutic combination for the treatment or prevention of a muscle cell associated genetic disorder, comprising: (a) antisense oligonucleotides specific for mutations in muscle cell-specific transcripts; (b) a third conjugate comprising a saponin covalently linked to a fourth ligand of an endocytic receptor on a muscle cell, the saponin being a triterpenoid 12,13-dehydrooleanane-type saponin. Further disclosed is a therapeutic combination comprising:

[0086] In a preferred embodiment, a therapeutic combination is provided, wherein the antisense oligonucleotide does not exceed 150 nt, preferably wherein the oligonucleotide is 5-150 nt in size, preferably 8-100 nt, and most preferably 10-50 nt.

[0087] Due to the synergistic nature of the endocytic receptor targeting and endosomal escape promoting activity of the saponins presented herein, it has been unexpectedly discovered that only small amounts of saponin are required to achieve efficient delivery of therapeutic nucleic acids, such as antisense oligonucleotides specific for mutations in muscle cell-specific transcripts, into muscle cells.

[0088] With this in mind, in a preferred embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination according to the present disclosure comprising 1-30 nM of saponin, preferably 3-25 nM, more preferably 5-20 nM, even more preferably about 7-15 nM, most preferably 8-12 nM, for example about 10 nM.

[0089] Typically, saponins suitable for application in the targeted saponin conjugates disclosed herein are those that exhibit endosomal escape promoting activity. As can be seen in Table 1, such saponins have a triterpene 12,13-dehydrooleanane skeleton, in which the basic structure of the triterpene skeleton is a pentacyclic C30 terpene skeleton (also called sapogenin or aglycone).

[0090] [Table 1]

[0091] [Table 2]

[0092] [Table 3]

[0093] [Table 4]

[0094] [Table 5]

[0095] [Table 6]

[0096] [Table 7]

[0097] [ka]

[0098] As can be seen in Table 1, many of the triterpenoid 12,13-dehydrooleanane-type saponins contain an aldehyde group at the C-23 position of the saponin aglycone core structure.

[0099] Without wishing to be bound by any theory, it has been observed that the presence of said aldehyde group in the aglycone core structure of saponin (also referred to herein as the "aglycone") is beneficial to the ability of saponin to stimulate and / or enhance endosomal escape of therapeutic nucleic acids contained in the conjugates of the invention.

[0100] Thus, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination according to the disclosure, wherein the saponin is an aldehyde group at the C-23 position of the saponin aglycone core structure, or A covalent bond at the C-23 position of the saponin aglycone core structure that covalently links the saponin within the first (or third) conjugate Including any of the following: Preferably, wherein the covalent bond at C-23 is a cleavable bond that undergoes cleavage under conditions present in an endosome or lysosome; More preferably, wherein the cleavable covalent bond at C-23 is adapted such that upon cleavage an aldehyde group is restored at C-23; Alternatively herein, the saponin used to prepare the conjugate comprises an aldehyde group at the C-23 position of the aglycone core structure of the saponin in its natural form, at least in an unconjugated state, e.g., as present in or extracted from its source plant material, e.g., prior to being covalently linked within a conjugate disclosed herein.

[0101] Such saponins may be covalently linked to a first (or third) ligand of an endocytic receptor on a muscle cell by any functional group present on the saponin suitable for conjugation as known in the art, or may be covalently linked by reacting the aldehyde group at C-23 of the aglycone core structure of the saponin such that the aldehyde group at C-23 is converted into a covalent bond at C-23 which covalently links the saponin into a first (or third) conjugate.

[0102] Without wishing to be bound by any theory, such free aldehyde groups have been observed to be beneficial for triterpenoid 12,13-dehydrooleanane-type saponins due to their endosomal escape stimulating properties and may be involved in promoting vesicle membrane destabilization.

[0103] Thus, to still further enhance the endosomal escape promoting properties of the saponin, and thus further improve escape of a therapeutic nucleic acid provided with the saponin to an endosomal compartment as part of the disclosed pharmaceutical compositions / therapeutic combinations, the covalent bond at the C-23 position can be selected such that its cleavage (e.g. in response to conditions present in mammalian endosomes or lysosomes) restores an aldehyde group at the C-23 position of the aglycone core structure of the saponin.

[0104] Examples of suitable bond types that can be designed to restore this aldehyde group include one or more of semicarbazone, hydrazone, imine, acetal, including 1,3-dioxolane, and / or oxime bonds. As shown in the Examples herein below, such functional design of the conjugates as disclosed herein has been successfully achieved with saponin, whereby the aldehyde group naturally present at C-23 of the saponin was converted to a semicarbazone or hydrazone covalent bond at C-23 of the saponin aglycone core structure that links the saponin within the conjugate. Such a bond at C-23 effectively releases the saponin from the conjugate in response to acidic conditions, with the release of the saponin restoring the aldehyde group at C-23.

[0105] In light of the above, the endosomal escape promoting properties of such saponins are also highly evident when the aldehyde group is replaced by a maleimide-containing moiety attached to the C-23 position by a cleavable covalent bond that is cleaved off under acidic conditions present, for example, in the endosomes and / or lysosomes of human cells, whereby the aldehyde group at the C-23 position of the saponin aglycone core structure is restored upon said cleavage under acidic conditions present in the endosomes and / or lysosomes of human cells.

[0106] In an advantageous embodiment, such cleavable covalent bond may be selected from a semicarbazone bond, a hydrazone bond or an imine bond.

[0107] For example, in possible embodiments, the maleimide-containing moiety can be part of a molecule that comprises or consists of 4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)piperazine-1-carbohydrazide, which is attached to the C-23 position of the aglycone core structure of the saponin upon formation of a semicarbazone bond (hereinafter referred to as SC-maleimide), or wherein the maleimide-containing moiety is part of a molecule that comprises or consists of N-ε-maleimidocaproic acid hydrazide, which is attached to the C-23 position of the aglycone core structure of the saponin upon formation of a hydrazone bond (hereinafter referred to as EMCH).

[0108] For the conjugates of the 12,13-dehydrooleanane type saponins of the present invention, which naturally contain an aldehyde group at C-23 in their natural or unconjugated form, saponins whose aglycon core structure is either queratic acid or gypsogenin are particularly suitable. With this in mind, it has been observed that the saponins particularly suitable for the conjugates of the present invention are 12,13-dehydrooleanane type saponins containing a queratic acid aglycon or gypsogenin aglycon core structure, or derivatives of said saponins in which the aldehyde group at C-23 of both of these aglycons has been converted to a covalent bond at C-23 when the C-23 aldehyde group of such aglycon core structure is used for conjugation.

[0109] An example of an unconjugated saponin with an aldehyde group at the C-23 position is depicted as saponin A and is exemplified by the following structure:

[0110] [ka]

[0111] However, it should be noted that saponins containing different 12,13-dehydrooleanane-type aglycone core structures have also been observed to exhibit sufficient endosomal escape-promoting properties, and therefore other aglycone structures listed in Table 1 may also be advantageous for purposes of the present disclosure.

[0112] Thus, in a further embodiment there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination according to the disclosure, wherein the aglycone core structure of the saponin is Quilacic acid; (and / or a quillic acid derivative in which the aldehyde group at C-23 of quillic acid has been converted to a covalent bond at C-23, preferably wherein said covalent bond is a bond that covalently links the saponin within the conjugate;) Gypsogenin; (and / or gypsogenin derivatives in which the aldehyde group at C-23 of gypsogenin has been converted to a covalent bond at C-23, preferably wherein said covalent bond is a bond that covalently links the saponin within the conjugate;) 2α-hydroxyoleanolic acid; 16α-hydroxyoleanolic acid; Hederagenin (23-hydroxyoleanolic acid); 16α,23-dihydroxyoleanolic acid; Protoestigenin-21(2-methylbut-2-enoate)-22-acetate; 23-Oxo-valingtogenol C-21,22-bis(2-methylbut-2-enoate); 23-Oxo-barringtogenol C-21(2-methylbut-2-enoate)-16,22-diacetate; 3,16,28-trihydroxyoleanan-12-ene; Gypsogenic acid; and Its derivatives One or more of the following is selected: Preferably, wherein the aglycone core structure of the saponin is Quilacic acid; (and / or quillic acid derivatives in which the aldehyde group at C-23 of quillic acid has been converted to a covalent bond at C-23, preferably wherein said covalent bond is a bond that covalently links the saponin within the conjugate); Gypsogenin; (and / or a gypsogenin derivative in which the aldehyde group at C-23 of gypsogenin has been converted to a covalent bond at C-23, preferably wherein said covalent bond is a bond that covalently links the saponin within the conjugate). Selected from; More preferably, wherein the aglycone core structure of the saponin is selected from quillaric acid (and / or a quillaric acid derivative in which the aldehyde group at C-23 of quillaric acid is converted to a covalent bond at C-23, preferably wherein said covalent bond is a bond that covalently links the saponin within the conjugate).

[0113] Saponins can include one or more sugar chains attached to an aglycone core structure. Preferred saponins of the compositions or conjugates of the present disclosure include a single chain (i.e., monodesmosidic type) or two chains (i.e., bidesmosidic type) attached to a triterpene 12,13-dehydrooleanane aglycone core structure, optionally containing an aldehyde group at the C-23 position.

[0114] Glycosylation has also been hypothesized to play a role in endosomal escape-promoting properties.

[0115] In light of the above, in a further embodiment there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination according to the disclosure, wherein the saponin sugar fraction comprises a sugar chain selected from any one of the sugar chains as listed in Group A or Group B as presented in Table 2 below.

[0116] [Table 8]

[0117] [Table 9]

[0118] For example, in an embodiment favoring the conjugation option, the saponin is a bidesmosidic saponin.

[0119] In related embodiments, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination according to the present disclosure, wherein the saponin is at least a bidesmosidic saponin comprising a first glycan selected from Group A and a second glycan selected from Group B; preferably, wherein the first glycan comprises a terminal glucuronic acid residue, and / or wherein the second glycan comprises at least four glycan residues in a branched arrangement; more preferably, wherein the first glycan is Gal-(1→2)-[Xyl-(1→3)]-GlcA, and / or wherein the branched second glycan of at least four glycan residues comprises a terminal fucose residue and / or a terminal rhamnose residue.

[0120] In an advantageous embodiment of the conjugate of the invention, the saponin comprises one or both of a first glycan attached to the C-3 or C-28 atom of the aglycone core structure, and preferably comprises one glycan attached to the C-3 atom and a second glycan attached to the C-28 atom of the aglycone core structure.

[0121] In such instances, when the saponin contained in the conjugate of the invention carries the two glycans, the first glycan is attached to the C-3 position of the aglycone core structure and the second glycan is typically attached to the C-28 position of the saponin aglycone core structure, although for some saponins lacking an aldehyde group at the C-23 position, the second glycan can be attached to said C-23 position (see Table 1).

[0122] Thus, in further related embodiments, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination according to the disclosure, wherein the saponin comprises a first glycan at the C-3 position of the saponin aglycone core structure and / or a second glycan at the C-28 position of the saponin aglycone core structure; Preferably, wherein the first glycan is a carbohydrate substituent at the C-3β-OH group of the saponin aglycone core structure, and / or wherein the second glycan is a carbohydrate substituent at the C-28-OH group of the saponin aglycone core structure.

[0123] In a particularly preferred embodiment, conjugates are provided in which the saponin is a triterpenoid 12,13-dehydrooleanane-type saponin which, at least in its unconjugated state, comprises an aldehyde group at the C-23 position of the saponin aglycone core structure and which comprises as a carbohydrate substituent at the C-3β-OH group of the saponin aglycone core a glycan selected from Group A and which comprises a terminal glucuronic acid residue, preferably Gal-(1→2)-[Xyl-(1→3)]-GlcA.

[0124] In the following embodiments, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination according to the present disclosure, wherein the first or third conjugate each comprises two or more molecules of saponin, preferably 2-32 molecules of saponin, even more preferably 4-16 molecules of saponin, and most preferably 4-8 molecules of saponin.

[0125] These molecules can be the same saponin or saponins of the same aglycone core structure and different glycosylation chains, or can even be a mixture of different 12,13-dehydrooleanane-type endosomal escape-promoting saponins, e.g., a mixture of different saponins selected from Table 1.

[0126] In another embodiment there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination according to the disclosure, wherein the saponin is a) List A: - Quillaja saponaria saponin mixture or saponins isolated from Quillaja saponaria, such as Quil-A, QS-17-api, QS-17-xyl, QS-21, QS-21A, QS-21B, QS-7-xyl; -Saponinum album saponin mixture or saponins isolated from Saponinum album; - Saponaria officinalis saponin mixture or saponins isolated from Saponaria officinalis; and Quillaja bark saponin mixtures or saponins isolated from Quillaja bark, such as Quil-A, QS-17-api, QS-17-xyl, QS-21, QS-21A, QS-21B, QS-7-xyl or b) List B: SA1641, gypsoside A, NP-017772, NP-017774, NP-017777, NP-017778, NP-018109, NP-017888, NP-017889, NP-018108, SO1658 and phytolaccagenin a saponin comprising a gypsogenin aglycone core structure selected from: c) List C: AG1856, AG1, AG2, agrostemoside E, GE1741, gypsophila saponin 1 (Gyp1), NP-017674, NP-017810, NP-003881, NP-017676, NP-017677, NP-017705, NP-017706, NP-017773, NP-017775, SA1657, saponarioside B, SO1542, SO1584, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862, SO1904, QS-17, QS-18, QS-21, QS-22, QS-23, QS-24, QS-25, QS-26, QS-27, QS-28, QS-29, QS-30, QS-31, QS-32, QS-33, QS-34, QS-35, QS-36, QS-37, QS-38, QS-39, QS-40, QS-41, QS-42, QS-43, QS-44, QS-45, QS-46, QS-47, QS-48, QS-49, QS-50, QS-51, QS-52, QS-53, QS-54, QS-55, QS-56, QS-57, QS-59, QS-60, QS-61, QS-62, QS-63, QS-64, QS-65, QS-70, QS-71, QS-72, QS-73, Q A-xylo, QS-21 B-apio and QS-21 B-xylo a saponin comprising a quinoline aglycone core structure selected from: d) List D: Escin Ia, escinate, α-hederin, AMA-1, AMR, AS6.2, AS64R, Assamsaponin F, Dipsacoside B, Esculentoside A, Macrantoidin A, NP-005236, NP-012672, Primular Acid 1, Saikosaponin A, Saikosaponin D, Tea Seed Saponin I and Tea Seed Saponin J A saponin having a 12,13-dehydrooleanane type aglycone core structure without an aldehyde group at the C-23 position of the aglycone, selected from One or more of the following: Preferably the saponin is any one or more of the saponins selected from lists A, B or C, more preferably from lists B or C, even more preferably from list C.

[0127] In particular embodiments, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination according to the disclosure, wherein the saponin is selected from the group consisting of AG1856, GE1741, saponin isolated from Quillaja saponaria, Quil-A, QS-17, QS-21, QS-7, SA1641, Saponaria officinalis, and the like. officinalis), saponarioside B, any one or more of SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862 and SO1904; preferably, wherein the saponin is any one or more of QS-21, SO1832, SO1861, SA1641 and GE1741; more preferably, wherein the saponin is QS-21, SO1832 or SO1861; and most preferably SO1861.

[0128] In more particular embodiments, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination according to the disclosure, wherein the saponin is derived from Saponaria officinalis (Saponaria officinalis). officinalis), preferably wherein the saponin is any one or more of saponariosides B, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862 and SO1904; more preferably wherein the saponin is any one or more of SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862 and SO1904; even more preferably wherein the saponin is any one or more of SO1832, SO1861 and SO1862; even more preferably wherein the saponin is SO1832 and SO1861; most preferably SO1861.

[0129] In particular embodiments, compositions may be provided comprising a muscle cell targeted saponin conjugate for therapeutic or prophylactic use as disclosed herein and therapeutic combinations of the present disclosure, wherein one, two or three, preferably one or two, more preferably one of the following: i. the aldehyde group on the aglycone core structure of at least one saponin, when present, is derivatized; ii. the carboxyl group of the glucuronic acid moiety on the first glycan of the at least one saponin is derivatized when present in the at least one saponin; and iii. At least one acetoxy (Me(CO)O-) group on the second glycan of at least one saponin, if present, is derivatized.

[0130] In more particular embodiments, compositions for therapeutic or prophylactic use and therapeutic combinations as disclosed herein may be provided, wherein at least one saponin is i. an aglycone core structure, -Reduction to alcohol; - conversion to a hydrazone bond via reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), where the maleimide group of EMCH is optionally derivatized by formation of a thioether bond with mercaptoethanol; - Conversion to a hydrazone bond via reaction with N-[β-maleimidopropionic acid] hydrazide (BMPH), where the maleimide group of BMPH is optionally derivatized by formation of a thioether bond with mercaptoethanol; or - Conversion to a hydrazone bond via reaction with N-[κ-maleimidoundecanoic acid]hydrazide (KMUH), where the maleimide group of KMUH is optionally derivatized by formation of a thioether bond with mercaptoethanol. an aglycone core structure containing an aldehyde group that has been derivatized with ii. a first glycan comprising a carboxyl group, preferably a carboxyl group of a glucuronic acid moiety, that has been derivatized by conversion to an amide bond through reaction with 2-amino-2-methyl-1,3-propanediol (AMPD) or N-(2-aminoethyl)maleimide (AEM); or iii. a second glycan containing an acetoxy group (Me(CO)O-) that has been derivatized by conversion to a hydroxyl group (HO-) by deacetylation; or iv. Any combination of two or three derivatizations i., ii. and / or iii., preferably any combination of two derivatizations i., ii. and / or iii. Includes.

[0131] In a specific embodiment, a composition is provided comprising a targeted saponin conjugate, wherein at least one saponin comprises a first glycan according to Group A and Group B of Table 2, respectively, and a second glycan, wherein the first glycan comprises two or more sugar moieties, and the second glycan comprises two or more sugar moieties, and wherein the aglycone core structure is preferably quillic acid or gypsogenin, more preferably quillic acid, and wherein one, two or three, preferably one or two of the following: i. the aldehyde group on the aglycone core structure is derivatized; ii. the carboxyl group of the glucuronic acid moiety in the first glycan is derivatized; and iii. At least one acetoxy (Me(CO)O-) group on the second glycan is derivatized.

[0132] An embodiment is a conjugate of the invention, wherein one, two or three, preferably one or two, more preferably one of the following: iv. the aldehyde group on the aglycone core structure of at least one saponin, when present, is derivatized; v. the carboxyl group of the glucuronic acid moiety on the first saccharide chain of the at least one saponin is derivatized when present in the at least one saponin; and At least one acetoxy (Me(CO)O-) group on the second glycan of at least one saponin, if present, is derivatized.

[0133] In a specific embodiment, a therapeutic combination or composition for therapeutic or prophylactic use as disclosed herein is provided, in which the aldehyde functional group at the C-23 position of the aglycone core structure of at least one saponin is covalently bound to a linker EMCH, which is covalently bound to a sulfhydryl group of an oligomeric or polymeric molecule of the covalent saponin conjugate, such as a sulfhydryl group of cysteine, via a thioether bond. The binding of the EMCH linker to the aldehyde group of the aglycone of the saponin results in the formation of a hydrazone bond. Such a hydrazone bond is a typical example of a bond that is cleavable under the acidic conditions inside endosomes and lysosomes. As explained above, the inventors have surprisingly realized that it is not essential for saponin-mediated endosomal escape that the saponin is present in a free form in the endosome or lysosome. The saponin contained in the first or third conjugate disclosed herein can also enhance the delivery of therapeutic nucleic acid from endosome / lysosome into the cytosol of the target muscle cell. The saponin coupled to the first or fourth ligand, respectively, contained in the first or third conjugate can be released from the conjugate of the present invention after delivery to the endosome or lysosome of the target muscle cell that exposes an endocytic receptor to which the ligand can bind. Thus, the saponin coupled to the first or fourth ligand in the conjugate is transferred from outside the cell into the endosome (or lysosome), where the saponin is released from the remainder of the conjugate upon pH-driven cleavage of the hydrazone bond. In the endosome (or lysosome), the free saponin can exert its stimulatory activity upon delivery of therapeutic nucleic acid, such as the ASO described above, to the cytosol of the muscle cell.

[0134] For the sake of completeness, with respect to nucleic acids or oligonucleotides that are advantageously part of the compositions and / or therapeutic combinations disclosed herein, the term oligonucleotide as used herein shall be understood to encompass both oligomers made of naturally occurring nucleotides and thus chemically oligonucleotides, as well as oligomers that include modified oligonucleotides or analogs thereof. For example, the synthetic oligomer may include a 2'-modified nucleoside that may be selected from 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAE0E), 2'-ON-methylacetamide (2'-O-NMA), locked nucleic acid (LNA), ethylene-bridged nucleic acid (ENA), and (S)-constrained ethyl-bridged nucleic acid (cEt), and the like. In light of this, in possible embodiments, the oligonucleotide may be structurally or functionally any of the following: deoxyribonucleic acid (DNA) oligomers, ribonucleic acid (RNA) oligomers, antisense oligonucleotides (ASOs, AONs), small interfering RNAs (siRNAs), anti-microRNAs (anti-miRNAs), DNA aptamers, RNA aptamers, mRNAs, minicircle DNAs, peptide nucleic acids (PNAs), phosphoramidate morpholino oligomers (PMOs), locked nucleic acids (LNAs), bridged nucleic acids (BNAs), 2'-deoxy-2'-fluoroarabinonucleic acids (FA ... The siRNA may be defined as any of the siRNAs or any other category known in the art, such as BNA-based siRNAs selected from BNA-based siRNAs, 2'-O-methoxyethyl-RNA (MOE), 3'-fluorohexitol nucleic acid (FHNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), BNA-based siRNAs and BNA-based antisense oligonucleotides (BNA-AONs), chemically modified siRNAs, metabolically stable siRNAs and chemically modified metabolically stable siRNAs.

[0135] From a functional point of view, in an advantageous embodiment, a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination according to the present disclosure is provided, wherein the oligonucleotide is an oligonucleotide designed to induce exon skipping.

[0136] In related embodiments, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination according to the present disclosure, wherein the oligonucleotide comprises or consists of any one of the following: morpholino phosphorodiamidate oligomer (PMO), 2'-O-methyl (2'-OMe) phosphorothioate RNA, 2'-O-methoxyethyl (2'-O-MOE) RNA {2'-O-methoxyethyl-RNA (MOE)}, locked or bridged nucleic acid (LNA or BNA), 2'-O,4'- Aminoethylene-bridged nucleic acid (BNANC), peptide nucleic acid (PNA), 2'-deoxy-2'-fluoroarabinonucleic acid (FANA), 3'-fluorohexitol nucleic acid (FHNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), silencing RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), antagomir (miRNA antagonist), aptamer RNA or aptamer DNA, single-stranded RNA or single-stranded DNA, double-stranded RNA (dsRNA) or double-stranded DNA;

[0137] In particularly preferred embodiments, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination according to the present disclosure, wherein the oligonucleotide comprises or consists of a morpholino phosphorodiamidate oligomer (PMO) or a 2'-O-methyl (2'-OMe) phosphorothioate RNA.

[0138] In particular and preferred specific embodiments for DMD, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination according to the present disclosure, wherein the oligonucleotide is designed to induce exon skipping of the human dystrophin gene transcript, preferably wherein exon skipping involves exon 51 skipping, or exon 53 skipping, or exon 45 skipping, preferably wherein the oligonucleotide is a 2'O-methyl-phosphorothioate antisense oligonucleotide or a phosphorodiamidate morpholino oligomer antisense oligonucleotide designed to induce exon 51 skipping, or exon 53 skipping, or exon 45 skipping.

[0139] In a very particular embodiment building on the previous embodiment, the oligonucleotide is selected from eteplirsen, drisapersen, golodirsen, viltolarsen and casimersen.

[0140] In an advantageous embodiment, a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination according to the present disclosure is provided that comprises two or more nucleic acids, preferably two or more different oligonucleotides, more preferably, where at least one of the two or more different oligonucleotides is an antisense oligonucleotide.Such combinations comprising two or more therapeutic nucleic acids are known in the art, for example for muscle wasting disorders, a combination approach based on two AONs for double exon skipping in myostatin and dystrophin was proposed for the management of Duchenne muscular dystrophy [Kemaladewi el al, 2011].

[0141] Now, regarding the detailed embodiment of the first or fourth ligand of the endocytic receptor on the muscle cell, it must be noted that endocytic receptors expressed on the surface of muscle cells are known, some of which, such as the transferrin receptor (CD71) or the muscle specific kinase (MuSK), are described in WO2018129384 or WO2020028857. Further examples of suitable receptors include muscle transmembrane transporters, such as GLUT4 or ENT2 (described among many others in Ebner, 2015) or for example the tetraspanin CD63 [Baik, 2021]. Indeed, many endocytic receptors present on the surface of muscle cells have been characterized to date, with the transferrin receptor (CD71) and possibly the insulin-like growth factor 1 (IGF-I) receptor (IGF1R) being the most studied. The ligands for these receptors can be selected from natural ligands such as transferrin (Tf), the natural ligand for CD71, or LDL, the ligand for the LDL receptor. Alternatively, the receptors can be non-naturally occurring ligands, such as various antibodies or binding fragments thereof, or alternatively synthetic ligands such as zymosan A, which binds to the endocytic mannose receptor, or synthetic fragments of naturally occurring ligands, such as fragments of IGF-I, the ligand for GF1R, or fragments of IGF-II, the ligand for CI-MPR (also known as IGF2R).

[0142] In a preferred embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein, wherein the endocytic receptor on a muscle cell to which the first ligand binds is selected from the transferrin receptor (CD71), the insulin-like growth factor 1 (IGF-I) receptor (IGF1R), the tetraspanin CD63; muscle-specific kinase (MuSK), the glucose transporter GLUT4, the cation-independent mannose 6-phosphate receptor (CI-MPR) and the LDL receptor.

[0143] Similarly, in another preferred embodiment, a therapeutic combination according to the present disclosure is provided, wherein the endocytic receptor on a muscle cell to which the fourth ligand binds is selected from the transferrin receptor (CD71), the insulin-like growth factor 1 (IGF-I) receptor (IGF1R), the tetraspanin CD63; the muscle-specific kinase (MuSK), the glucose transporter GLUT4, the cation-independent mannose 6-phosphate receptor (CI-MPR), and the LDL receptor.

[0144] In a further preferred embodiment, the present disclosure provides a composition for the therapeutic or prophylactic agent disclosed herein, wherein the first ligand is Insulin-like growth factor 1 (IGF-I) or a fragment thereof; Insulin-like growth factor 2 (IGF-II) or a fragment thereof Mannose 6-phosphate, Transferrin (Tf), Zymosan A, and An antibody or a binding fragment thereof specific for binding to an endocytosis receptor, the endocytosis receptor being preferably selected from the group consisting of the transferrin receptor (CD71), the insulin-like growth factor 1 (IGF-I) receptor (IGF1R), the tetraspanin CD63, the muscle-specific kinase (MuSK), the glucose transporter GLUT4, the cation-independent mannose 6-phosphate receptor (CI-MPR) and the LDL receptor. Select one of the following: Preferably, wherein the first ligand is an antibody or binding fragment thereof specific for binding to the transferrin receptor.

[0145] Similarly, in a further preferred embodiment, a therapeutic combination according to the present disclosure is provided, wherein the fourth ligand is Insulin-like growth factor 1 (IGF-I) or a fragment thereof; Insulin-like growth factor 2 (IGF-II) or a fragment thereof Mannose 6-phosphate, Transferrin (Tf), Zymosan A, and An antibody or a binding fragment thereof specific for binding to an endocytosis receptor, the endocytosis receptor being preferably selected from the group consisting of the transferrin receptor (CD71), the insulin-like growth factor 1 (IGF-I) receptor (IGF1R), the tetraspanin CD63, the muscle-specific kinase (MuSK), the glucose transporter GLUT4, the cation-independent mannose 6-phosphate receptor (CI-MPR) and the LDL receptor. Select one of the following: Preferably, wherein the fourth ligand is an antibody or binding fragment thereof specific for binding to the transferrin receptor.

[0146] In a related particularly advantageous embodiment, the first or fourth ligand is a monoclonal antibody specific for binding to the transferrin receptor, or a Fab' fragment, or at least one single domain antibody, and even more preferably, wherein the first or fourth ligand is a monoclonal antibody specific for binding to the transferrin receptor.

[0147] In a further advantageous embodiment, the first or fourth ligand is a monoclonal antibody, such as a humanized or human monoclonal antibody, an IgG, a molecule comprising or consisting of a single domain antibody, at least one VHH domain, preferably a camelid VH, a variable heavy chain novel antigen receptor (VNAR) domain, a Fab, scFv, Fv, dAb, F(ab)2 and an Fcab fragment.

[0148] In certain embodiments, it may be advantageous to include an additional ligand in the saponin conjugate for improved targeting to muscle cells or to target specific subtypes of muscle cells.

[0149] Thus, in a further advantageous embodiment, a composition for therapeutic or prophylactic use as disclosed herein according to the present disclosure is provided, wherein the first conjugate comprises a further third ligand, preferably wherein the further third ligand is an antibody or a binding fragment thereof specific for a cell surface molecule, optionally a cell surface molecule which is a further endocytic receptor on a muscle cell.

[0150] Similarly, in another embodiment there is provided a therapeutic combination according to the present disclosure, wherein the third conjugate comprises an additional sixth ligand, preferably wherein the additional sixth ligand is an antibody or binding fragment thereof specific for a cell surface molecule, optionally a cell surface molecule which is an additional endocytic receptor on a muscle cell.

[0151] As already briefly mentioned above, in certain advantageous embodiments, it can also be envisaged that the nucleic acids provided in the compositions and / or therapeutic combinations disclosed herein, optionally being antisense oligonucleotides, are targeted to muscle cells by conjugation with at least one further ligand molecule.

[0152] With this in mind, in a further embodiment there is provided a composition for therapeutic or prophylactic use as disclosed herein, wherein the nucleic acid is comprised in a second conjugate, wherein the nucleic acid is covalently linked to a second ligand; preferably, wherein the second ligand is a ligand of an endocytic receptor on a muscle cell; more preferably, wherein the second ligand is different from the first ligand of the covalently linked first conjugate comprising saponin, and even more preferably, wherein the second ligand is a ligand of an endocytic receptor on a muscle cell that is different from the endocytic receptor on the muscle cell to which the first ligand binds.

[0153] Similarly, in a possible alternative embodiment, a therapeutic combination is provided wherein an antisense oligonucleotide is covalently linked to a fifth ligand of a fourth conjugate; preferably, wherein the fifth ligand is a ligand of an endocytic receptor on a muscle cell; more preferably, wherein the fifth ligand is different from the fourth ligand of a covalently linked third conjugate that comprises saponin, and even more preferably, wherein the fifth ligand is a ligand of an endocytic receptor on a muscle cell that is different from the endocytic receptor on the muscle cell to which the fourth ligand binds.

[0154] In the example where two ligand-targeted conjugates are present in a composition or a therapeutic combination, advantageous combinations of ligands can be envisaged to optimise the targeting strategy, potentially even for a selected muscle cell type or subtype. By ligand combinations, it is meant a combination of two or more different ligands that together ensure effective targeting to the cells of interest, preferably with no or only minimal cross-interference, possibly functioning synergistically and preferably not competing with each other for, e.g., binding sites or epitopes on their possible common target endocytic receptors or on their different target receptors.

[0155] In a possible embodiment, the first and second ligands of the compositions described herein or similarly the fourth and fifth ligands of the combinations disclosed herein can potentially be the same ligand. This can occur, for example, when no or few competing events are expected for the binding of those two-ligand conjugates (e.g., one is, for example, a first ligand-saponin conjugate and the second is a second ligand-ASO conjugate), which can occur depending on the dose and the abundance and distribution of endocytic receptors that the ligands present in both conjugates target in parallel. An example of such a situation is a therapeutic composition in which the fourth and fifth ligands are the same, for example, a monoclonal antibody, for example, a monoclonal antibody specific for CD71.

[0156] Alternatively, in an advantageous embodiment, the first and second ligands of the compositions described herein, or similarly the fourth and fifth ligands of the combinations disclosed herein, may be different ligands of the same endocytic receptor. Such a situation is advantageous, especially when two such ligands target epitopes on their common target receptor that are spatially well separated from each other, since less competition for binding sites on the target receptor may be expected. An example of such a combination for the first and second ligands, or similarly the fourth and fifth ligands, may be a combination of two different antibodies targeting CD71, for example one monoclonal IgG and the other a single domain VHH. An alternative example also directed against CD71 may involve, for example, one ligand being transferrin or a fragment thereof, and the other ligand being a CD71 targeting antibody. Another example may be when one ligand of the combination is an IGF1R specific antibody, while the other ligand is, for example, IGF-I or a receptor-binding synthetic peptide derived therefrom.

[0157] In another embodiment, the first and second ligands of the compositions described herein or similarly the fourth and fifth ligands of the combinations disclosed herein may be different ligands, each specific for a different endocytic receptor. Possible exemplary combinations of such ligands include, for example, a combination comprising a ligand of the transferrin receptor (CD71) and a ligand of the insulin-like growth factor 1 (IGF-I) receptor; or a second combination comprising a ligand of the transferrin receptor (CD71) and a ligand of the tetraspanin CD63; or a further multi-ligand combination of a ligand of the transferrin receptor (CD71), a ligand of the insulin-like growth factor 1 (IGF-I) receptor, a ligand of the tetraspanin CD63 and / or a ligand of the muscle-specific kinase (MuSK). However, many other combinations may be possible, such as those involving multi-ligand combinations with the same ligand, different ligands of the same endocytic receptor, and / or ligands of different endocytic receptors, combinations that will require testing with respect to specific compositions or therapeutic combinations as disclosed herein.

[0158] Thus, in an advantageous embodiment, a composition for therapeutic or prophylactic use as disclosed herein is provided, wherein the combination of the first and second ligand is one of the following combinations of ligands: Ligand for the transferrin receptor (CD71) and the insulin-like growth factor 1 (IGF-I) receptor, Ligand for the transferrin receptor (CD71) and the tetraspanin CD63; · Ligand for the transferrin receptor (CD71) and for the muscle specific kinase (MuSK); Ligand for transferrin receptor (CD71) and cation-independent mannose 6-phosphate receptor (CI-MPR) two ligands of the transferrin receptor (CD71), one ligand being transferrin and the other ligand being an antibody or binding fragment thereof specific for binding to the transferrin receptor (CD71); Two ligands of the LDL receptor, one of which is or comprises LDL and the other of which is an antibody or a binding fragment thereof specific for binding to the LDL receptor. Selected from; Preferably, at least one of the first and second ligands in the combination is an antibody or a binding fragment thereof, and optionally, at least one of the first and second ligands in the combination is transferrin (Tf) or insulin-like growth factor 1 (IGF-I).

[0159] Likewise, in a further embodiment, a therapeutic combination according to the present disclosure is provided, wherein the combination of the fourth ligand of the third conjugate and the fifth ligand of the fourth conjugate is the following combination of ligands: Ligand for the transferrin receptor (CD71) and the insulin-like growth factor 1 (IGF-I) receptor, Ligand for the transferrin receptor (CD71) and the tetraspanin CD63; · Ligand for the transferrin receptor (CD71) and for the muscle specific kinase (MuSK); Ligand for the transferrin receptor (CD71) and ligand for the cation-independent mannose 6-phosphate receptor (CI-MPR), two ligands of the transferrin receptor (CD71), one ligand being transferrin and the other ligand being an antibody or binding fragment thereof specific for binding to the transferrin receptor (CD71); Two ligands of the LDL receptor, one of which is or comprises LDL and the other of which is an antibody or a binding fragment thereof specific for binding to the LDL receptor. Selected from; Preferably, wherein at least one of the fourth and fifth ligands in the combination is an antibody or a binding fragment thereof, and optionally, wherein at least one of the fourth and fifth ligands in the combination is transferrin (Tf) or insulin-like growth factor 1 (IGF-I).

[0160] Usually, the compatibility of two different ligands in a combination should be expected, which means that in theory, it should not make a difference whether the ligand of an effective combination is conjugated to a saponin as the first ligand or to an oligonucleotide as the second ligand.However, sometimes, due to affinity, spatial considerations and / or non-covalent interactions between a particular ligand and the molecule to which it is conjugated, it may be preferable for the first or second ligand to be preferentially one specific ligand rather than the other from a particular combination, for example.However, in principle, when a particular combination of ligands is preferred, any one of them can be assigned as the first ligand, as the second ligand or even as an additional third ligand for the composition as disclosed herein.

[0161] In particular embodiments, therapeutic combinations are provided, wherein the first ligand is the same as the fourth ligand, and / or the second ligand is the same as the fifth ligand, and / or the third ligand is the same as the sixth ligand, preferably, wherein the first conjugate is the same as the third conjugate, and / or the second conjugate is the same as the fourth conjugate, more preferably, the first and third conjugates are the same and the second and fourth conjugates are the same.

[0162] In further possible embodiments, as well as targeted saponin conjugates as described herein, targeted nucleic acid or oligonucleotide conjugates may also be provided which comprise two or more nucleic acid or oligonucleotide molecules, optionally 2 to 16 molecules or 2 to 8 molecules, optionally 2, 3, 4, 5 or 6 molecules.

[0163] In particular embodiments, compositions for use according to the present disclosure are provided, wherein the second ligand is conjugated to 2-5 molecules of nucleic acid per molecule of the second ligand; preferably 3-4 molecules of nucleic acid per molecule of the second ligand; more preferably, wherein the second ligand is conjugated to an average of 4 molecules of nucleic acid per molecule of the second ligand.

[0164] Similarly, in an alternative embodiment, a therapeutic combination according to the present disclosure is provided, wherein the fifth ligand of the fourth conjugate is conjugated to 2 to 5 molecules of antisense oligonucleotide per molecule of the fifth ligand; preferably 3 to 4 molecules of antisense oligonucleotide per molecule of the fifth ligand; more preferably, wherein the fifth ligand is conjugated to an average of 4 molecules of antisense oligonucleotide per molecule of the fifth ligand.

[0165] In light of clinical practice, in a further preferred embodiment there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination according to the present disclosure, wherein the two or more nucleic acid or oligonucleotide molecules are two or more different oligonucleotides conjugated together, respectively as part of a second conjugate or as part of a third conjugate, and wherein at least one (preferably a plurality) of the two or more different oligonucleotides is an antisense oligonucleotide.

[0166] Regarding the covalent linking option of the conjugate, many different embodiments are possible, some preferred ones being those involving the use of a conditionally cleavable bond as already briefly mentioned above in connection with some advantageous saponins.

[0167] Thus, in a possible embodiment, a composition for use according to the present disclosure is provided, wherein the first ligand of the first conjugate comprises a chain of amino acid residues comprising at least one cysteine ​​residue and / or at least one lysine residue, and wherein the covalent linkage to the first ligand by the saponin in the first conjugate comprises a covalent bond to at least one cysteine ​​residue and / or at least one lysine residue, and / or optionally, wherein the second ligand of the second conjugate also comprises a chain of amino acid residues comprising at least one cysteine ​​residue and / or at least one lysine residue, and wherein the covalent linkage to the second ligand by the nucleic acid comprises a covalent bond to at least one cysteine ​​residue and / or at least one lysine residue; preferably, wherein two or more molecules of saponin are linked to one molecule of the first ligand via distinct cysteine ​​residues and / or distinct lysine residues, and / or optionally wherein two or more molecules of nucleic acid are linked to one molecule of a second ligand via distinct cysteine ​​and / or distinct lysine residues; more preferably, wherein the first ligand and / or optionally also the second ligand comprises a chain of amino acid residues comprising a multi-cysteine ​​repeat, which is optionally a tetracysteine ​​repeat represented by the sequence HRWCCPGCCKTF (SEQ ID NO: 4), and wherein the covalent linkage to the first ligand by the saponin in the first conjugate or to the second ligand by the nucleic acid in the second conjugate, respectively, comprises a covalent bond to any one or more of the cysteine ​​residues of the multi-cysteine ​​repeat; most preferably, wherein two or more molecules of saponin are linked to one molecule of a first ligand via distinct cysteine ​​residues of the multi-cysteine ​​repeat, and / or optionally, wherein two or more molecules of nucleic acid are linked to one molecule of a second ligand via distinct cysteine ​​residues of the multi-cysteine ​​repeat.

[0168] In another embodiment, a composition for use according to the present disclosure is provided, wherein the covalent linkage of the saponin to the first ligand in the first conjugate is via a first linker to which the saponin is covalently attached; preferably, wherein the first linker comprises a covalent bond selected from any one or more of a semicarbazone bond, an imine bond, a hydrazone bond, an imine bond, an acetal bond including a 1,3-dioxolane bond, a ketal bond, an ester bond, an oxime bond, a disulfide bond, a thioether bond, an amide bond, a peptide bond and an ester bond, preferably a hydrazone bond or a semicarbazone bond; more preferably, wherein the saponin is an aldehyde group at the C-23 position of the saponin aglycone core structure, or A covalent bond at the C-23 position of the aglycone core structure of the saponin, which covalently links the saponin in the first conjugate via the first linker and is included as part of the first linker, preferably the covalent bond at the C-23 position is a cleavable bond that undergoes cleavage under conditions present in an endosome or lysosome, more preferably the cleavable covalent bond at the C-23 position is adapted to restore an aldehyde group at the C-23 position upon cleavage. Contains any of the following; or a saponin which, at least in its unconjugated state, comprises an aldehyde group at the C-23 position of its aglycone core structure, said aldehyde group participating in the formation of a covalent bond with a first linker.

[0169] In related embodiments, such compositions are provided, wherein the first linker is a cleavable linker that undergoes cleavage under acidic, reducing, enzymatic and / or light-induced conditions; Preferably, wherein the first linker is Bonds that undergo cleavage under acidic conditions, such as semicarbazone bonds, hydrazone bonds, imine bonds, acetal bonds including 1,3-dioxolane bonds, ketal bonds, ester bonds and / or oxime bonds; proteolytically sensitive bonds, e.g. amide or peptide bonds, which are subject to proteolysis, preferably by cathepsin B; Bonds that are redox cleavable, such as disulfide bonds, or that are susceptible to thiol exchange reactions, such as thioether bonds and a cleavable bond selected from Preferably, the bond is one that is subject to cleavage in vivo under acidic conditions present in the endosomes and / or lysosomes of human cells, preferably at a pH of 4.0 to 6.5, more preferably at a pH of ≦5.5; More preferably, it is an acid-sensitive bond selected from any one or more of a semicarbazone bond, a hydrazone 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, it is selected from a semicarbazone bond and a hydrazone bond; most preferably, it is a hydrazone bond.

[0170] In a further and advantageous embodiment, a composition for use according to the present disclosure is provided, wherein the covalent linkage of the nucleic acid in the second conjugate to the second ligand is via a second linker to which the nucleic acid is covalently attached; preferably, wherein the second linker comprises or consists of the linker 3-(2-pyridyldithio) succinimidyl propionate (SPDP); optionally, wherein the second linker covalently links the nucleic acid to a lysine residue or a glycan residue, preferably a lysine residue contained in the second ligand, preferably a partially trimmed glycan.

[0171] In related embodiments, such compositions are provided, wherein the second linker is a cleavable linker that undergoes cleavage under acidic, reducing, enzymatic and / or light-induced conditions; Preferably, wherein the second linker is Bonds that undergo cleavage under acidic conditions, such as semicarbazone bonds, hydrazone bonds, imine bonds, acetal bonds including 1,3-dioxolane bonds, ketal bonds, ester bonds and / or oxime bonds; proteolytically sensitive bonds, e.g. amide or peptide bonds, which are subject to proteolysis, preferably by cathepsin B; Bonds that are redox cleavable, such as disulfide bonds, or that are susceptible to thiol exchange reactions, such as thioether bonds and a cleavable bond selected from More preferably, wherein the second linker is Bonds that are subject to cleavage under acidic conditions, such as semicarbazone bonds and hydrazone bonds Bonds that are easily cleaved under reducing conditions, such as disulfide bonds The cleavable bond is selected from:

[0172] In related embodiments, such compositions are provided, wherein the second linker is a cleavable linker that undergoes cleavage under acidic, reducing, enzymatic and / or light-induced conditions; Preferably, wherein the second linker is Semicarbazone bonds, hydrazone bonds, and other bonds that are subject to cleavage under acidic conditions and preferably an acid-sensitive bond that undergoes cleavage in vivo under acidic conditions present in the endosomes and / or lysosomes of human cells, preferably at a pH of 4.0 to 6.5, more preferably at a pH of ≦5.5; more preferably a bond selected from any one or more of a semicarbazone bond, a hydrazone 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 a semicarbazone bond and a hydrazone bond; and most preferably a hydrazone bond.

[0173] In certain embodiments, when the saponin is conjugated by a covalent bond (preferably an acid-sensitive bond) at the C-23 position of the saponin aglycone core structure, said covalent bond at the C-23 position may advantageously be selected or adapted such that upon cleavage (e.g. under acidic conditions) an aldehyde group at the C-23 position is restored. Advantageously, such a covalent bond may be selected from any one or more of a semicarbazone bond, a hydrazone bond, an imine bond, an acetal bond including a 1,3-dioxolane bond and / or an oxime bond, preferably either a semicarbazone bond or a hydrazone bond.

[0174] Similarly, in possible embodiments, there is provided a therapeutic combination according to the present disclosure, wherein the fourth ligand of the third conjugate comprising a saponin comprises a chain of amino acid residues comprising at least one cysteine ​​residue and / or at least one lysine residue, wherein the covalent linkage of the fourth ligand by the saponin in the third conjugate comprises a covalent bond to at least one cysteine ​​residue and / or at least one lysine residue, and / or optionally, wherein the fifth ligand of the fourth conjugate comprising an antisense oligonucleotide also comprises a chain of amino acid residues comprising at least one cysteine ​​residue and / or at least one lysine residue, and wherein the covalent linkage of the fifth ligand by the antisense oligonucleotide comprises a covalent bond to at least one cysteine ​​residue and / or at least one lysine residue; preferably, wherein two or more molecules of saponin are linked to one molecule of the fourth ligand via distinct cysteine ​​residues and / or distinct lysine residues, and / or optionally wherein two or more molecules of the antisense oligonucleotide are linked to one molecule of the fifth ligand via distinct cysteine ​​and / or distinct lysine residues; more preferably, wherein the fourth ligand and / or optionally also the fifth ligand comprises a chain of amino acid residues including a multi-cysteine ​​repeat, which is optionally a tetracysteine ​​repeat represented by the sequence HRWCCPGCCKTF (SEQ ID NO: 4), and wherein the covalent linkage to the fourth ligand by the saponin in the third conjugate or to the fifth ligand by the antisense oligonucleotide in the fourth conjugate, respectively, comprises a covalent bond to any one or more of the cysteine ​​residues of the multi-cysteine ​​repeat; most preferably, wherein two or more molecules of the saponin are linked to one molecule of the fourth ligand via distinct cysteine ​​residues of the multi-cysteine ​​repeat, and / or optionally, wherein two or more molecules of the antisense oligonucleotide are linked to one molecule of the fifth ligand via distinct cysteine ​​residues of the multi-cysteine ​​repeat.

[0175] In another embodiment, a therapeutic combination is provided, wherein the covalent linkage of the saponin to the fourth ligand in the third conjugate is via a third linker to which the saponin is covalently attached; preferably, wherein the third linker comprises a covalent bond selected from any one or more of a semicarbazone bond, an imine bond, a hydrazone bond, an acetal bond including a 1,3-dioxolane bond, a ketal bond, an ester bond, an oxime bond, a thioether bond, an amide bond, a peptide bond and an ester bond, preferably a hydrazone bond or a semicarbazone bond; more preferably, wherein the saponin is an aldehyde group at the C-23 position of the saponin aglycone core structure, or A covalent bond at the C-23 position of the aglycone core structure of the saponin, which covalently links the saponin in the first conjugate via the first linker and is included as part of the first linker, preferably the covalent bond at the C-23 position is a cleavable bond that undergoes cleavage under conditions present in an endosome or lysosome, more preferably the cleavable covalent bond at the C-23 position is adapted to restore an aldehyde group at the C-23 position upon cleavage. Contains any of the following; Alternatively, the saponin used to prepare the conjugate is a saponin that, at least in its unconjugated state, contains an aldehyde group at the C-23 position of the aglycone core structure of the saponin, said aldehyde group participating in the formation of a covalent bond with a third linker.

[0176] In a related embodiment, a therapeutic combination is provided, wherein the third linker is a cleavable linker that undergoes cleavage under acidic, reducing, enzymatic and / or light-induced conditions; Preferably, wherein the third linker is Bonds that undergo cleavage under acidic conditions, such as semicarbazone bonds, hydrazone bonds, imine bonds, acetal bonds including 1,3-dioxolane bonds, ketal bonds, ester bonds and / or oxime bonds; proteolytically sensitive bonds, e.g. amide or peptide bonds, which are subject to proteolysis, preferably by cathepsin B; Bonds that are redox cleavable, such as disulfide bonds, or that are susceptible to thiol exchange reactions, such as thioether bonds and a cleavable bond selected from Preferably, it is an acid-sensitive bond that undergoes cleavage in vivo under acidic conditions present in the endosomes and / or lysosomes of human cells, preferably at pH 4.0 to 6.5, more preferably at pH ≦ 5.5; more preferably, it is a bond selected from a semicarbazone bond and a hydrazone bond; and most preferably, it is a hydrazone bond.

[0177] In a further and advantageous embodiment, a therapeutic combination is provided, wherein the covalent linkage of the antisense oligonucleotide to the fifth ligand is via a fourth linker to which the nucleic acid is covalently attached; preferably, wherein the fourth linker comprises or consists of the linker 3-(2-pyridyldithio) succinimidyl propionate (SPDP); optionally, wherein the fourth linker covalently links the nucleic acid to a lysine residue or a glycan residue, preferably a lysine residue contained in the fifth ligand, preferably a partially trimmed glycan.

[0178] In related embodiments, such therapeutic combinations are provided, wherein the fourth linker is a cleavable linker that undergoes cleavage under acidic, reducing, enzymatic and / or light-induced conditions; Preferably, wherein the fourth linker is Bonds that undergo cleavage under acidic conditions, such as semicarbazone bonds, hydrazone bonds, imine bonds, acetal bonds including 1,3-dioxolane bonds, ketal bonds, ester bonds and / or oxime bonds; proteolytically sensitive bonds, e.g. amide or peptide bonds, which are subject to proteolysis, preferably by cathepsin B; Bonds that are redox cleavable, such as disulfide bonds, or that are susceptible to thiol exchange reactions, such as thioether bonds and a cleavable bond selected from More preferably, wherein the second linker is Bonds that are subject to cleavage under acidic conditions, such as semicarbazone bonds and hydrazone bonds Bonds that are easily cleaved under reducing conditions, such as disulfide bonds comprising a cleavable bond selected from: More preferably, it is an acid-sensitive bond that undergoes cleavage in vivo under acidic conditions present in the endosomes and / or lysosomes of human cells, preferably at pH 4.0 to 6.5, more preferably at pH ≦ 5.5; more preferably, it is a bond selected from a semicarbazone bond and a hydrazone bond; and most preferably, it is a hydrazone bond.

[0179] As already explained above, saponins containing an aldehyde group at the C-23 position of the aglycone are particularly preferred due to the potency of the endosomal escape promoting activity they exhibit towards nucleic acids such as oligonucleotides. Thus, preferred saponins for the first or third conjugates are those that contain or form an aldehyde group at the C-23 position of the aglycone core structure of the saponin under the acidic conditions present in the endosomes and / or lysosomes of human cells.

[0180] For example, when the saponins of Groups B and C described above are covalently linked in the conjugate by linker chemistry involving an aldehyde group (e.g., formation of a hydrazone or semicarbazone bond), it is preferred that the aldehyde group is reformed (restored) in the endosome or lysosome when the conjugate is endocytosed and the saponin is cleaved away from the remainder of the first or third conjugate by cleavage of the cleavable bond. Examples of such saponins suitable for this purpose are listed in Table 1, such as the saponins of Groups A-C, in particular Groups B and C, as outlined herein above. A particularly advantageous example of a saponin from Table 1 is SO1861.

[0181] As explained above, various different exemplary embodiments of the disclosed various different saponin-containing conjugates and / or nucleic acid-containing conjugates can be envisaged, and thus various different conjugation modes of the saponin and optionally also the nucleic acid with the targeting ligand and / or oligomeric or polymeric structures (further referred to as scaffolds, e.g. PEG-based), respectively. In light of the above-described embodiments, this conjugation can be either by direct covalent bonding of at least two molecules contained in the conjugate, or via a linker, such as the first or third linker (for linking the saponin to the ligand) or the second or fourth linker (for linking the nucleic acid to the ligand) described above. The use of a linker can establish a covalent bond by the saponin and optionally also by the nucleic acid (preferably an oligonucleotide such as an ASO or PMO) in the composition and / or combination of the present invention to the ligand (e.g. immunoglobulin, mAb, sdAb, VHH, etc.), i.e. to its respective targeting ligand. As explained above, in possible embodiments, such linkers may be stable under the conditions present in mammalian (e.g. human) endosomes / lysosomes or unstable (i.e. cleavable) under said conditions, the latter meaning that such linkers are cleaved in response to said conditions, thereby releasing at least the saponin (and also the nucleic acid, if of targeted type) covalently linked by such cleavable linker from its respective targeting ligand.

[0182] Numerous examples of cleavable first or third linkers covalently linking a saponin to a ligand are described above, including the more detailed embodiment of a cleavable first linker attached to a saponin via an acid-sensitive bond at the C-23 position of the saponin aglycone core, which acid-sensitive bond is preferably established by reaction of an aldehyde group at the C-23 position of the aglycone core of such saponin, and is configured to restore said group under acidic conditions present in mammalian (e.g. human) endosomes / lysosomes.

[0183] Alternatively, when the receptor internalization rate is not a limiting factor, in another possible embodiment, the first or third linker covalently linking the saponin to the ligand in the composition / combination of the present invention may be a stable linker, which may be linked to the saponin by reacting, for example, preferably and if present, via a glucuronic acid group with a glucuronic acid unit in the first glycan attached to the C3β-OH group of the aglycon core structure of the saponin. As a result of such reaction, a stable first linker may be created that comprises a stable (i.e. non-cleavable) bond to the first glycan attached to the C3β-OH group of the aglycon core structure of the saponin, which covalently links the saponin to a targeting ligand (e.g. an immunoglobulin such as a mAb, sdAb, VHH, etc.) or to a scaffold, if present. In light of this, a possible embodiment is a saponin conjugate as provided, where the saponin belongs to the saponins comprising a glucuronic acid unit in a first saccharide chain at the C3β-OH group of the aglycon core structure of the saponin, where upon reaction of the glucuronic acid unit, the linker is preferably covalently attached via an amide bond created in the first saccharide chain attached to the C3β-OH group of the aglycon core structure of the saponin, more preferably to an amine group present in the ligand (such as an amine group of a lysine or the N-terminus of a proteinaceous ligand such as an immunoglobulin) or to a scaffold if present in addition. The glucuronic acid functional group is particularly advantageous since upon reaction, it may establish a covalent linkage of the saponin with the ligand or with the scaffold of the saponin conjugate of the invention, either via a direct covalent bond or a linker, where the linker is a stable linker, but can also be designed to be a cleavable linker.

[0184] The choice between a cleavable first linker and a stable first linker is entirely up to the skilled artisan and can be made, for example, depending on the choice of ligand and the desired release rate of any of the distinct molecules contained in the conjugate as disclosed herein. The saponin conjugated via a cleavable first linker or a stable first linker can be part of any embodiment as disclosed herein, for example, an embodiment of a pharmaceutical composition / therapeutic combination for use of the present invention, in which a nucleic acid (e.g., an oligonucleotide, preferably a PMO or an ASO) is also targeted and is linked to its targeting ligand via either a cleavable second linker or a stable second linker. Although examples of both stable and cleavable second linkers have been provided above, it is highly likely that both will be used for the conjugation of nucleic acids within the nucleic acid-containing conjugates of the compositions / combinations of the present invention. The choice between them will be highly dependent on the type of nucleic acid used and the intended therapy. For example, the stable linker can be very easy to use for the conjugation of only one strand of the therapeutic nucleic acid that is designed to act in a single-stranded form in cells.The two strands of such therapeutic nucleic acid can be selected to dissociate in response to the conditions present in endosomes / lysosomes, thus releasing the therapeutic strand from the conjugate and entering the cytosol in a manner that is promoted by the presence of the endosomal escape-promoting saponin described herein.For double-stranded nucleic acids, in some embodiments, a cleavable second linker can be advantageous, which can be considered to be either conjugated to a ligand or conjugated to a scaffold.

[0185] In a specific embodiment, a composition for use according to the present disclosure is provided, wherein the saponin is or comprises at least one molecule of SO1861, the nucleic acid is drisapersen or eteplirsen, and the first ligand is an anti-CD71 antibody or binding fragment thereof, and preferably the second ligand is an anti-CD71 antibody or binding fragment thereof.

[0186] Similarly, in a specific embodiment, a therapeutic combination is provided, wherein the saponin is or comprises at least one molecule of SO1861, the antisense oligonucleotide is drisapersen or eteplirsen, and the fourth ligand is an anti-CD71 antibody or binding fragment thereof, and preferably the fifth ligand is an anti-CD71 antibody or binding fragment thereof.

[0187] To construct complex covalent conjugates, molecular scaffolds including oligomeric or polymeric structures can be used, for example to contain a large or fixed number of ligands such as antibodies, or multiple nucleic acid molecules, or especially saponin molecules. One advantage of this is that the scaffold can be designed so that it contains a fixed number of molecules, such as saponins. For example, the scaffold can contain exactly one saponin molecule, but can also contain several (e.g., 2, 3, or 4) saponins or a relatively constant and fixed number of saponins in a large number (e.g., 10, 20, or 100). In some cases, such oligomeric or polymeric structures will appear as either linear, branched or cyclic polymers, oligomers, dendrimers, dendrons (e.g. either G2, G3, G4 or G5 dendrons for a maximum covalent attachment of 4, 8, 16 or 32 saponin moieties, respectively), dendronized polymers, dendronized oligomers or assemblies of either pure or mixed forms of these structures, made of poly(amines), e.g. polyethyleneimine and poly(amidoamines) or alternatively polyethylene glycol, poly(esters) such as poly(lactides), poly(lactams), polylactide-co-glycolide copolymers, poly(dextrins), or peptides or proteins, or natural and / or artificial polyamino acids, e.g. poly-lysine, DNA polymers such as DNA containing 2-100 nucleotides, stabilized RNA polymers or PNA (peptide nucleic acid) polymers containing e.g. 2-200 nucleotides. Preferably, such scaffolds may be made of oligomeric and / or polymeric structures such as dendrimers, dendrons, dendronized polymers, dendronized oligomers, DNA, e.g., nucleic acids of 2-200, polyethylene glycol, oligoethylene glycol (OEG), such as OEG3, OEG4 and OEG5.

[0188] Thus, in an advantageous embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination according to the present disclosure, wherein the first linker or the third linker, respectively, further comprises an oligomeric or polymeric structure which is either a dendron, such as a polyamidoamine (PAMAM) dendrimer, or a polyethylene glycol, such as any of PEG3 to PEG30; preferably the polymeric or oligomeric structure is any one of PEG4 to PEG12 or any one of G2 dendron, G3 dendron, G4 dendron and G5 dendron, more preferably G2 dendron or G3 dendron or PEG3 to PEG30.

[0189] The structures forming such oligomeric / polymeric scaffolds advantageously lack intrinsic biological activity. Typically, the scaffolds are made of inert molecules to avoid potential health risks. Depending on the number of selected saponins to be incorporated into the specific embodiment of the muscle-targeting conjugates presented herein, the type and size or length of the oligomeric / polymeric structure can be appropriately selected. In other words, the number of saponins to be coupled to the ligand and nucleic acid as part of the conjugate can determine the selection of a suitable oligomeric or polymeric structure that carries a sufficient number of binding sites to couple the desired number of saponins and provide a covalent saponin-binding structure therewith. For example, the length of the OEG or the size of the dendron or polylysine molecule determines the maximum number of saponins that can be covalently linked to such oligomeric or polymeric structures that may be included as part of the first linker in the conjugate.

[0190] In an advantageous embodiment, a conjugate comprising such a scaffold may contain a defined number or range of saponins covalently linked thereto, rather than a random number. This is particularly advantageous in terms of obtaining marketing approval during drug development. Defined number in this context means that the conjugate may contain a predefined number of saponins. This is achieved, for example, by designing a scaffold comprising an oligomeric / polymeric structure with a certain number of groups to which one or more saponins can associate. Under ideal circumstances, each of these groups will associate with one saponin molecule, thus resulting in a conjugate containing a defined number of saponins. For example, it is envisaged to provide a standard set of scaffolds including 2, 4, 8, 16, 32, 64, etc.

[0191] In certain embodiments of the conjugates of the invention, for example, for non-ideal binding situations where not all groups present in the oligomer / polymer will be associated with a saponin molecule, scaffolds may be provided where the number of saponin molecules will be defined as a range, such as, for example, 2-4 saponin molecules per scaffold, 3-6 saponin molecules per scaffold, 4-8 saponin molecules per scaffold, 6-8 saponin molecules per scaffold, 6-12 saponin molecules per scaffold, etc.

[0192] Such a first linker comprising any number of saponins attached to an oligomeric or polymeric molecule may in some embodiments function as a carrier (support, scaffold) for multiple saponin moieties, which bind to ligands and nucleic acids, thus forming specific embodiments of the muscle cell targeting therapeutic conjugates disclosed herein. In advantageous embodiments, such an oligomeric or polymeric molecule-containing linker loaded with saponin molecules may be attached to the remainder of the muscle cell targeting conjugate, preferably by a cleavable bond.

[0193] In a further possible embodiment, there is provided a composition for use according to the present disclosure for use in intravenous or subcutaneous administration to a human subject.

[0194] In a further highly probable embodiment, there is provided a composition for use according to the present disclosure and / or a therapeutic combination according to the present disclosure comprising a pharma- ceutically acceptable excipient and / or a pharma-ceutically acceptable diluent.

[0195] In a further embodiment, a kit is provided comprising components (a) and (b) of the therapeutic combination of the present disclosure, optionally wherein components (a) and (b) are provided in separate vials or in a mixture suitable for intravenous or subcutaneous or intramuscular injection.

[0196] Finally, in a further possible embodiment, there is provided a therapeutic combination or kit of the present disclosure for use as a medicine. EXAMPLES

[0197] material: SO1861 was isolated and purified from a crude plant extract obtained from soapwort (Saponaria officinalis L) by Extrasynthese, France and / or Analyticon Discovery GmbH.

[0198] Antisense oligonucleotides with the sequence 5'-UCAAGGAAGAUGGCAUUUCU-3' [SEQ ID NO: 1] and ASOs with the same sequence but with thiol modifications (DMD-ASO and 5'-thiol-DMD-ASO, respectively) were custom-ordered and purchased from Hanugen Therapeutics Pvt Ltd. PMOs with the sequence 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (DMD-PMO or DMD-PMO(1)) [SEQ ID NO: 2] and PMOs with the same sequence but with disulfide amide modifications (5'-disulfide amide-DMD-PMO or 5'-disulfide amide-DMD-PMO(1)) were custom-ordered and purchased from Gene Tools, LLC. The PMO (DMD-PMO(1)) of sequence 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' [SEQ ID NO:2] and one with a disulfide amide modification at the 3' (3'-disulfide amide-DMD-PMO(1)) were custom ordered from Gene Tools. The PMO (M23D) of sequence 5'-GGCCAAACCTCGGCTTACCTGAAAT-3' [SEQ ID NO:3] and one with a disulfide amide modification at the same sequence (3'-disulfide amide-M23D) were custom ordered from Gene Tools, LLC. The PMO (DMD-PMO(2)) of sequence 5'-GTGTCACCAGAGTAACAGTCTGAGTAGGAG-3' [SEQ ID NO:16] and one with a disulfide amide modification at the 3' (3'-disulfide amide-DMD-PMO(2)) were custom ordered from Gene Tools. The PMO with the sequence 5'-GGCAGTTTCCTTAGTAACCACAGGTTGTGT-3' (DMD-PMO(3)) [SEQ ID NO: 17] and with a disulfide amide modification at the 3' (3'-disulfide amide-DMD-PMO(3)) were custom ordered and purchased from Gene Tools. The PMO with the sequence 5'-GTTGCCTCCGGTTCTGAAGGTGTTC-3' (DMD-PMO(4)) [SEQ ID NO: 18] and with a disulfide amide modification at the 3' (3'-disulfide amide-DMD-PMO(4)) were custom ordered and purchased from Gene Tools.PMO of sequence 5'-CCTCCGGTTCTGAAGGTGTTC-3' (DMD-PMO(5)) [SEQ ID NO: 19] and one with a disulfide amide modification at 3' (3'-disulfide amide-DMD-PMO(5)) were custom purchased from Gene Tools.

[0199] Anti-CD71 antibody (clone OKT9) targeting human CD71 (hCD71) and mouse targeting anti-CD71 antibody (clone R17 217.1.3) (mCD71) were both purchased from BioXCell. Anti-CD63 antibody (clone H5C6) targeting human CD63 (hCD63) and mouse targeting anti-CD63 antibody (clone NVG-2) (mCD63) were both purchased from Biolegend. IGF-1 ligand was purchased from PeproTech.

[0200] Tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 98%, Sigma-Aldrich), 5,5-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent, 99%, Sigma-Aldrich), Zeba™ spin desalting columns (2 mL, Thermo-Fisher), NuPAGE™ 4-12% Bis-Tris protein gel (Thermo-Fisher), NuPAGE™ MES SDS running buffer (Thermo-Fisher), Novex™ Sharp prestained protein standards (Thermo-Fisher), PageBlue™ protein staining solution (Thermo-Fisher), Pierce™ BCA protein assay kit (Thermo-Fisher), N-ethylmaleimide (NEM, 98%, Sigma-Aldrich), 1,4-dithiothreitol (DTT, 98%, Sigma-Aldrich), Sephadex G25 (GE Healthcare), Sephadex G50M (GE Healthcare), Superdex 200P (GE Healthcare), isopropyl alcohol (IPA, 99.6%, VWR), tris(hydroxymethyl)aminomethane (Tris, 99%, Sigma-Aldrich), tris(hydroxymethyl)aminomethane hydrochloride (Tris.HCL, Sigma-Aldrich), L-histidine (99%, Sigma-Aldrich), D-(+)-trehalose dehydrate (99%, Sigma-Aldrich), polyethylene glycol sorbitan monolaurate (TWEEN 20, Sigma-Aldrich), Dulbecco's phosphate buffered saline (DPBS, Thermo-Fisher), guanidine hydrochloride (99%, Sigma-Aldrich), ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA-Na2, 99%, Sigma-Aldrich), sterile filtered 0.2 μm and 0.45 μm (Sartorius), Vivaspin T4 and T15 concentrators (Sartorius), Superdex 200PG (GE Healthcare), tetra(ethylene glycol), dimethyl sulfoxide (DMSO, 99%, Sigma-Aldrich), N-(2-aminoethyl)maleimide trifluoroacetate (AEM, 98%, Sigma-Aldrich), L-cysteine ​​(98.5%, Sigma-Aldrich), deionized water (DI) was freshly taken from a laboratory ultrapure water system (MilliQ, Merck), nickel-nitrilotriacetic acid agarose (Ni-NTA agarose, Protino), glycine (99.5%, VWR), 5,5-dithiobis(2-nitrobenzoic acid) (Ellman's reagent, DTNB, 98%, Sigma-Aldrich), S-acetylmercaptosuccinic anhydride fluorescein (SAMSA reagent, Invitrogen) sodium bicarbonate (99.7%, Sigma-Aldrich), sodium carbonate (99.9%, Sigma-Aldrich), Sephadex PD MiniTrap desalting columns (GE Healthcare) with G-25 resin, PD10 G25 desalting columns (GE Healthcare), 0.Zeba spin desalting columns of 5, 2, 5 and 10 mL (Thermo-Fisher), Vivaspin centrifugal filters T4 10 kDa MWCO, T4 100 kDa MWCO and T15 (Sartorius), Biosep s3000 aSEC columns (Phenomenex), Vivacell ultrafiltration units 10 and 30 kDa MWCO (Sartorius), Nalgene Rapid-Flow filters (Thermo-Fisher), dichloromethane (Sigma-Aldrich), methanol (Sigma-Aldrich), diethyl ether (Sigma-Aldrich), acetonitrile (Sigma-Aldrich), pyridine 2-thione (Sigma-Aldrich), goat anti-human IgG-HRP (Southern Biotech), goat anti-human κ-HRP (Southern Biotech), Biotech), concentrated Tris (Thermo-Fisher), MOPS running buffer (20×, Thermo-Fisher), LDS sample buffer (4×, Thermo-Fisher), TBS blocking buffer (Thermo-Fisher), Tris (tris(hydroxymethyl)aminomethane, Merck), TrisHCl (Sigma-Aldrich), Minisart RC15 0.2 μm filters (Sartorius), Minisart 0.45 μm filter (Sartorius), PD Minitrap G25 (Cytiva), TNBS (2,4,6-trinitrobenzenesulfonic acid, Sigma-Aldrich), sodium dodecyl sulfate (SDS, Sigma-Aldrich), SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, Thermo-Fisher), THPP (tris(hydroxypropyl)phosphine, Sigma-Aldrich), DBCO-NHS (CAS 1353016-71-3, BroadPharm), PEG4-SPDP (2-pyridyldithiol-tetraoxatetradecane-N-hydroxysuccinimide, Thermo-Fisher), Novex™ TBE-Urea gel, 15% (Thermo-Fisher), TBE buffer (ris-Borat-EDTA, Thermo-Fisher), GlyCLICK™ (10 mg) azide activation kit (Genovis) and an immobilized GlycINATOR™ column (from the GlyCLICK™ azide activation kit) (Genovis) were used.

[0201] [Table 10]

[0202] [Table 11]

[0203] Abbreviation Ab antibody Ac Acetyl AON Antisense Oligonucleotides ASO Antisense Oligonucleotides BCA Bicinchoninic Acid BGG Bovine gamma globulin aSEC Analytical Size Exclusion Chromatography DAR Drug-Antibody Ratio DBCO Dibenzocyclooctyne DBCO-NHS Dibenzocyclooctyne-N-hydroxysuccinimide ester DCM Dichloromethane DIPEA N,N-Diisopropylethylamine DMEM Dulbecco's Modified Eagle's Medium DMF N,N-Dimethylformamide DMSO Dimethyl sulfoxide DPBS Dulbecco's Phosphate Buffered Saline DTME Dithiobismaleimideethane DTNB 5,5'-Dithiobis-(2-nitrobenzoic acid) DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid EDCI.HCl 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EMCH.TFA N-(ε-Maleimidocaproic acid) hydrazide, trifluoroacetate Equiv. EtBr Ethidium bromide FBS Fetal Bovine Serum GalT Galactose-1-phosphate uridylyltransferase HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate HRP Horseradish Peroxidase IPA Isopropyl Alcohol LC-MS Liquid Chromatography-Mass Spectrometry LDS Lithium dodecyl sulfate LRMS low resolution mass spectrometry NEM N-ethylmaleimide NHS N-hydroxysuccinimide ester mAb Monoclonal Antibody min MOPS 3-(morpholin-4-yl)propane-1-sulfonic acid MPLC Medium Pressure Liquid Chromatography MWCO Molecular Weight Cutoff NMM 4-Methylmorpholine PBS Phosphate Buffered Saline PBS-T Phosphate-buffered saline containing Tween-20 PEG4-SPDP 2-pyridyldithiol-tetraoxatetradecane-N-hydroxysuccinimide PMO Phosphorodiamidate Morpholino Oligomers PDT Pyridine 2-thione rpm Revolutions per minute RT room temperature rt retention time SDS Sodium dodecyl sulfate SEC Size Exclusion Chromatography SMCC 4-(N-maleimidomethyl)cyclohexane-1-carboxylate succinimidyl TBEU (Tris-hydroxymethyl)-aminomethane)-boron-EDTA-urea (REA) TBS Tris-buffered saline TCEP Tris(2-carboxyethyl)phosphine hydrochloride TCO4-NHS trans-cyclooctene-N-hydroxysuccinimide Temp Temperature TFA Trifluoroacetic acid THPP Tris(hydroxypropyl)phosphine TMB 3,3',5,5'-Tetramethylbenzidine TNBS 2,4,6-trinitrobenzenesulfonic acid Tris Tris(hydroxymethyl)aminomethane UDP-GalNAz Uridine diphosphate-N-azidoacetylmannosamine

[0204] Methods (as carried out in Examples 1-5) SO1861-maleimide, SO1861-NHS synthesis SO1861 is derived from soap plant (Saponaria officinalis L) (Extrasynthese, France and / or Analyticon Discovery GmbH, Germany) and was coupled to the respective handle by Symeres (NL) according to methods known in the art.

[0205] Conjugation of SO1861 to antibodies and proteins Custom production of IGF-1-SO1861, mCD71-SO1861 and hCD71-SO1861 was carried out by Fleet Bioprocessing (UK).

[0206] Conjugation of 5'-disulfide amide-DMD-PMO, 5'-thiol-DMD-ASO and 3'-disulfide amide-M23D to antibodies Custom production of mCD71-M23D, mCD71-M23D-SO1861 and hCD71-DMD-ASO, hCD71-DMD-PMO, hCD71-DMD-ASO-SO1861 and hCD71-DMD-PMO-SO1861 was carried out by Fleet Bioprocessing (UK).

[0207] Analysis and preparation methods LC-MS method 1 Instrument: Waters IClass; Binary Pump: UPIBSM, SM: UPISMFTN SO attached; UPCMA, PDA: UPPDATC, 210-320 nm, SQD: ACQ-SQD2 ESI, mass range depends on product molecular weight: neg or neg / pos in the range of 1500-2400 or 2000-3000; ELSD: Gas pressure 40 psi, Drift tube temperature: 50°C; Column: Acquity C18, 50 x 2.1 mm, 1.7 μm Temperature: 60°C, Flow rate: 0.6 mL / min, Linear gradient depends on product polarity: A t0=2%A, t 5.0min = 50%A, t 6.0min =98%A B t0=2%A, t 5.0min = 98%A, t 6.0min =98%A Post time: 1.0 min, eluent A: acetonitrile, eluent B: 10 mM ammonium bicarbonate in water (pH = 9.5).

[0208] LC-MS method 2 Instrument: Waters IClass; Binary Pump: UPIBSM, SM: UPISMFTN SO attached; UPCMA, PDA: UPPDATC, 210-320 nm, SQD: ACQ-SQD2 ESI, mass range depends on product molecular weight: pos / neg 100-800 or neg 2000-3000; ELSD: Gas pressure 40 psi, Drift tube temperature: 50°C; Column: Waters XSelect™ CSH C18, 50 x 2.1 mm, 2.5 μm, Temperature: 25°C, Flow rate: 0.5 mL / min, Gradient: t 0min = 5%A, t 2.0min = 98%A, t 2.7min = 98% A, post time: 0.3 min, eluent A: acetonitrile, eluent B: 10 mM ammonium bicarbonate in water (pH = 9.5).

[0209] LC-MS method 3 Instrument: Waters IClass; Binary Pump: UPIBSM, SM: UPISMFTN SO attached; UPCMA, PDA: UPPDATC, 210-320 nm, SQD: ACQ-SQD2 ESI, mass range depends on product molecular weight pos / neg 105-800, 500-1200 or 1500-2500; ELSD: Gas pressure 40 psi, Drift tube temperature: 50°C; Column: Waters XSelect™ CSH C18, 50 x 2.1 mm, 2.5 μm, Temperature: 40°C, Flow rate: 0.5 mL / min, Gradient: t 0min = 5%A, t 2.0min = 98%A, t 2.7min = 98% A, post time: 0.3 min, eluent A: 0.1% formic acid in acetonitrile, eluent B: 0.1% formic acid in water.

[0210] LC-MS method 4 Equipment: Waters IClass; Binary Pump: UPIBSM, SM: UPISMFTN SO attached; UPCMA, PDA: UPPDATC, 210-320 nm; SQD: ACQ-SQD2 ESI; Mass range depends on product molecular weight: pos / neg 100-800 or neg 2000-3000; ELSD: Gas pressure 40 psi, Drift tube temperature: 50 °C Column: Waters Acquity Shield RP18, 50 × 2.1 mm, 1.7 μm, Temperature: 25 °C, Flow rate: 0.5 mL / min, Gradient: t 0min = 5%A, t 2.0min = 98%A, t 2.7min = 98% A, post time: 0.3 min, eluent A: acetonitrile, eluent B: 10 mM ammonium bicarbonate in water (pH = 9.5).

[0211] Preparative MP-LC method 1 Machine: Revelis™ Preparative MPLC; Column: Waters XSelect™ CSH C18 (145×25 mm, 10 μm); Flow rate: 40 mL / min; Column temperature: Room temperature; Eluent A: 10 mM ammonium bicarbonate in water pH=9.0; Eluent B: 99% acetonitrile + 1% 10 mM ammonium bicarbonate in water; Gradient: A t 0min = 5% B, t 1min = 5% B, t 2min = 10% B, t 17min =50%B,t 18min =100%B,t 23min =100%B A t 0min = 5% B, t 1min = 5% B, t 2min = 20% B, t 17min =60%B,t 18min =100%B,t 23min = 100% B; detection UV: 210, 235, 254 nm and ELSD.

[0212] Preparative MP-LC method 2 Model: Reveleris (trademark) fractionation MPLC; Column: Phenomenex LUNA C18(3) (150×25mm, 10μm); Flow rate: 40 mL / min; Column temperature: room temperature; Eluent A: 0.1% (v / v) formic acid in water, Eluent B: 0.1% (v / v) formic acid in acetonitrile; Gradient: A t 0min = 5%B, t 1min = 5%B, t 2min = 20%B, t 17min = 60%B, t 18min = 100%B, t 23min = 100%B B t 0min = 2%B, t 1min = 2%B, t 2min = 2%B, t 17min = 30%B, t 18min = 100%B, t 23min = 100%B C t 0min = 5%B, t 1min = 5%B, t 2min = 10%B, t 17min = 50%B, t 18min = 100%B, t 23min = 100%B D t 0min = 5%B, t 1min = 5%B, t 2min = 5%B, t 17min = 40%B, t 18min = 100%B, t 23min = 100%B; Detection UV: 210, 235, 254nm and ELSD.

[0213] Preparative LC-MS method 3 MS model: Agilent Technologies G6130B quadrupole; HPLC model: Agilent Technologies 1290 preparative LC; Column: Waters XSelect (trademark) CSH (C18, 150×19mm, 10μm); Flow rate: 25 mL / min; Column temperature: room temperature; Eluent A: 100% acetonitrile; Eluent B: 10 mM ammonium bicarbonate in water pH = 9.0; Gradient: A t0=20%A, t 2.5min = 20%A, t 11min = 60%A, t 13min = 100%A, t 17min =100%A B t0=5%A, t 2.5min = 5%A, t 11min = 40%A, t 13min = 100%A, t 17min = 100% A; detection: DAD (210 nm); detection: MSD (ESI pos / neg) mass range: 100-800; fraction collection based on DAD.

[0214] Preparative LC-MS method 4 MS model: Agilent Technologies G6130B quadrupole; HPLC model: Agilent Technologies 1290 preparative LC; Column: Waters XBridge Protein (C4, 150 x 19 mm, 10 μm); Flow rate: 25 mL / min; Column temperature: room temperature; Eluent A: 100% acetonitrile; Eluent B: 10 mM ammonium bicarbonate in water pH = 9.0; Gradient: A t0=2%A, t 2.5min = 2%A, t 11min = 30% A, t 13min = 100%A, t 17min =100%A B t0=10%A, t 2.5min = 10%A, t 11min = 50%A, t 13min = 100%A, t 17min =100%A C t0=5%A, t 2.5min = 5%A, t 11min = 40%A, t 13min = 100%A, t 17min = 100%A; Detection: DAD (210 nm); Detection: MSD (ESI pos / neg) Mass range: 100-800; Fraction collection based on DAD

[0215] Flash Chromatography Grace Reveleris X2™ C-815 Flash; Solvent delivery system: self-priming 3-piston pump, 4 independent channels for up to 4 solvents in one run, automatic line switching when solvent is empty; Maximum pump flow rate 250ml / min; Maximum pressure 50 bar (725psi); Detection: UV 200-400nm, combination of up to 4 UV signals and full UV range scan, ELSD; Column size: 4-330g for Luer device, 750g-max 3000g with optional holder.

[0216] UV-visible spectrophotometry Concentrations were determined using either a Thermo Nanodrop 2000 spectrophotometer or a Perkin Elmer Lambda 365 spectrophotometer and the following mass extinction coefficient (EC) values: An experimentally determined molar ε495=58,700 M-1 cm-1 and Rz280:495=0.428 were used for SAMSA-Fluorescein. M23D-SS-amide; mass EC265 = 259,210 M-1 cm-1 Ellman's Reagent (TNB); Molar EC412 = 14,150 M-1 cm-1 DMD-PMO; Molar EC265 = 318,050 135,027 M-1 cm-1 DMD-ASO; Molar EC265 = 310,000 252,512 M-1 cm-1 Pyridine 2-thione (PDT); Molar ε363 = 8,080 M-1 cm-1

[0217] TNBS assay Glycine standards (0, 2.5, 5, 10, 15 and 20 μg / ml) were freshly prepared using DPBS pH 7.5. TNBS assay reagent was prepared by combining TNBS (40 μl) with DPBS pH 7.5 (9.96 ml). 10% w / v SDS prepared using DI water. For the assay, 60 μl of each sample (singlecate) and standard (triplicate) were plated out. TNBS reagent (60 μl) was added to each well and incubated for 3 hours at 37° C. and 600 rpm on a plate shaker. Then, 50 μl of 10% SDS and 25 μl 1M HCl were added and the plate was analyzed at 340 nm. SO1861-hydrazone-NHS incorporation was determined by depletion of lysine concentration of the conjugate compared to the unmodified protein.

[0218] SEC The conjugates were analyzed by SEC using an Akta purifier 10 system and a Biosep SEC-s3000 column eluted with DPBS:IPA (85:15). Conjugate purity was determined by integration of the conjugate peak compared to impurities / aggregate forms.

[0219] SDS-PAGE and Western blotting Native proteins and conjugates were analyzed by SDS-PAGE under heat-denaturing non-reducing and reducing conditions against a protein ladder using 4-12% Bis-Tris gels and MOPS as running buffer (200 V, ca. 40 min). Samples were prepared at 0.5 mg / mL with LDS sample buffer and MOPS running buffer as diluents. For reduced samples, DTT was added to a final concentration of 50 mM. Samples were heat-treated at 90-95 °C for 2 min and 5 μg (10 μl) was added to each well. Protein ladder (10 μl) was loaded without pretreatment. An empty column was filled with 1× LDS sample buffer (10 μl). After the gel was run, it was washed three times with DI water (100 mL) with shaking (15 min, 200 rpm). Coomassie staining was performed by incubating the gel with PAGEBlue protein stain (30 mL) with shaking (60 min, 200 rpm). Excess staining solution was removed, rinsed twice with DI water (100 ml), and destained with DI water (100 ml) (60 min, 200 rpm). The resulting gels were imaged and processed using ImageJ (Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, Maryland, USA) and MyCurveFit (two-point correlation of protein ladders).

[0220] Western blotting From SDS-PAGE, the gel was transferred to a nitrocellulose membrane using freshly prepared transfer buffer using an X-Cell Blot module with the following setup ((-)BP-BP-FP-gel-NC-BP-BP-BP(+)) and conditions (30V, 60min). BP-blotting pad; FP-filter pad; NC-nitrocellulose membrane. Afterwards, the NC was washed twice with PBS-T (100ml) with shaking (5min, 200rpm), non-specific sites were blocked with blocking buffer (50ml) with shaking (30min, 200rpm), and then active sites were labeled with a combination of goat anti-human κ-HRP (1:2000) and goat anti-human IgG-HRP (1:2000) (50ml) diluted in blocking buffer with shaking (30min, 200rpm). The NCs were then washed once with PBS-T (100 ml) with shaking (5 min, 200 rpm) and the conjugated antibodies were detected with freshly prepared and freshly filtered CN / DAB substrate (25 ml). Colour development was observed visually and stopped after 2 min by washing the NCs with water and the resulting blots were photographed.

[0221] TBEU-PAGE Oligonucleotide conjugates and oligonucleotide standards were analyzed by TBE-urea PAGE under heat-denaturing, non-reducing conditions against oligo ladders using 15% TBE-urea gels and TBE as running buffer (180V, approximately 60 min). Samples were prepared at 0.5mg / ml and standards were prepared at 50-5μg / ml, respectively, all containing TBE-urea sample buffer and purified H2O as diluent. Samples and standards were heat treated at 70°C for 3 min and 10μl was added to each well to equal 5μg of protein and conjugate samples and 0.5 / 0.2 / 0.1 / 0.5μg (DMD-ASO) or 0.2 / 0.1 / 0.05μg (DMD-PMO) oligonucleotides per lane. Oligo ladders reconstituted at 0.1μg / band / ml in TE pH 7.5 (2μl) were loaded without pretreatment. After the gel was run, it was stained with freshly prepared ethidium bromide solution (1 μg / mL) with shaking (40 min, 200 rpm). The resulting gel was visualized by UV epi-illumination (254 nm) and imaged and processed using ImageJ (Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, Maryland, USA).

[0222] mCD71-M23D An aliquot of mCD71 (42.9 mg, 4.20 ml) was buffer exchanged into DPBS pH 7.5 and standardized to 2.5 mg / ml. To an aliquot of mCD71 (34.4 mg, 0.23 μmol, 2.53 mg / ml) was added an aliquot of freshly prepared SMCC solution (2.0 mg / ml, 3.53 molar equivalents, 0.81 μmol), the mixture was vortexed briefly, and then incubated at 20° C. for 60 min with roller mixing. After incubation, the reaction was quenched by adding an aliquot of freshly prepared glycine solution (2.0 mg / ml, approximately 20 molar equivalents, 4.05 μmol), the mixture was vortexed briefly, and then incubated at 20° C. for >15 min with end-over-end mixing. The conjugate was purified on a Superdex 200 column eluted with TBS pH 7.5 and analyzed by UV-Vis to give purified mCD71-SMCC (31.7 mg, yield: 96%, 0.942 mg / ml, SMCC to mCD71 ratio=2.1).

[0223] Separately, an aliquot of M23D-SS-amide (17.2 mg, 1.99 μmol, 10.0 mg / ml) reconstituted with TBS pH 7.5 was added to freshly prepared THPP solution (50 mg / ml, 10 molar equivalents, 19.9 μmol, 82.8 μl) and the mixture was vortexed briefly and then incubated with end-over-end mixing for 60 min at 37° C. After incubation, the oligo was purified through a PD10 Sephadex G25M column eluted with TBS pH 7.5 to give M23D-SH (14.8 mg, yield: 86%, thiol to M23D ratio=0.98).

[0224] To an aliquot of mCD71-SMCC (31.7 mg, 0.21 μmol, 0.942 mg / ml) was added an aliquot of M23D-SH (4.122 mg / ml, 4.0 molar equivalents, 0.85 μmol, 1.771 ml), the mixture was vortexed briefly and then incubated at 20° C. with end-over-end mixing. After approximately 72 hours, the conjugate mixture was concentrated and purified on a Superdex 200PG column eluted with DPBS pH 7.5 to obtain purified mCD71-M23D conjugate. An aliquot was analyzed by BCA colorimetric assay and the conjugate was assigned a new EC value, then concentrated and standardized to 2.5 mg / ml, 0.2 μm filtered, and then distributed into aliquots for characterization and product testing. The result was an mCD71-M23D conjugate (total yield=25.7 mg, 73%, M23D to mCD71 ratio=1.2).

[0225] mCD71-SO1861 For the generation of mCD71-SO1861, either SO1861-SC-maleimide or SO1861-EMCH were used to generate two different mCD71-SO1861 conjugates. The procedure is exemplarily described for the conjugation between mCD71 and SO1861-EMCH.

[0226] An aliquot of mCD71 (55.1 mg, 0.37 μmol, 8.10 mg / ml, 6.80 ml) was standardized to 5 mg / ml in DPBS pH 7.5, then 30 μl / ml (330 μl) of premixed concentrated Tris / Tris.HCl / EDTA containing concentrated Tris (127 mg / ml, 1.05 M), concentrated Tris.HCl (623 mg / ml, 3.95 M) and concentrated EDTA.2Na.2H2O (95 mg / ml, 0.26 M) in a 1:1:1 v / v combination was added to give a 50 mM TBS, 2.5 mM EDTA buffer pH approx. 7.5. To mCD71 (50 mg, 0.33 μmol, 5.044 mg / ml) was added an aliquot of freshly prepared TCEP solution (2.00 mg / ml, 2.74 molar equivalents, 0.912 μmol), the mixture was vortexed briefly, then incubated at 20° C. for 210 min with end-over-end mixing. After incubation (before adding SO1861-EMCH), mCD71-SH was dispensed for multiple conjugation and a 1.0 mg (0.201 ml) aliquot was removed and purified by gel filtration using Zeba spin desalting columns in TBS pH 7.5. This aliquot was characterized by UV-vis analysis and Ellman's assay (3.248 mg / ml, thiol to mCD71 ratio = 3.97). To an aliquot of mCD71-SH (42 mg, 0.28 μmol, 4.978 mg / ml) was added an aliquot of freshly prepared SO1861-EMCH solution (2.0 mg / ml, 8 molar equivalents, 2.24 μmol, 2.32 ml), the mixture was vortexed briefly, and then incubated for 120 min at 20° C. In addition to this conjugation reaction, two aliquots of desalted mCD71-SH (0.25 mg, 0.077 ml, 1.67 nmol) were reacted with NEM (8.00 equivalents, 134 nmol, 6.7 μl of a 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) as positive and negative controls, respectively, for 120 min at 20° C. After incubation, an approximately 0.4 mg aliquot of the mCD71-SO8161 mixture was removed, purified by gel filtration using Zeba spin desalting columns in TBS pH 7.5, and characterized in parallel with positive and negative controls by Ellman's assay to obtain SO1861 uptake.The reaction was quenched by adding an aliquot of freshly prepared NEM solution (5 molar equivalents, 1.40 μmol, 70.1 μl of a 2.5 mg / ml solution) to the bulk mCD71-SO1861 mixture. The conjugate was purified by Superdex 200 column eluted with DPBS pH 7.5 to obtain purified mCD71-SO1861 conjugate. The product was concentrated, then standardized to 2.5 mg / ml, 0.2 μm filtered, and then divided into aliquots for characterization, product testing, and further conjugation. The result was mCD71-SO1861 conjugate (total yield=37.3 mg, 89%, SO1861 to mCD71 ratio=3.8).

[0227] IGF-1-SO1861 IGF-1 (4 mg) was dissolved in DPBS pH 7.5 (1.60 ml). To IGF-1 (3.88 mg, 0.51 μmol, 4.821 mg / ml) was added an aliquot of freshly prepared SO1861-hydrazone-NHS solution (2.0 mg / ml, 5 molar equivalents, 2.53 μmol, 3.80 ml), the mixture was vortexed briefly, and then incubated at 20° C. for 60 min. After conjugation, a freshly prepared aliquot of glycine solution (25 equivalents, 13 μmol, 95 μl of a 10 mg / ml solution) was added, and the conjugate was then purified by elution with DPBS pH 7.5 on a Zeba 10 ml 7K MWCO spin desalting column to obtain purified IGF-1-SO1861 conjugate. Aliquots were analyzed by BCA colorimetric assay to confirm the new EC value, then concentrated by centrifugal filtration (3,000 g, 20° C., 10 min intervals), standardized to 2.5 mg / ml, 0.2 μm filtered, and distributed into aliquots for characterization and customer testing. The result was IGF-1-SO1861 conjugate (total yield=2.83 mg, 73%, SO1861 to IGF-1 ratio=2.7).

[0228] hCD71-DMD-ASO An aliquot of hCD71 (60 mg, 4.20 ml) was buffer exchanged into DPBS pH 7.5 and standardized to 2.5 mg / ml. To an aliquot of hCD71 (58 mg, 0.38 μmol, 2.53 mg / ml) was added an aliquot of freshly prepared PEG4-SPDP solution (10 mg / ml, 10 molar equivalents, 3.8 μmol), the mixture was vortexed briefly, and then incubated at 20° C. for 60 min with end-over-end mixing. After incubation, the reaction was quenched by adding an aliquot of freshly prepared glycine solution (2.0 mg / ml, 50 molar equivalents, 19 μmol), the mixture was vortexed briefly, and then incubated at 20° C. for >15 min with end-over-end mixing. The conjugate was purified by Zeba 40K spin desalting column eluted with TBS pH 7.5 and analyzed by UV-Vis to give purified hCD71-PEG4-SPDP (51.3 mg, yield: 88%, 0.95 mg / ml, PEG4-SPDP to hCD71 ratio = 4.5).

[0229] Separately, an aliquot of DMD-ASO-SH (14.4 mg, 2 μmol, 10.0 mg / ml) reconstituted with TBS pH 7.5 was added to freshly prepared THPP solution (50 mg / ml, 10 molar equivalents, 20 μmol, 82.8 μl) and the mixture was vortexed briefly and then incubated with end-over-end mixing for 60 min at 37° C. After incubation, the oligos were purified through a PD10 Sephadex G25M column eluted with TBS pH 7.5 to yield reduced DMD-ASO-SH (13.2 mg, yield: 92%, thiol to DMD-ASO ratio=0.91).

[0230] To an aliquot of hCD71-PEG4-SPDP (25 mg, 0.16 μmol, 0.95 mg / ml), an aliquot of DMD-ASO-SH (4 mg / ml, 4.0 molar equivalents, 0.65 μmol, 1.17 ml) was added, the mixture was vortexed briefly, and then incubated at 20°C with end-over-end mixing. The progress of the reaction was measured by the amount of PDT transferred. After 16 hours, the conjugate mixture was concentrated and purified on a Superdex 200PG column eluted with DPBS pH 7.5 to obtain the purified hCD71-DMD-ASO conjugate. An aliquot was analyzed by BCA colorimetric assay, the conjugate was assigned a new EC value, then concentrated and standardized to 2.5 mg / ml, 0.2 μm filtered, and then distributed into aliquots for characterization and product testing. The result was a hCD71-DMD-ASO conjugate (total yield=23.1 mg, 82%, DMD-ASO to hCD71 ratio=3.5). In a second synthesis, the hCD71-DMD-ASO conjugate was synthesized in the same manner as above, DMD-ASO to hCD71 ratio=2.1).

[0231] hCD71-DMD-PMO An aliquot of hCD71 (60 mg, 4.20 ml) was buffer exchanged into DPBS pH 7.5 and standardized to 2.5 mg / ml. To an aliquot of hCD71 (58 mg, 0.38 μmol, 2.53 mg / ml) was added an aliquot of freshly prepared PEG4-SPDP solution (10 mg / ml, 10 molar equivalents, 3.8 μmol), the mixture was vortexed briefly, and then incubated at 20° C. for 60 min with end-over-end mixing. After incubation, the reaction was quenched by adding an aliquot of freshly prepared glycine solution (2.0 mg / ml, 50 molar equivalents, 19 μmol), the mixture was vortexed briefly, and then incubated at 20° C. for >15 min with end-over-end mixing. The conjugate was purified by Zeba 40K spin desalting column eluted with TBS pH 7.5 and analyzed by UV-Vis to give purified hCD71-PEG4-SPDP (51.3 mg, yield: 88%, 0.95 mg / ml, PEG4-SPDP to hCD71 ratio = 4.1).

[0232] Separately, an aliquot of DMD-PMO-SS-amide reconstituted with TBS pH 7.5 (20.2 mg, 2 μmol, 10.0 mg / ml) was added to freshly prepared THPP solution (50 mg / ml, 10 molar equivalents, 20 μmol, 82.8 μl) and the mixture was vortexed briefly and then incubated with end-over-end mixing for 60 min at 37° C. After incubation, the oligo was purified on a PD10 Sephadex G25M column eluted with TBS pH 7.5 to give DMD-PMO-SH (16.4 mg, yield: 81%, thiol to DMD-PMO ratio=0.97).

[0233] To an aliquot of hCD71-PEG4-SPDP (25 mg, 0.16 μmol, 0.95 mg / ml), an aliquot of DMD-PMO-SH (4.1 mg / ml, 4.0 molar equivalents, 0.65 μmol, 1.59 ml) was added, the mixture was vortexed briefly, and then incubated at 20° C. with end-over-end mixing. The progress of the reaction was measured by the amount of PDT transferred. After approximately 16 hours, the conjugate mixture was concentrated and purified on a Superdex 200PG column eluted with DPBS pH 7.5 to obtain the purified hCD71-DMD-PMO conjugate. An aliquot was analyzed by BCA colorimetric assay, the conjugate was assigned a new EC value, then concentrated and standardized to 2.5 mg / ml, 0.2 μm filtered, and then distributed into aliquots for characterization and product testing. The result was a hCD71-DMD-PMO conjugate (total yield=28.2 mg, 92%, DMD-PMO to hCD71 ratio=3.9). In a second synthesis, a hCD71-DMD-PMO conjugate was synthesized in the same manner, and DMD-PMO to hCD71 ratio=3.2).

[0234] hCD71-SO1861 As-supplied hCD71 (55.1 mg, 0.37 μmol, 8.10 mg / ml, 6.80 ml) was buffer exchanged by elution into TBS pH 7.5 using Zeba spin desalting columns and standardized to 3 mg / ml. To hCD71 (50 mg, 0.33 μmol, 5.044 mg / ml) was added an aliquot of freshly prepared TCEP solution (2.00 mg / ml, 3 molar equivalents, 1 μmol), the mixture was vortexed briefly, and then incubated at 20°C for 210 min with end-over-end mixing. After incubation (before adding SO1861-SC-maleimide), a 1.0 mg (0.201 ml) aliquot of the hCD71 solution was removed and purified by gel filtration using Zeba spin desalting columns in TBS pH 7.5. The aliquot was characterized by UV-Vis analysis and Ellman's assay (2.23 mg / ml, thiol to hCD71 ratio = 4.35). To an aliquot of bulk hCD71-SH (42 mg, 0.28 μmol, 4.978 mg / ml) was added an aliquot of freshly prepared SO1861-SC-maleimide solution (2.0 mg / ml, 8 molar equivalents, 2.24 μmol, 2.32 ml), the mixture was vortexed briefly, and then incubated at 20°C for 120 min with end-over-end mixing. Besides this conjugation reaction, two desalted aliquots of hCD71-SH (0.25 mg, 0.077 ml, 1.67 nmol) were reacted with NEM (8.00 equivalents, 13.4 nmol, 6.7 μl of 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) as positive and negative controls, respectively, at 20°C for 120 min. After incubation, an approximately 0.4 mg aliquot of the hCD71-SO8161 mixture was removed and purified by gel filtration using Zeba spin desalting columns in TBS pH 7.5, and characterized in parallel with the positive and negative controls by Ellman's assay to obtain SO1861 incorporation. The reaction was quenched by adding an aliquot of freshly prepared NEM solution (5 molar equivalents, 1.4 μmol of 2.5 mg / ml solution) to the bulk hCD71-SO1861 mixture. The conjugate was purified through a Zeba 40K MWCO spin desalting column eluted with DPBS pH 7.5 to give purified hCD71-SO1861 conjugate.An aliquot of the product was analyzed by BCA colorimetric assay to confirm the new EC280 value. The product was then standardized to 2.5 mg / ml, 0.2 μm filtered, and then divided into aliquots for characterization, product testing, and further conjugation. The result was hCD71-SO1861 conjugate (total yield=37.8 mg, 89%, SO1861 to hCD71 ratio=4.2).

[0235] Cell culture (human) Immobilized human myoblasts from a non-DMD donor (KM155) and a DMD-affected donor (DM8036) were cultured in supplement-packed skeletal muscle cell growth medium (PromoCell, Germany) further supplemented with 15% fetal bovine serum (Gibco, United Kingdom) and 0.5% gentamicin (Sigma-Aldrich, USA) according to the manufacturer's instructions. For differentiation, cells were seeded on a 0.5% gelatin-coated surface and, at approximately 70-80% confluence, the growth medium was replaced with DMEM (Gibco) supplemented with 2% FBS (Gibco), 2% GlutaMAX, 1% glucose (Sigma-Aldrich) and 0.5% gentamicin. Treatments were initiated after at least 3 days, but not longer than 5 days of differentiation (based on the presence of differentiated myotubes).

[0236] Cell culture and in vitro experiments (mouse) Mouse myoblast cell line C2C12 was maintained in 10% FBS DMEM medium + Pen / Strep and seeded in maintenance medium (10% FBS in DMEM medium + Pen / Strep) at 240,000 cells per well (cpw) in 24-well plates or 40,000 cpw in 96-well plates (wp) and incubated at 37°C with 5% CCO2. 24 hours after seeding, cells were switched to differentiation medium (2% horse serum in DMEM) and incubated for 3 days, after which the medium was changed. After another 24 hours, the medium was changed again and compounds were added and incubated for 48 hours. Differentiation medium was then changed (no compounds) and cells were incubated for another 24 hours. After a total of 72 hours of treatment, cells at 24wp were harvested for exon skipping analysis and cell viability was assessed at 96wp.

[0237] Exon skipping analysis and quantification (human) RNA was isolated with TRIsure isolation reagent (Bioline) and chloroform extraction of RNA from the aqueous phase; isopropanol precipitation were performed as known to those skilled in the art. 1000 ng of total RNA was used for cDNA synthesis and diluted in an appropriate amount of RNase-free water to yield 8 μl RNA dilution. The priming premix contained 1 μl dNTP mix (10 mM each) and 1 μl specific reverse primer (for KM155, h53R 5'-CTCCGGTTCTGAAGGTGTTC-3' [SEQ ID NO: 5]; for DM8036, h55R 5'-ATCCTGTAGGACATTGGCAGTT-3' [SEQ ID NO: 6]). The mixture was heated at 70°C for 5 min and then cooled on ice for at least 1 min. A reaction mixture containing 0.5 μl rRNasin (Promega), 4.0 μl RT buffer, 1.0 μl Tetro RT (Bioline) and 4.5 μl RNase-free water was prepared and added to the chilled mixture to give a total volume of 20 μl for each reaction. RT-PCR was carried out at 42° C. for 60 min, then at 85° C. for 5 min and chilled on ice. For skip analysis, the nested PCR procedure was followed. For this purpose, in the first PCR, 3 μl cDNA was added to a mixture of 2.5 μl 10× SuperTaq PCR buffer, 0.5 μl dNTP mix (10 mM each), 0.125 μl Taq DNA polymerase TAQ-RO (5 U / μl; Roche) 16.875 μl RNase-free water and 1 μl (10 pmol / μl) of each primer flanking the targeted exon: for KM155, h48F 5′-AAAAGACCTTGGGCAGCTTG-3′ [SEQ ID NO: 7] and h53R 5′-CTCCGGTTCTGAAGGTGTTC-3′ [SEQ ID NO: 5]; for DM8036, h47F2 5′-TGAAACTGGAGGACCCGTG-3′ [SEQ ID NO: 8] and h54R 5′-CCAAGAGGCATTGATATTCTC-3′ [SEQ ID NO: 9] were used. The samples were subjected to a PCR run at 94°C for 5 min, followed by 25 cycles of 94°C for 40 s, 60°C for 40 s, 72°C for 180 s, followed by 72°C for 7 min.For the second PCR, 1.5 μl PCR1 sample was added to a mixture of 5 μl 10× SuperTaq PCR buffer, 1 μl dNTP mix (10 mM each), 0.25 μl Taq DNA polymerase TAQ-RO (5 U / μl; Roche), 38.25 μl RNase-free water, and 2 μl (10 pmol / μl) of each primer flanking the targeted exon: for KM155, h49F 5′-CCAGCCACTCAGCCAGTG-3′ [SEQ ID NO: 10] and h52R2 5′-TTCTTCCAACTGGGGACGC-3′ [SEQ ID NO: 11]; for DM8036, h47F 5′-CCCATAAGCCCAGAAGAGC-3′ [SEQ ID NO: 12] and h53R 5′-CTCCGGTTCTGAAGGTGTTC-3′ [SEQ ID NO: 13] were used. These samples were subjected to a PCR run at 94°C for 5 min, followed by 32 cycles of 94°C for 40 s, 60°C for 40 s, 72°C for 60 s, followed by 72°C for 7 min. Exon skipping levels were quantified using the Ultra Sensitivity NGS kit (Agilent) on a Femto Pulse system according to the manufacturer's instructions. Alternatively, specific PCR fragments were analyzed using a Bioanalyzer 2100 with a DNA1000 chip (Lab-on-a-Chip; Agilent) or separated on a 2% agarose gel run at 120 V. Gels were imaged and band intensities quantified using ImageJ. The expected non-skipped products are 408 bp (KM155) and 475 bp (DM8036) in size, respectively, and the skipped products are 175 bp (KM155) or 242 bp (DM8036).

[0238] Exon skipping analysis and quantification (mouse in vitro and in vivo) For analysis of skips from mouse cell lines, cells were harvested and RNA was isolated using 0.5ml TRIzol™ Reagent (Thermo Scientific) for each sample according to the manufacturer's instructions. For analysis of tissues, 30-50mg of frozen tissue was first minced and mRNA was isolated using TRIzol™ Reagent (Thermo Scientific) and TissueLyser LT (Qiagen) according to the manufacturer's instructions. For cDNA synthesis per sample, 0.5µg RNA in 5.0µl was added with 10.0µl ddH2O, 4µl 5x iScript™ Reaction Mix and 1.0µl iScript™ Reverse Transcriptase (BioRad) to yield a total volume of 20.0µl per reaction. RT-PCR was performed at 25°C for 5 min, 46°C for 60 min and 95°C for 2 min. For skip analysis, SapphireAmp™ Fast PCR Master Mix (TakaraBio) was used according to the manufacturer's instructions. For this purpose, 9.7 μl RNase-free water, 12.5 μl 2× Master Mix, 0.4 μl 10 μM FW primer 5′-ACCCAGTCTACCACCCTATC-3′ (SEQ ID NO: 14) and 0.4 μl 10 μM RV primer 5′-CTCTTTATCTTCTGCCCACCTT-3′ (SEQ ID NO: 15) were added to a PCR tube, mixed, and then 2 μl cDNA (50 ng) was added to give a total volume of 25 μl. The samples were subjected to a PCR run at 94° C. for 1 min, 35 cycles of 98° C. for 5 s, 55° C. for 5 s, 72° C. for 5 s, followed by 72° C. for 1 min. Samples were mixed with 2 μl 6× loading buffer, then 16 μl was loaded onto a 2% agarose gel and run at 80 V for 60-90 min. Gels were imaged and band intensities were quantified with a ChemiDoc™ XRS+ system and Image Lab™ software (BioRad). The expected non-skipped product is 788 bp in size, and the skipped product is 575 bp, respectively.

[0239] Cell viability (in vitro in mice) After treatment, cell viability was determined by CellTiter-Glo™ 2.0 assay performed according to the manufacturer's instructions (Promega). Luminescence signal was measured on a SpectraMax ID5 plate reader (Molecular Devices). For quantification, the cell viability of treated / untreated cells was calculated by subtracting the background signal of the "medium only" wells from all other wells and then dividing the background corrected signal of the treated wells by the background corrected signal of the untreated wells (×100).

[0240] In vivo studies Nine male CD-1 mice per group, aged 6-7 weeks at dosing, were given a single intravenous (iv) injection of the compound listed in Table A2 or vehicle. During the treatment period, animals were weighed periodically (the day before dosing and twice weekly after dosing) and any clinical findings were recorded. On days 4, 14 and 28, three mice per group were sacrificed by exsanguination and samples from various different tissues and organs were removed for analysis. Serum was prepared and analyzed for ALT (AU480, Beckman Coulter) and creatinine levels (colorimetric method, Beckman Coulter). Tissues were stored in RNALater and flash frozen until time of analysis. Dystrophin skip levels were determined in heart, diaphragm and gastrocnemius samples.

[0241] [Table 12]

[0242] Results (Examples 1 to 5) Example 1. DMD-PMO+SO1861 and DMD-ASO+SO1861 DMD-ASO (a 2'O-methyl-phosphorothioate antisense oligonucleotide with the same sequence and chemical modifications as drisapersen that induces exon 51 skipping of human dystrophin) and DMD-PMO (a phosphorodiamidate morpholino oligomer antisense oligonucleotide with the same sequence but without the 5'-modification as eteplirsen that induces exon 51 skipping of human dystrophin) were evaluated for exon skipping activity in differentiated human myotubes derived from a non-DMD (healthy) donor (KM155) in combination with the endosomal escape promoter SO1861-EMCH (4 μM). Surprisingly, this revealed a strong promotion of exon skipping of exon 51 after 72 h only in combination with SO1861 (Table A3): exposure to 20 μM DMD-ASO alone showed no skipping activity, whereas 0.08 μM DMD-ASO+SO1861-EMCH already revealed 40% exon skipping, pointing to a greater than 250-fold improvement (Figure 1A). Similarly, exposure to 20 μM DMD-PMO resulted in 4% skipping, whereas 1.25 μM DMD-ASO+SO1861-EMCH already achieved 5% skipping and 20 μM achieved 34%, pointing to a 16-fold increase in potency for the non-targeted oligo in conjunction with the non-targeted SO1861-EMCH (Figure 1B).

[0243] In conclusion, coadministration of SO1861-EMCH potently promoted on-target delivery of DMD-ASO and DMD-PMO and induced significant exon 51 skipping at exposure concentrations 1-2 orders of magnitude lower compared to conditions in which SO1861-EMCH was absent.

[0244] [Table 13]

[0245] Example 2. hCD71-DMD-ASO + SO1861-EMCH or hCD71-DMD-PMO + SO1861-EMCH or mCD71-M23D + SO1861-EMCH DMD-ASO-SH and DMD-PMO-SS-amide were conjugated to PEG4-SPDP-modified human anti-CD71 monoclonal antibody (hCD71-PEG4-SPDP, FIG. 2A) to generate hCD71-DMD-ASO (DAR2.1) and hCD71-DMD-PMO (DAR3.2), as shown in FIG. 2B and FIG. 2C, respectively. The resulting compounds were tested for promoting cytoplasmic DMD oligo delivery and promoting dystrophin exon 51 skipping in differentiated human myotubes from non-DMD donors (KM155) and DMD-affected donors (DM8036) either without or in combination with 4 μM SO1861-EMCH. This revealed that in combination with 4 μM SO1861-EMCH, it potently promoted exon skipping of exon 51 at significantly lower concentrations compared to hCD71-DMD-ASO and hCD71-DMD-PMO alone, respectively, after 72 h of treatment (Tables A4 and A5): hCD71-DMD-ASO induced a low skipping rate in KM155 at 2181 nM (3%) (Figure 3A, left panel), which was an improvement compared to the non-targeted DMD-ASO (Table A3), whereas hCD71-DMD-ASO+SO1861 further increased skipping to 18-24% already at 18.2-109 nM in KM155 (Figure 3A, right panel). Similarly, hCD71-DMD-PMO resulted in no skipping (0%) at 2022 nM in KM155 (Figure 3B, left panel), whereas exon skipping was visible at concentrations of 2.8–16.9 nM hCD71-DMD-PMO when SO1861-EMCH was added (Figure 3B, right panel). More importantly, in differentiated myotubes from a DMD-affected donor (DM8036), only a 17% exon 51 skipping rate was observed at 363 nM hCD71-DMD-ASO (Figure 4A, left panel), whereas, surprisingly, a comparable skipping rate (15%) already occurred at a 720-fold lower concentration of 0.50 nM hCD71-DMD-ASO + 4 μM SO1861-EMCH. The skipping efficiency was significantly increased up to 64–68% with hCD71-DMD-ASO at exposure concentrations of only 18.2–109 nM (Figure 4A, right panel).Similarly, in DM8036 myotubes, hCD71-DMD-PMO alone reached a skip rate of 21% at 337 nM (Figure 4B, left panel), whereas already at exposure concentrations of 0.08–0.47 nM hCD71-DMD-PMO+4 μM SO1861-EMCH clearly produced measurable skipping (7–13%), which increased to a maximum of 33% at 101 nM (Figure 4B, right panel), realizing an improvement of 1–2 orders of magnitude.

[0246] Taken together, this demonstrates that co-administration of the targeted oligonucleotides hCD71-DMD-ASO and hCD71-DMD-PMO with SO1861-EMCH significantly improves on-target cytoplasmic delivery, specifically in relevant cell systems such as differentiated myotubes from DMD-affected donors.

[0247] [Table 14]

[0248] [Table 15]

[0249] Next, we conjugated M23D-SS-amide (a phosphorodiamidate morpholino oligomer antisense oligonucleotide that induces exon 23 skipping of mouse dystrophin) to an anti-CD71 monoclonal antibody targeting mouse CD71 modified with an SMCC linker (mCD71-SMCC, Figure 2D) to generate mCD71-M23D(DAR1.2) (Figure 2E). Co-administration of mCD71-M23D + 8 μM SO1861-SC-Mal was tested in differentiated C2C12 myotubes, revealing a strong promotion of exon skipping by mCD71-M23D at at least one order of magnitude lower concentrations in combination with SO1861-SC-Mal (clear band up to 27 nM, Figure 5, right panel), whereas mCD71-M23D alone showed no activity at any tested concentration up to 433 nM (Figure 5, left panel).

[0250] This indicates that co-administration of the SO1861 compound is a broadly applicable cross-species useful method for increasing the potency of targeted PMOs with a variety of different targeting sequences (various different exons, various different sequences).

[0251] Example 3. mCD71-SO1861+M23D or IGF-1-SO1861+M23D Two different conjugates, mCD71-SO1861(DAR3.8), were generated by conjugating either SO1861-EMCH or SO1861-SC-maleimide (Figure 6A) to a TCEP-treated anti-CD71 monoclonal antibody targeting mouse CD71 (mCD71-SH, Figure 6B) (Figure 6C). Each of these conjugates was co-administered with a constant concentration of 500 nM M23D in the concentration range of 0-720 nM into C2C12 differentiated myotubes. This revealed that the combinations mCD71-SO1861 (SO1861-EMCH) + 500 nM M23D (12% skip rate at 710 nM and 8% skip rate at 142 nM) (Figure 7A, left panel) and mCD71-SO1861 (SO1861-SC-Mal) + 500 nM M23D (19% skip rate at 710 nM and 10% skip rate at 142 nM) potently promoted M23D efficacy in exon 23 skipping to the same extent, whereas 500 nM M23D alone (0 nM conjugate) only showed a 5% skip rate (Figure 7A, right panel). The data indicate that the addition of the mCD71-SO1861-Mal conjugate significantly increases the on-target effect mediated by M23D, i.e. exon skipping, independent of the conjugation and linker.

[0252] Next, IGF-1 ligand was conjugated to SO1861-hydrazone-NHS via lysine to generate IGF-1-SO1861 (DAR2.7), as shown in Figure 6D. IGF-1-SO1861 was tested in C2C12 differentiated myotubes at concentrations ranging from 0 to 3333 nM and co-administered with 500 nM M23D. This revealed that the combination of IGF-1-SO1861 + 500 nM M23D promoted M23D cytoplasmic delivery, i.e., dystrophin exon 23 skipping (19% skip rate at 3333 nM, 11% skip rate at 667 nM, and 8% skip rate at 133 nM) (Figure 7B).

[0253] Taken together, these data indicate that antibody and non-antibody ligand conjugated, i.e. targeted, SO1861 in a co-administration setting leads to a significant efficacy enhancement, i.e. on-target delivery of the PMO payload in muscle cells.

[0254] Example 4. hCD71-DMD-ASO-SO1861 or hCD71-DMD-PMO-SO1861 SO1861-SC-Mal was conjugated to TCEP-treated anti-CD71 monoclonal antibody targeting human CD71 (hCD71-SH) via cysteine, and PEG4-SPDP was conjugated to lysine to generate hCD71-SO1861-PEG4-SPDP. DMD-ASO-SH or DMD-PMO-SH (as previously described in Figures 2B and 2C) were also conjugated to anti-CD71 monoclonal antibody targeting human CD71 (hCD71) to generate hCD71-DMD-ASO (DAR2.2) and hCD71-DMD-PMO (DAR3.1). The conjugates were tested in differentiated human myotubes from non-DMD donors (KM155) and DMD-affected donors (DM8036), including controls. As expected, these treatments revealed no or very little exon skipping in KM155 myotubes at concentrations ranging from 0 to 2.4% 72 hours after treatment, ranging from 0.084 nM to 651 nM for hCD71-DMD-ASO (Figure 8A, left panel; Table A6) and 0.078 nM to 610 nM hCD71-DMD-PMO (Figure 8B, left panel; Table A6). However, when 4 μM SO1861-SC-Mal was co-administered with either hCD71-DMD-ASO (Figure 8A, right panel; Table A7) or hCD71-DMD-PMO (Figure 8B, right panel; Table A7), a strong promotion of exon skipping was observed in differentiated human myotubes: already at about 0.5 nM, 0-5% and 0-4% exon skipping was observed for hCD71-DMD-ASO and hCD71-DMD-PMO, respectively, which increased to 29-40% for 109 nM hCD71-DMD-ASO and 4-5% for 102 nM hCD71-DMD-PMO, respectively, constituting an improvement of several orders of magnitude in potency compared to conditions without SO1861-SC-Mal.

[0255] More relevantly, in differentiated myotubes from DMD-affected donors, treatment with the targeted conjugates hCD71-DMD-ASO and hCD71-DMD-PMO also revealed skip rates up to 7-11% at 72 h post-treatment with 651 nM hCD71-DMD-ASO (Figure 8A, left panel; Table A7) and 610 nM hCD71-DMD-PMO (Figure 8B, left panel; Table A7), respectively. Strikingly, however, coadministration of either hCD71-DMD-ASO or hCD71-DMD-PMO with 4 μM SO1861-SC-Mal potently promoted exon skipping in differentiated human myotubes from DMD-affected donors: 92-96% skipping rates were observed with 109 nM hCD71-DMD-ASO+SO1861-SC-Mal (Figure 9A, right panel) and 37-57% skipping rates with 102 nM hCD71-DMD-PMO+SO1861-SC-Mal (Figure 9B, right panel). The effect was still measurable down to at least 0.50 nM hCD71-DMD-ASO, and skipping was observed even at 0.013 nM for hCD71-DMD-PMO+SO1861-SC-Mal.

[0256] [Table 16]

[0257] [Table 17]

[0258] Example 5. mCD71-M23D (in vivo efficacy) CD-1 male mice received a single injection of mCD71-M23D(DAR1.2) (Figure 2). In addition, a vehicle control group was included.

[0259] As Figure 10 (A-C) shows, animals receiving vehicle (group 1) or mCD71-M23D (2.80 mg / kg PMO; group 2) did not demonstrate exon 23 skipping in any of the tissues examined or at any time point (days 4, 14, or 28).

[0260] The data indicate that the conjugate in the absence of targeted SO1861 does not achieve any skipping at the doses tested.

[0261] In vivo tolerance of mCD71-M23D in CD-1 mice Conjugate mCD71-M23D (Figure 2) was administered as detailed in Table A2. During the course of the study, one animal (out of the remaining six in Group 2) treated with mCD71-M23D was found dead on day 11, and biomarker data were missing for one of three mice on day 28. At the time of sacrifice on day 14, two mice (out of three) administered mCD71-M23D in Group 2 exhibited renal abnormalities and elevated serum creatinine (Figure 11A). No significant or sustained changes in the renal biomarker ALT were evident (Figure 11B). Of note, ALT levels were comparable to vehicle controls (Group 1) on days 14 and 28 and beyond.

[0262] Methods (as carried out in Examples 6-7) SO1861 SO1861 is derived from Saponaria officinalis L (Extrasynthese, France) and was coupled to the respective handles according to methods known in the art by Symeres (NL).

[0263] Synthesis of DBCO-(M23D)2 The synthesis of DBCO-(M23D)2 was carried out by Symeres (NL).

[0264] Conjugation of SO1861 with antibodies Custom conjugate production of mCD63-SO1861 was carried out by Abzena (UK).

[0265] Conjugation of DBCO-(M23D)2 and antibodies Custom conjugate production of mCD71-M23D and mCD63-M23D was carried out by Abzena (UK).

[0266] Analysis and preparation methods Analysis method SEC method 1 Reaction analyzer: Analytical SEC instrument DIONEX Ultimate 3000 UPLC (DIONEX 6); Column: Waters Protein BEH SEC column, 200 Å, 1.7 μm, 4.6 mm × 150 mm; Mobile phase: Buffer A (0.2 M potassium phosphate buffer, pH 6.8, 0.2 M KCl, 15% isopropanol in ultrapure water); Method: Isocratic Buffer A for 10 min; Flow rate: 0.35 ml / min; Run time: 10 min; Detection UV: 214 nm, 248 nm, 260 nm and 280 nm; Column oven: 30 °C; Autosampler: Ambient; Injection volume: 10 μL; Sample preparation: The final sample was analyzed by diluting the sample to 1.0 mg / ml with DPBS.

[0267] LC-MS method 1 Mass spectrometry (LC-MS) instrument: XEVO-G2XS TOF; column: Agilent Poroshell 300SB-C3 guard, 5 μm RP column; mobile phase: buffer A (water, 0.1% formic acid) buffer B (MeCN, 0.1% formic acid); injection method: 0–2.0 min, 10% B 2.0–7.0 min, 10–80% B 7.0–8.0 min, 100% B 8.0–8.01 min, 10% B 8.01-10.0 min, 10% B; MS method: Capillary voltage: 3.0 kV, Cone voltage: 120 V, Cone temperature: 140 °C, Desolvation temperature: 450 °C,: Flow rate: 0.4 ml / min: Run time: 10 min: Detection: TIC; Column oven: 60 °C; Autosampler: ambient; Injection volume: 10 µL; Sample preparation: a. Final samples, intermediates and reaction mixtures were analyzed by diluting the samples to 0.05 mg / ml with DPBS.

[0268] LC-MS method 2 Instrument: Agilent 1260 Infinity II, 1260 G7112B binary pump, 1260 G7167A multisampler, 1290MCT G7116B column compartment 1260 G7115A DAD (210, 220 and 210-320 nm), PDA (210-320 nm), G6130B MSD (ESI pos / neg) mass range 90-1500, Column: XSelect CSH C18 (30×2.1 mm 3.5 μm) Flow rate: 1 ml / min, Column temperature: 40°C, Eluent A: 0.1% formic acid in water, Eluent B: 0.1% formic acid in acetonitrile, Gradient: t 0min = 5% B, t 1.6min =98%B,t 3min = 98% B, post run: 1.3 min.

[0269] LC-MS method 3 Instrument: Agilent 1260 Infinity II, 1260 G7112B binary pump, 1260 G7167A multisampler, 1260MCT G7116A column compartment 1260 G7115A DAD (210, 220 and 210-320 nm), PDA (210-320 nm), G6130B MSD (ESI pos / neg) mass range 90-1500, Column: XSelect CSH C18 (30 x 2.1 mm 3.5 μm), Flow rate: 1 ml / min, Column temperature: 25 °C, Eluent A: 10 mM ammonium bicarbonate in water (pH 9.5); Eluent B: acetonitrile, Gradient: t 0min = 5% B, t 1.6min =98%B,t 3min = 98% B, post run: 1.2 min.

[0270] LC-MS method 4 Instrument: Agilent 1260 Infinity II, 1260 G7112B binary pump, 1260 G7167A multisampler, 1290MCT G7116B column compartment 1260 G7115A DAD (210-320 nm, 210 and 220 nm), PDA (210-320 nm), G6130B MSD (ESI pos / neg) mass range 90-1500; Column: Waters C4 BEH (50×2.1 mm 3.5 μm); Flow rate: 1 ml / min; Column temperature: 40°C; Eluent A: 0.1% formic acid in water; Eluent B: 0.1% formic acid in acetonitrile; Gradient: A t 0min = 5% B, t 2.5min =98%B,t 4min =98%B B t 0min = 5% B, t 0.05min = 5% B, t 5min =98%B,t 6min =98%B Post run: 1.5 minutes.

[0271] Preparative separation method Preparative MP-LC method 1 Machine: Buchi Reveleris™ Preparative MPLC; Column: Waters XSelect™ CSH C18 (145×25 mm, 10 μm); Flow rate: 40 ml / min; Column temperature: room temperature; Eluent A: 10 mM ammonium bicarbonate in water pH=9.0; Eluent B: 99% acetonitrile in water + 1% 10 mM ammonium bicarbonate; Gradient: t 0min =50%B,t 4min =50%B,t 16min =100%B,t 21min = 100% B; detection UV: 220, 254, 270 nm; UV-based fraction collection.

[0272] Preparative MP-LC method 2 Machine: Buchi Reveleris™ Preparative MPLC; Column: Phenomenex LUNA C18(3) (150×25 mm, 10 μm); Flow rate: 40 ml / min; Column temperature: room temperature; Eluent A: 0.1% (v / v) formic acid in water, Eluent B: 0.1% (v / v) formic acid in acetonitrile; Gradient: t 0min =5%B,t 1min =5%B,t 2min =20%B,t 17min =60%B,t 18min =100%B,t 23min = 100% B; detection UV: 220, 240, 280 nm; UV-based fraction collection.

[0273] Preparative LC-MS method MS model: Agilent Technologies G6120AA quadrupole; HPLC model: Agilent Technologies 1200 preparative LC; Column: Waters XBridge Protein (C4, 150×19 mm, 10μ); Flow rate: 25 ml / min; Column temperature: room temperature; Eluent A: 0.1% formic acid in water; Eluent B: 100% acetonitrile; Gradient: t0=10%A,t 2.5min =10%A,t 11min =50%A,t 13min =100%A,t 17min = 100% A; detection: DAD (220-320 nm); detection: MSD (ESI pos / neg) mass range: 100-1000; fraction collection based on DAD.

[0274] Flash Chromatography Grace Reveleris X2™ C-815 Flash; Solvent delivery system: self-priming 3-piston pump, 4 independent channels for up to 4 solvents in one run, automatic line switching when solvent is empty; Maximum pump flow rate 250ml / min; Maximum pressure 50 bar (725psi); Detection: UV 200-400nm, combination of up to 4 UV signals and full UV range scan, ELSD; Column size: 4-330g for Luer device, 750g-max 3000g with optional holder.

[0275] DBCO-(M23D)2 synthesis Intermediate 1 (N-[2-(4-{2-azatricyclo[10.4.0.0 4 , 9 ]Hexadeca-1(12),4(9),5,7,13,15-hexaen-10-yn-2-yl}-N-(2-{[(tert-butoxy)carbonyl]amino}ethyl)-4-oxobutanamido)ethyl]carbamate tert-butyl To a solution of DBCO acid (50.0 mg, 0.164 mmol) in DMF (1.00 ml) was added DIPEA (34.0 μL, 0.195 mmol) and HATU (62.3 mg, 0.164 mmol) and the mixture was stirred for 15 min. Then, di-tert-butyl (azanediylbis(ethane-2,1-diyl))dicarbamate (59.6 mg, 0.197 mmol) was added and the reaction mixture was stirred at room temperature. After 30 min, the reaction mixture was added to water (10.0 ml). The resulting thick suspension was centrifuged (5000 RPM, 3 min) to give a clear solution with a solid on top. The solution was removed by pipette and the solid was dissolved in acetonitrile (10.0 ml). The resulting solution was concentrated in vacuo. The residue was purified by flash chromatography (DCM-methanol / DCM (1 / 9, v / v) gradient 100:0 increasing to 0:50) to give the title product (90.0 mg, 93%) as a colorless solidified oil. 100% purity based on LC-MS. LRMS(m / z):591[M+H] 1+ LC-MS rt(min): 2.12 1

[0276] Intermediate 2 (N,N-bis(2-azaniumylethyl)-4-{2-azatricyclo[10.4.0.0 4 , 9 ]Hexadeca-1(12),4(9),5,7,13,-15-hexaen-10-yn-2-yl}-4-oxobutanamide ditrifluoroacetate N-[2-(4-{2-azatricyclo[10.4.0.0 4 , 9tert-Butyl]hexadeca-1(12),4(9),5,7,13,15-hexaen-10-yn-2-yl}-N-(2-{[(tert-butoxy)carbonyl]amino}ethyl)-4-oxobutanamido)ethyl]carbamate (90.0 mg, 0.152 mol) was dissolved in DCM (2.00 ml) and cooled to 0° C. Then TFA (2.00 ml, 26.0 mmol) was added and the reaction mixture was stirred at 0° C. for 40 min. The reaction mixture was allowed to reach room temperature over 10 min. Next, the reaction mixture was cooled to 0° C. and diluted with toluene (5 ml). The resulting solution was concentrated in vacuo and co-evaporated with DCM (2×5 ml) to give the crude title product as a slightly pinkish oil, which was used directly in the next step. Purity 88% based on LC-MS. LRMS(m / z):196[M+2] 2+ ,391[M+1] 1+ LC-MS rt(min): 1.17 (LC-MS method 2)

[0277] Intermediate 3 N-[2-(4-{2-azatricyclo[10.4.0.0 4 , 9 ]Hexadeca-1(12),4(9),5,-7,13,15-hexaen-10-yn-2-yl}-N-[2-({[(1S,4E)-cyclooct-4-en-1-yloxy]carbonyl}amino)ethyl]-4-oxobutanamido)ethyl]carbamic acid (1S,4E)-cyclooct-4-en-1-yl Crude N,N-bis(2-azaniumylethyl)-4-{2-azatricyclo[10.4.0.0 4 , 9To a solution of ]hexadeca-1(12),4(9),-5,7,13,15-hexaen-10-yn-2-yl}-4-oxobutanamide ditrifluoroacetate (0.152 mmol) was added TCO4-NHS carbonate (102 mg, 0.380 mmol) and the mixture was stirred at room temperature. After 30 min, the reaction mixture was subjected to preparative MP-LC (preparative MP-LC method 1). The fractions corresponding to the product were combined and immediately pooled, frozen and lyophilized overnight to give the title compound (90.0 mg, 85%) as a slightly brownish oil. Purity 99% based on LC-MS. LRMS(m / z):696[M+1] 1+ LC-MS rt(min): 2.35 (LC-MS method 3)

[0278] Intermediate 4 2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]-N-[2-(pyridin-2-yldisulfanyl)ethyl]acetamide To a solution of methyltetrazine-NHS ester (50.0 mg, 0.153 mmol) in DMF (800 μL) was added 2-(2-pyridinyldisulfanyl)ethanamine hydrochloride (40.8 mg, 0.183 mmol) and DIPEA (53.0 μL, 0.306 mmol). The reaction mixture was stirred at room temperature. After 2 h, the reaction mixture was subjected to preparative MP-LC (preparative MP-LC method 2). The fractions corresponding to the product were combined and immediately pooled, frozen and lyophilized overnight to give the title compound (53.6 mg, 88%) as a pink solid. 100% purity based on LC-MS. LRMS(m / z):399[M+1] 1+ LC-MS rt(min): 1.87 (LC-MS method 3)

[0279] Intermediate 5: M23D-mTz To a solution of M23D-DSA (294 mg, 34.0 μmol) in water (15.0 ml) was added DTT (100 mg, 648 μmol). The reaction mixture was stirred at room temperature. After 2 hours, the reaction mixture was divided into 6 equal fractions and poured into acetonitrile (6×45 ml). The resulting suspension was shaken and allowed to stand for 30 minutes. The suspension was then centrifuged (7830 RPM, 20 min). The solution was decanted and the residue was treated with acetonitrile (20 ml per vial). The resulting suspension was centrifuged (7830 RPM, 3 min). The residue was dissolved in water (total volume 10.0 ml) and the solutions were combined. A solution of 2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]-N-[2-(pyridin-2-yldisulfanyl)ethyl]acetamide (54.2 mg, 136 μmol) in acetonitrile (4.00 ml) was then added. The reaction mixture was stirred at room temperature. After 2 hours, the reaction mixture was aliquoted and poured into acetonitrile (6×45 ml). The resulting suspension was shaken and centrifuged (7830 RPM, 3 min). The solution was decanted and the residue was dissolved in water / acetonitrile (6×2 ml, 1 / 1, v / v). The resulting solution was poured into acetonitrile (6×20 ml). The suspension was then centrifuged (7830 RPM, 3 min). The solution was decanted and the residue was dissolved in water / acetonitrile (total volume 15 ml, 1 / 1, v / v) and lyophilized overnight to give the title compound (340 mg, quantitative yield) as a pink solid, 99% purity based on LC-MS. LRMS(m / z):1468[M+6] 6+ , 1259[M+7] 7+ , 1102[M+8] 8+ , 979[M+9] 9+ , 881[M+10] 10+ LC-MS rt(min): 1.75 (LC-MS method 4A)

[0280] DBCO-(M23D)2 A solution of M23D-mTz (16.4 mg, 1.86 μmol) in water (1.00 ml) and acetonitrile (0.400 ml) was diluted with N-[2-(4-{2-azatricyclo[10.4.0.0 4 , 9 A stock solution of (1S,4E)-cyclooct-4-en-1-yl]hexadeca-1(12),4(9),5,7,13,15-hexaen-10-yn-2-yl}-N-[2-({[(1S,4E)-cyclooct-4-en-1-yloxy]carbonyl}amino)ethyl]-4-oxobutanamido)ethyl]carbamate (0.67 mg, 0.964 μmol) was added and the reaction mixture was stirred at room temperature. After each addition, the LC-MS 3A The progress of the reaction was monitored by RT. In this way, complete conversion to the title product was observed. Stock solutions were added as follows; 400 μl, 10 min-100 μl, 10 min-100 μl, 10 min-25 μl, 10 min-25 μl, 10 min-25 μl. The reaction mixture was then subjected to preparative LC-MS. Fractions corresponding to the product were combined and immediately pooled, frozen and lyophilized overnight to give the title compound (10.5 mg, 62%) as a white solid. 100% purity (broad peak) based on LC-MS. LRMS(m / z):1142[M+16] 16+ , 1075[M+17] 17+ , 1015[M+18] 18+ , containing multiple m / z values ​​of known fragments LC-MS rt(min): 2.84 (LC-MS method 4B)

[0281] Conjugation of mouse anti-CD63 (mCD63) mAb with DBCO-(M23D)2 1. Preparation of mCD63 mCD63 was buffer exchanged into TBS using Vivaspin (50 kDa MWCO) to a final concentration of 10.0 mg / ml.

[0282] 2. Carbohydrate modification on the antibody Fc domain An immobilized GlycINATOR™ column (GlyCLICK™ Azide Activation Kit, from Genovis) was equilibrated and prepared according to the vendor's instructions. The sample was then loaded onto the column, the medium was resuspended, and the mixture was incubated and mixed at room temperature for 1 hour. The column was then centrifuged and the sample was eluted.

[0283] UDP-GalNAz (from GlyCLICK™ Azide Activation Kit, Genovis) was reconstituted in TBS according to the supplier's instructions and transferred to the pooled eluate together with GalT (from GlyCLICK™ Azide Activation Kit, Genovis). This mixture was incubated overnight at 30° C. in the dark. The reaction was then loaded onto a preconditioned desalting column (according to the supplier's instructions) and centrifuged to collect the flow-through containing the azide-modified mAb. This was stored at 4° C. in the dark until further use in conjugation.

[0284] 3. Conjugation with DBCO-(M23D)2 mCD63 was buffer exchanged into DPBS using Vivaspin (50 kDa MWCO) to a final concentration of 10.0 mg / ml DBCO-(M23D)2 (5.0 equiv., 1 mM in DPBS, pH 7.4) and added to the mAb solution. The reaction mixture was incubated at 37° C. for 24 hours and then directly purified by preparative SEC (HiLoad 26 / 600 Superdex 200 pg, DPBS). The conjugate was characterized by SEC-UV (DAR determination). The pooled fractions were concentrated using Vivaspin as above to a final concentration of >10.0 mg / ml, sterile filtered through a 0.22 μm filter unit, and stored at 4° C. until further use.

[0285] [Table 18]

[0286] Conjugation of mouse anti-CD63 (mCD63) mAb with SO1861 1. Preparation of mCD63 mCD63 was buffer exchanged into DPBS+5 mM EDTA, pH 7.4 using Vivaspin (50 kDa MWCO) to a final concentration of 8.0 mg / ml.

[0287] 2. Conjugation of mCD63 mCD63 was preincubated at 37° C. for about 15 min, followed by the addition of TCEP (3.8 eq.). The reaction mixture was diluted to 6.5 mg / ml and then incubated at 37° C. for 1 h. The reduction of mCD63 was monitored by modified LC-MS. The reaction was equilibrated at 22° C. and SO1861 (8.0 eq.) was added. The reaction was monitored by modified LC-MS, and when the reaction was complete, it was quenched with DTT (100 eq.).

[0288] 3. Purification of mCD63 Conjugates The reaction mixture was directly purified by P2 desalting column using DPBS. The conjugate was characterized by SEC and modified LC-MS (DAR determination, final extinction coefficient), then concentrated to >10 mg / ml using Vivaspin (50 kDa MWCO), sterile filtered through 0.22 μm filter unit and stored at +4° C. until further use.

[0289] [Table 19]

[0290] Conjugation of mouse anti-CD71 (mCD71) mAb with DBCO-(M23D)2 1. Preparation of mCD71 mCD71 was buffer exchanged into TBS using Vivaspin (50 kDa MWCO) to a final concentration of 10.0 mg / ml.

[0291] 2. Carbohydrate modification on the antibody Fc domain An immobilized GlycINATOR™ column (GlyCLICK™ Azide Activation Kit, from Genovis) was equilibrated and prepared according to the vendor's instructions. The sample was then loaded onto the column, the medium was resuspended, and the mixture was incubated and mixed at room temperature for 1 hour. The column was then centrifuged and the sample was eluted.

[0292] UDP-GalNAz (GlyCLICK™ Azide Activation Kit, from Genovis) was reconstituted in TBS according to the supplier's instructions and transferred to the pooled eluate together with GalT (GlyCLICK™ Azide Activation Kit, from Genovis). The mixture was incubated overnight at 30° C. in the dark. The reaction was then loaded onto a preconditioned desalting column (following the supplier's instructions) and centrifuged to collect the flow-through containing the azide-modified mCD71. It was stored at 4° C. in the dark until further use in conjugation.

[0293] 3. Conjugation with DBCO-(M23D)2 mCD71 was buffer exchanged into DPBS using Vivaspin (50 kDa MWCO) to a final concentration of 10.0 mg / ml. DBCO-(M23D)2 (5.0 eq., 1 mM in DPBS, pH 7.4) was added to the mCD71 solution and the reaction mixture was incubated at 37°C for 24 hours and then directly purified by preparative SEC (HiLoad 26 / 600 Superdex 200 pg, DPBS). The conjugate was characterized by SEC-UV (DAR determination). The pooled fractions were concentrated using Vivaspin as above to a final concentration of >10.0 mg / ml, sterile filtered through 0.22 μm filter units and stored at 4°C until further use.

[0294] [Table 20]

[0295] Cell culture and in vitro experiments (mouse) Mouse myoblast cell line C2C12 was maintained in 10% FBS DMEM medium + Pen / Strep and plated in maintenance medium (10% FBS in DMEM medium + Pen / Strep) at 240,000 cells per well (cpw) in 24-well plates or 40,000 cpw in 96-well plates (wp) and incubated at 37°C with 5% CO2. 24 hours after plating, cells were switched to differentiation medium (2% horse serum in DMEM) and incubated for 3 days before changing the medium. After another 24 hours, the medium was changed again and compounds were added and incubated for 48 hours. After a total of 48 hours of treatment, cells at 24wp were harvested for exon skipping analysis and cell viability was assessed at 96wp.

[0296] Exon skipping analysis and quantification (mouse in vitro) The experiments were carried out as described above in "Methods" (as carried out in Examples 1-5).

[0297] Cell viability (mouse in vitro) The experiments were carried out as described above in "Methods" (as carried out in Examples 1-5).

[0298] Results (Examples 6 to 7) Example 6. mCD71-M23D+SO1861-SC-Mal or mCD63-M23D+SO1861-SC-Mal (in vitro) DBCO-(M23D)2, a branched scaffold carrying two M23D (phosphorodiamidate morpholino oligomer antisense oligonucleotide that induces exon 23 skipping of mouse dystrophin) oligonucleotide payloads, was generated as shown in Figure 12. DBCO-(M23D)2 was conjugated to either an anti-CD71 monoclonal antibody targeting mouse CD71 or an anti-CD63 monoclonal antibody targeting mouse CD63 to generate mCD71-M23D(DAR3.5) and mCD63-M23D(DAR3.4), respectively (see Figure 13 for conjugation procedure). Either mCD71-M23D or mCD63-M23D conjugates were co-administered with a fixed concentration of 8 μM of the endosomal escape promoter SO1861-SC-Mal and tested for dystrophin exon 23 skipping in differentiated C2C12 mouse myotubes after 48 h of treatment. A strong promotion of exon 23 skipping by co-administration of 8 μM SO1861-SC-Mal was evident for both mCD71-M23D (clear band down to 0.2 nM, at least three orders of magnitude improvement) (FIG. 14A, right panel) and mCD63-M23D (clear band down to 6.0 nM, two orders of magnitude improvement) (FIG. 14B, right panel). mCD71-M23D alone showed no activity at any concentration tested up to 758 nM (Figure 14A, left panel), and mCD63-M23D alone showed only a 2% skip rate at 755 nM (Figure 14B, left panel). Treatment did not affect cell viability as determined by CTG assay. These data indicate that coadministration of SO1861-SC-Mal improves CD71- and CD63-targeted M23D delivery in myotubes by at least 2-3 orders of magnitude.

[0299] Example 7. mCD63-SC-SO1861+M23D or mCD71-M23D (in vitro) SO1861-SC-Mal was conjugated to an anti-CD63 monoclonal antibody targeting mouse CD63 to generate mCD63-SC-SO1861(DAR4.1) (see FIG. 15 for conjugation procedure). mCD63-SC-SO1861 was co-administered with a fixed concentration of 500 nM M23D (phosphorodiamidate morpholino oligomer antisense oligonucleotide that induces exon 23 skipping of mouse dystrophin) into differentiated C2C12 mouse myotubes. This revealed a promotion of exon 23 skipping with 28% skipping rate at 662 nM mCD63-SC-SO1861 and 2% skipping rate at 1.6 nM after 48 hours of treatment (FIG. 16A), while 500 nM M23D alone showed no exon skipping activity (FIG. 16C). These data demonstrate that mCD63-SC-SO1861 induces on-target enhanced cytoplasmic delivery of M23D, thereby induced enhanced exon 23 skipping.

[0300] Next, mCD63-SC-SO1861 (Figure 15) was co-administered with a fixed concentration of 80 nM mCD71-M23D (Figure 13). Treatment with 80 nM mCD71-M23D alone did not result in exon skipping (Figure 16C). Co-administration of mCD63-SC-SO1861 with 80 nM mCD71-M23D revealed enhanced exon 23 skipping with 10% skipping rate at 662 nM mCD63-SC-SO1861 and still 4% skipping rate at 5.3 nM after 48 h of treatment (Figure 16B).

[0301] Taken together, these data indicate that combining SO1861 conjugated with ligand 1 (i.e., targeted SO1861) with a PMO payload conjugated with ligand 2 (i.e., targeted M23D) leads to significant potency enhancement (an example of a targeted two-component system).

[0302] Methods (as carried out in Examples 8-9) SO1861-SC-Maleimide SO1861 is derived from Saponaria officinalis L (Extrasynthese, France) and was coupled to the respective handles according to methods known in the art by Symeres (NL).

[0303] Conjugation of SO1861 with antibodies Custom conjugate production of hCD71-SC-SO1861 and hCD63-SC-SO1861 was carried out by Fleet Bioprocessing (UK).

[0304] Conjugation of 5'-thiol-DMD-ASO, 5'-disulfide amide-DMD-PMO (1) and 3'-disulfide amide-DMD-PMO (1, 2, 3, 4 and 5) with antibodies Custom conjugate production of hCD71-5'-SS-DMD-ASO, hCD71-5'-SS-DMD-PMO(1), hCD71-3'-SS-DMD-PMO(1), hCD71-3'-SS-DMD-PMO(2), hCD71-3'-SS-DMD-PMO(3), hCD71-3'-SS-DMD-PMO(4), hCD71-3'-SS-DMD-PMO(5) and hCD63-5'-SS-DMD-ASO was carried out by Fleet Bioprocessing (UK).

[0305] Analysis and preparation methods Conjugates were characterized by UV-Vis spectrophotometry, BCA colorimetric assay, analytical SEC, SDS-PAGE, Western blotting and urea-PAGE gel electrophoresis. Prior to conjugation with SO1861-SC-maleimide, reduced mAb-SH (either hCD71-SH or hCD63-SH) was analyzed by UV-Vis spectrophotometry and Ellman's assay.

[0306] UV-visible spectrophotometry Concentrations were determined using either a Thermo Nanodrop 2000 spectrophotometer or a Perkin Elmer Lambda 365 spectrophotometer with the following mass extinction coefficient (EC) values: An experimentally determined molar ε495=58,700 M-1 cm-1 and Rz280:495=0.428 were used for SAMSA-Fluorescein. Ellman's reagent (TNB); Molar ε412 = 14,150 M-1 cm-1 Pyridine 2-thione (PDT); Molar ε363 = 8,080 M-1 cm-1 In addition, DMD oligonucleotide incorporation was measured by UV-visible spectrophotometry and BCA colorimetric assay as follows: ε 265 Determined using literature values: DMD-PMO(1); Molar ε363 = 318,050 (mg / ml)-1 cm-1 DMD-PMO(2); Molar ε363 = 318,120 (mg / ml)-1 cm-1 DMD-PMO(3); Molar ε363 = 308,180 (mg / ml)-1 cm-1 DMD-PMO(4); Molar ε363 = 247,710 (mg / ml)-1 cm-1 DMD-PMO(5); Molar ε363 = 207,890 (mg / ml)-1 cm-1 DMD-ASO; Molar ε363 = 310,00 (mg / ml)-1 cm-1

[0307] SEC The conjugates were analyzed by SEC using an Akta purifier 10 system and a Biosep SEC-s3000 column eluted with DPBS:IPA (85:15). Conjugate purity was determined by integration of the conjugate peak compared to impurities / aggregate forms.

[0308] SDS-PAGE Native proteins and conjugates were analyzed by SDS-PAGE under heat-denaturing non-reducing and reducing conditions against a protein ladder using 4-12% Bis-Tris gels and MOPS as running buffer (200 V, ca. 40 min). Samples were prepared at 0.5 mg / mL with LDS sample buffer and MOPS running buffer as diluents. For reduced samples, DTT was added to a final concentration of 50 mM. Samples were heat-treated at 90-95 °C for 15 min and 2.5 μg (5 μL) was added to each well. Protein ladder (10 μL) was loaded without pretreatment. An empty column was filled with 1× LDS sample buffer (10 μL). After the gel was run, it was washed three times with DI water (100 mL) with shaking (15 min, 200 rpm). Coomassie staining was performed by incubating the gel with PAGEBlue protein stain (30 mL) with shaking (60 min, 200 rpm). Excess staining solution was removed, rinsed twice with DI water (100 ml), and destained with DI water (100 ml) (60 min, 200 rpm). The resulting gels were imaged and processed using ImageJ (Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, Maryland, USA) and MyCurveFit (two-point correlation of protein ladders).

[0309] Western blotting From SDS-PAGE, the gel was transferred to a nitrocellulose membrane using freshly prepared transfer buffer using an X-Cell Blot module with the following setup ((-)BP-BP-FP-gel-NC-BP-BP-BP(+)) and conditions (30V, 60min). BP-blotting pad; FP-filter pad; NC-nitrocellulose membrane. Later, NC was washed twice with PBS-T (100ml) with shaking (5min, 200rpm), non-specific sites were blocked with blocking buffer (50ml) with shaking (30min, 200rpm), and then active sites were labeled with a combination of goat anti-human κ-HRP (1:2000) and goat anti-human IgG-HRP (1:2000) (50ml) diluted in blocking buffer with shaking (30min, 200rpm). The NCs were then washed once with PBS-T (100 ml) with shaking (5 min, 200 rpm) and the complexed antibodies were detected with Ultra TMB-blotting solution (25 ml). The color development was observed visually and stopped by washing the NCs with water, and the resulting blots were photographed.

[0310] Urea-PAGE gel electrophoresis This characterization of the conjugates was performed under denaturing, non-reducing conditions with EtBr or SYBR Green staining in comparison to oligonucleotide standards and oligonucleotide standard ladders (report residue "free" oligonucleotides).

[0311] hAb-SC-SO1861 A summary of the conjugations of hAb-SO1861, including hCD71-SC-SO1861 and hCD63-SC-SO1861, is given below, where the quantities given in italics in brackets are shown as examples for hCD71-SC-SO1861.

[0312] An aliquot of hAb was buffer exchanged into TBS pH 7.5 and normalized to 5 mg / ml. An aliquot of hAb (95.8 mg, 6.39 x 10 -4mmol, 4.94 mg / ml) with an aliquot of freshly prepared TCEP (3.18 equiv., 2.03 × 10 -3 A 1.0 mg aliquot (0.203 ml) of the reaction mixture was removed after incubation, purified on a Zeba 7K spin desalting column eluted with TBS pH 7.5, and characterized by UV-vis and Ellman assay (3.407 mg / ml, SH to hAb ratio = 4.8). To this bulk reaction, an aliquot of SO1861-SC-maleimide (6 mol equivalents, 3.79 x 10 -3 2 × 0.25 mg of desalted hAb-SH (1.67 × 10 mmol, 8.3 mg, 4.14 ml) was added with gentle stirring, the mixture was vortexed briefly, and then incubated for 120 min at 20 °C. -6 mmol, 0.073 ml) was dispensed and NEM (8 mol equivalents, 1.34 × 10 -5 10 mmol, 6.7 μl, 0.25 mg / ml) or TBS pH 7.5 (6.7 μl) and then incubated in parallel with bulk conjugation as positive and negative reaction controls, respectively. After incubation, 0.5 mg aliquots (approximately 0.100 ml) of the hAb-SC-SO1861 mixture were removed and purified by Zeba 7K spin desalting column eluted with TBS pH 7.5 and characterized in parallel with positive and negative controls by Ellman's assay to obtain SO1861 incorporation. After reaction, the bulk hAb-SC-SO1861 mixture was diluted with an aliquot of freshly prepared NEM solution (5 mol equivalents, 3.16 × 10 -3The reaction was quenched by the addition of 100 mmol of 2.5 mg / ml solution (158 μl of 2.5 mg / ml solution). The quenched reaction mixture was purified using a sanitized 2.6×40 cm Superdex 200 column eluted with DPBS pH 7.5. The purified hAb-SC-SO1861 was collected and analyzed by UV-Vis spectrophotometry. It was then concentrated to >2.5 mg / ml using Vivaspin 20 centrifugal filters, standardized to 2.5 mg / ml, and 0.2 μm filtered under laminar flow. The product was distributed into aliquots for product testing, characterization, and further conjugation work. The results were as follows: hCD71-SC-SO1861 (total yield = 78.4 mg, 82%, SO1861 to hCD71 ratio = 4.0) hCD63-SC-SO1861 conjugate (total yield = 58.8 mg, SO1861 to hCD63 ratio = 4.8).

[0313] hAb-DMD-oligonucleotide The conjugation of hAb-targeted DMD oligonucleotides, including hCD71-5'-SS-DMD-ASO, hCD71-5'-SS-DMD-PMO(1), hCD71-3'-SS-DMD-PMO(1-5) and hCD63-5'-SS-DMD-ASO, is outlined below, with the amounts given in italics within the brackets being shown as an example for hCD71-5'-SS-DMD-PMO(1).

[0314] An aliquot of hAb was buffer exchanged into DPBS pH 7.5 and standardized to 2.5mg / ml. To an aliquot of hAb (hCD71, 20.0mg, 1.33x10-4mmol, 2.5mg / ml) was added an aliquot of freshly prepared PEG4-SPDP solution (10.0mg / ml, 10.0 molar equivalents, 1.33x10-3mmol, 0.075ml), the mixture was vortexed briefly and then incubated at 20°C for 60min with end-over-end mixing. After incubation, the reaction was quenched by adding an aliquot of freshly prepared glycine solution (10mg / ml, 50 molar equivalents, 6.65x10-3mmol, 8.1μl), the mixture was vortexed briefly and then incubated at 20°C for >15min with end-over-end mixing. The conjugate was purified (eluted with TBS pH 7.5 using a Zeba 40K spin desalting column, filtered at 0.45 μm) and then analyzed by UV-Vis to obtain purified hAb-SPDP (hCD71-SPDP, 21.2 mg, 2.04 mg / ml, SPDP to hCD71 ratio=3.9). The hAb-SPDP was used immediately.

[0315] Separately, the desired DMD oligonucleotides (DMD-PMO(1)-5'-amide, 20.2 mg, 1.99 x 10-3 mmol, 10.00 mg / ml) were reconstituted using TBS pH 7.5, pooled into a single aliquot, and analyzed by UV-Vis to determine ε 280 , ε 260 and the ratio ε 260 / ε 280 To this aliquot was added an aliquot of freshly prepared THPP solution (50 mg / ml, 10 molar equivalents, 1.99×10-2 mmol, 83 μl), the mixture was vortexed briefly, and then incubated at 37° C. for 60 min with end-over-end mixing. After incubation, the oligonucleotide was purified using a PD10 Sephadex G25 column and eluted with TBS pH 7.5 to yield the reduced DMD oligonucleotide-SH (DMD-PMO(1)-5'-SH, 16.4 mg, 81%, thiol to DMD-PMO(1) ratio=1.02).

[0316] An aliquot of hAb-SPDP (hCD71-SPDP, 10.2 mg, 6.79×10-5 mmol, 2.04 mg / ml) was added with an aliquot of oligonucleotide-SH (DMD-PMO(1)-SH, 2.73 mg / ml, 8.0 molar equivalents, 5.43×10-4 mmol, 2.01 ml), the mixture was vortexed briefly, and then incubated overnight at 20° C. with end-over-end mixing. After approximately 16 hours, the conjugate mixture was analyzed by UV-Vis to confirm incorporation by transfer of PDT, and then purified using a sanitized 2.6×60 cm Superdex 200PG column eluted with DPBS pH 7.5. The conjugate was analyzed by UV-Vis and BCA colorimetric assay to confirm fresh ε 280 was assigned. The material was concentrated (using Vivacell 100 (30K MWCO) to the highest concentration achievable) and then distributed into aliquots for product testing and characterization. The result, as an example, was hCD71-5'-SS-DMD-PMO(1) conjugate (total yield = 65%; DMD-PMO(1) to hCD71 ratio = 2.2). Other results were as follows: hCD71-3'-SS-DMD-PMO(1) (total yield = 58%, DMD-PMO(1) to hCD71 ratio = 2.1) hCD71-3'-SS-DMD-PMO(2) (total yield = 70%, DMD-PMO(2) to hCD71 ratio = 3.0) hCD71-3'-SS-DMD-PMO(3) (total yield = 65%, DMD-PMO(3) to hCD71 ratio = 2.6) hCD71-3'-SS-DMD-PMO(4) (total yield = 68%, DMD-PMO(4) to hCD71 ratio = 2.3) hCD71-3'-SS-DMD-PMO(5) (total yield = 67%, DMD-PMO(5) to hCD71 ratio = 2.1) hCD71-5'-SS-DMD-ASO (overall yield = 76%, DMD-ASO to hCD71 ratio = 2.1) hCD63-5'-SS-DMD-ASO (total yield = 60%, DMD-ASO to hCD63 ratio = 2.3)

[0317] Cell culture (human) Immortalized human myoblasts from a non-DMD donor (KM155) were cultured as described in Methods above (as carried out in Examples 1-5).

[0318] Human myoblasts from a fixed DMD-affected donor (KM1328) were cultured in homemade growth medium containing 80 ml 199 medium (Thermo Fisher Scientific) and 320 ml DMEM (Thermo Fisher Scientific) supplemented with 20% fetal bovine serum (Gibco, United Kingdom), 50 μg / ml gentamicin (Thermo Fisher Scientific), 25 μg / ml fetuin (Thermo Fisher Scientific), 5 ng / ml human epidermal growth factor (Thermo Fisher Scientific), 0.5 ng / ml basic fibroblast growth factor (Thermo Fisher Scientific), 5 μg / ml insulin (Sigma) and 0.2 μg / ml dexamethasone (Sigma). For differentiation, cells were seeded onto Matrigel-coated surfaces, which were prepared by incubation with 0.1 mg Corning™ Matrigel™ basement membrane matrix (Corning) per ml DMEM (Gibco) for 60 min at 37° C. At 100% confluence, growth medium was replaced with DMEM (Gibco) supplemented with 2% fetal bovine serum (Gibco), 10 μg / ml insulin (Sigma) and 50 μg / ml gentamicin (Thermo Fisher Scientific). After at least 3 days, but no longer than 5 days of differentiation, treatment was initiated based on the presence of differentiated myotubes).

[0319] Exon skipping analysis and quantification (human) RNA was isolated with TRIsure isolation reagent (Bioline) and chloroform extraction of RNA from the aqueous phase; isopropanol precipitation were performed as known to those skilled in the art. 1000 ng of total RNA was used for cDNA synthesis and diluted in an appropriate amount of RNase-free water to yield 8 μl RNA dilution.

[0320] For exon 51 and exon 53 skipping analysis in KM155 myotubes, the priming premix contained 1 μl dNTP mix (10 mM each) and 1 μl specific reverse primer (for KM155, exon 51: h53R 5′-CTCCGGTTCTGAAGGTGTTC-3′ [SEQ ID NO: 5]; exon 53: h55R 5′-ATCCTGTAGGACATTGGCAGTT-3 [SEQ ID NO: 6]). This mixture was heated at 70° C. for 5 min and then cooled on ice for at least 1 min. 0.5 μl rRNasin (Promega), 4.0 μl 5× RT buffer (Promega), 1.0 μl M-MLV RT (Promega), and 4.5 μl Reaction mixtures containing RNase-free water were prepared and added to the cooled mixtures to give a total volume of 20 μl for each reaction. RT-PCR was performed for 60 min at 42°C, then 5 min at 85°C and cooled on ice. For skip analysis, the nested PCR approach was followed. For this purpose, in the first PCR, 3 μl cDNA was added to a mixture of 2.5 μl 10× SuperTaq PCR buffer, 0.5 μl dNTP mix (10 mM each), 0.125 μl Taq DNA polymerase TAQ-RO (5 U / μl; Roche), 16.875 μl RNase-free water and 1 μl (10 pmol / μl) of each primer flanking the targeted exon. The following primers were used: for KM155, exon 51: h48F 5′-AAAAGACCTTGGGCAGCTTG-3′ [SEQ ID NO: 7] and h53R exon 53: h50F 5'-AGGAAGTTAGAAGATCTGAGC-3' [SEQ ID NO:20] and h55R 5'-ATCCTGTAGGACATTGGCAGTT-3' [SEQ ID NO:6]. These samples were subjected to a PCR run at 94°C for 5 minutes, followed by 25 cycles of 94°C for 40 seconds, 60°C for 40 seconds, 72°C for 180 seconds, followed by 72°C for 7 minutes.For the second PCR, 1.5 μl PCR1 sample was added to 5 μl 10× SuperTaq PCR buffer, 1 μl dNTP mix (10 mM each), 0.25 μl Taq DNA polymerase TAQ-RO (5 U / μl; Roche), 38.25 μl RNase-free water and 2 μl (10 pmol / μl) of a mixture of each primer flanking the targeted exon. The following primers were used: for KM155, exon 51: h49F 5′-CCAGCCACTCAGCCAGTG-3′ [SEQ ID NO: 10] and h52R2 5′-TTCTTCCAACTGGGGACGC-3′ [SEQ ID NO: 11], exon 53: h52F 5′-CCCCAGTTGGAAGAACTCATT-3′ [SEQ ID NO: 21] and h54R 5′-CCAAGAGGCATTGATATTCTC-3′ [SEQ ID NO: 9]. These samples were subjected to a PCR run at 94°C for 5 min, followed by 32 cycles of 94°C for 40 s, 60°C for 40 s, and 72°C for 60 s, followed by 72°C for 7 min. Exon skipping levels were quantified using the Femto Pulse system (Agilent) with the Ultra Sensitivity NGS kit according to the manufacturer's instructions. Alternatively, specific PCR fragments were analyzed using a Bioanalyzer 2100 with a DNA1000 chip (Lab-on-a-Chip; Agilent). For exon 51 skipping, the expected non-skipped product is 408 bp in size (KM155) and the skipped product is 175 bp (KM155). For exon 53 skipping, the expected non-skipped product is 438 bp in size (KM155) and the skipped product is 226 bp (KM155).

[0321] For exon 51 skipping analysis in KM1328 myotubes, the priming premix contained 1 μl dNTP mix (10 mM each) and 1 μl specific reverse primer (for KM1328, exon 51: h57R 5'-TCTGAACTGCTGGAAAGTCG-3' [SEQ ID NO: 22]). This mixture was heated at 70°C for 5 min and then cooled on ice for at least 1 min. Reaction mixtures containing 0.5 μl rRNasin (Promega), 4.0 μl 5x RT buffer (Promega), 1.0 μl M-MLV RT (Promega) and 4.5 μl RNase-free water were prepared and added to the cooled mixtures to yield a total volume of 20 μl for each reaction. RT-PCR was performed at 42°C for 60 min, then 70°C for 10 min and cooled on ice. For skipping analysis, the nested PCR approach was followed. For this purpose, in the first PCR, 3 μl cDNA was added to a mixture of 2.5 μl 10× SuperTaq PCR buffer, 0.5 μl dNTP mix (10 mM each), 0.125 μl Taq DNA polymerase TAQ-RO (5 U / μl; Roche), 16.875 μl RNase-free water and 1 μl (10 pmol / μl) of each primer flanking the targeted exon. The following primers were used: for KM1328, exon 51: h48F 5′-AAAAGACCTTGGGCAGCTTG-3′ [SEQ ID NO: 7] and h57R 5′-TCTGAACTGCTGGAAAGTCG-3′ [SEQ ID NO: 22]. The samples were subjected to a PCR run at 94° C. for 5 min, followed by 25 cycles of 94° C. for 40 s, 60° C. for 40 s, 72° C. for 180 s, followed by 72° C. for 7 min. For the second PCR, 1.5 μl PCR1 sample was added to a mixture of 5 μl 10× SuperTaq PCR buffer, 1 μl dNTP mix (10 mM each), 0.25 μl Taq DNA polymerase TAQ-RO (5 U / μl; Roche), 38.25 μl RNase-free water, and 2 μl (10 pmol / μl) of each primer flanking the targeted exon.The following primers were used: for KM1328, exon 51: h49F2 5'-AAACTGAAATAGCAGTTCAAGC-3' [SEQ ID NO: 23] and h54R 5'-CCAAGAGGCATTGATATTCTC-3' [SEQ ID NO: 9]. The samples were subjected to a PCR run at 94°C for 5 min, followed by 32 cycles of 94°C for 40 s, 60°C for 40 s, 72°C for 90 s, followed by 72°C for 7 min. Exon skipping levels were quantified by analyzing specific PCR fragments using a Bioanalyzer 2100 with a DNA1000 chip (Lab-on-a-Chip; Agilent). For exon 51 skipping, the expected non-skipped product is 793 bp in size (KM1328) and the skipped product is 560 bp (KM1328).

[0322] Results (Examples 8 to 9) Example 8. hCD71-5'-SS-DMD-ASO + SO1861-SC-Mal or hCD71-5'-SS-DMD-PMO (1) + SO1861-SC-Mal or hCD71-3'-SS-DMD-PMO (1, 2, 3, 4 or 5) + SO1861-SC-Mal (in vitro) Anti-CD71 monoclonal antibodies targeting human CD71 were conjugated to DMD-ASO-SH (an active 2'O-methyl-phosphorothioate antisense oligonucleotide with the same sequence and chemical modifications as drisapersen that induces exon 51 skipping of human dystrophin) or DMD-PMO(1)-SH (an active phosphorodiamidate morpholino oligomer [PMO] antisense oligonucleotide with the same sequence and chemical modifications as eteplirsen that induces exon 51 skipping of human dystrophin) via 5' disulfide bond formation to generate hCD71-5'-SS-DMD-ASO (DAR2.1) and hCD71-5'-SS-DMD-PMO(1) (DAR2.2), respectively (see Figures 17A-D for conjugation procedure). The resulting compounds were tested for dystrophin exon 51 skipping in differentiated human myotubes from a non-DMD donor (KM155) either without or in combination with 4 μM endosomal escape promoter SO1861-SC-Mal. This revealed that hCD71-5'-SS-DMD-ASO alone showed low exon 51 skipping rates (2.6%) even at 600 nM (Figure 18A, left panel; Table A8), but in combination with 4 μM SO1861-SC-Mal, it strongly promoted exon 51 skipping after 72 h of treatment, revealing exon 51 skipping rates of 13.4 to 43.6% already at 0.46 to 100 nM hCD71-5'-SS-DMD-ASO+SO1861-SC-Mal (Figure 18A, right panel; Table A9). Similarly, exposure to hCD71-5'-SS-DMD-PMO(1) induced minimal exon 51 skipping (0.4-1.1%) at concentrations ranging from 16.6 to 600 nM (Figure 18B, left panel; Table A8), but increased exon 51 skipping to 10.4-12.5% ​​at 6-fold lower exposure concentrations of 2.78-100 nM hCD71-5'-SS-DMD-PMO(1)+SO1861-SC-Mal (Figure 18B, right panel; Table A9), a marked improvement in efficacy compared to conditions without SO1861-SC-Mal.Thus, coadministration of SO1861-SC-Mal with hCD71-5'-SS-DMD-ASO and hCD71-5'-SS-DMD-PMO(1), a targeted DMD oligonucleotide conjugated to hCD71 at the 5', improved on-target delivery and induced significant exon 51 skipping.

[0323] Next, targeted DMD-PMOs 3'-conjugated to anti-hCD71 were tested for exon 51 skipping activity in human myotubes. Anti-CD71 monoclonal antibodies targeting human CD71 were conjugated to DMD-PMO(1)-SH, DMD-PMO(2)-SH (an active PMO antisense oligonucleotide inducing exon 51 skipping of human dystrophin, as described in Echigoya et al. (2017) ), or DMD-PMO(3)-SH (an active PMO antisense oligonucleotide inducing exon 51 skipping of human dystrophin, as described in Echigoya et al. (2017) ). hCD71-3'-SS-DMD-PMO(1) (DAR2.1), hCD71-3'-SS-DMD-PMO(2) (DAR3.0) and hCD71-3'-SS-DMD-PMO(3) (DAR2.6) were conjugated to hCD71-3'-SS-DMD-PMO(1) (DAR2.1), hCD71-3'-SS-DMD-PMO(2) (DAR3.0) and hCD71-3'-SS-DMD-PMO(3) (DAR2.6), respectively (see Figures 17A-D for conjugation procedures). The resulting compounds were tested for dystrophin exon 51 skipping in differentiated human myotubes from a non-DMD donor (KM155) either without or in combination with 4 μM SO1861-SC-Mal. As shown for hCD71-5'-SS-DMD-PMO(1), this revealed enhanced exon 51 skipping for hCD71-3'-SS-DMD-PMO(1) in combination with 4 μM SO1861-SC-Mal after 72 h of treatment: exposure to hCD71-3'-SS-DMD-PMO(1) alone resulted in very little exon 51 skipping (0.4-2.2%) at 0.077-600 nM (Figure 19A, left panel; Table A8), whereas exposure concentrations of 2.78-100 nM of hCD71-3'-SS-DMD-PMO(1)+SO1861-SC-Mal increased exon 51 skipping to 8.2-9.7% (Figure 19A, right panel; Table A9).More strikingly, exon 51 skipping was strongly promoted for hCD71-3'-SS-DMD-PMO(2) in combination with 4 μM SO1861-SC-Mal: exposure to hCD71-3'-SS-DMD-PMO(2)+SO1861-SC-Mal revealed exon 51 skipping already at 0.013 nM conjugate (7.8%), which increased to 75.6-80.4% at 2.78-100 nM conjugate (Figure 19B, right panel; Table A9), whereas exposure to hCD71-3'-SS-DMD-PMO(2) alone resulted in only 5.7% exon 51 skipping even at 600 nM conjugate (Figure 19B, left panel; Table A8), a four-order improvement compared to conditions without SO1861-SC-Mal. Moreover, exposure to hCD71-3'-SS-DMD-PMO(3) in combination with 4 μM SO1861-SC-Mal strongly promoted exon 51 skipping: hCD71-3'-SS-DMD-PMO(3)+SO1861-SC-Mal revealed exon 51 skipping (9.6%) already at 0.077 nM conjugate, which increased to 28.3-29.2% at 2.78-100 nM (Figure 19C, right panel; Table A9), whereas exposure to hCD71-3'-SS-DMD-PMO(2) alone resulted in no exon 51 skipping (0.0%) at all concentrations tested (up to 600 nM) (Figure 19C, left panel; Table A8). Thus, coadministration of SO1861-SC-Mal with hCD71-3'-SS-DMD-PMO(1), hCD71-3'-SS-DMD-PMO(2), and hCD71-3'-DMD-PMO(3), targeted DMD oligonucleotides conjugated to hCD71 at the 3', improved on-target delivery and induced significant exon 51 skipping.

[0324] Targeted DMD-PMOs inducing exon 53 skipping of human dystrophin were also tested in human myotubes. DMD-PMO(4)-SH (an active PMO antisense oligonucleotide with the same sequence and chemical modification as golodirsen, which induces exon 53 skipping of human dystrophin) or DMD-PMO(5)-SH (an active PMO antisense oligonucleotide with the same sequence and chemical modification as viltolarsen, which induces exon 53 skipping of human dystrophin) were conjugated to an anti-CD71 monoclonal antibody targeting human CD71 via 3' disulfide bond formation to generate hCD71-3'-SS-DMD-PMO(4) (DAR2.3) and hCD71-3'-SS-DMD-PMO(5) (DAR2.1), respectively (see Figures 17A-D for conjugation procedure). The resulting compounds were tested for dystrophin exon 53 skipping in differentiated human myotubes from a non-DMD donor (KM155) either without or in combination with 4 μM SO1861-SC-Mal, revealing that only in combination with SO1861-SC-Mal exon 53 skipping was promoted after 72 h of treatment: exposure to 600 nM hCD71-3'-SS-DMD-PMO(4) alone resulted in only a 0.4% exon 53 skipping rate (Figure 20A, left panel; Table A8), whereas 2.78 nM hCD71-3'-SS-DMD-PMO(4) + SO1861-SC-Mal already resulted in a 5.4% exon 53 skipping rate (Figure 20A, right panel; Table A9). Similarly, exposure to 600 nM hCD71-3'-SS-DMD-PMO(5) alone resulted in an exon 53 skipping rate of 0.3% (Figure 20B, left panel; Table A8), whereas exposure to 2.78 nM hCD71-3'-SS-DMD-PMO(5) + SO1861-SC-Mal already resulted in an exon 53 skipping rate of 6.0% (Figure 20B, right panel; Table A9), which increased to 8.4% with 100 nM hCD71-3'-SS-DMD-PMO(5), achieving an improvement of 1-2 orders of magnitude.These data indicate that coadministration of SO1861-SC-Mal with hCD71-3'-SS-DMD-PMO (4) and hCD71-3'-SS-DMD-PMO (5), targeted DMD oligonucleotides conjugated to hCD71 at the 3', enhances their on-target delivery and induces exon 53 skipping.

[0325] Taken together, these data indicate that co-administration of SO1861-SC-Mal is broadly applicable to effectively increase the potency in human myotubes of hCD71-targeted DMD oligonucleotides of a variety of different targeting sequences (various different exons, various different sequences) and conjugation methods (either 5' or 3').

[0326] [Table 21]

[0327] [Table 22]

[0328] Example 9. hCD71-5'-SS-DMD-ASO+hCD63-SC-SO1861 or hCD63-5'-SS-DMD-ASO+hCD71-SC-SO1861 (in vitro) DMD-ASO-SH was conjugated to either an anti-CD71 monoclonal antibody targeting human CD71 or an anti-CD63 monoclonal antibody targeting human CD63 to generate hCD71-5'-SS-DMD-ASO (DAR2.1) and hCD63-5'-SS-DMD-ASO (DAR2.3), respectively (see Figures 17A-D for conjugation procedure). SO1861-SC-Mal was also conjugated to either an anti-CD71 monoclonal antibody targeting human CD71 or an anti-CD63 monoclonal antibody targeting human CD63 to generate hCD71-SC-SO1861 (DAR4.0) and hCD63-SC-SO1861 (DAR4.8), respectively (see Figure 21 for conjugation procedure). To differentiated human myotubes from a non-DMD (healthy) donor (KM155), hCD71-5'-SS-DMD-ASO and hCD63-5'-SS-DMD-PMO were co-administered with a fixed concentration of 100 nM hCD63-SC-SO1861 or hCD71-SC-SO1861, respectively. Strikingly, co-administration of hCD71-5'-SS-DMD-ASO with 100 nM hCD63-SC-SO1861 revealed enhanced exon 51 skipping, with an exon skipping rate of 28.7% already at 0.46 nM hCD71-5'-SS-DMD-ASO, which increased to exon skipping rates of 50.0-68.1% at 16.6-100 nM hCD71-5'-SS-DMD-ASO (Figure 22A; Table A10). Coadministration of hCD63-5'-SS-DMD-ASO with 100 nM hCD71-SC-SO1861 revealed a 29.2% exon skipping rate at 16.6 nM and enhanced exon 51 skipping (Figure 22B; Table A10). These data indicate that hCD63-SC-SO1861 and hCD71-SC-SO1861 induce enhanced on-target cytoplasmic delivery of hCD71-targeted or hCD63-targeted DMD-ASO, thereby induced enhanced exon 51 skipping.

[0329] Next, we switched between titrated conjugates and co-administered conjugates at a fixed concentration. Differentiated human muscle from a non-DMD (healthy) donor (KM155) was treated with hCD63-SC-SO1861 in combination with a fixed concentration of 55.6 nM hCD71-5'-SS-DMD-ASO. This revealed that exon 51 skipping was promoted with 72.6% exon skipping rate at 16.6 nM hCD63-SC-SO1861, 53.1% exon skipping rate at 2.78 nM, and still 21.0% exon skipping rate at 0.46 nM (Figure 23A; Table A11). More strikingly and relevantly, in differentiated myotubes from a DMD-affected donor (KM1328), co-administration of hCD63-SC-SO1861 with a constant concentration of 55.6 nM hCD71-5'-SS-DMD-ASO already resulted in a 53.8% skip rate at 0.46 nM hCD63-SC-SO1861, which increased to a 93.4% skip rate at 16.6 nM and a 97.0% skip rate at 600 nM hCD63-SC-SO1861 (Figure 23B, Table A11).

[0330] Taken together, these data indicate that combining a ligand 1-conjugated oligonucleotide payload (i.e., either hCD71- or hCD63-targeted DMD-ASO) with a ligand 2-conjugated SO1861 (i.e., either hCD71- or hCD63-targeted SO1861) or vice versa, i.e., combining a ligand 2-conjugated oligonucleotide payload with a ligand 1-conjugated SO1861, leads to a significant enhancement of efficacy in disease-relevant cell lines, such as human myotubes, specifically differentiated myotubes from DMD-affected donors (an example of a two-target, two-component system).

[0331] [Table 23]

[0332] [Table 24]

[0333] References Cardamone M,et al.,2008 - Cardamone M;Darras BT;Ryan MM,2008,’Inherited myopathies and muscular dystrophies’,Seminars in Neurology,vol.28,pp.250 - 259,http: / / dx.doi.org / 10.1055 / s-2008-1062269 Shadrin et al,2016 - Striated muscle function,regeneration,and repair,Cell Mol Life Sci.2016 November;73(22):4175-4202.doi:10.1007 / s00018-016-2285-z. Dumont NA,Wang YX,Rudnicki MA.Intrinsic and extrinsic mechanisms regulating satellite cell function.Development.2015;142(9):1572-1581,doi:10.1242 / dev.114223 Uygur and Lee,Mechanisms of Cardiac Regeneration.Dev Cell.2016 Feb 22;Volume 36(4):pp.362-374,doi:10.1016 / j.devcel.2016.01.018 Slordal L,Spigset O.Heart failure induced by non-cardiac drugs,Drug Saf 2006;Volume 29(7):pp.567-86,doi:10.2165 / 00002018-200629070-00003 Ebner et al.,Strategies for Skeletal Muscle Targeting in Drug Discovery,Current Pharmaceutical Design 2015,Volume 21(10),pp.1327-1336,DOI:10.2174 / 1381612820666140929095755 Gonzalez-Jamett et al.,Hereditary Myopathies,2018,DOI:10.5772 / intechopen.76076,www.intechopen.com / chapters / 60283 Savarese et al.,Is Gene-Size an Issue for the Diagnosis of Skeletal Muscle Disorders?,2020,Volume 7(3),pp.203-216,DOI:10.3233 / JND-190459 Duan et al.,Duchenne muscular dystrophy,Nat Rev Dis Primers,2021 Feb 18;Volume 7(1):13,doi:10.1038 / s41572-021-00248-3 Bladen,C.L.et al.,The TREAT-NMD Duchenne muscular dystrophy registries:conception,design,and utilization by industry and academia.Hum.Mutat.34,1449-1457 (2013),DOI:10.1002 / humu.22390. Goemans,N.et al.,A randomized placebo-controlled phase 3 trial of an antisense oligonucleotide,drisapersen,in Duchenne muscular dystrophy.Neuromuscul.Disord.Volume 28(1),pp.4-15 (2018),doi:10.1016 / j.nmd.2017.10.004. Mendell,J.R.et al.,Assessment of systemic delivery of rAAVrh74.MHCK7.micro-dystrophin in children with duchenne muscular dystrophy:a nonrandomized controlled trial.JAMA Neurol.Volume 77(9),pp.1122-1131 (2020),doi:10.1001 / jamaneurol.2020.1484. Mendell,J.R.et al,A phase I / IIa follistatin gene therapy trial for Becker muscular dystrophy,Mol.Ther.Volume 23(1),pp.192-201 (2015),doi:10.1038 / mt.2014.200. Wasala,N.B.et al.,Single SERCA2a therapy ameliorated dilated cardiomyopathy for 18 months in a mouse model of Duchenne muscular dystrophy,Mol.Ther.Volume 28(3),pp.845-854 (2020),doi:10.1016 / j.ymthe.2019.12.011 Aranega,et al.,Review MiRNAs and Muscle Regeneration:Therapeutic Targets in Duchenne Muscular Dystrophy,Int J Mol Sci.(2021 Apr),Volume 22(8):4236,doi:10.3390 / ijms22084236 Chemello,et al.,Correction of muscular dystrophies by CRISPR gene editing,J.Clin.Invest.Volume 130(6),pp.2766-2776 (2020),doi:10.1172 / JCI136873 Nelson,et al.,Genome engineering:a new approach to gene therapy for neuromuscular disorders,Nat.Rev.Neurol.Volume 13(11),pp.647-661 (2017),doi:10.1038 / nrneurol.2017.126 Cerro-Herreros et al.,Therapeutic Potential of AntagomiR-23b for Treating Myotonic Dystrophy,Mol Ther Nucleic Acids,2020 Sep 4;Volume 21,pp.837-849,doi.org / 10.1016 / j.omtn.2020.07.021 Bladen,C.L.et al.,The TREAT-NMD DMD global database:analysis of more than 7000 Duchenne muscular dystrophy mutations.Hum.Mutat.Volume 36(4),pp.395-402 (2015),doi:10.1002 / humu.22758 Hanahan and Weinberg,Hallmarks of cancer:the next generation,Cell (2011,Mar 4);Volume 144(5),pp.646-674,doi:10.1016 / j.cell.2011.02.013. Wang et al.,Saponins as Natural Adjuvant for Antisense Morpholino Oligonucleotides Delivery In Vitro and in mdx Mice,Molecular Therapy:Nucleic Acids,2018,Volume 11,pp.192-202,doi:10.1016 / j.omtn.2018.02.004 Wang et al.,Saponins enhance exon skipping of 2’-O-methyl phosphorothioate oligonucleotide in vitro and in vivo,Drug Design,Development and Therapy Volume 12:pp.3705-3715 (2018),DOI:10.2147 / DDDT.S179008 Kemaladewi et al,Dual exon skipping in myostatin and dystrophin for Duchenne muscular dystrophy,BMC Medical Genomics,Volume 4,36 (2011),DOI:10.1186 / 1755-8794-4-36 Baik et al,Cell type-selective targeted delivery of a recombinant lysosomal enzyme for enzyme therapies,Molecular Therapy,Volume 29(12),pp.3512-3524 (2021),DOI:10.1016 / j.ymthe.2021.08.020 Echigoya et al.,Quantitative antisense screening and optimization for exon 51 skipping in Duchenne muscular dystrophy,Molecular Therapy,Volume 25(11),pp.2561 - 2572 (2017),DOI:10.1016 / j.ymthe.2017.07.014

Claims

1. 1. A pharmaceutical composition for use in the treatment of a muscle wasting disorder, comprising: Therapeutic nucleic acids, and A covalently linked first conjugate comprising a saponin and a first ligand for an endocytic receptor on a muscle cell. wherein the saponin is a triterpenoid 12,13-dehydrooleanane-type saponin.

2. 2. The composition for use according to claim 1, wherein the muscle wasting disorder is a muscle cell-related genetic disorder.

3. 2. The composition for use of claim 1, wherein said treatment of said muscle wasting disorder involves antisense therapy.

4. The saponin is an aldehyde group at the C-23 position of the aglycone core structure of the saponin; or a covalent bond at the C-23 position of the aglycone core structure of the saponin, which covalently links the saponin within the first conjugate; 2. The composition for use according to claim 1, comprising any one of:

5. The saponin aglycone core structure is Quila acid; Gypsogenin; 2α-hydroxyoleanolic acid; 16α-hydroxyoleanolic acid; Hederagenin (23-hydroxyoleanolic acid); 16α,23-dihydroxyoleanolic acid; Protoestigenin-21(2-methylbut-2-enoate)-22-acetate; 23-oxo-valingutogenol C-21,22-bis(2-methylbut-2-enoate); 23-Oxo-barringtogenol C-21(2-methylbut-2-enoate)-16,22-diacetate; 3,16,28-trihydroxyoleanan-12-ene; and Gypsogenic acid 2. The composition for use according to claim 1, selected from any one or more of:

6. the first conjugate comprises two or more molecules of the saponin; and / or 2. The composition for use according to claim 1, wherein the first conjugate comprises 1 to 16 molecules of the saponin per molecule of the first ligand.

7. The saponin is a) List A: Quillaja saponaria saponin mixtures or saponins isolated from Quillaja saponaria, such as Quil-A, QS-17-api, QS-17-xyl, QS-21, QS-21A, QS-21B, QS-7-xyl; - Saponinum album saponin mixture or saponins isolated from Saponinum album; - Saponaria officinalis saponin mixture or saponin isolated from Saponaria officinalis; and Quillaja bark saponin mixtures or saponins isolated from quillaja bark, such as Quil-A, QS-17-api, QS-17-xyl, QS-21, QS-21A, QS-21B, QS-7-xyl or a saponin selected from any one or more of: b) List B: SA1641, gypsoside A, NP-017772, NP-017774, NP-017777, NP-017778, NP-018109, NP-017888, NP-017889, NP-018108, SO1658 and phytolaccagenin a saponin containing a gypsogenin aglycone core structure selected from: c) List C: AG1856, AG1, AG2, agrostemoside E, GE1741, gypsophila saponin 1 (Gyp1), NP-017674, NP-017810, NP-003881, NP-017676, NP-017677, NP-017705, NP-017706, NP-017773, NP-017775, SA1657, SO1542, SO1584, SO1674, SO1700, SO1730, SO1772, SO1832, saponariosides B, 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 a saponin comprising a chiral acid aglycone core structure selected from: d) List D: Escin Ia, escinate, α-hederin, AMA-1, AMR, AS6.2, AS64R, assamsaponin F, dipsacoside B, esculentoside A, macrantoidin A, NP-005236, NP-012672, primulic acid 1, saikosaponin A, saikosaponin D, tea seed saponin I, and tea seed saponin J A saponin having a 12,13-dehydrooleanane-type aglycone core structure without an aldehyde group at the C-23 position of the aglycone, selected from and / or 2. The composition for use according to claim 1, wherein the saponin is any one or more of AG1856, GE1741, saponin isolated from Quillaja saponaria, Quil-A, QS-17, QS-21, QS-7, SA1641, saponin isolated from Saponaria officinalis, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, saponarioside B, SO1832, SO1861, SO1862 and SO1904.

8. the endocytic receptor on muscle cells to which the ligand binds is selected from the transferrin receptor (CD71), insulin-like growth factor 1 (IGF-I) receptor (IGF1R), tetraspanin CD63; muscle-specific kinase (MuSK), glucose transporter GLUT4, cation-independent mannose 6-phosphate receptor (CI-MPR), and LDL receptor; and / or The first ligand is Insulin-like growth factor 1 (IGF-I) or a fragment thereof; Insulin-like growth factor 2 (IGF-II) or a fragment thereof; mannose hexaphosphate, Transferrin (Tf), Zymosan A, and An antibody or a binding fragment thereof specific for binding to the endocytosis receptor, wherein the endocytosis receptor is preferably selected from the group consisting of transferrin receptor (CD71), insulin-like growth factor 1 (IGF-I) receptor (IGF1R), tetraspanin CD63, muscle-specific kinase (MuSK), glucose transporter GLUT4, cation-independent mannose-6-phosphate receptor (CI-MPR), and LDL receptor.

2. The composition for use according to claim 1, wherein the composition is selected from any one of the following:

9. The composition for use of claim 1 , wherein the therapeutic nucleic acid is an oligonucleotide, defined as a nucleic acid not exceeding 150 nt.

10. The oligonucleotide may be selected from the group consisting of morpholino phosphorodiamidate oligomers (PMO), 2'-O-methyl (2'-OMe) phosphorothioate RNA, 2'-O-methoxyethyl (2'-O-MOE) RNA {2'-O-methoxyethyl-RNA (MOE)}, locked / bridged nucleic acids (BNA), 2'-O,4'-aminoethylene bridged nucleic acids (BNANC), peptide nucleic acids (PNA), 2'-deoxy-2'-fluoroarabinonucleic acid (FANA), 3'-fluoro-2'-aminoethylene bridged nucleic acids (BNANC ...

10. The composition for use according to claim 9, comprising or consisting of any one of orohexitol nucleic acid (FHNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), silencing RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), antagomir (miRNA antagonist), aptamer RNA or DNA, single-stranded RNA or DNA, double-stranded RNA (dsRNA) or double-stranded DNA.

11. the therapeutic nucleic acid is contained in a second conjugate, the therapeutic nucleic acid being covalently linked to a second ligand; and / or The combination of the first ligand and the second ligand is the following combination of ligands: Ligands for the transferrin receptor (CD71) and insulin-like growth factor 1 (IGF-I) receptor, Ligand for the transferrin receptor (CD71) and the tetraspanin CD63; Ligands for the transferrin receptor (CD71) and muscle-specific kinase (MuSK); Ligands for the transferrin receptor (CD71) and the cation-independent mannose 6-phosphate receptor (CI-MPR) - two ligands of the transferrin receptor (CD71), one ligand being transferrin and the other ligand being an antibody or binding fragment thereof specific for binding to the transferrin receptor (CD71); - two ligands of the LDL receptor, one of which is or comprises LDL and the other of which is an antibody or a binding fragment thereof specific for binding to said LDL receptor; The composition for use according to claim 1, selected from:

12. the covalent linkage of the saponin to the first ligand in the first conjugate is via a first linker to which the saponin is covalently attached; and / or 2. The composition for use according to claim 1, wherein the first linker is a cleavable linker that undergoes cleavage under acidic, reducing, enzymatic and / or photoinduced conditions.

13. A composition for use as described in claim 1, wherein the saponin is or comprises at least one molecule of SO1861, the therapeutic nucleic acid is drisapersen or eteplirsen, and the first ligand is an anti-CD71 antibody or a binding fragment thereof.

14. 10. The composition for use according to claim 1, for use in intravenous or subcutaneous administration to a human subject.

15. 2. The composition for use according to claim 1, comprising a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent.

Images