Compositions Comprising Therapeutic Nucleic Acids and Saponins for Treating Muscle Wasting Disorders - Patent application

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. With the latter aspect in mind, herein disclosed are pharmaceutical compositions and advantageous components thereof 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 an endosomal escape-promoting saponin in the pharmaceutical composition disclosed herein, comprising a therapeutic nucleic acid and, optionally, a muscle cell targeting ligand conjugated thereto. As demonstrated for the first time herein, this type of saponin surprisingly not only retains its endosomal escape-promoting properties in fully differentiated muscle cells, but can also be successfully delivered thereto together with a therapeutic nucleic acid, even in the unconjugated state.

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. With the latter aspect in mind, herein disclosed are pharmaceutical compositions and advantageous components thereof 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 an endosomal escape-promoting saponin in the pharmaceutical composition disclosed herein, comprising a therapeutic nucleic acid and, optionally, a muscle cell targeting ligand conjugated thereto. As demonstrated for the first time herein, this type of saponin surprisingly not only retains its endosomal escape-promoting properties in fully differentiated muscle cells, but can also be successfully delivered thereto together with a therapeutic nucleic acid, even in the unconjugated state. [Background technology]

[0002] Muscle wasting disorders are a major cause of human disease worldwide and can be caused by underlying genetic pathologies, as seen in various muscular dystrophies or congenital myopathies, among others [Cardamone, 2008], or can be age-related, such as the age-related muscle loss known as sarcopenia, or can 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 thanks to the presence of resident muscle stem cells called satellite cells (SCs), which activate, proliferate, and fuse upon injury to repair damaged or newly form 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. Moreover, these cells have a resilience, characterized in particular by contractility, extensibility, and elasticity, which are key features required for their primary function in muscle tissue, namely, to generate tension, thereby generating the force that contracts the muscle cells, thereby generating voluntary or involuntary movements of various body parts.

[0005] In light of this, it is also very well known that, despite the widespread acceptance 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 particularly 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 those affected by acquired, advanced or genetic muscle wasting disorders. In fact, the majority of them have no available drugs and palliative treatments are often the only available solution to alleviate their suffering.

[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. Thus, congenital myopathies are considered to be caused by genetic defects in the contractile apparatus within muscle cells 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 large forces by generating tension in a synchronized and controlled manner.

[0010] This structural and functional complexity of the muscle cell, including its intracellular contractile apparatus, the protein web that underpins and explains the specialized functions and organization of the sarcolemma, and its outer multicomponent scaffold, is the product of a large muscle cell-specific proteome.

[0011] A substantial portion of this proteome is large structural proteins translated from purely muscle cell-specific transcripts that arise from often very large, multi-exonic genes that are prone to extensive alternative splicing events [Savarese, 2020]. Indeed, a variety of mutations scattered along some of the largest genes in the human genome, including DMD, TTN, NEB, and RYR1, among others, are recognized as underlying causes of some of the best-characterized myocyte-related genetic disorders.

[0012] 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, and ultimately leading to death, usually due to myocardial or respiratory failure. DMD is recessive and X-linked (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 how affected the reading frame of the transcript is. 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.

[0013] 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 specifically bind 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 ultimately was 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.

[0014] 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.

[0015] Thousands of different mutations of DMD have been found in DMD or BMD patients [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 the COL6A gene encoding laminin α2 chain / collagen 6A); or familial dilated cardiomyopathy (disease gene: LMNA / lamin A / C), as well as nemaline myopathy (nemaline myopathy) among others. These include congenital myopathies, including myopathy (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 myopathy (disease genes: TPM3 / alpha tropomyosin-3, CTA / skeletal alpha actin, RYR1 / ryanodine receptor channel), as well as any syndrome involving mutations, for example, in the TTN gene (titin). Thus, due to the variability of mutations, even for 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.

[0016] 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 in turn 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 failure of many nucleic acid-based therapeutics in clinical trials, such as the DMD exon-skipping ASO drisapersen.

[0017] 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 genetic 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.

[0018] For a comprehensive review of the various different drug delivery approaches to striated muscle cells, DC Ebner et al., 2015, Curr Pharm Des, 21(10):1327-36. doi:10.2174 / 1381612820666140929095755 can be referred to, 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 WO 2018 / 129384 or WO 2020 / 028857. Summary of the Invention [Problem to be solved by the invention]

[0019] 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]

[0020] To address this need, the present inventors have developed and herein described novel pharmaceutical compositions comprising muscle cell-targeted therapeutic nucleic acids in combination with triterpenoid saponins of the 12,13-dehydrooleanane type, such specific saponin types being described, for example, in multicomponent conjugates disclosed in WO 2020 / 126620, where they were described as having endosomal escape-promoting activity for various antibody-drug conjugates (ADCs) in several cancer cell types.

[0021] Furthermore, in the context of tumor cells, such saponins are mentioned, for example, in WO2020126609, which describes the silencing of the HSP27 gene in various tumor models by combining saponin with BNAs for silencing HSP27 or by combining saponin in a conjugate of a monoclonal antibody directed against a tumor cell marker with BNAs for silencing HSP27.

[0022] However, terminally differentiated muscle cells, specifically cardiomyocytes, differ significantly from genetically unstable, constantly dividing tumor cells in terms of metabolism, as well as in terms of 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 quite different from the healthy tight junction-rich blood vessels that supply muscle tissue.

[0023] Despite these differences, we observed that combining muscle endocytosis receptor ligand-conjugated ASOs with 12,13-dehydrooleanane-type triterpenoid saponins had an unexpectedly robust effect on exon skipping efficiency in human and mouse DMD transcripts. Without wishing to be bound by any theory, we hypothesize that these findings indicate that this specific group of endosomal escape-promoting saponins has a highly potent ability to stimulate efficient exit of therapeutic nucleic acids from muscle cell endosomes into the appropriate muscle cell internal compartment (a highly desirable, but poorly understood, phenomenon for therapeutics, termed endosomal escape).

[0024] 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.

[0025] In summary, to address the shortcomings of the prior art, a novel pharmaceutical composition for use in the treatment or prevention of muscle wasting disorders, in particular muscle cell-associated genetic disorders, is presented herein, comprising an endosomal escape-promoting saponin of the 12,13-dehydrooleanane type and a therapeutic nucleic acid, advantageously conjugated with a ligand for targeting a muscle-specific endocytic receptor. The inventors have observed that the saponins described herein have a unique ability to efficiently deliver therapeutic nucleic acids to striated muscle cells, presumably by facilitating endosomal escape of the nucleic acid specifically in the target muscle cells. The findings presented herein pave the way for the development of novel, potentially less therapeutically burdensome and therefore safer, therapeutic methods for patients suffering from muscle wasting disorders. These and other advantages are further presented in the following sections.

[0026] In light of the above promising findings, disclosed herein are improved biologically active compounds and pharmaceutical compositions comprising a nucleic acid and a free form (i.e., not conjugated to a macromolecule) endocytic escape-promoting 12,13-dehydrooleanane-type saponin bearing an aldehyde group at the C-23 position of the saponin aglycone core structure.

[0027] 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.

[0028] The innovative concepts presented herein are described with reference to detailed embodiments, which are to be considered 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.

[0029] One of the objectives of some embodiments of the present disclosure is to provide a solution to the insufficient delivery of nucleic acids to the appropriate compartments of their target cells and the associated non-specificity problems encountered when administering nucleic acid-based therapeutics to human patients suffering from muscle wasting disorders and in need of such therapeutics.

[0030] A further object of some of these embodiments is to provide a solution to the problem of poor safety profiles of current nucleic acid-based drugs when administered to human patients in need thereof, particularly in excessive doses that induce side effects.

[0031] Yet a further object 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 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.

[0032] 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 Nucleic acids, and saponin, In a composition comprising This is achieved by providing a composition, wherein the saponin is a triterpenoid 12,13-dehydrooleanane-type saponin that contains an aldehyde group at the C-23 position of the saponin aglycone core structure under conditions present in the endosomes and / or lysosomes of human cells.

[0033] In a further aspect, at least one of the above objects is achieved by a therapeutic combination for the treatment or prevention of a muscle cell associated genetic disorder, comprising: (a) a nucleic acid, and (b) Saponin In a therapeutic combination comprising This is achieved by providing a therapeutic combination, wherein the saponin is a triterpenoid 12,13-dehydrooleanane-type saponin that contains an aldehyde group at the C-23 position of the saponin aglycone core structure under conditions present in the endosomes and / or lysosomes of human cells.

[0034] In further aspects, there are provided further embodiments of the compositions for therapeutic or prophylactic use disclosed herein and / or of the therapeutic combinations according to the present disclosure, which further address one or more of the above mentioned objectives.

[0035] In particularly advantageous aspects, various different embodiments of the present disclosure are provided that include advantageous components, such as those selected from a preferred subtype of endosomal escape-promoting saponin, a different therapeutic nucleic acid, such as an antisense oligonucleotide, configured to induce skipping of a defective exon of a wasting muscle cell disorder-associated gene transcript, and an advantageous ligand or combination thereof for targeting said nucleic acid to an endocytic receptor on a muscle cell, as well as a covalent linker for connecting the ligand to the nucleic acid, preferably configured to be cleavable under conditions present in a human endosome.

[0036] These and other aspects of the present disclosure are presented in detail in subsequent sections.

[0037] definition The term "saponin" has its usual established meaning and refers herein 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 may 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 a conjugate comprising 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.

[0038] 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 a covalent bond 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. Saponin derivatives in the context of the present invention should be understood as saponin functional derivatives. "Functionality" in the context of saponin derivatives is understood as the ability or activity of the saponin or saponin derivative to promote endosomal escape of effector molecules that contact a cell together with the saponin or saponin derivative.

[0039] 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.

[0040] 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.

[0041] The term "Api / Xyl-" or "Api- or Xyl-" in the context of glycan names has its ordinary scientific meaning and refers herein to glycans that either contain an apiose (Api) moiety or a xylose (Xyl) moiety.

[0042] 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 appropriate 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).

[0043] 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.

[0044] 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 as the 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.

[0045] 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.

[0046] 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. according to the definition provided above, 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.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.

[0047] As used herein, the term "endocytic receptor on a muscle cell" should be understood to refer to a surface molecule whose specific ligand is accessible from the outside or surface of the muscle cell sarcolemma and may be a receptor or transporter that 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 can 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 that 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.

[0048] 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, and any conjugate of an antibody or antibody fragment thereof, such as an ...

[0049] 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. Patent No. 5,693,780 and Kabat EA et al, (1991), supra. In some embodiments, the VH domain comprises an amino acid sequence 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).

[0050] The term "single domain antibody", or abbreviated "sdAb" or "nanobody", has its usual scientific meaning and is used herein to refer to an antibody fragment consisting of a single monomeric variable antibody domain, unless it refers to two or more monomeric variable antibody domains, such as, for example, 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 referred to as a 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 5,000 Daltons, even more preferably less than 2,000 Daltons, and most preferably less than 1,500 Daltons.

[0051] 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 includes at least one or more atoms.

[0052] 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 a conjugate as disclosed herein can include one or more nucleic acid or oligonucleotide molecules and one or more ligands that bind to an endocytic receptor present on the surface of a muscle cell, preferably an antibody or a binding fragment thereof, such as an IgG, a monoclonal antibody (mAb), a VHH domain or another nanobody type, a bivalent nanobody molecule comprising two single domain antibodies, etc. In some embodiments, 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.

[0053] 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.

[0054] 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 any direction from the stated reference value (greater or less than), unless otherwise stated or otherwise clear from the context (except where such number would exceed 100% of possible values).

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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".

[0059] 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.

[0060] 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.

[0061] "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%).

[0062] 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%).

[0063] 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%).

[0064] 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, also known as Quillaja saponin, is a fraction 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.

[0065] 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.

[0066] 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]

[0067] [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] Generation of hCD71-DMD-ASO with hCD71-PEG4-SPDP precursor [Figure 2C] Preparation of hCD71-DMD-PMO with hCD71-PEG4-SPDP precursor [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 7 [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 6] SO1861-EMCH and SO1861-SC-maleimide, schematic representation. [Figure 7] 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 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 DMD-affected donor (DM8036). [Figure 9A] 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 9B] 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 9C] 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 10] (A) Serum creatinine and (B) serum ALT analysis on days 4, 14, and 28 from a single dose study with mCD71-M23D (Group 2) and vehicle control (Group 1). [Figure 11A] Synthesis of Intermediate 3 (via Intermediates 1 and 2) [Figure 11B] Synthesis of intermediate 4 [Figure 11C] To synthesize intermediate 5 by coupling intermediate 4 with M23D-SH (reduced form). [Figure 11D]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 12A] 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 12B] 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 12C] Schematic representation of the conjugation procedure for mAb-M23D, including mCD71-M23D and mCD63-M23D. Legend explaining the symbolically represented glycan residues. [Figure 12D] Schematic representation of the conjugation procedure for mAb-M23D, including mCD71-M23D and mCD63-M23D. Legend explaining the symbolically represented molecules. [Figure 13] 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 14A] Schematic representation of the conjugation procedure for hAb-DMD-oligos, including hCD71-5'-SS-DMD-ASO, hCD71-5'-SS-DMD-PMO (1), and hCD71-3'-SS-DMD-PMO (1-5), 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 14B]Schematic representation of the conjugation procedure of hAb-DMD-oligos, including hCD71-5'-SS-DMD-ASO, hCD71-5'-SS-DMD-PMO (1), and hCD71-3'-SS-DMD-PMO (1-5), involving activation of protected DMD-oligos. Note: For clarity of the schematic representation, hAb-DMD-oligos are indicated with DAR4. [Figure 14C] Schematic representation of the conjugation procedure of hAb-DMD-oligos, including hCD71-5'-SS-DMD-ASO, hCD71-5'-SS-DMD-PMO (1), and hCD71-3'-SS-DMD-PMO (1-5), involving disulfide bond formation between activated DMD-oligo-SH and hAb-PEG4-SPDP. Note: For clarity of the schematic representation, hAb-DMD-oligos are indicated with DAR4. [Figure 14D] Schematic representation of the conjugation procedure of hAb-DMD-oligos, including hCD71-5'-SS-DMD-ASO, hCD71-5'-SS-DMD-PMO (1), and hCD71-3'-SS-DMD-PMO (1-5), with reference to the figure legend. Note: For clarity of the schematic representation, hAb-DMD-oligos are indicated with DAR4. [Figure 15] 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 16A] 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 16B]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 16C] 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 17] 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). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] Disclosed herein are improved biologically active pharmaceutical compositions comprising an endocytosis escape-promoting saponin and a therapeutic nucleic acid, preferably linked to a ligand for targeting a muscle cell surface endocytic receptor. The compositions disclosed herein are particularly advantageous 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.

[0069] 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.

[0070] One of several objects of the embodiments of the present disclosure is to provide a solution to the problem of delivery inefficiencies encountered when administering nucleic acid-based therapeutics to human patients suffering from muscle wasting disorders and in need of such therapeutics. Another of several objects of the present embodiments 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 their insufficient ability to reach and / or enter diseased muscle cells with little to no off-target activity against non-diseased cells.

[0071] 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 by endocytosis receptor-mediated endocytosis.

[0072] 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.

[0073] 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.

[0074] 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 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.

[0075] As will be demonstrated by the data presented herein, inclusion of triterpenoid 12,13-dehydrooleanane-type saponins, which contain an aldehyde group at the C-23 position of the saponin aglycone core structure, in the therapeutic compositions of the present disclosure appeared to stimulate efficient exit of therapeutic nucleic acids from muscle cell endosomes to the appropriate internal muscle cell compartment.

[0076] 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 saponin In a composition comprising The composition is provided, wherein the saponin is a triterpenoid 12,13-dehydrooleanane-type saponin that contains an aldehyde group at the C-23 position of the saponin aglycone core structure under acidic conditions present in the endosomes and / or lysosomes of human cells.

[0077] In other words, in a general aspect the invention provides a pharmaceutical composition for use in the treatment or prevention of a muscle wasting disorder comprising: Nucleic acids, and saponin, In a composition comprising Saponin is attached to the C-23 position of the saponin aglycone core structure. An aldehyde group; or an acid-sensitive cleavable covalent bond adapted to be cleaved under acidic conditions, such that said cleavage generates or restores an aldehyde group at the C-23 position of the saponin aglycone core structure; and a triterpenoid 12,13-dehydrooleanane type saponin containing any one of the following: Furthermore, the composition is a triterpenoid 12,13-dehydrooleanane-type saponin that contains an aldehyde group at the C-23 position of the saponin aglycone core structure under the acidic conditions present in the endosomes and / or lysosomes of human cells.

[0078] In the next general aspect, the present invention also provides a therapeutic combination, e.g. for the treatment or prevention of a muscle cell-related genetic disorder, comprising: (a) a nucleic acid, and (b) Saponin In a therapeutic combination comprising Also provided is a therapeutic combination, wherein the saponin is a triterpenoid 12,13-dehydrooleanane-type saponin that contains an aldehyde group at the C-23 position of the saponin aglycone core structure under the acidic conditions present in the endosomes and / or lysosomes of human cells.

[0079] Thus, in the next general aspect, the present invention provides a therapeutic combination, preferably for the treatment or prevention of a muscle cell associated genetic disorder, comprising: (a) a nucleic acid, and (b) Saponin In a therapeutic combination comprising At the C-23 position of the saponin aglycone core structure, An aldehyde group; or an acid-sensitive cleavable covalent bond adapted to be cleaved under acidic conditions, such that said cleavage generates or restores an aldehyde group at the C-23 position of the saponin aglycone core structure; A triterpenoid 12,13-dehydrooleanane type saponin comprising any one of Thus, there is provided a therapeutic combination that is a triterpenoid 12,13-dehydrooleanane-type saponin that contains an aldehyde group at the C-23 position of the saponin aglycone core structure under the acidic conditions present in the endosomes and / or lysosomes of human cells.

[0080] As used herein, the context will understand that the nucleic acids forming part of the compositions disclosed herein for therapeutic purposes and therapeutic combinations are selected to have therapeutic activity for treating or preventing the selected muscle wasting disorder. In other words, the nucleic acid as contained in the compositions / combinations 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, based on either his knowledge of the mutations that cause such disorders or the genetic mutation screening results of a given patient, which therapeutic nucleic acid should be included in a new composition / combination as disclosed herein to perform an improved treatment.

[0081] 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.

[0082] In a particularly advantageous embodiment, a composition for therapeutic or prophylactic use as disclosed herein is provided, wherein the treatment or prevention of a muscle wasting disorder preferably involves antisense therapy involving exon skipping.

[0083] Saponins suitable for application in the compositions and therapeutic combinations disclosed herein have a triterpene 12,13-dehydrooleanane-type backbone in which the basic triterpene structure is a pentacyclic C30 terpene backbone (also called sapogenin or aglycone) and further contains an aldehyde group at the C-23 position in its native, underivatized state. Examples of such known saponins are shown in Table 1.

[0084] [Table 1]

[0085] [Table 2]

[0086] [Table 3]

[0087] [Table 4]

[0088] [Table 5]

[0089] [ka]

[0090] A notable feature of these saponins is an aldehyde group located at the C-23 position of the saponin aglycone core structure. 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 the saponin (also referred to herein as the "aglycone") is particularly beneficial to the ability of the saponin to stimulate and / or enhance endosomal escape of therapeutic nucleic acids contained in the conjugates of the invention.

[0091] It appears particularly beneficial for the endosomal escape promoting properties that the aldehyde group at the C-23 position of the saponin aglycone core structure becomes a free aldehyde group once inside the endosome, and thus, to the preferred saponins disclosed herein, whose native forms are shown in Table 1, in principle any chemical modification can be applied that uncaps or restores the free aldehyde group at that position at the C-23 position of the saponin aglycone core structure under the acidic conditions present in the endosomes and / or lysosomes of human cells.

[0092] As disclosed herein, such chemical modifications include converting or replacing the aldehyde group at C-23 with an acid-sensitive cleavable covalent bond adapted to be cleaved under acidic conditions, such that said cleavage generates or restores an aldehyde group at C-23 of the saponin aglycone core structure, resulting in an aldehyde-capped (or aldehyde-protected) saponin derivative that contains an aldehyde group at C-23 of the saponin aglycone core structure after entry into the endosomal compartment of a mammalian (e.g., human) cell. For such designed bonds, restoration of the aldehyde group at C-23 is a result of acidity causing cleavage of said acid-sensitive covalent bond in response to the acidic conditions present in mammalian / human endosomal and / or lysosomal compartments.

[0093] In light of the above, in a further embodiment, a composition or therapeutic combination for therapeutic or prophylactic use as disclosed herein is disclosed, wherein the acid sensitive cleavable covalent bond is 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 the acid sensitive cleavable covalent bond is either a semicarbazone bond or a hydrazone bond.

[0094] In advantageous embodiments, such cleavable covalent bonds may be selected from semicarbazone bonds, hydrazone bonds, or imine bonds.

[0095] For example, it has been observed that when the aldehyde group is replaced by a maleimide-containing moiety attached to the C-23 position with a cleavable covalent bond that is cleaved off under acidic conditions present 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, the endosomal escape promoting properties of such saponins are still highly evident.

[0096] Thus, in the following embodiments, compositions or therapeutic combinations for therapeutic or prophylactic use as disclosed herein are provided, wherein an acid-sensitive, cleavable covalent bond links a maleimide-containing moiety to the C-23 position of the saponin aglycone core structure.

[0097] For example, in further 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 saponin aglycone core structure 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 saponin aglycone core structure upon formation of a hydrazone bond (hereinafter referred to as EMCH).

[0098] Thus, in possible embodiments, compositions or therapeutic combinations for therapeutic or prophylactic use as disclosed herein are disclosed, wherein the aldehyde group at C-23 position of the saponin aglycone core structure is either a free aldehyde group or an aldehyde group substituted by a maleimide-containing moiety attached to said C-23 position with a cleavable covalent bond that is cleaved away under acidic conditions present in the endosomes and / or lysosomes of human cells, such that said cleavage under acidic conditions present in the endosomes and / or lysosomes of human cells restores the aldehyde group at C-23 position of said saponin aglycone core structure; preferably, wherein the cleavable covalent bond is selected from a semicarbazone bond, a hydrazone bond, or an imine bond; more preferably, selected from a semicarbazone bond and a hydrazone bond; most preferably, a hydrazone bond.

[0099] In another embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination of the disclosure, wherein the maleimide-containing moiety is part of a molecule comprising or consisting 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 saponin aglycone core structure upon formation of a semicarbazone bond (hereinafter referred to as SC-maleimide), or wherein the maleimide-containing moiety is part of a molecule comprising or consisting of N-ε-maleimidocaproic acid hydrazide which is attached to the C-23 position of the saponin aglycone core structure upon formation of a hydrazone bond (hereinafter referred to as EMCH).

[0100] In further specific embodiments, then, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination of the present disclosure, wherein the saponin is a free saponin, defined as consisting of one or more saponin molecules that lack direct or indirect covalent conjugation to a macromolecule, such as a nucleic acid or a ligand.

[0101] In a preferred embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination of the present disclosure, wherein the composition comprises 2-6 μM saponin, preferably 3-5 μM, more preferably 3.5-4.5 μM, and most preferably about 4 μM saponin.

[0102] Many of the known 12,13-dehydrooleanane type saponins that naturally contain an aldehyde group at the C-23 position in their native or unconjugated form are saponins whose aglycone core structures are either queratic acid or gypsogenin. An exemplary such saponin is depicted as saponin A and is illustrated by the following structure:

[0103] [ka]

[0104] With this in mind, it has been observed that saponins containing a quilacic acid aglycone or a gypsogenin aglycone core structure are particularly suitable for the purposes of the present disclosure.

[0105] Thus, in a further embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination of the present disclosure, wherein the saponin aglycone core structure is selected from any one or more of quillaric acid, gypsogenin, and derivatives thereof, preferably wherein the saponin aglycone core structure is quillaric acid or gypsogenin, more preferably quillaric acid.

[0106] In light of the above, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination of the present disclosure, wherein the saponin aglycone core structure is -Quiras acid, · quilacic acid derivatives in which the aldehyde group at C-23 of quilacic acid is converted into an acid-sensitive cleavable covalent bond at C-23 of quilacic acid; Gypsogenin, and Gypsogenin derivatives in which the aldehyde group at C-23 of gypsogenin has been converted into an acid-sensitive cleavable covalent bond at C-23 of gypsogenin Selected from one or more of the following: Preferably herein the saponin aglycone core structure is quillaric acid or a quillaric acid derivative in which the aldehyde group at the C-23 position of quillaric acid has been converted to an acid-sensitive cleavable covalent bond at the C-23 position of quillaric acid.

[0107] Saponins can include one or more sugar chains attached to an aglycone core structure. Preferred saponins of the compositions or therapeutic combinations 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 that contains an aldehyde group at the C-23 position.

[0108] Glycosylation has also been hypothesized to play a role in endosomal escape-promoting properties.

[0109] 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 of the disclosure, wherein the saponin is at least a bidesmosidic saponin comprising a first glycan comprising a terminal glucuronic acid residue and a second glycan comprising at least four saccharide residues in a branched arrangement; preferably wherein the first glycan is Gal-(1→2)-[Xyl-(1→3)]-GlcA, and / or wherein the branched second glycan of at least four saccharide residues comprises a terminal fucose residue and / or a terminal rhamnose residue.

[0110] In related embodiments, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination of the present 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.

[0111] In another embodiment there is provided a composition as disclosed herein for therapeutic or prophylactic use or a therapeutic combination of the disclosure, wherein 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 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 containing a chiral acid aglycone core structure selected from Any one or more of the following: Preferably the saponin is one or more of the saponins selected from list B or C, more preferably from list C.

[0112] In particular embodiments, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination of the present 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, officinalis), any one or more of saponins, saponarioside B, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862 and SO1904; preferably, wherein the saponin is any one or more of QS-21, SO1832, AG1856, SO1861, SA1641 and GE1741; more preferably, wherein the saponin is QS-21, SO1832 or SO1861; and most preferably SO1861.

[0113] In more particular embodiments, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination of the present 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; and most preferably SO1861.

[0114] In further detailed embodiments, a composition or therapeutic combination as disclosed herein may be prepared with a suitable triterpenoid 12,13-dehydrooleanane type saponin comprising an aldehyde group at the C-23 position of the saponin aglycone core structure, wherein one or 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.

[0115] In more particular embodiments, there can be provided a composition or therapeutic combination as disclosed, wherein the at least one triterpenoid 12,13-dehydrooleanane-type saponin comprising an aldehyde group at the C-23 position of the saponin aglycone core structure further comprises: 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 of the derivatizations i., ii. and / or iii. Includes.

[0116] In specific embodiments, compositions and therapeutic combinations are provided, wherein at least one saponin comprises a first glycan and a second glycan, wherein the first glycan comprises two or more sugar moieties, the second glycan comprises two or more sugar moieties, and wherein the aglycone core structure is quillic acid or gypsogenin, more preferably quillic acid, and wherein one, two or three, preferably one or two of the following are present: 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.

[0117] 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.

[0118] In a specific embodiment, a composition or therapeutic combination according to the present disclosure is provided, wherein the aldehyde functional group at the C-23 position of the saponin aglycone core structure is covalently bound to an EMCH cap. The binding of EMCH to the aldehyde group of the saponin aglycone results in the formation of a hydrazone bond. Such a hydrazone bond is a typical example of a cleavable bond that restores the aldehyde group at the C-23 position of the saponin aglycone core structure under the acidic conditions inside endosomes and lysosomes.

[0119] The development of the advantageous compositions presented herein is based on the surprising realization that, by virtue of the inclusion of an endosomal escape-promoting saponin in the compositions of the invention, nucleic acids can be delivered to muscle cells with improved efficiency, thereby aiding in the treatment and / or prevention of muscle wasting disorders.

[0120] With this in mind, in an advantageous embodiment, the nucleic acid is a therapeutic nucleic acid adapted to a genomic mutation within a mutated transcript or gene that is affected in 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.

[0121] 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 can 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 disproportion myopathy) or advantageously the TTN gene (titin).

[0122] In possible embodiments, a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination of the present disclosure is provided, wherein the nucleic acid is any one of the following: morpholino phosphorodiamidate oligomers (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 (BNA), 2'-O,4'-aminoethylene bridged nucleic acid (BNANC), peptide nucleic acid (PNA), 2'-deoxy-2'-fluoroarabinonucleic acid (FANA), 3'-fluoro-2'-aminoethyl ... The nucleic acid oligonucleotide may comprise or consist of a 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; preferably wherein the nucleic acid oligonucleotide comprises or consists of a morpholino phosphorodiamidate oligomer (PMO) or 2'-O-methyl (2'-OMe) phosphorothioate RNA.

[0123] In a further embodiment there is provided a composition or therapeutic combination for therapeutic or prophylactic use as disclosed herein, wherein the nucleic acid 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; More preferably, wherein the nucleic acid 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, Even more preferably wherein the nucleic acid is selected from eteplirsen, drisapersen, golodirsen, viltolarsen, and casimersen.

[0124] In a particularly advantageous embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination of the present disclosure, wherein the nucleic acid is an oligonucleotide, defined as a nucleic acid not exceeding 150 nt, preferably wherein the oligonucleotide is 5-150 nt in size, preferably 8-100 nt, most preferably 10-50 nt. Preferably, the oligonucleotide is an antisense oligonucleotide, even more preferably a mutation-specific antisense oligonucleotide, most preferably an oligonucleotide designed to induce exon skipping.

[0125] As used herein, the term oligonucleotide shall be understood to include both oligomers made of naturally occurring nucleotides and thus chemically oligonucleotides, as well as oligomers including modified oligonucleotides and their analogs.For example, synthetic oligomers may include 2'-modified nucleosides, which 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), etc. In light of this, in possible embodiments, the oligonucleotide may be structurally or functionally selected from 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 (FAN ... A), 2'-O-methoxyethyl-RNA (MOE), 3'-fluorohexitol nucleic acid (FHNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), BNA-based siRNA, and BNA-based antisense oligonucleotide (BNA-AON), chemically modified siRNA, metabolically stable siRNA, and chemically modified metabolically stable siRNA, or any other category known in the art.

[0126] From a functional point of view, in an advantageous embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination of the present disclosure, wherein the oligonucleotide is an antisense oligonucleotide, preferably a mutation-specific antisense oligonucleotide, most preferably an oligonucleotide designed to induce exon skipping.

[0127] In related embodiments, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination of the present disclosure, wherein the oligonucleotide comprises or consists of any one of the following: morpholino phosphorodiamidate oligomers (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 (BNA), 2'-O,4'-amino Ethylene-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.

[0128] In a particularly preferred embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate 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.

[0129] 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 of the present disclosure, wherein the oligonucleotide is designed to induce exon skipping of the human dystrophin gene transcript, preferably wherein the exon skipping involves exon 51 skipping or exon 53 skipping or exon 45 skipping, more 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.

[0130] In a more detailed embodiment building on the immediately preceding embodiment, the oligonucleotide is selected from eteplirsen, drisapersen, golodirsen, viltolarsen, and casimersen.

[0131] In an advantageous embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination of the present disclosure, wherein the composition comprises two or more different nucleic acids, the two or more different nucleic acids are preferably two or more different oligonucleotides, more preferably wherein at least one of the two or more different oligonucleotides is an antisense oligonucleotide.

[0132] Such combinations of two or more therapeutic nucleic acids are known in the art, for example for muscle wasting disorders, a combinatorial approach based on two AONs for double exon skipping in myostatin and dystrophin has been proposed for the management of Duchenne muscular dystrophy [Kemaladewi el al, 2011].

[0133] In particularly preferred embodiments, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination of the present disclosure, wherein the nucleic acid is conjugated to a ligand of an endocytic receptor on muscle cells.

[0134] Regarding the ligands of endocytic receptors on muscle cells, it must be noted that many 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, to date many endocytic receptors present on the surface of muscle cells have been characterized, with the transferrin receptor (CD71) and possibly the insulin-like growth factor 1 (IGF-I) receptor (IGF1R) being the most studied. The ligands of these receptors can be selected from natural ligands, such as transferrin (Tf), the natural ligand of CD71, or LDL, the ligand of the LDL receptor. Alternatively, the receptor may be a non-naturally occurring ligand, such as various antibodies or binding fragments thereof, or alternatively, a synthetic ligand such as zymosan A, which binds to the endocytic mannose receptor, or a synthetic fragment of a naturally occurring ligand, such as a fragment of IGF-I, which is a ligand for IGF1R, or a fragment of IGF-II, which is a ligand for CI-MPR (also known as IGF2R).

[0135] In a preferred embodiment, there is provided a therapeutic or prophylactic use as disclosed herein or a therapeutic combination of the present disclosure, wherein the endocytic receptor on muscle cells to which the ligand conjugated to the nucleic acid binds is selected from the transferrin receptor (CD71), the insulin-like growth factor 1 (IGF-1) receptor (IGF1R), the tetraspanin CD63; the muscle-specific kinase (MuSK), the glucose transporter GLUT4, the cation-independent mannose 6-phosphate receptor (CI-MPR).

[0136] In a further preferred embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination of the present disclosure, wherein the ligand conjugated to the nucleic acid 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 (IGF-IR), 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 ligand is an antibody or binding fragment thereof specific for binding to the transferrin receptor, more preferably wherein the ligand is a monoclonal antibody or a Fab' fragment or at least one single domain antibody specific for binding to the transferrin receptor, and even more preferably wherein the ligand is a monoclonal antibody specific for binding to the transferrin receptor.

[0137] In a related particularly advantageous embodiment, the ligand conjugated to the nucleic acid is a monoclonal antibody or a Fab' fragment or at least one single domain antibody specific for binding to the transferrin receptor, and even more preferably wherein the ligand is a monoclonal antibody specific for binding to the transferrin receptor.

[0138] In a further advantageous embodiment, the ligand conjugated to the nucleic acid is a monoclonal antibody, such as a humanized or human monoclonal antibody, an IgG, a molecule comprising or consisting of a single domain antibody, a molecule having at least one V HH domain, preferably camelid V H , variable heavy chain novel antigen receptor (VNAR) domains, Fab, scFv, Fv, dAb, F(ab)2 and Fcab fragments.

[0139] In certain advantageous embodiments, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination of the present disclosure, wherein the ligand is conjugated to 2-5 molecules of nucleic acid per molecule of ligand; preferably 3-4 molecules of nucleic acid per molecule of ligand; more preferably wherein the ligand is conjugated to an average of 4 molecules of nucleic acid per molecule of ligand.

[0140] In certain embodiments, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination of the present disclosure, wherein the ligand 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 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 nucleic acid molecules are linked to one ligand molecule via distinct cysteine ​​residues and / or distinct lysine residues; More preferably, wherein the ligand comprises a chain of amino acid residues comprising a multi-cysteine ​​repeat, optionally a tetracysteine ​​repeat represented by the sequence HRWCCPGCCKTF (SEQ ID NO: 4), and wherein the covalent linkage to the ligand by the nucleic acid comprises a covalent bond to any one or more of the cysteine ​​residues of the multi-cysteine ​​repeat; Most preferably, herein, two or more nucleic acid molecules are linked to one ligand molecule via distinct cysteine ​​residues of the multi-cysteine ​​repeat.

[0141] In a related embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination of the present disclosure, wherein the covalent linkage of the nucleic acid to the ligand is via a linker to which the nucleic acid is covalently attached; Preferably, the linker herein comprises or consists of the linker 3-(2-pyridyldithio)succinimidyl propionate (SPDP); optionally, the linker herein covalently links the nucleic acid to a lysine residue of the ligand or to a glycan residue, preferably a partially trimmed glycan.

[0142] In certain applications, it may be advantageous if one or more of the nucleic acid molecules, e.g., oligonucleotide molecules, are linked to the ligand via a cleavable bond, where the cleavable bond is susceptible to cleavage, e.g., under acidic, reducing, enzymatic and / or light-induced conditions. Cleavable bonds that are susceptible to cleavage under acidic conditions present in the endosomes and / or lysosomes of human cells are preferred.

[0143] In light of the above, in a related advantageous embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination of the present disclosure, wherein the linker is a cleavable linker that undergoes cleavage under acidic, reducing, enzymatic and / or light-induced conditions; Preferably, the 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 comprising a cleavable bond selected from: More preferably, wherein the linker is Bonds that undergo cleavage under acidic conditions, such as semicarbazone bonds, hydrazone bonds, or imine bonds; and / or bonds that are susceptible to proteolysis, e.g., by cathepsin B, and / or Bonds that are easily cleaved under reducing conditions, such as disulfide bonds The cleavable bond is selected from:

[0144] Advantageously, the bond is an acid-sensitive bond, i.e., it will undergo in vivo cleavage under acidic conditions present in the endosomes and / or lysosomes of human cells, preferably at pH≦6.5, preferably at pH≦6, more preferably at pH≦5.5, more preferably it is an acid-sensitive bond selected from a semicarbazone bond and a hydrazone bond; most preferably it is a hydrazone bond.

[0145] Such cleavable bonds are preferably less susceptible to cleavage, or are cleaved only to a small extent, when the nucleic acid ligand conjugate is present outside the endosomes and lysosomes of a cell, such as when the nucleic acid ligand conjugate is outside the cell or in an endocytic vesicle after the conjugate has engaged an endocytic receptor by binding of the ligand to its target endocytic receptor on a muscle cell. For example, the cleavable bond is preferably less susceptible to cleavage when the conjugate is present in the circulation of a human subject and / or extracellularly in an organ of a human subject, as compared to the susceptibility of the bond to cleavage when the conjugate is present in an endosome or lysosome of a target cell, herein a muscle cell.

[0146] In particular embodiments, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the present disclosure, wherein the linker is directly or indirectly covalently linked to the ligand.

[0147] In further particular embodiments, there is provided a composition as disclosed herein for therapeutic or prophylactic use or a therapeutic combination of the disclosure, wherein the saponin is or comprises at least one molecule of either SO1861, SO1861-EMCH, or SO1861-SC-maleimide, preferably SO1861-EMCH or SO1861-SC-maleimide, the nucleic acid is drisapersen or eteplirsen, and the ligand conjugated to the nucleic acid is an anti-CD71 antibody or a binding fragment thereof. For the therapeutic or prophylactic uses disclosed herein, the use is for intravenous or subcutaneous or intramuscular administration to a human subject, preferably for intramuscular administration.

[0148] In another embodiment, the compositions disclosed herein for therapeutic or prophylactic use, the use is for intravenous or subcutaneous or intramuscular administration to a human subject, preferably for intramuscular administration.

[0149] In yet another embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a therapeutic combination of the present disclosure comprising a pharma- ceutically acceptable excipient and / or a pharma-ceutically acceptable diluent.

[0150] In a related aspect, there is further provided a kit comprising components (a) and (b) of the therapeutic combination, optionally wherein components (a) and (b) are in separate vials, preferably wherein components (a) and (b) are provided in a mixture suitable for subcutaneous or intramuscular injection.

[0151] Finally, in particular embodiments, the therapeutic combinations and / or kits according to the present disclosure are provided for use as medicines. EXAMPLES

[0152] SO1861 was isolated and purified from a crude plant extract obtained from soapwort (Saponaria officinalis L) by Extrasynthese, France and / or Analyticon Discovery GmbH, Germany.

[0153] An antisense oligonucleotide with the sequence 5'-UCAAGGAAGAUGGCAUUUCU-3' [SEQ ID NO: 1] and an ASO with the same sequence but a thiol modification (DMD-ASO and 5'-thiol-DMD-ASO, respectively) were custom-ordered and purchased from Hanugen Therapeutics Pvt Ltd. A PMO with the sequence 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (DMD-PMO or DMD-PMO(1)) [SEQ ID NO: 2] and a PMO with the same sequence but a disulfide amide modification (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.

[0154] Anti-CD71 antibody (clone OKT9) targeting human CD71 (hCD71) and anti-CD71 antibody (clone R17 217.1.3) targeting mouse (mCD71) were both purchased from BioXCell. Anti-CD63 antibody (clone NVG-2) targeting mouse (mCD63) was purchased from Biolegend. IGF-1 ligand was purchased from PeproTech.

[0155] 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.The following reagents were used: Zeba spin desalting columns 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.

[0156] [Table 6]

[0157] 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

[0158] Methods of Examples 1 to 4 SO1861-maleimide, SO1861-NHS synthesis SO1861 is derived from Saponaria officinalis L (Analyticon Discovery GmbH, Germany) and was coupled to the respective handle by Symeres (The Netherlands) according to methods known in the art.

[0159] 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).

[0160] 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).

[0161] 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).

[0162] 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.

[0163] 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).

[0164] 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.

[0165] Preparative MP-LC method 2 Model: Reveleris (trademark) separation MPLC; KRARA: Phenomenex LUNA C18(3) (150×25mm, 10μm); flow rate: 40mL / min; KURAL temperature: room temperature; dissolution solution A: 0.1% (v / v) acid in water, eluate B: 0.1% (v / v) acid in Asterton; 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; UV output: 210, 235, 254nm and ELSD.

[0166] Fractional LC-MS method 3 MS model: Agilent Technologies G6130B quadrupole; HPLC model: Agilent Technologies 1290 fractionation LC; cartridge: Waters XSelect (trademark) CSH (C18, 150×19 mm, 10 μm); flow rate: 25 mL / min; cartridge temperature: room temperature; eluent A: 100% acelite; eluent B: 10 mM bicarbonate ammonium hydroxide in water, pH=9.0; graphene: 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.

[0167] 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

[0168] 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.

[0169] 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

[0170] 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.

[0171] 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.

[0172] 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).

[0173] 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.

[0174] 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).

[0175] 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).

[0176] 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).

[0177] 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).

[0178] 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).

[0179] 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).

[0180] 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).

[0181] 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).

[0182] 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).

[0183] 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).

[0184] Cell culture (human) Immobilized human myoblasts from a non-DMD donor (KM155) and a DMD 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, and 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).

[0185] 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.

[0186] 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 yield a total volume of 20 μl for each reaction. RT-PCR was performed at 42° C. for 60 min, then at 85° C. for 5 min and chilled on ice. For skip analysis, a 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, and the skipped products are 175 bp (KM155) or 242 bp (DM8036), respectively.

[0187] 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 to 10.0μl ddH2O, 4.0μl 5x iScript™ Reaction Mix and 1.0μl iScript™ Reverse Transcriptase (BioRad) to yield a total volume of 20.0μl for each 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, and 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 produce 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, and 72° C. for 5 s, followed by 1 min at 72° C. 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.

[0188] 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).

[0189] 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 thereafter) 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 analysis. Dystrophin skip levels were determined in heart, diaphragm, and gastrocnemius samples.

[0190] [Table 7]

[0191] Method of Example 5 SO1861-SC-Maleimide SO1861 is derived from Saponaria officinalis L (Extrasynthese, France) and was coupled to the respective handles by Symeres (The Netherlands) according to methods known in the art.

[0192] Synthesis of DBCO-(M23D)2 The synthesis of DBCO-(M23D)2 was carried out by Symeres (The Netherlands).

[0193] Conjugation of DBCO-(M23D)2 and antibodies Custom conjugate production of mCD71-M23D and mCD63-M23D was carried out by Abzena (UK).

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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 with a 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

[0204] 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 a period of 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 coevaporated 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)

[0205] 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)

[0206] 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)

[0207] 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)

[0208] 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)

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] [Table 8]

[0214] 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.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] [Table 9]

[0219] 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 an additional 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.

[0220] Exon skipping analysis and quantification (mouse in vitro) Performed as described above in "Methods" (as carried out in Examples 1-4).

[0221] Cell viability (mouse in vitro) Performed as described above in "Methods" (as carried out in Examples 1-4).

[0222] Method of Example 6 SO1861-SC-Maleimide SO1861 is derived from Saponaria officinalis L (Extrasynthese, France) and was coupled to the respective handles by Symeres (The Netherlands) according to methods known in the art.

[0223] 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), and hCD71-3'-SS-DMD-PMO(5) was carried out by Fleet Bioprocessing (UK).

[0224] 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

[0225] 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.

[0226] 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).

[0227] 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.

[0228] 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).

[0229] hAb-DMD-oligonucleotide The conjugation of hAb-targeted DMD oligonucleotides, including hCD71-5'-SS-DMD-ASO, hCD71-5'-SS-DMD-PMO(1), and hCD71-3'-SS-DMD-PMO(1-5), is outlined below, with the amounts shown in italics within the brackets being shown as an example for hCD71-5'-SS-DMD-PMO(1).

[0230] 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.

[0231] 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).

[0232] 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)

[0233] Cell culture (human) Immortalized human myoblasts from a non-DMD donor (KM155) were cultured as described above in "Methods" (as carried out in Examples 1-4).

[0234] 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, exon 51: h53R 5'-CTCCGGTTCTGAAGGTGTTC-3' [SEQ ID NO: 5]; exon 53: 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. 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 with the Ultra Sensitivity NGS kit (Agilent) 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).

[0235] result 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 % 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 34% at 20 μM, pointing to a 16-fold increase in potency for the non-targeted oligo in conjunction with the non-targeted SO1861-EMCH (Figure 1B).

[0236] 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.

[0237] [Table 10]

[0238] Example 2. hCD71-DMD-ASO + SO1861-EMCH or hCD71-DMD-PMO + SO1861-EMCH or mCD71-M23D + SO1861-EMCH DMD-ASO-SH and DMD-PMO-SH 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 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 333 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, also 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.

[0239] 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.

[0240] [Table 11]

[0241] [Table 12]

[0242] 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).

[0243] 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).

[0244] Example 3. hCD71-DMD-ASO+SO1861-SC-Mal or hCD71-DMD-PMO+SO1861-SC-Mal DMD-ASO-SH or DMD-PMO-SH were conjugated (as previously described in Figures 2B and 2C) to an 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 72 hours post-treatment, ranging from 0 to 2.4%, at concentrations ranging from 0.084 nM to 651 nM for hCD71-DMD-ASO (Figure 7A, left panel; Table A6) and 0.078 nM to 610 nM hCD71-DMD-PMO (Figure 7B, left panel; Table A6). However, when 4 μM SO1861-SC-Mal was co-administered with either hCD71-DMD-ASO (Figure 7A, right panel; Table A7) or hCD71-DMD-PMO (Figure 7B, right panel; Table A7), a strong promotion of exon skipping was observed in differentiated human myotubes: already at about 0.5 nM, exon skipping rates of 0-5% and 0-4% were 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.

[0245] More relevantly, in differentiated myotubes from DMD-affected donors, treatment with the targeted conjugates hCD71-DMD-ASO and hCD71-DMD-PMO again revealed skip rates of up to 7–11% at 72 h post-treatment with 651 nM hCD71-DMD-ASO (Figure 7A, left panel; Table A7) and 610 nM hCD71-DMD-PMO (Figure 7B, 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 8A, right panel) and 37-57% skipping rates with 102 nM hCD71-DMD-PMO+SO1861-SC-Mal (Figure 8B, 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.

[0246] [Table 13]

[0247] [Table 14]

[0248] Example 4. 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.

[0249] As Figure 9 (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).

[0250] The data indicate that the conjugate in the absence of targeted SO1861 does not achieve any skipping at the doses tested.

[0251] 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 was also 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 showed renal abnormalities and elevated serum creatinine (Figure 10A). No significant or sustained changes in the renal biomarker ALT were evident (Figure 10B). Of note, ALT levels were comparable to vehicle controls (Group 1) on days 14 and 28 and beyond.

[0252] Example 5. 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 11. 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 12 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. 13A, right panel) and mCD63-M23D (clear band down to 6.0 nM, two orders of magnitude improvement) (FIG. 13B, right panel). mCD71-M23D alone showed no activity at any concentration tested up to 758 nM (Figure 13A, left panel), and mCD63-M23D alone showed only a 2% skip rate at 755 nM (Figure 13B, 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.

[0253] Example 6. 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 as eteplirsen but without the 5'-modification 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 14A-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 15A, 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 15A, 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 15B, 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 15B, 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.

[0254] 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 14A-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 16A, 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 16A, 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 16B, 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 16B, 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 S16C, 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 S16C, 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), i.e., targeted DMD oligonucleotides conjugated to hCD71 at the 3', improved on-target delivery and induced significant exon 51 skipping.

[0255] 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 14A-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 17A, 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 17A, 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 17B, 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 17B, 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.

[0256] 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').

[0257] [Table 15]

[0258] [Table 16]

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Claims

1. 1. A pharmaceutical composition for use in the treatment or prevention of a muscle wasting disorder, comprising: nucleic acids, and saponin In a composition comprising The composition, wherein the saponin is a triterpenoid 12,13-dehydrooleanane-type saponin comprising an acid-sensitive cleavable covalent bond at the C-23 position of the saponin aglycone core structure adapted to be cleaved under acidic conditions, such that upon said cleavage an aldehyde group is generated or restored at the C-23 position of the saponin aglycone core structure.

2. the muscle wasting disorder is a muscle cell-related genetic disorder or a congenital myopathy or muscular dystrophy; Or, 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 1B, congenital muscular dystrophy; or familial dilated cardiomyopathy; or Duchenne muscular dystrophy. A composition for use according to claim 1.

3. 3. The composition for use according to claim 1 or 2, wherein said treatment or prevention of said muscle wasting disorder involves antisense therapy or involves exon skipping.

4. A composition for use according to claim 1 or 2, wherein a maleimide-containing moiety is attached to the C-23 position by the cleavable covalent bond.

5. 5. The composition for use of claim 4, wherein the maleimide-containing moiety is part of a molecule comprising or consisting of 4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)piperazine-1-carbohydrazide which attaches to the C-23 position of the saponin aglycone core structure upon formation of a semicarbazone bond (hereinafter referred to as SC-maleimide), or wherein the maleimide-containing moiety is part of a molecule comprising or consisting of N-ε-maleimidocaproic acid hydrazide which attaches to the C-23 position of the saponin aglycone core structure upon formation of a hydrazone bond (hereinafter referred to as EMCH).

6. The composition for use according to claim 1 or 2, wherein the cleavable covalent bond is selected from a semicarbazone bond, a hydrazone bond, or a hydrazide bond.

7. 3. The composition for use according to claim 1 or 2, wherein the saponin aglycone core structure corresponds to the core structure of a saponin selected from quillaric acid or gypsogenin.

8. The saponin is at least a bidesmosidic saponin comprising a first sugar chain containing a terminal glucuronic acid residue and a second sugar chain containing at least four sugar residues in a branched arrangement; or the first sugar chain is Gal-(1→2)-[Xyl-(1→3)]-GlcA, and / or the branched second sugar chain of at least four sugar residues comprises a terminal fucose residue and / or a terminal rhamnose residue; or the saponin comprises the first sugar chain at the C-3 position of the saponin aglycone core structure and / or the second sugar chain at the C-28 position of the saponin aglycone core structure; or wherein the first sugar chain is a carbohydrate substituent at the C-3β-OH group of the saponin aglycone core structure, and / or the second sugar chain is a carbohydrate substituent at the C-28-OH group of the saponin aglycone core structure.

9. 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, saponariosid B, SO1542, SO1584, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862, SO1904, QS-7, QS-7 api, QS-17, QS-18, QS-21 A-apio, QS-21 A-xylo, QS-21 B-apio and QS-21 B-xylo A saponin containing a chiral acid aglycone core structure selected from 3. The composition for use according to claim 1 or 2, wherein the composition is any one or more of the following:

10. 3. The composition for use according to claim 1 or 2, 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, saponarioside B, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862 and SO1904.

11. 3. The composition for use according to claim 1 or 2, wherein the nucleic acid is an oligonucleotide, defined as a nucleic acid not exceeding 150 nt, or the oligonucleotide is 5-150 nt, or 8-100 nt, or 10-50 nt in size, or the oligonucleotide is an antisense oligonucleotide, or a mutation-specific antisense oligonucleotide, or an oligonucleotide designed to induce exon skipping.

12. the nucleic acid is 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 nucleic acid or bridged nucleic acid (BNA), 2'-O,4'-aminoethylene bridged nucleic acid (BNANC), peptide nucleic acid (PNA), 2'-deoxy-2'-fluoroarabinonucleic acid (FANA), 3. A composition for use according to claim 1 or 2, comprising or consisting of 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 DNA, single-stranded RNA or DNA, double-stranded RNA (dsRNA) or double-stranded DNA.

13. the nucleic acid is designed to induce exon skipping of a human dystrophin gene transcript, or the exon skipping involves exon 51 skipping, exon 53 skipping, or exon 45 skipping; or the nucleic acid is a 2'O-methyl-phosphorothioate antisense oligonucleotide or a phosphorodiamidate morpholino oligomer antisense oligonucleotide designed to induce exon 51 skipping, exon 53 skipping, or exon 45 skipping; Or the composition for use according to claim 1 or 2, wherein the nucleic acid is selected from eteplirsen, drisapersen, golodirsen, viltolarsen, and casimersen.

14. the nucleic acid is conjugated to a ligand for an endocytic receptor on a muscle cell; or the endocytic receptor on muscle cells to which the ligand binds is selected from the transferrin receptor (CD71), insulin-like growth factor 1 (IGF-1) receptor (IGF1R), tetraspanin CD63; muscle-specific kinase (MuSK), glucose transporter GLUT4, cation-independent mannose 6-phosphate receptor (CI-MPR), and / or The 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 binding fragment thereof specific for binding to the endocytosis receptor, wherein the endocytosis receptor is optionally selected from transferrin receptor (CD71), insulin-like growth factor 1 (IGF-I) receptor (IGF-IR), tetraspanin CD63, muscle-specific kinase (MuSK), glucose transporter GLUT4, cation-independent mannose-6-phosphate receptor (CI-MPR), and LDL receptor; selected from any one of: Optionally, the ligand is an antibody or binding fragment thereof specific for binding to the transferrin receptor; 3. The composition for use of claim 1 or 2, optionally wherein the ligand is a monoclonal antibody or Fab' fragment or at least one single domain antibody specific for binding to the transferrin receptor, or wherein the ligand is a monoclonal antibody specific for binding to the transferrin receptor.

15. 3. A composition for use according to claim 1 or 2, for use in intravenous or subcutaneous administration to a human subject.

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