Targeted saponin-nucleic acid conjugates for the treatment of muscle wasting disorders

Abstract

The present invention relates to the field of treatment and prevention of muscle wasting disorders, in particular those involving genetic factors that can be targeted by delivery of therapeutic nucleic acids to muscle cells. 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 a muscle cell targeting ligand. 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 also appears to successfully reach and enter muscle cells in vivo via an endothelial transcytosis pathway and subsequent endocytic trapping in the sarcolemma.

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 a muscle cell targeting ligand. 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 also appears to successfully reach and enter muscle cells in vivo via an endothelial transcytosis pathway and subsequent endocytic trapping in the sarcolemma. [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, but not limited to, other muscular dystrophies, including facioscapulohumeral muscular dystrophy (disease gene: DUX4 / double homeobox 4), myotonic dystrophy type 1 (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 / merosin or either 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 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 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. Regarding transporter-based and antibody-based targeting techniques, delivery of nucleic acids by targeting endocytosis (or internalization) receptors on the muscle cell surface through ligand mediation is shown, for example, in WO2018129384 or WO2020028857. Summary of the Invention [Problem to be solved by the invention]

[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, which have been characterized and reported, for example, in WO2020126620, as possessing endosomal escape-promoting activity against various antibody-drug conjugates (ADCs) in several cancer cell types.

[0021] In the context of tumor cells, such saponins are further disclosed in WO2020126626, WO2020126620, WO2020126627, WO2020126064 and WO2020126609, which describe silencing of the HSP27 gene in tumor models by conjugation of a monoclonal antibody directed against a tumor cell marker, a saponin and a BNA to silence HSP27. Next, WO 2020126627, WO 2020126064, WO 2020126604, WO 2020126600 and WO 2020126609 describe silencing the HSP27 gene in tumor models by a combination of a first conjugate of a monoclonal antibody directed against a tumor cell marker with saponin and a second conjugate of a monoclonal antibody directed against a tumor cell marker with a BNA for silencing HSP27. Finally, WO 2020126609 describes silencing the HSP27 gene in tumor models by a combination of a saponin with a BNA for silencing HSP27 or a combination of a saponin with a conjugate of a monoclonal antibody directed against a tumor cell marker with a BNA 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, the inventors 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 both human and mouse DMD transcripts in vitro and in vivo, respectively. Without wishing to be bound by any theory, the inventors hypothesize that these findings indicate that the specific group of endosomal escape-promoting saponins is not only capable of stimulating efficient exit of therapeutic nucleic acids from muscle cell endosomes to the appropriate muscle cell internal compartment (in a highly desirable, yet poorly understood, phenomenon termed endosomal escape for therapeutics), but also, unexpectedly, when formulated with the ligand and ASO, successfully undergoes in vivo endothelial cell transcytosis from the blood to the external environment of the muscle cell, which does not appear to result in any loss of payload-containing cargo via endosomal escape to the internal compartment of the endothelial cell.

[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. Thus, the concentrations of both saponins as well as nucleic acids as used by Wang et al. to induce exon skipping activity in their DMD model system significantly exceeded the concentrations of the respective components in the novel compositions as presented in the remainder of this specification.

[0025] In summary, to address the shortcomings of the prior art, novel pharmaceutical compositions for use in the treatment or prevention of muscle wasting disorders, in particular muscle cell-associated genetic disorders, as well as novel muscle-specific endocytic receptor-targeted therapeutic nucleic acid conjugates with covalently linked 12,13-dehydrooleanane-type endosomal escape-promoting saponins for the delivery of therapeutic nucleic acids to muscle cells, which, as observed by the inventors, have a unique ability to efficiently deliver therapeutic nucleic acids to striated muscle cells in vivo, presumably by facilitating specific endosomal escape in the target muscle cells, and also, unexpectedly, by successfully undergoing endothelial transcytosis from the blood into said cells. The findings presented herein pave the way for the development of novel, less therapeutically burdensome and therefore safer, therapeutic methods for patients suffering from muscle wasting disorders. These and other advantages are further presented in the following sections.

[0026] Disclosed herein are improved biologically active compounds and pharmaceutical compositions comprising covalently linked conjugates of a therapeutic nucleic acid, a muscle cell surface endocytic receptor targeting ligand, and an endocytic escape-promoting saponin. The conjugates disclosed herein have particular advantages in that they exhibit highly desirable properties such as enhanced and efficient delivery of therapeutic nucleic acids, such as antisense oligonucleotides, to differentiated muscle cells, particularly striated muscle cells, including cardiomyocytes.

[0027] 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.

[0028] One of the objectives of several embodiments of the present disclosure is to provide a solution to the problem of non-specificity faced when administering nucleic acid-based therapeutics to human patients suffering from muscle wasting disorders and in need of such therapeutics.

[0029] A further objective of the present embodiment 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.

[0030] 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.

[0031] 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 saponin, Nucleic acids, and Ligands for endocytic receptors on muscle cells In a composition comprising a covalently linked conjugate comprising This is achieved by providing a composition, wherein the saponin is an endosomal escape promoting triterpenoid 12,13-dehydrooleanane type saponin.

[0032] In a further aspect, at least one of the above objects is achieved by providing a covalently linked conjugate for delivery of a therapeutic nucleic acid into a muscle cell, comprising: saponin, Nucleic acids, and Ligands for endocytic receptors on muscle cells In a conjugate comprising This is achieved by providing a conjugate, wherein the saponin is an endosomal escape-promoting triterpenoid 12,13-dehydrooleanane-type saponin, and the conjugate comprises 1 to 16 molecules of saponin and 1 to 5 molecules of nucleic acid per one molecule of ligand.

[0033] In further aspects, there are provided further embodiments of the compositions for therapeutic or prophylactic use as disclosed herein and / or of the conjugates according to the present disclosure, which further address one or more of the above mentioned objectives.

[0034] In particularly advantageous aspects, various different embodiments of the present disclosure are provided that include advantageous conjugates comprising one selected from an advantageous endosomal escape-promoting saponin, a variety of different therapeutic nucleic acids, such as, for example, antisense oligonucleotides configured to induce skipping of a defective exon of a wasting muscle cell disorder associated gene transcript, an advantageous ligand or combination thereof to target an endocytic receptor on a muscle cell, and an advantageous covalent linker to join any of the above together, possibly also configured to be cleavable under conditions present in human endosomes.

[0035] These and other aspects of the present disclosure are presented in detail in subsequent sections.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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).

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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 ...

[0048] 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).

[0049] 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 5000 Daltons, even more preferably less than 2000 Daltons, and most preferably less than 1500 Daltons.

[0050] As used herein, the term "covalently linked" refers to the characteristic that two or more molecules are joined together by at least one covalent bond, i.e., directly linked, or through a chain of covalent bonds, i.e., through a linker that contains at least one or more atoms.

[0051] 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 saponins or saponin molecules, one or more nucleic acid or oligonucleotide molecules, 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 different molecules, such as three, 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.

[0052] 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.

[0053] 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).

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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".

[0058] 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.

[0059] 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.

[0060] "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%).

[0061] 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%).

[0062] 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%).

[0063] 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.

[0064] 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.

[0065] 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]

[0066] [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 9 [Figure 4] Exon skipping using (A) hCD71-DMD-ASO without (left panel) or with (right panel) co-administration of SO1861-EMCH and (B) hCD71-DMD-PMO without (left panel) or with (right panel) co-administration of SO1861-EMCH in differentiated human myotubes from a DMD-affected donor (DM8036); note, in (A, right panel), the first sample at 0.013 nM (asterisk) indicates an empty lane. [Diagram 5] Exon skipping using mCD71-M23D PMO without (left panel) or with (right panel) co-administration of SO1861-SC-Mal in differentiated mouse C2C12 myotubes. [Figure 6A]Synthesis of mCD71-SO1861 using either SO1861-EMCH or SO1861-SC-maleimide resulting in an intact mAb conjugated to SO1861 via the interchain cysteines, held together by a variety of forces as known in the art; Note: the spacing between the heavy chains has been expanded in these figures for clarity of schematic representation. [Figure 6B] Generation of an mCD71-SH intermediate was used to obtain the synthesis of mCD71-SO1861, in which the intact mAb is conjugated to SO1861 via the interchain cysteines and held together by a variety of forces as known in the art; Note: For clarity of schematic representation, the spacing between the heavy chains has been expanded in these figures. [Figure 6C] Synthesis of mCD71-SO1861 was used to obtain an intact mAb conjugated to SO1861 via the interchain cysteines, held together by a variety of forces as known in the art. Synthesis of mCD71-SO1861; Note: For clarity of schematic representation, the spacing between the heavy chains has been expanded in these figures. [Figure 6D] IGF-1 ligand was conjugated to SO1861-hydrazone-NHS to generate IGF-1-SO1861 [Figure 7] Exon skipping using (A) mCD71-SO1861 (combined with SO1861-EMCH) + M23D (left panel) and mCD71-SO1861 (combined with SO1861-SC-Mal) + M23D (right panel), and (B) IGF-1-SO1861 + M23D (left panel) and control (right panel) in differentiated mouse C2C12 myotubes. [Figure 8A] Synthesis of hCD71-DMD-ASO-SO1861 and hCD71-DMD-PMO-SO1861, interchain disulfide reduction of hCD71 resulting in intact mAb conjugated to SO1861 through the interchain cysteines and held together by various forces as known in the art; Note: the spacing between heavy chains has been expanded in these figures for clarity of schematic representation. [Figure 8B] Synthesis of hCD71-DMD-ASO-SO1861 and hCD71-DMD-PMO-SO1861, resulting in intact mAb conjugated to SO1861 through the interchain cysteines and held together by various forces as known in the art. Synthesis of hCD71-SO1861; Note: For clarity of schematic representation, the spacing between heavy chains has been expanded in these figures. [Figure 8C] Synthesis of hCD71-DMD-ASO-SO1861 and hCD71-DMD-PMO-SO1861, resulting in intact mAb conjugated to SO1861 through the interchain cysteines and held together by various forces as known in the art; Synthesis of hCD71-SO1861-PEG4-SPDP; Note: For clarity of schematic representation, the spacing between heavy chains has been expanded in these figures. [Figure 8D] Synthesis of hCD71-DMD-ASO-SO1861 and hCD71-DMD-PMO-SO1861, resulting in intact mAb conjugated to SO1861 through the interchain cysteines and held together by various forces as known in the art. Synthesis of hCD71-DMD-ASO-SO1861; Note: For clarity of schematic representation, the spacing between heavy chains has been expanded in these figures. [Figure 8E] Synthesis of hCD71-DMD-ASO-SO1861 and hCD71-DMD-PMO-SO1861, resulting in intact mAb conjugated to SO1861 through the interchain cysteines and held together by various forces as known in the art. Synthesis of hCD71-DMD-PMO-SO1861; Note: For clarity of schematic representation, the spacing between heavy chains has been expanded in these figures. [Figure 8F] Synthesis of mCD71-M23D-SMCC, resulting in an intact mAb conjugated to SO1861 through the interchain cysteines, held together by various forces as known in the art; Note: For clarity of schematic representation, the spacing between the heavy chains has been expanded in these figures. [Figure 8G]Synthesis of mCD71-M23D-SO1861, resulting in an intact mAb conjugated to SO1861 via the interchain cysteines and held together by various forces as known in the art; Note: For clarity of schematic representation, the spacing between the heavy chains has been expanded in these figures. [Figure 9A] Exon skipping assessment using hCD71-DMD-ASO (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 9B] Exon skipping assessment using 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 9C] Exon skipping assessment using hCD71-DMD-ASO-SO1861 1 component in differentiated human myotubes from a non-DMD (healthy) donor (KM155) [Figure 10A] Exon skipping assessment using hCD71-DMD-ASO without (left panel) or with (right panel) co-administration of SO1861-SC-Mal in differentiated human myotubes from a DMD-affected donor (DM8036). [Figure 10B] Exon skipping assessment using hCD71-DMD-PMO without (left panel) or with (right panel) co-administration of SO1861-SC-Mal in differentiated human myotubes from a DMD-affected donor (DM8036). [Figure 10C] Exon skipping assessment using hCD71-DMD-ASO-SO1861 1 component in differentiated human myotubes from a DMD-affected donor (DM8036) [Figure 11] Assessment of exon skipping using mCD71-M23D-SO1861 in differentiated mouse C2C12 myotubes [Figure 12A]Exon skipping analysis of mice from single dose mCD71-M23D (group 2) or single dose mCD71-M23D-SO1861 (three dose groups, groups 3, 4, 5) and vehicle control (group 1) in gastrocnemius muscle on days 4, 14, and 28 after treatment; note, in FIG. 12A, group 4 showing empty lanes, PCR amplification of sample 3 on day 4 (asterisk) initially failed, but technical repeats of cDNA synthesis / PCR showed a full-length product and no measurable skipping in this animal. [Figure 12B] Exon skipping analysis of mice from single dose mCD71-M23D (group 2) or single dose mCD71-M23D-SO1861 (three dose groups, groups 3, 4, and 5) and vehicle control (group 1) in the diaphragm on days 4, 14, and 28 after treatment. [Figure 12C] Exon skipping analysis of mice from single dose mCD71-M23D (group 2) or single dose mCD71-M23D-SO1861 (three dose groups, groups 3, 4, 5) and vehicle control (group 1) in hearts 4, 14, and 28 days after treatment. [Figure 13] Densitometric quantification of mean exon skipping in mice (N=3 per group, per time point, except for gastrocnemius group 4, day 4, where N=2) from single-dose mCD71-M23D (group 2) or single-dose mCD71-M23D-SO1861 (three dose groups, groups 3, 4, and 5) in (A) gastrocnemius, (B) diaphragm, and (C) heart on days 4, 14, and 28 post-treatment; (A-C) quantification of band intensity by ChemiDoc™ XRS+ system and Image Lab™ software (BioRad). [Figure 14A] Serum creatinine analysis on days 4, 14, and 28 from single dose studies with mCD71-M23D (group 2) or single dose mCD71-M23D-SO1861 (three dose groups: group 3, group 4, group 5) and vehicle control groups (N=3 per dose / time point except for mCD71-M23D, where N=2 on day 28). [Figure 14B]Serum ALT analysis on days 4, 14, and 28 from single dose studies with mCD71-M23D (group 2) or single dose mCD71-M23D-SO1861 (three dose groups: group 3, group 4, group 5) and vehicle control groups (N=3 per dose / time point except for mCD71-M23D, where N=2 on day 28). [Figure 15A] Schematic representation of possible conjugates of the invention. Shown is an IgG antibody covalently conjugated with four saponin molecules "S" attached to either the light chain or hinge region of the antibody (both options are depicted diagrammatically), and with four effector nucleic acid molecules "NA" covalently attached to the constant domain of the antibody heavy chain. [Figure 15B] Schematic representation of possible embodiments of the conjugates of the invention. Shown is an IgG antibody covalently conjugated with four trivalent linkers, each linker covalently bound to a saponin and covalently bound to a nucleic acid molecule. The trivalent linkers are covalently bound to the antibody, for example, by the hinge region or by the light chain (both options shown). [Figure 15C] Schematic representation of possible embodiments of the conjugates of the present invention. Shown is a single domain antibody covalently conjugated to two trivalent linkers, each linker covalently bound to a saponin and covalently bound to a nucleic acid molecule. [Figure 15D] Schematic representation of possible embodiments of the conjugates of the present invention. Shown is a single domain antibody covalently conjugated to a trivalent linker, which is covalently bound to a saponin and covalently bound to a nucleic acid molecule. [Figure 15E] Schematic representation of a possible embodiment of the conjugate of the invention. Shown is an IgG antibody covalently conjugated with four saponin molecules "S" attached to the hinge region of the antibody, and with four effector nucleic acid molecules "NA" covalently attached to the constant domain of the antibody heavy chain by glycan remodeling and click chemistry. [Figure 16A]Synthesis of DBCO-(M23D)2 via a synthetic scheme involving the synthesis of intermediate 3 (via intermediates 1 and 2). [Figure 16B] Synthesis of DBCO-(M23D)2 via a synthetic scheme involving the synthesis of intermediate 4. [Figure 16C] Synthesis of DBCO-(M23D)2 via a synthetic scheme involving coupling of intermediate 4 with M23D-SH (reduced form) to achieve synthesis of intermediate 5. [Figure 16D] 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 17A] 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 17B] 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 17C] Schematic representation of the conjugation procedure for mAb-M23D, including mCD71-M23D and mCD63-M23D. Legend explaining the symbolically represented glycan residues. [Figure 17D] Schematic representation of the conjugation procedure for mAb-M23D, including mCD71-M23D and mCD63-M23D. Legend explaining the symbolically represented molecules. [Figure 18]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 19] Schematic representation of the conjugation procedure of mAb-SO1861, such as mCD63-SC-SO1861. Conjugation between mAb and SO1861 on the reduced interchain disulfide bond results in mAb-(SO1861)4. Note: For clarity of the schematic representation, mAb-SO1861 is shown with DAR4. [Figure 20A] Exon 23 skipping analysis of mCD63-SC-SO1861+M23D in differentiated mouse C2C12 myotubes. [Figure 20B] Exon 23 skipping analysis of mCD63-SC-SO1861+mCD71-M23D in differentiated mouse C2C12 myotubes. [Figure 20C] Exon skipping in differentiated mouse C2C12 myotubes using M23D, mCD63-SC-SO1861, or mCD71-M23D alone as a control. [Figure 21A] Schematic representation of the conjugation procedure of mAb-M23D-SO1861, such as mCD63-M23D-SC-SO1861. (A) Conjugation between mAb and SO1861 on reduced interchain disulfide dibonds, resulting in mAb-(SO1861)4. Note: For clarity of the schematic representation, mAb-M23D-SO1861 is shown with DAR4 / 4. For an explanation of the symbolically represented glycan residues or the symbolically represented molecule, see Figure 17C and Figure 17D, respectively. [Figure 21B]Schematic representation of the conjugation procedure for mAb-M23D-SO1861, such as mCD63-M23D-SC-SO1861. (B) Preparation of trimmed and azide-modified mAb-(SO1861)4 glycan, following FIG. 21A. Note: For clarity of the schematic representation, mAb-M23D-SO1861 is shown with DAR4 / 4. For an explanation of the symbolically represented glycan residues or the symbolically represented molecule, see FIG. 17C and FIG. 17D, respectively. [Figure 21C] Schematic representation of the conjugation procedure of mAb-M23D-SO1861, such as mCD63-M23D-SC-SO1861. Conjugation between trimmed mAb-(SO1861)4 glycan and DBCO-(M23D)2 via strain-promoted azide-alkyne click reaction to give mAb-(SO1861)4-(M23D)4. Note: For clarity of the schematic representation, mAb-M23D-SO1861 is shown as DAR4 / 4. For an explanation of the symbolically represented glycan residues or the symbolically represented molecule, see Figure 17C and Figure 17D, respectively. [Figure 22] Assessment of exon 23 skipping using (A) mCD71-M23D-EMCH-SO1861(2) or (B) mCD63-M23D-SC-SO1861 in differentiated mouse C2C12 myotubes. [Figure 23A] Schematic representation of the conjugation procedure for hAb-DMD-oligos, including hCD71-5'-SS-DMD-ASO, hCD71-5'-SS-DMD-PMO (1), hCD71-3'-SS-DMD-PMO (1-5), and hCD63-5'-SS-DMD-ASO, involving hAb functionalization with PEG4-SPDP at activated lysine (Lys) residues. Note: For clarity of the schematic representation, hAb-DMD-oligos are denoted with DAR4. [Figure 23B]Schematic representation of the conjugation procedure for hAb-DMD-oligos, including hCD71-5'-SS-DMD-ASO, hCD71-5'-SS-DMD-PMO (1), hCD71-3'-SS-DMD-PMO (1-5), and hCD63-5'-SS-DMD-ASO, involving activation of protected DMD-oligos. Note: For clarity of the schematic representation, hAb-DMD-oligos are indicated with DAR4. [Figure 23C] Schematic representation of the conjugation procedure of hAb-DMD-oligos, including hCD71-5'-SS-DMD-ASO, hCD71-5'-SS-DMD-PMO (1), hCD71-3'-SS-DMD-PMO (1-5), and hCD63-5'-SS-DMD-ASO, involving disulfide bond formation between activated DMD-oligo-SH and hAb-PEG4-SPDP. Note: For clarity of the schematic representation, hAb-DMD-oligo is indicated with DAR4. [Figure 23D] Schematic representation of the conjugation procedure of hAb-DMD-oligos, including hCD71-5'-SS-DMD-ASO, hCD71-5'-SS-DMD-PMO (1), hCD71-3'-SS-DMD-PMO (1-5), and hCD63-5'-SS-DMD-ASO, involved in the figure legend. Note: For clarity of the schematic representation, hAb-DMD-oligos are indicated with DAR4. [Figure 24] 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 25A] 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 25B]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 25C] 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 26] Exon 53 skipping analysis of (A) hCD71-3'-SS-DMD-PMO(4) (DAR2.3) without (left panel) or with (right panel) co-administration of SO1861-SC-Mal, and (B) hCD71-3'-SS-DMD-PMO(5) (DAR2.1) without (left panel) or with (right panel) co-administration of SO1861-SC-Mal in differentiated human myotubes from a non-DMD (healthy) donor (KM155). [Figure 27] Schematic representation of the conjugation procedure of hAb-SO1861, including hCD71-SC-SO1861 and hCD63-SC-SO1861. Conjugation between hAb and SO1861 on the interchain disulfide bond results in hAb-(SO1861)4. Note: For clarity of the schematic representation, hAb-SO1861 is shown with DAR4. [Figure 28] Exon 51 skipping analysis of (A) hCD71-5'-SS-DMD-ASO (DAR2.1) with co-administration of hCD63-SC-SO1861 (DAR4.8) and (B) hCD63-5'-SS-DMD-ASO (DAR2.3) with co-administration of hCD71-SC-SO1861 (DAR4.0) in differentiated human myotubes from a non-DMD (healthy) donor (KM155). [Figure 29]Exon 51 skipping analysis of hCD63-SC-SO1861 (DAR4.8) with co-administration of hCD71-5'-SS-DMD-ASO (DAR2.1) in (A) differentiated human myotubes from a non-DMD (healthy) donor (KM155) and (B) differentiated human myotubes from a DMD-affected donor (KM1328). [Figure 30A] Schematic representation of the conjugation procedure of hAb-DMD-oligo-SO1861, including hCD71-5'-SS-DMD-ASO-SC-SO1861, hCD63-5'-SS-DMD-ASO-SC-SO1861, hCD71-3'-SS-DMD-PMO(1-5)-SC-SO1861 and hCD63-3'-SS-DMD-PMO(1-4)-SC-SO1861. Conjugation between hAb and SO1861 on the interchain disulfide bond, resulting in hAb-(SO1861)4, followed by hAb functionalization with PEG4-SPDP at activated lysine (Lys) residues. Note: For clarity of the schematic representation, hAb-DMD-oligo-SO1861 is shown with DAR4 / 4. [Figure 30B] Schematic representation of the conjugation procedure of hAb-DMD-oligo-SO1861, including hCD71-5'-SS-DMD-ASO-SC-SO1861, hCD63-5'-SS-DMD-ASO-SC-SO1861, hCD71-3'-SS-DMD-PMO(1-5)-SC-SO1861 and hCD63-3'-SS-DMD-PMO(1-4)-SC-SO1861. Disulfide bond formation between activated DMD-oligo-SH and hAb-((SO1861)4-(SPDP)4 gives hAb-(SO1861)4-(DMD-oligo)4. Note: For clarity of the schematic representation, hAb-DMD-oligo-SO1861 is shown with DAR4 / 4. [Figure 30C]Schematic representation of the conjugation procedure for hAb-DMD-oligo-SO1861, including hCD71-5'-SS-DMD-ASO-SC-SO1861, hCD63-5'-SS-DMD-ASO-SC-SO1861, hCD71-3'-SS-DMD-PMO(1-5)-SC-SO1861 and hCD63-3'-SS-DMD-PMO(1-4)-SC-SO1861. Figure legend. Note: For clarity of the schematic representation, hAb-DMD-oligo-SO1861 is shown with DAR4 / 4. [Figure 31A] Exon 51 skipping analysis of hCD71-5'-SS-DMD-ASO-SC-SO1861 (DAR2.3 / 4.0) (left panel) and hCD63-5'SS-DMD-ASO-SC-SO1861 (DAR2.2 / 4.8) (right panel) in differentiated human myotubes from a DMD-affected donor (KM1328). [Figure 31B] Exon 51 skipping analysis of hCD71-3'-SS-DMD-PMO(1)-SC-SO1861 (DAR 2.4 / 4.0) (left panel) and hCD63-3'-SS-DMD-PMO(1)-SC-SO1861 (DAR 2.7 / 4.8) (right panel) in differentiated human myotubes from a DMD-affected donor (KM1328). [Figure 31C] Exon 51 skipping analysis of hCD71-3'-SS-DMD-PMO(2)-SC-SO1861 (DAR 3.1 / 4.0) (left panel) and hCD63-3'-SS-DMD-PMO(2)-SC-SO1861 (DAR 2.7 / 4.8) (right panel) in differentiated human myotubes from a DMD-affected donor (KM1328). [Figure 31D] Exon 51 skipping analysis of hCD71-3'-SS-DMD-PMO(3)-SC-SO1861 (DAR 2.5 / 4.0) (left panel) and hCD63-3'-SS-DMD-PMO(3)-SC-SO1861 (DAR 2.8 / 4.8) (right panel) in differentiated human myotubes from a DMD-affected donor (KM1328). [Diagram 32]Exon 53 skipping analysis of (A) hCD71-3'-SS-DMD-PMO(4)-SC-SO1861 (DAR 2.0 / 4.0) (left panel) and hCD63-3'-SS-DMD-PMO(4)-SC-SO1861 (DAR 2.4 / 4.8) (right panel), and (B) hCD71-3'-SS-DMD-PMO(5)-SC-SO1861 (DAR 2.0 / 4.0) in differentiated human myotubes from a DMD-affected donor (KM1328). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0067] Disclosed herein are improved biologically active pharmaceutical compositions comprising a covalently linked conjugate of a therapeutic nucleic acid, a muscle cell surface endocytic receptor targeting ligand, and an endocytic escape-promoting saponin. The conjugates disclosed herein have the particular advantage of exhibiting 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.

[0068] 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.

[0069] One of the several objects of the embodiments of the present disclosure is to provide a solution to the problem of non-specificity faced when administering nucleic acid-based therapeutics to human patients suffering from muscle wasting disorders and in need of such therapeutics. Another of the several objects of the present embodiments is to provide a solution to the problem of insufficient safety profile of current nucleic acid-based drugs when administered to human patients in need thereof, particularly when administered in excessive doses that induce side effects. Yet another of the 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 insufficient ability to reach and / or enter diseased muscle cells with little to no off-target activity against non-diseased cells.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] As will be demonstrated by the data presented herein, inclusion of triterpenoid 12,13-dehydrooleanane-type saponins in the therapeutic compositions and conjugates of the present disclosure not only appeared to stimulate efficient escape of therapeutic nucleic acids from muscle cell endosomes to the appropriate internal muscle cell compartment, but also, unexpectedly, did not impede endothelial cell transcytosis in vivo from the blood to the external environment of the muscle cell, thereby enabling efficient in vivo delivery of the saponin-containing therapeutic compositions and conjugates of the present disclosure to muscle cells following intravenous delivery.

[0075] 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: saponin, Nucleic acids, and Ligands for endocytic receptors on muscle cells In a composition comprising a covalently linked conjugate comprising The composition is provided wherein the saponin is a triterpenoid 12,13-dehydrooleanane-type saponin.

[0076] As used herein, the term "covalently linked conjugate", in the context of a covalently linked conjugate comprising a saponin, a nucleic acid, and a ligand for an endocytic receptor on a muscle cell, should be taken to refer to a conjugate in which the saponin, nucleic acid, and ligand are covalently linked together.

[0077] As used herein, the context will understand that the nucleic acid forming part of the composition disclosed herein for therapeutic purposes is selected to have a therapeutic activity for treating or preventing the selected muscle wasting disorder. In other words, the nucleic acid as comprised in the conjugate as disclosed herein will be a therapeutic nucleic acid for one disorder, whereas 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 potential therapeutic nucleic acid and will be able to decide, based on either his knowledge of the mutations that cause such disorders or on the results of genetic mutation screening of a given patient, which therapeutic nucleic acid should be included in the new conjugate as disclosed herein to perform an improved treatment.

[0078] 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.

[0079] 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.

[0080] Interestingly, it has been observed that favorable nucleic acid delivery properties can be correlated with the number of nucleic acid and saponin molecules conjugated together per molecule of endocytic receptor targeting ligand present in the conjugate.

[0081] In light of the above observations, in an advantageous embodiment there is provided a composition for therapeutic or prophylactic use as disclosed herein, wherein the conjugate comprises 1 to 16 molecules of saponin and 1 to 5 molecules of nucleic acid per molecule of ligand.

[0082] In another advantageous embodiment, provided herein is a covalently linked conjugate for delivery of a therapeutic nucleic acid into muscle cells, comprising: saponin, Nucleic acids, and Ligands for endocytic receptors on muscle cells In a conjugate comprising The saponin is a triterpenoid 12,13-dehydrooleanane type saponin, and Further disclosed is a conjugate, wherein the conjugate comprises 1-16 molecules of saponin and 1-5 molecules of nucleic acid per molecule of ligand.

[0083] In a related aspect, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the disclosure, wherein the conjugate comprises 2 to 8 molecules of saponin per molecule of ligand; Preferably, 3 to 6 molecules of saponin per molecule of ligand; More preferably, 4 to 5 molecules of saponin per molecule of ligand. Includes; Most preferably, the conjugate contains an average of 4 to 4.5 molecules of saponin per molecule of ligand.

[0084] In a further related aspect, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the disclosure, wherein the conjugate comprises 2-5 molecules of nucleic acid per molecule of ligand; Preferably, 3 to 4 molecules of nucleic acid per one molecule of ligand. Includes; More preferably, the conjugate contains an average of four molecules of nucleic acid per molecule of ligand.

[0085] Typically, saponins suitable for application in the conjugates disclosed herein are those that exhibit endosomal escape promoting activity. As can be seen in Table 1, such saponins have a triterpene 12,13-dehydrooleanane skeleton, in which the basic structure of the triterpene skeleton is a pentacyclic C30 terpene skeleton (also called sapogenin or aglycone).

[0086] [Table 1]

[0087] [Table 2]

[0088] [Table 3]

[0089] [Table 4]

[0090] [Table 5]

[0091] [Table 6]

[0092] [Table 7]

[0093] [ka]

[0094] As can be seen from Table 1, many of the triterpenoid 12,13-dehydrooleanane-type saponins contain an aldehyde group at the C-23 position of the saponin aglycone core structure.

[0095] Without wishing to be bound by any theory, it has been observed that the presence of said aldehyde group in the aglycone core structure of saponin (also referred to herein as the "aglycone") is beneficial to the ability of saponin to stimulate and / or enhance endosomal escape of therapeutic nucleic acids contained in the conjugates of the invention.

[0096] Thus, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the disclosure, wherein the saponin is an aldehyde group at the C-23 position of the saponin aglycone core structure, or A covalent bond at the C-23 position of the saponin aglycone core structure that covalently links the saponins within the conjugate Including any of the following: Preferably, wherein the covalent bond at C-23 is a cleavable bond that undergoes cleavage under conditions present in an endosome or lysosome; More preferably, wherein the cleavable covalent bond at C-23 is adapted such that upon cleavage an aldehyde group is restored at C-23; Alternatively, the saponin used to prepare the conjugates herein, at least in an unconjugated state, e.g., prior to being covalently linked within a conjugate disclosed herein, comprises an aldehyde group at the C-23 position of the saponin aglycone core structure in its natural form, e.g., as present in or extracted from its source plant material.

[0097] Such saponins may be covalently linked to the remainder of the conjugate (which may include nucleic acids and ligands for endocytic receptors on muscle cells) by any functional group present on the saponin suitable for conjugation as known in the art, or may be covalently linked by reacting the aldehyde group at C-23 of the saponin aglycone core structure such that the reaction converts the aldehyde group at C-23 into a covalent bond at C-23 that covalently links the saponin to the remainder of the conjugate.

[0098] Without wishing to be bound by any theory, such free aldehyde groups have been observed to be beneficial for triterpenoid 12,13-dehydrooleanane-type saponins due to their endosomal escape stimulating properties and may be involved in promoting vesicle membrane destabilization.

[0099] Thus, to still further enhance the endosomal escape promoting properties of the saponin, and thus to further improve escape of a therapeutic nucleic acid present with the saponin in an endosomal compartment as part of a disclosed conjugate, the covalent bond at the C-23 position can be selected such that its cleavage (e.g., in response to conditions present in a mammalian endosome or lysosome) restores an aldehyde group at the C-23 position of the saponin aglycone core structure.

[0100] Examples of suitable bond types that can be designed to restore this aldehyde group include one or more of semicarbazone bonds, hydrazone bonds, imine bonds, acetal bonds, including 1,3-dioxolane bonds, and / or oxime bonds. As shown in the Examples herein below, such functional design of the conjugates as disclosed herein has been successfully achieved with saponin, whereby the aldehyde group naturally present at C-23 of the saponin was converted to a semicarbazone or hydrazone covalent bond at C-23 of the saponin aglycone core structure that links the saponin within the conjugate. Such a bond at C-23 effectively releases the saponin from the conjugate in response to acidic conditions, with the release of the saponin restoring the aldehyde group at C-23.

[0101] In light of the above, the endosomal escape promoting properties of such saponins are also highly evident when the aldehyde group is replaced by a maleimide-containing moiety attached to the C-23 position by a cleavable covalent bond that is cleaved off under acidic conditions present, for example, in the endosomes and / or lysosomes of human cells, whereby the aldehyde group at the C-23 position of the saponin aglycone core structure is restored upon said cleavage under acidic conditions present in the endosomes and / or lysosomes of human cells.

[0102] In advantageous embodiments, such cleavable covalent bonds may be selected from semicarbazone bonds, hydrazone bonds, or imine bonds.

[0103] For example, in possible embodiments, the maleimide-containing moiety can be part of a molecule that comprises or consists of 4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)piperazine-1-carbohydrazide, which is attached to the C-23 position of the 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).

[0104] For the conjugates of the 12,13-dehydrooleanane type saponins of the present invention, which naturally contain an aldehyde group at C-23 in their natural or unconjugated form, saponins whose aglycon core structure is either queratic acid or gypsogenin are particularly suitable. With this in mind, it has been observed that the saponins particularly suitable for the conjugates of the present invention are 12,13-dehydrooleanane type saponins containing a queratic acid aglycon or gypsogenin aglycon core structure, or derivatives of said saponins in which the aldehyde group at C-23 of both aglycons has been converted to a covalent bond at C-23, when the C-23 aldehyde group of such aglycon core structure is used for conjugation.

[0105] An example of an unconjugated saponin with an aldehyde group at the C-23 position is depicted as saponin A and is exemplified by the following structure:

[0106] [ka]

[0107] It should be noted, however, that saponins containing different 12,13-dehydrooleanane-type aglycone core structures have also been observed to exhibit sufficient endosomal escape-promoting properties, and therefore other aglycone structures listed in Table 1 may also be advantageous for purposes of the present disclosure.

[0108] Thus, in a further embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the disclosure, wherein the saponin aglycone core structure is Quilacic acid; (and / or a quilacic acid derivative in which the aldehyde group at C-23 of quilacic acid has been converted to a covalent bond at C-23, preferably wherein said covalent bond is a bond that covalently links the saponin within the conjugate;) Gypsogenin; (and / or gypsogenin derivatives in which the aldehyde group at C-23 of gypsogenin has been converted to a covalent bond at C-23, preferably wherein said covalent bond is a bond that covalently links the saponin within the conjugate;) 2α-hydroxyoleanolic acid; 16α-hydroxyoleanolic acid; Hederagenin (23-hydroxyoleanolic acid); 16α,23-dihydroxyoleanolic acid; Protoestigenin-21(2-methylbut-2-enoate)-22-acetate; 23-Oxo-valingtogenol C-21,22-bis(2-methylbut-2-enoate); 23-Oxo-barringtogenol C-21(2-methylbut-2-enoate)-16,22-diacetate; 3,16,28-trihydroxyoleanan-12-ene; Gypsogenic acid; and Its derivatives One or more of the following is selected: Preferably herein the saponin aglycone core structure is Quilacic acid; (and / or quillic acid derivatives in which the aldehyde group at C-23 of quillic acid has been converted to a covalent bond at C-23, preferably wherein said covalent bond is a bond that covalently links the saponin within the conjugate); Gypsogenin; (and / or a gypsogenin derivative in which the aldehyde group at C-23 of gypsogenin has been converted to a covalent bond at C-23, preferably wherein said covalent bond is a bond that covalently links the saponin within the conjugate). Selected from; More preferably herein, the saponin aglycone core structure is selected from quillaric acid (and / or a quillaric acid derivative in which the aldehyde group at C-23 of quillaric acid is converted to a covalent bond at C-23, preferably wherein said covalent bond is a bond that covalently links the saponin within the conjugate).

[0109] Saponins can include one or more sugar chains attached to an aglycone core structure. Preferred saponins of the compositions or conjugates of the present disclosure include a single chain (i.e., monodesmosidic type) or two chains (i.e., bidesmosidic type) attached to a triterpene 12,13-dehydrooleanane aglycone core structure, optionally containing an aldehyde group at the C-23 position.

[0110] Glycosylation has also been hypothesized to play a role in endosomal escape-promoting properties.

[0111] In light of the above, in a further embodiment there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the disclosure, wherein the saponin sugar fraction comprises a sugar chain selected from any one of the sugar chains as listed in Group A or Group B as presented in Table 2 below.

[0112] [Table 8]

[0113] [Table 9]

[0114] [Table 10]

[0115] For example, in an embodiment advantageous for the conjugation option, the saponin is a bidesmosidic saponin.

[0116] In a related embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the present disclosure, wherein the saponin is at least a bidesmosidic saponin comprising a first glycan selected from group A and a second glycan selected from group B; preferably wherein the first glycan comprises a terminal glucuronic acid residue, and / or wherein the second glycan comprises at least four glycan residues in a branched arrangement; more preferably wherein the first glycan is Gal-(1→2)-[Xyl-(1→3)]-GlcA, and / or wherein the branched second glycan of at least four glycan residues comprises a terminal fucose residue and / or a terminal rhamnose residue.

[0117] In an advantageous embodiment of the conjugate of the invention, the saponin comprises one or both of a first glycan attached to the C-3 or C-28 atom of the aglycone core structure, and preferably comprises one glycan attached to the C-3 atom and a second glycan attached to the C-28 atom of the aglycone core structure.

[0118] In such an example, when the saponin included in the conjugate of the invention carries the two glycans, the first glycan is attached to the C-3 position of the aglycone core structure and the second glycan is typically attached to the C-28 position of the saponin aglycone core structure, although for some saponins lacking an aldehyde group at the C-23 position, the second glycan can be attached to said C-23 position (see Table 1).

[0119] Thus, in further related embodiments, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the disclosure, wherein the saponin comprises a first glycan at the C-3 position of the saponin aglycone core structure and / or a second glycan at the C-28 position of the saponin aglycone core structure; Preferably, wherein the first glycan is a carbohydrate substituent at the C-3β-OH group of the saponin aglycone core structure, and / or wherein the second glycan is a carbohydrate substituent at the C-28-OH group of the saponin aglycone core structure.

[0120] In a particularly preferred embodiment, conjugates are provided in which the saponin is a triterpenoid 12,13-dehydrooleanane-type saponin which, at least in its unconjugated state, comprises an aldehyde group at the C-23 position of the saponin aglycone core structure and which comprises as a carbohydrate substituent at the C-3β-OH group of the saponin aglycone core a glycan selected from Group A and which comprises a terminal glucuronic acid residue, preferably Gal-(1→2)-[Xyl-(1→3)]-GlcA.

[0121] In the following embodiments, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the present disclosure, wherein the conjugate comprises two or more molecules of saponin, preferably 2-32 molecules of saponin, even more preferably 4-16 molecules of saponin, and most preferably 4-8 molecules of saponin.

[0122] These molecules may be the same saponin, or saponins of the same aglycone core structure and different glycosylation chains, or may even be a mixture of different 12,13-dehydrooleanane-type endosomal escape-promoting saponins, e.g., a mixture of different saponins selected from Table 1.

[0123] In another embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the present 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 comprising a quinoline aglycone core structure selected from: d) List D: Escin Ia, escinate, α-hederin, AMA-1, AMR, AS6.2, AS64R, Assamsaponin F, Dipsacoside B, Esculentoside A, Macrantoidin A, NP-005236, NP-012672, Primular Acid 1, Saikosaponin A, Saikosaponin D, Tea Seed Saponin I and Tea Seed Saponin J A saponin having a 12,13-dehydrooleanane type aglycone core structure without an aldehyde group at the C-23 position of the aglycone, selected from One or more of the following: Preferably the saponin is any one or more of the saponins selected from lists A, B or C, more preferably from lists B or C, even more preferably from list C.

[0124] In particular embodiments, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the disclosure, wherein the saponin is selected from the group consisting of AG1856, GE1741, saponin isolated from Quillaja saponaria, Quil-A, QS-17, QS-21, QS-7, SA1641, Saponaria officinalis, 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, SO1861, SA1641 and GE1741; more preferably, wherein the saponin is QS-21, SO1832 or SO1861; and most preferably SO1861.

[0125] In more particular embodiments, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the disclosure, wherein the saponin is derived from Saponaria officinalis (Saponaria officinalis). officinalis), preferably wherein the saponin is any one or more of saponariosides B, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862 and SO1904; more preferably wherein the saponin is any one or more of SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862 and SO1904; even more preferably wherein the saponin is any one or more of SO1832, SO1861 and SO1862; even more preferably wherein the saponin is SO1832 and SO1861; and most preferably SO1861.

[0126] In particular embodiments, there may be provided a conjugate as disclosed herein for therapeutic or prophylactic use and compositions comprising same, wherein one, two or three, preferably one or two, more preferably one of the following, namely: 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 saccharide chain 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.

[0127] In more particular embodiments, there may be provided a conjugate as disclosed herein for therapeutic or prophylactic use and compositions comprising the same, wherein the at least one saponin is i. an aglycone core structure, -Reduction to alcohol; - conversion to a hydrazone bond via reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), where the maleimide group of EMCH is optionally derivatized by formation of a thioether bond with mercaptoethanol; - Conversion to a hydrazone bond via reaction with N-[β-maleimidopropionic acid] hydrazide (BMPH), where the maleimide group of BMPH is optionally derivatized by formation of a thioether bond with mercaptoethanol; or - Conversion to a hydrazone bond via reaction with N-[κ-maleimidoundecanoic acid]hydrazide (KMUH), where the maleimide group of KMUH is optionally derivatized by formation of a thioether bond with mercaptoethanol. an aglycone core structure containing an aldehyde group that has been derivatized with ii. a first glycan comprising a carboxyl group, preferably a carboxyl group of a glucuronic acid moiety, that has been derivatized by conversion to an amide bond through reaction with 2-amino-2-methyl-1,3-propanediol (AMPD) or N-(2-aminoethyl)maleimide (AEM); or iii. a second glycan containing an acetoxy group (Me(CO)O-) that has been derivatized by conversion to a hydroxyl group (HO-) by deacetylation; or iv. Any combination of two or three derivatizations i., ii. and / or iii., preferably any combination of two of the derivatizations i., ii. and / or iii. Includes.

[0128] In a specific embodiment, conjugates and compositions comprising the same are provided, wherein at least one saponin comprises a first glycan according to Group A and Group B of Table 2, respectively, and a second glycan, wherein the first glycan comprises two or more sugar moieties, and the second glycan comprises two or more sugar moieties, and wherein the aglycone core structure is preferably quillic acid or gypsogenin, more preferably quillic acid, and wherein one, two or three, preferably one or two of the following: i. the aldehyde group on the aglycone core structure is derivatized; ii. the carboxyl group of the glucuronic acid moiety in the first glycan is derivatized; and iii. At least one acetoxy (Me(CO)O-) group on the second glycan is derivatized.

[0129] 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 glycan 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.

[0130] In a specific embodiment, a conjugate or composition for therapeutic or prophylactic use as disclosed herein is provided, in which the aldehyde functional group at the C-23 position of the aglycone core structure of at least one saponin is covalently bound to a linker EMCH, which is covalently bound to a sulfhydryl group of an oligomeric or polymeric molecule of the covalent saponin conjugate, such as a sulfhydryl group of cysteine, via a thioether bond. The binding of the EMCH linker to the aldehyde group of the aglycone of the saponin results in the formation of a hydrazone bond. Such a hydrazone bond is a typical example of a bond that can be cleaved under the acidic conditions inside endosomes and lysosomes. The saponin coupled to the ligand contained in the conjugate or the nucleic acid contained in the conjugate, etc., can be released from the conjugate of the present invention after delivery to the endosome or lysosome of the target muscle cell that exposes an endocytic receptor to which the ligand can bind. Thus, the saponin coupled to the ligand and nucleic acid in the conjugate is transferred from the outside of the cell to the endosome (or lysosome), where it is released from the remainder of the conjugate upon pH-driven cleavage of the hydrazone bond. In the endosome (or lysosome), the free saponin can exert its stimulatory activity upon delivery of the therapeutic nucleic acid contained in the conjugate of the invention into the cytosol of the muscle cell. As explained above, the inventors have surprisingly recognized that it is not essential for saponin-mediated endosomal escape that the saponin be present in a free form in the endosome or lysosome. The saponin contained in the conjugates disclosed herein can also enhance the exit of the therapeutic nucleic acid from the endosome / lysosome into the cytosol of the target muscle cell.

[0131] The development of the advantageous compositions presented herein was based on the surprising realization that, thanks to the inclusion of an endosomal escape-promoting saponin in the conjugates of the present invention, any nucleic acid can be delivered to muscle cells with improved and high efficiency, thus helping in the treatment and / or prevention of muscle wasting disorders.

[0132] With this in mind, in an advantageous embodiment, the nucleic acid is a therapeutic nucleic acid adapted to target a genomic mutation within a mutated transcript or gene 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.

[0133] In a detailed embodiment, such a gene target is mutated human dystrophin underlying 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, such as, but not limited to, DUX4 / double homeobox 4 underlying facioscapulohumeral muscular dystrophy, or DMPK underlying myotonic dystrophy type 1, or EMD / emerin and LMNA / lamin A / C underlying Emery-Dreifuss muscular dystrophy, or MYOT / myotilin, LMNA / lamin A / C underlying limb-girdle muscular dystrophy 1. Further examples, either LAMA2 / merosin or COL6A genes encoding laminin alpha 2 chain / collagen 6A that 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).

[0134] In a particularly advantageous embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the present disclosure, wherein the nucleic acid is an oligonucleotide, defined as a nucleic acid not exceeding 150 nt, preferably wherein the oligonucleotide is between 5 and 150 nt in size, preferably between 8 and 100 nt, most preferably between 10 and 50 nt.

[0135] 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.

[0136] 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 conjugate according to the present disclosure, wherein the oligonucleotide is an antisense oligonucleotide, more preferably a mutation-specific antisense oligonucleotide, most preferably an oligonucleotide designed to induce exon skipping.

[0137] In related embodiments, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the disclosure, wherein the oligonucleotide comprises or consists of any one of the following: morpholino phosphorodiamidate oligomer (PMO), 2'-O-methyl (2'-OMe) phosphorothioate RNA, 2'-O-methoxyethyl (2'-O-MOE) RNA {2'-O-methoxyethyl-RNA (MOE)}, locked or bridged nucleic acid (LNA or BNA), 2'-O,4'- Aminoethylene-bridged nucleic acid (BNANC), peptide nucleic acid (PNA), 2'-deoxy-2'-fluoroarabinonucleic acid (FANA), 3'-fluorohexitol nucleic acid (FHNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), silencing RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), antagomir (miRNA antagonist), aptamer RNA or aptamer DNA, single-stranded RNA or single-stranded DNA, double-stranded RNA (dsRNA) or double-stranded DNA;

[0138] 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.

[0139] In particular and preferred specific embodiments for DMD, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the disclosure, wherein the oligonucleotide is designed to induce exon skipping of the human dystrophin gene transcript, preferably wherein exon skipping involves exon 51 skipping or exon 53 skipping or exon 45 skipping, preferably wherein the oligonucleotide is a 2'O-methyl-phosphorothioate antisense oligonucleotide or a phosphorodiamidate morpholino oligomer antisense oligonucleotide designed to induce exon 51 skipping or exon 53 skipping or exon 45 skipping.

[0140] In a more detailed embodiment building on the previous embodiment, the oligonucleotide is selected from eteplirsen, drisapersen, golodirsen, viltolarsen, and casimersen.

[0141] In an advantageous embodiment, a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the present disclosure is provided, wherein the conjugate comprises two or more molecules of nucleic acid. Such therapeutic combinations of two or more therapeutic nucleic acids are known in the art, for muscle wasting disorders, for example, a combination approach based on two AONs for double exon skipping in myostatin and dystrophin was proposed for the management of Duchenne muscular dystrophy [Kemaladewi el al, 2011].

[0142] In a further advantageous embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the present disclosure, wherein preferably the two or more molecules of nucleic acid are 2 to 16 molecules, more preferably 2 to 8 molecules, and optionally 2, 3, 4, 5, or 6 molecules.

[0143] In light of clinical practice, in a 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 two or more molecules of nucleic acid are two or more oligonucleotides; optionally two or more different oligonucleotides, wherein at least one of the two or more different oligonucleotides is an antisense oligonucleotide.

[0144] Regarding detailed embodiments regarding the ligands of endocytic receptors on muscle cells, it must be noted that endocytic receptors expressed on the surface of muscle cells are known, some of which, such as the transferrin receptor (CD71) or muscle-specific kinase (MuSK), are described in WO2018129384 or WO2020028857. Further examples of suitable receptors include 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 GF1R, or a fragment of IGF-II, which is a ligand for CI-MPR (also known as IGF2R).

[0145] In a preferred embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the disclosure, wherein the endocytic receptor on muscle cells to which the ligand binds is selected from the transferrin receptor (CD71), the insulin-like growth factor 1 (IGF-I) receptor (IGF1R), the tetraspanin CD63; the muscle-specific kinase (MuSK), the glucose transporter GLUT4, the cation-independent mannose 6-phosphate receptor (CI-MPR), and the LDL receptor.

[0146] In a further 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 ligand is Insulin-like growth factor 1 (IGF-I) or a fragment thereof; Insulin-like growth factor 2 (IGF-II) or a fragment thereof Mannose 6-phosphate Transferrin (Tf), Zymosan A, and An antibody or a binding fragment thereof specific for binding to an endocytosis receptor, the endocytosis receptor being preferably selected from the transferrin receptor (CD71), the insulin-like growth factor 1 (IGF-I) receptor (IGF1R), the tetraspanin CD63, the muscle-specific kinase (MuSK), the glucose transporter GLUT4, the cation-independent mannose 6-phosphate receptor (CI-MPR), and the LDL receptor. Select one of the following: Preferably, the ligand herein is an antibody or binding fragment thereof specific for binding to the transferrin receptor.

[0147] In a related particularly advantageous embodiment, 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.

[0148] In a further advantageous embodiment, the ligand is a monoclonal antibody, such as a humanized or human monoclonal antibody, an IgG, a molecule comprising or consisting of a single domain antibody, at least one VHH domain, preferably a camelid VH, a variable heavy chain novel antigen receptor (VNAR) domain, a Fab, scFv, Fv, dAb, F(ab)2 and an Fcab fragment.

[0149] In a specific embodiment, the conjugate of the invention is provided with the proviso that the ligand of the endocytic receptor on muscle cells is not a ligand of CD71 or MuSK, preferably with the proviso that the ligand is not a CD71-binding antibody or a CD71-binding fragment thereof, or a MuSK-binding antibody or a MuSK-binding fragment thereof.

[0150] As explained above, in a further advantageous embodiment, a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the disclosure is provided, wherein the covalent linkage of the saponin in the conjugate is via a first linker to which the saponin is covalently attached; preferably, wherein the first linker comprises a covalent bond selected from any one or more of a semicarbazone bond, a hydrazone bond, an imine bond, an acetal bond including a 1,3-dioxolane bond, a ketal bond, an ester bond, an oxime bond, a disulfide bond, a thioether bond, an amide bond, a peptide bond and an ester bond, preferably selected from any one or more of a semicarbazone bond, a hydrazone bond, an imine bond, a disulfide bond, a thioether bond, an amide bond, a peptide bond and an ester bond, more preferably a hydrazone bond or a semicarbazone bond; even more preferably, wherein the saponin is an aldehyde group at the C-23 position of the saponin aglycone core structure, or A covalent bond at the C-23 position of the saponin aglycone core structure that covalently links the saponin in the first conjugate via the first linker and that is included as part of the first linker, preferably the covalent bond at the C-23 position is a cleavable bond that will undergo cleavage under conditions present in an endosome or lysosome, more preferably the cleavable covalent bond at the C-23 position is adapted for cleavage to restore an aldehyde group at the C-23 position. Contains any of the following; Alternatively, the saponin used in preparing the conjugate herein is a saponin that, at least in the unconjugated state, comprises an aldehyde group at the C-23 position of the saponin aglycone core structure, and wherein after preparation of the conjugate, said aldehyde group is involved in forming a covalent bond with a first linker.

[0151] Thus, in a related advantageous embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the disclosure, wherein the first linker is a cleavable linker that will undergo cleavage under acidic, reducing, enzymatic and / or light-induced conditions; Preferably, wherein the first linker is bonds that undergo cleavage under acidic conditions, such as semicarbazone bonds, hydrazone bonds, imine bonds, acetal bonds including 1,3-dioxolane bonds, ketal bonds, ester bonds, and / or oxime bonds; proteolytically sensitive bonds, e.g. amide or peptide bonds, which are subject to proteolysis, preferably by cathepsin B; Bonds that are redox cleavable, such as disulfide bonds, or that are susceptible to thiol exchange reactions, such as thioether bonds comprising a cleavable bond selected from: More preferably wherein the first linker is Semicarbazone bond, or hydrazone bonds, which are subject to cleavage under acidic conditions, 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:

[0152] Preferably, such bonds are acid-sensitive bonds that undergo cleavage under the acidic conditions present in the endosomes and / or lysosomes of human cells, preferably at a pH of 4.0 to 6.5, more preferably at a pH ≦ 5.5.

[0153] In advantageous embodiments, the acid sensitive 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, a ketal bond, an ester bond, and / or an oxime bond, even more preferably selected from a semicarbazone bond and a hydrazone bond; most preferably a hydrazone bond.

[0154] In certain embodiments, when the saponin is conjugated by a covalent bond (preferably an acid-sensitive bond) at the C-23 position of the saponin aglycone core structure, said covalent bond at the C-23 position may advantageously be selected or adapted such that upon cleavage (e.g. under acidic conditions) an aldehyde group at the C-23 position is restored. Advantageously, such a covalent bond may be selected from any one or more of a semicarbazone bond, a hydrazone bond, an imine bond, an acetal bond including a 1,3-dioxolane bond, and / or an oxime bond, and is preferably either a semicarbazone bond or a hydrazone bond.

[0155] Preferably, such cleavable bonds are not easily cleaved or are only slightly cleaved when the conjugate is present outside the endosomes and lysosomes of a cell, such as when the conjugate is outside the cell or in an endocytic vesicle after binding to an endocytic receptor. Preferably, the bond is efficiently cleaved in vivo 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.

[0156] 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, compared to the susceptibility of the bond when the conjugate is present in the endosome or lysosome of a target cell that has endocytosed the conjugate. Thus, cleavable bonds that 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 (i.e., acid-sensitive bonds), are preferred, more preferably bonds selected from semicarbazone bonds and hydrazone bonds; most preferably hydrazone bonds. Once the saponin is cleaved away from the remainder of the conjugate, the saponin exerts its endosomal escape-promoting activity on further molecules present with the saponin in the endosome, such as the nucleic acid or oligonucleotide of the conjugate of the present disclosure, in an enhanced manner.

[0157] Saponins containing an aldehyde group at the C-23 position of the aglycone are particularly preferred because such saponins have potent endosomal escape promoting activity towards nucleic acids such as oligonucleotides.Thus, preferred for the conjugates are saponins that contain or form 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.

[0158] For example, when the Group B and Group C saponins described above are covalently attached in the conjugate via linker chemistry involving an aldehyde group (e.g., formation of a hydrazone or semicarbazone bond), it is preferred that the aldehyde group is reformed (i.e. restored) in the endosome or lysosome upon endocytosis of the conjugate and cleavage of the saponin away from the remainder of the conjugate by cleavage of the cleavable bond. Examples of such saponins suitable for this purpose are listed in Table 1, e.g., the saponins of Groups A-C, in particular Groups B and C, as outlined herein above.

[0159] To construct complex covalent conjugates, for example to include a large or fixed number of ligands such as antibodies, or multiple nucleic acid molecules, or especially saponin molecules, a molecular scaffold comprising an oligomeric or polymeric structure can be used. One advantage of this is that the scaffold can be designed so that it includes a fixed number of molecules, such as saponins. For example, the scaffold can include exactly one saponin molecule, but can also include several (e.g., 2, 3, or 4) saponins or a relatively constant and fixed number of saponins in a large number (e.g., 10, 20, or 100). In some cases, such oligomeric or polymeric structures will appear as either linear, branched or cyclic polymers, oligomers, dendrimers, dendrons (e.g. either G2, G3, G4 or G5 dendrons for a maximum covalent attachment of 4, 8, 16 or 32 saponin moieties, respectively), dendronized polymers, dendronized oligomers or assemblies of either pure or mixed forms of these structures, made of poly(amines), e.g. polyethyleneimine and poly(amidoamines), or alternatively polyethylene glycol, poly(esters), such as poly(lactides), poly(lactams), polylactide-co-glycolide copolymers, poly(dextrins), or peptides or proteins, or natural and / or artificial polyamino acids, e.g. poly-lysine, DNA polymers, such as DNA containing 2-100 nucleotides, stabilized RNA polymers or PNA (peptide nucleic acid) polymers, e.g. containing 2-200 nucleotides. Preferably, such scaffolds may be made of oligomeric and / or polymeric structures such as dendrimers, dendrons, dendronized polymers, dendronized oligomers, DNA, e.g., nucleic acids of 2-200, polyethylene glycol, oligoethylene glycol (OEG), such as OEG3, OEG4 and OEG5.

[0160] Thus, in an advantageous embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the present disclosure, wherein the first linker further comprises any oligomeric or polymeric structure which is a dendron, such as a polyamidoamine (PAMAM) dendrimer, or a polyethylene glycol, such as any of PEG3 to PEG30; preferably the polymeric or oligomeric structure is any one of PEG4 to PEG12 or any one of G2 dendron, G3 dendron, G4 dendron and G5 dendron, more preferably G2 dendron or G3 dendron or PEG3 to PEG30.

[0161] The structures forming such oligomeric / polymeric scaffolds advantageously lack intrinsic biological activity. Typically, the scaffolds are made of inert molecules to avoid potential health risks. Depending on the number of selected saponins to be incorporated into the specific embodiment of the muscle-targeting conjugates presented herein, the type and size or length of the oligomeric / polymeric structure can be appropriately selected. In other words, the number of saponins to be coupled to the ligand and nucleic acid as part of the conjugate can determine the selection of a suitable oligomeric or polymeric structure that carries a sufficient number of binding sites to couple the desired number of saponins and provide a covalent saponin-binding structure therewith. For example, the length of the OEG or the size of the dendron or polylysine molecule determines the maximum number of saponins that can be covalently linked to such oligomeric or polymeric structures that may be included as part of the first linker in the conjugate.

[0162] In an advantageous embodiment, a conjugate comprising such a scaffold may contain a defined number or range of saponins covalently linked thereto, rather than a random number. This is particularly advantageous in terms of obtaining marketing approval during drug development. In this context, a defined number means that the conjugate may contain a predefined number of saponins. This is achieved, for example, by designing a scaffold comprising an oligomeric / polymeric structure with a certain number of groups to which one or more saponins can associate. Under ideal circumstances, each of these groups will associate with one saponin molecule, thus resulting in a conjugate containing a defined number of saponins. For example, it is envisioned to provide a standard set of scaffolds including 2, 4, 8, 16, 32, 64, etc.

[0163] In certain embodiments of the conjugates of the invention, for example, for non-ideal binding situations where not all groups present in the oligomer / polymer will be associated with a saponin molecule, scaffolds may be provided where the number of saponin molecules will be defined as a range, such as, for example, 2-4 saponin molecules per scaffold, 3-6 saponin molecules per scaffold, 4-8 saponin molecules per scaffold, 6-8 saponin molecules per scaffold, 6-12 saponin molecules per scaffold, etc.

[0164] Such a first linker comprising any number of saponins attached to an oligomeric or polymeric molecule may in some embodiments act as a carrier (support, scaffold) for multiple saponin moieties, which in turn bind to ligands and nucleic acids, thus forming certain embodiments of the muscle cell targeting therapeutic conjugates disclosed herein. In advantageous embodiments, such oligomeric or polymeric molecule-containing linkers loaded with saponin molecules may be attached to the remainder of the muscle cell targeting conjugate, preferably by a cleavable bond.

[0165] In another advantageous embodiment, a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the present disclosure is provided, wherein the covalent linkage of the nucleic acid within the conjugate is achieved by a second linker to which the nucleic acid is covalently attached; preferably, wherein the second linker comprises or consists of the linker 3-(2-pyridyldithio) succinimidyl propionate (SPDP); optionally, wherein the second linker covalently links the nucleic acid to a lysine residue, preferably a lysine residue contained in a ligand, or to a glycan residue, preferably a partially trimmed glycan.

[0166] Similar to saponin conjugation, it may also be advantageous if one or more of the nucleic acid molecules, such as oligonucleotide molecules, particularly double-stranded ones such as siRNA, are linked by a cleavable bond in the conjugate, where the cleavable bond is cleaved, for example, under acidic conditions, reducing conditions, enzymatic conditions and / or light-induced conditions.Cleavable bonds that are cleaved under the acidic conditions present in the endosomes and / or lysosomes of human cells are preferred.

[0167] 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 conjugate according to the disclosure, wherein the second linker is a cleavable linker that will undergo cleavage under acidic, reducing, enzymatic and / or light-induced conditions; Preferably, wherein the second linker is bonds that undergo cleavage under acidic conditions, such as semicarbazone bonds, hydrazone bonds, imine bonds, acetal bonds including 1,3-dioxolane bonds, ketal bonds, ester bonds, and / or oxime bonds; proteolytically sensitive bonds, e.g. amide or peptide bonds, which are subject to proteolysis, preferably by cathepsin B; Bonds that are redox cleavable, such as disulfide bonds, or that are susceptible to thiol exchange reactions, such as thioether bonds comprising a cleavable bond selected from: More preferably wherein the first linker is Bonds that are subject to cleavage under acidic conditions, such as semicarbazone bonds or hydrazone 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:

[0168] Advantageously, the bond is acid sensitive, 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 any one or more of a semicarbazone bond, a hydrazone bond, an imine bond, an acetal bond including a 1,3-dioxolane bond, a ketal bond, an ester bond and / or an oxime bond, even more preferably it is an acid sensitive bond selected from a semicarbazone bond and a hydrazone bond; most preferably it is a hydrazone bond.

[0169] Such cleavable bonds are preferably less susceptible to cleavage, or cleaved only to a small extent, when the conjugate is present outside the endosomes and lysosomes of a cell, such as when the conjugate is outside the cell or in an endocytic vesicle after the conjugate has engaged with 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 in an endosome or lysosome of a target cell, herein a muscle cell.

[0170] In particular embodiments, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the disclosure, wherein the first linker and / or wherein the second linker is directly or indirectly covalently linked to the ligand.

[0171] In a further equally advantageous embodiment, a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the present disclosure is provided, wherein the covalent linkage of the ligand within the conjugate is achieved by a third linker to which the ligand is covalently attached; preferably, 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 third linker comprises a covalent bond made with said at least one cysteine ​​residue or said at least one lysine residue; 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 third linker comprises a covalent bond with any one or more of the cysteine ​​residues of the multi-cysteine ​​repeat; optionally, wherein two or more third linkers are linked to one molecule of the ligand by binding each of the two or more third linkers to a distinct cysteine ​​residue of the multi-cysteine ​​repeat comprised in the ligand.

[0172] In a further possible embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the present disclosure, wherein the conjugate comprises a central linker for achieving a covalent link between at least one molecule of saponin, at least one molecule of nucleic acid and at least one molecule of ligand, wherein said covalent linkage is achieved either directly or via a first linker, a second linker and / or a third linker, respectively; preferably wherein the central linker is a trifunctional linker.

[0173] For example, in related embodiments, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the present disclosure, wherein the central linker, in its unconjugated form, has the structure A:

[0174] [ka] is a trifunctional linker represented by

[0175] In a possible embodiment related to the above-mentioned embodiments, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the disclosure, wherein the conjugate comprises 1 to 4 trifunctional linkers for each molecule of ligand contained in the conjugate, more preferably 1 to 2 trifunctional linkers, and most preferably an average of 1.2 to 1.8 trifunctional linkers.

[0176] In another related embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the disclosure, wherein the trifunctional linker, in its conjugated form, has the structure B:

[0177] [ka] (In the formula, S is at least one saponin molecule preferably selected from group A, B or C; L1 is a first linker to which the at least one molecule of saponin is covalently attached; E is at least one molecule of nucleic acid; L2 is a second linker to which the at least one molecule of nucleic acid is covalently attached; A is at least one molecule of a ligand, preferably an antibody or a binding fragment thereof, optionally one or more single domain antibodies; and L3 is a third linker to which the at least one molecule of ligand is covalently attached; where L1, L2 and L3 are the same or different. It is represented by:

[0178] In particular embodiments, L1 may optionally comprise an oligomeric or polymeric structure as described herein above for specific embodiments, preferably comprising PEG, more preferably selected from PEG3 to PEG30, or a structure comprising a G2, G3 or G4 dendron.

[0179] In a particular embodiment, a conjugate is provided, in which at least one saponin is covalently bound via a thioether bond to a sulfhydryl group in one of the at least one ligand, preferably an antibody or binding fragment thereof, and / or in one of the at least one nucleic acid, preferably an ASO, preferably via N-ε-maleimidocaproic hydrazide (EMCH) covalently bound to an aldehyde group at C23 position of the aglycone core structure of the saponin and covalently bound to a sulfhydryl group in the ligand, such as a sulfhydryl group of cysteine.

[0180] In another detailed embodiment, a conjugate is provided, wherein at least one saponin is a bidesmosidic triterpene saponin or derivative thereof belonging to the 12,13-dehydrooleanane type, optionally bearing an aldehyde functional group at the C23 position and comprising a glucuronic acid unit in a first saccharide chain attached to the C3β-OH group of the aglycone core structure of the saponin, wherein the saponin is covalently bound to an amino acid residue of a ligand and / or at least one nucleic acid via the carboxyl group of the glucuronic acid unit in the first saccharide chain, preferably via a linker, wherein the amino acid residue is preferably selected from cysteine ​​and lysine.

[0181] Various different schematic representations of possible embodiments of the therapeutic muscle cell targeting conjugates of the present invention are depicted and described in FIG. 15 and its legend.

[0182] As explained above, various different exemplary embodiments of the conjugates for targeting muscle cells disclosed herein can be envisaged, according to which various different modes of conjugation of the saponin and / or the nucleic acid (preferably an oligonucleotide) to the ligand and / or the oligomeric or polymeric structure (further referred to as the scaffold) can be envisaged. In light of the above described embodiments, this conjugation can be either by direct covalent bonding of at least two molecules contained in the conjugate, or via a linker, such as the first linker, the second linker and / or the third linker described above. A linker can be used to establish the covalent bond of at least one molecule or at least one molecule contained in the conjugate to the rest of the conjugate, or more linkers can be used for this purpose. In particular embodiments, each of the distinct distinct molecules comprised in the conjugate (i.e., the distinct distinct molecules are saponins, nucleic acids, and ligands) may be covalently linked to the remainder of the conjugate by its own linker (i.e., a first, second, or third linker, respectively), for example by being attached to a common scaffold. As explained above, in possible embodiments, the linkers may be stable under the conditions present in mammalian (e.g., human) endosomes / lysosomes, or may be unstable (i.e., cleavable) under said conditions, meaning that they are cleaved in response to said conditions, thus releasing the molecules covalently linked via such cleavable linkers from the remainder of the conjugate.

[0183] A number of examples of cleavable first linkers that covalently link saponins within a conjugate are described above, including the more detailed embodiment of a cleavable first linker attached to a saponin at the C-23 position of the saponin aglycone core with an acid-sensitive bond, which acid-sensitive bond is preferably established by reacting an aldehyde group at the C-23 position of such saponin aglycone core, and is configured to replenish said group under acidic conditions present in mammalian (e.g., human) endosomes / lysosomes.

[0184] Alternatively, when the receptor internalization rate is not a limiting factor, in another possible embodiment, the first linker covalently linking the saponin in the conjugate of the invention may be a stable linker, which can be linked to the saponin by reacting, for example, preferably and if present, via a glucuronic acid group, with a glucuronic acid unit in the first glycan attached to the C3β-OH group of the aglycon core structure of the saponin. As a result of such reaction, a stable first linker can be created that includes a stable (i.e., non-cleavable) bond to the first glycan attached to the C3β-OH group of the aglycon core structure of the saponin, which covalently links the saponin to, for example, a ligand (e.g., an immunoglobulin such as a mAb, sdAb, VHH, etc.) or to a scaffold in the conjugate. In light of this, a possible embodiment is the conjugate as provided, where the saponin belongs to the saponins comprising a glucuronic acid unit in the first saccharide chain at the C3β-OH group of the aglycon core structure of the saponin, where the glucuronic acid unit has been reacted to a linker covalently, preferably via an amide bond created in the first saccharide chain attached to the C3β-OH group of the aglycon core structure of the saponin, more preferably to an amine group present in a ligand (such as an amine group of a lysine or the N-terminus of a protein ligand such as an immunoglobulin) or to a scaffold. The glucuronic acid functional group is particularly advantageous since it can be reacted to establish a covalent linkage, either via a direct covalent bond or via a linker, between the saponin and the ligand or the scaffold of the conjugate of the invention, where the linker is a stable linker, but can also be designed to be a cleavable linker.

[0185] The choice between a cleavable first linker and a stable first linker is entirely up to the skilled artisan and can be made, for example, depending on the choice of ligand and the desired release rate of any of the distinct molecules contained in the conjugate. Saponins conjugated via a cleavable first linker or a stable first linker can be part of any embodiment of the conjugate of the present invention, for example, an embodiment in which a nucleic acid (e.g., an oligonucleotide, preferably a PMO or an ASO) is linked via either a cleavable second linker or a stable second linker. Although examples of both stable second linkers and cleavable second linkers have been provided above, it is highly likely that both will be used for the conjugation of nucleic acids within the conjugates of the present invention. The choice between them will be highly dependent on the type of nucleic acid used and the intended therapy. For example, a stable linker can be very easily used for the conjugation of only one of the strands of a therapeutic nucleic acid that will be designed to act in a single-stranded form in the cell. The two strands of such therapeutic nucleic acids can be selected to dissociate in response to conditions present in endosomes / lysosomes, thus releasing the therapeutic strand from the conjugate and entering the cytosol in a manner facilitated by the presence of an endosomal escape-promoting saponin as described herein.Double-stranded nucleic acids may in some embodiments be advantageously provided with a cleavable second linker, which may be considered either conjugated to a ligand or conjugated to a scaffold.

[0186] In further particular embodiments, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the disclosure, wherein the saponin is or comprises at least one molecule of SO1861, the nucleic acid is drisapersen or eteplirsen, and the ligand is an anti-CD71 antibody or a binding fragment thereof.

[0187] In another embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein for use in intravenous or subcutaneous administration to a human subject.

[0188] In yet another embodiment, there is provided a composition for therapeutic or prophylactic use as disclosed herein or a conjugate according to the present disclosure comprising a pharma- ceutically acceptable excipient and / or a pharma-ceutically acceptable diluent.

[0189] In a particular aspect, there is provided a composition or conjugate according to the present disclosure for use as a medicament. EXAMPLES

[0190] material: SO1861 was isolated and purified from a crude plant extract obtained from soapwort (Saponaria officinalis L) by Extrasynthese, France and / or Analyticon Discovery GmbH, Germany.

[0191] 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.

[0192] Anti-CD71 antibody (clone OKT9) targeting human CD71 (hCD71) and mouse targeting anti-CD71 antibody (clone R17 217.1.3) (mCD71) were both purchased from BioXCell. Anti-CD63 antibody (clone H5C6) targeting human CD63 (hCD63) and mouse targeting anti-CD63 antibody (clone NVG-2) (mCD63) were both purchased from Biolegend. IGF-1 ligand was purchased from PeproTech.

[0193] 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.

[0194] [Table 11]

[0195] [Table 12]

[0196] 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

[0197] Methods (as carried out in Examples 1-6): SO1861-maleimide, SO1861-NHS synthesis SO1861 is derived from soap plant (Saponaria officinalis L) (Extrasynthese, France and / or Analyticon Discovery GmbH, Germany) and was coupled to the respective handle by Symeres (NL) according to methods known in the art.

[0198] Conjugation of SO1861 to antibodies and proteins Custom production of IGF-1-SO1861, mCD71-SO1861 and hCD71-SO1861 was carried out by Fleet Bioprocessing (UK).

[0199] 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).

[0200] 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).

[0201] 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).

[0202] 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.

[0203] 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).

[0204] 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.

[0205] Preparative MP-LC method 2 Model: Reveleris (trademark) fractionation 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, elution solution 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.

[0206] 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.

[0207] 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

[0208] 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.

[0209] 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

[0210] 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.

[0211] 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.

[0212] 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).

[0213] 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.

[0214] 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).

[0215] 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).

[0216] 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).

[0217] 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).

[0218] mCD71-SO1861 For the generation of mCD71-SO1861, either SO1861-SC-maleimide or SO1861-EMCH were used to generate two different mCD71-SO1861 conjugates. The procedure is exemplarily described for the conjugation between mCD71 and SO1861-EMCH.

[0219] An aliquot of mCD71 (55.1 mg, 0.37 μmol, 8.10 mg / ml, 6.80 ml) was standardized to 5 mg / ml in DPBS pH 7.5, then 30 μl / ml (330 μl) of premixed concentrated Tris / Tris.HCl / EDTA containing concentrated Tris (127 mg / ml, 1.05 M), concentrated Tris.HCl (623 mg / ml, 3.95 M) and concentrated EDTA.2Na.2H2O (95 mg / ml, 0.26 M) in a 1:1:1 v / v combination was added to give a 50 mM TBS, 2.5 mM EDTA buffer pH approx. 7.5. To mCD71 (50 mg, 0.33 μmol, 5.044 mg / ml) was added an aliquot of freshly prepared TCEP solution (2.00 mg / ml, 2.74 molar equivalents, 0.912 μmol), the mixture was vortexed briefly, then incubated at 20° C. for 210 min with end-over-end mixing. After incubation (before adding SO1861-EMCH), mCD71-SH was dispensed for multiple conjugation and a 1.0 mg (0.201 ml) aliquot was removed and purified by gel filtration using Zeba spin desalting columns in TBS pH 7.5. This aliquot was characterized by UV-vis analysis and Ellman's assay (3.248 mg / ml, thiol to mCD71 ratio = 3.97). To an aliquot of mCD71-SH (42 mg, 0.28 μmol, 4.978 mg / ml) was added an aliquot of freshly prepared SO1861-EMCH solution (2.0 mg / ml, 8 molar equivalents, 2.24 μmol, 2.32 ml), the mixture was vortexed briefly, and then incubated for 120 min at 20° C. In addition to this conjugation reaction, two aliquots of desalted mCD71-SH (0.25 mg, 0.077 ml, 1.67 nmol) were reacted with NEM (8.00 equivalents, 134 nmol, 6.7 μl of a 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) as positive and negative controls, respectively, for 120 min at 20° C. After incubation, an approximately 0.4 mg aliquot of the mCD71-SO8161 mixture was removed, purified by gel filtration using Zeba spin desalting columns in TBS pH 7.5, and characterized in parallel with positive and negative controls by Ellman's assay to obtain SO1861 uptake.The reaction was quenched by adding an aliquot of freshly prepared NEM solution (5 molar equivalents, 1.40 μmol, 70.1 μl of a 2.5 mg / ml solution) to the bulk mCD71-SO1861 mixture. The conjugate was purified by Superdex 200 column eluted with DPBS pH 7.5 to obtain purified mCD71-SO1861 conjugate. The product was concentrated, then standardized to 2.5 mg / ml, 0.2 μm filtered, and then divided into aliquots for characterization, product testing, and further conjugation. The result was mCD71-SO1861 conjugate (total yield=37.3 mg, 89%, SO1861 to mCD71 ratio=3.8).

[0220] IGF-1-SO1861 IGF-1 (4 mg) was dissolved in DPBS pH 7.5 (1.60 ml). To IGF-1 (3.88 mg, 0.51 μmol, 4.821 mg / ml) was added an aliquot of freshly prepared SO1861-hydrazone-NHS solution (2.0 mg / ml, 5 molar equivalents, 2.53 μmol, 3.80 ml), the mixture was vortexed briefly, and then incubated at 20° C. for 60 min. After conjugation, a freshly prepared aliquot of glycine solution (25 equivalents, 13 μmol, 95 μl of a 10 mg / ml solution) was added, and the conjugate was then purified by elution with DPBS pH 7.5 on a Zeba 10 ml 7K MWCO spin desalting column to obtain purified IGF-1-SO1861 conjugate. Aliquots were analyzed by BCA colorimetric assay to confirm the new EC value, then concentrated by centrifugal filtration (3,000 g, 20° C., 10 min intervals), standardized to 2.5 mg / ml, 0.2 μm filtered, and distributed into aliquots for characterization and customer testing. The result was IGF-1-SO1861 conjugate (total yield=2.83 mg, 73%, SO1861 to IGF-1 ratio=2.7).

[0221] mCD71-M23D-SO1861 To an aliquot of mCD71-SO1861 at DAR3.8 (20 mg, 0.126 μmol, 2.53 mg / ml) was added an aliquot of freshly prepared SMCC solution (2.0 mg / ml, 3.53 molar equivalents, 0.447 μmol), the mixture was vortexed briefly, and then incubated with end-over-end mixing for 60 min at 20° C. After incubation, the reaction was quenched by adding an aliquot of freshly prepared glycine solution (2.0 mg / ml, approximately 20 molar equivalents, 2.5 μmol), the mixture was vortexed briefly, and then incubated with end-over-end mixing for >15 min at 20° C. The conjugate was purified by Superdex 200 column eluted with TBS pH 7.5 and analyzed by UV-Vis to give purified mCD71-SO1861-SMCC (18.6 mg, yield: 93%, 0.942 mg / ml, SMCC to mCD71 ratio=2.6).

[0222] 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).

[0223] To an aliquot of mCD71-SO1861-SMCC (17.2 mg, 0.11 μmol, 0.95 mg / ml) was added an aliquot of M23D-SH (4.1 mg / ml, 4.0 molar equivalents, 0.44 μmol, 0.86 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-SO1861-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 the mCD71-SO1861-M23D conjugate (total yield=15.2 mg, 79%, ratio of M23D to mCD71-SO1861=1.6).

[0224] 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).

[0225] 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).

[0226] 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).

[0227] 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).

[0228] 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).

[0229] 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).

[0230] hCD71-SO1861 As-supplied hCD71 (55.1 mg, 0.37 μmol, 8.10 mg / ml, 6.80 ml) was buffer exchanged by elution into TBS pH 7.5 using Zeba spin desalting columns and standardized to 3 mg / ml. To hCD71 (50 mg, 0.33 μmol, 5.044 mg / ml) was added an aliquot of freshly prepared TCEP solution (2.00 mg / ml, 3 molar equivalents, 1 μmol), the mixture was vortexed briefly, and then incubated at 20°C for 210 min with end-over-end mixing. After incubation (before adding SO1861-SC-maleimide), a 1.0 mg (0.201 ml) aliquot of the hCD71 solution was removed and purified by gel filtration using Zeba spin desalting columns in TBS pH 7.5. The aliquot was characterized by UV-Vis analysis and Ellman's assay (2.23 mg / ml, thiol to hCD71 ratio = 4.35). To an aliquot of bulk hCD71-SH (42 mg, 0.28 μmol, 4.978 mg / ml) was added an aliquot of freshly prepared SO1861-SC-maleimide solution (2.0 mg / ml, 8 molar equivalents, 2.24 μmol, 2.32 ml), the mixture was vortexed briefly, and then incubated at 20°C for 120 min with end-over-end mixing. Besides this conjugation reaction, two desalted aliquots of hCD71-SH (0.25 mg, 0.077 ml, 1.67 nmol) were reacted with NEM (8.00 equivalents, 13.4 nmol, 6.7 μl of 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) as positive and negative controls, respectively, at 20°C for 120 min. After incubation, an approximately 0.4 mg aliquot of the hCD71-SO8161 mixture was removed and purified by gel filtration using Zeba spin desalting columns in TBS pH 7.5, and characterized in parallel with the positive and negative controls by Ellman's assay to obtain SO1861 incorporation. The reaction was quenched by adding an aliquot of freshly prepared NEM solution (5 molar equivalents, 1.4 μmol of 2.5 mg / ml solution) to the bulk hCD71-SO1861 mixture. The conjugate was purified through a Zeba 40K MWCO spin desalting column eluted with DPBS pH 7.5 to give purified hCD71-SO1861 conjugate.An aliquot of the product was analyzed by BCA colorimetric assay to confirm the new EC280 value. The product was then standardized to 2.5 mg / ml, 0.2 μm filtered, and then divided into aliquots for characterization, product testing, and further conjugation. The result was hCD71-SO1861 conjugate (total yield=37.8 mg, 89%, SO1861 to hCD71 ratio=4.2).

[0231] hCD71-DMD-PMO-SO1861 To an aliquot of hCD71-SO1861 at DAR4.2 in DPBS pH 7.5 (20 mg, 0.128 μmol, 2.53 mg / ml) was added an aliquot of freshly prepared PEG4-SPDP solution (10 mg / ml, 10 molar equivalents, 1.28 μmol), the mixture was vortexed briefly, and then incubated with end-over-end mixing for 60 min at 20° C. After incubation, the reaction was quenched by adding an aliquot of freshly prepared glycine solution (2.0 mg / ml, 50 molar equivalents, 6.4 μmol), the mixture was vortexed briefly, and then incubated with end-over-end mixing for >15 min at 20° C. 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-SO1861-PEG4-SPDP (17.2 mg, yield: 84%, 0.93 mg / ml, PEG4-SPDP to hCD71 ratio = 3.7).

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

[0233] To an aliquot of hCD71-SO1861-PEG4-SPDP (15 mg, 95 nmol, 0.98 mg / ml), an aliquot of DMD-PMO-SH (4 mg / ml, 4.0 molar equivalents, 0.38 μmol, 0.95 ml) was added, the mixture was vortexed briefly, and then incubated at 20° C. with end-over-end mixing. After 72 hours, the conjugate mixture was concentrated and purified on a Superdex 200PG column eluted with DPBS pH 7.5 to obtain purified hCD71-DMD-PMO-SO1861 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-SO1861 conjugate (total yield=11.8 mg, 49%, ratio of DMD-PMO to hCD71-SO1861=2.6).

[0234] hCD71-DMD-ASO-SO1861 To an aliquot of hCD71-SO1861 at DAR4.2 in DPBS pH 7.5 (20 mg, 0.128 μmol, 2.53 mg / ml) was added an aliquot of freshly prepared PEG4-SPDP solution (10 mg / ml, 10 molar equivalents, 1.28 μmol), the mixture was vortexed briefly, and then incubated with end-over-end mixing for 60 min at 20° C. After incubation, the reaction was quenched by adding an aliquot of freshly prepared glycine solution (2.0 mg / ml, 50 molar equivalents, 6.4 μmol), the mixture was vortexed briefly, and then incubated with end-over-end mixing for >15 min at 20° C. 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-SO1861-PEG4-SPDP (17.2 mg, yield: 84%, 0.93 mg / ml, PEG4-SPDP to hCD71 ratio = 3.7).

[0235] 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).

[0236] To an aliquot of hCD71-SO1861-PEG4-SPDP (15 mg, 95 nmol, 0.98 mg / ml), an aliquot of DMD-ASO-SH (4 mg / ml, 4.0 molar equivalents, 0.38 μmol, 0.68 ml) was added, the mixture was vortexed briefly, and then incubated at 20° C. with end-over-end mixing. After 72 hours, the conjugate mixture was concentrated and purified on a Superdex 200PG column eluted with DPBS pH 7.5 to obtain purified hCD71-DMD-ASO-SO1861 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 hCD71-DMD-ASO-SO1861 conjugate (total yield = 14.3 mg, 84%, DMD-ASO to hCD71-SO1861 ratio = 2.1).

[0237] 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).

[0238] 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.

[0239] 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.

[0240] 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, 0.4 μl 10 μM FW primer 5′-ACCCAGTCTACCACCCTATC-3′ [SEQ ID NO: 14], and 0.4 μl 10 μM RV primer 5′-CTCTTTATCTTCTGCCCACCTT-3′ [SEQ ID NO: 15] were added to a PCR tube, mixed, and then 2 μl cDNA (50 ng) was added to give a total volume of 25 μl. The samples were subjected to a PCR run at 94° C. for 1 min, 35 cycles of 98° C. for 5 s, 55° C. for 5 s, 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.

[0241] 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).

[0242] In vivo studies Nine male CD-1 mice per group, aged 6-7 weeks at dosing, received a single intravenous (iv) injection of the compounds listed in Table A2 or vehicle. To allow for comparison of efficacy, doses were standardized to provide similar PMO doses in groups 2 and 3. 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 time of analysis. Dystrophin skip levels were determined in heart, diaphragm, and gastrocnemius samples.

[0243] [Table 13]

[0244] Results (Examples 1 to 6) Example 1. DMD-PMO+SO1861 and DMD-ASO+SO1861 DMD-ASO (a 2'O-methyl-phosphorothioate antisense oligonucleotide with the same sequence and chemical modifications as drisapersen that induces exon 51 skipping of human dystrophin) and DMD-PMO (a phosphorodiamidate morpholino oligomer antisense oligonucleotide with the same sequence but without the 5'-modification as eteplirsen that induces exon 51 skipping of human dystrophin) were evaluated for exon skipping activity in differentiated human myotubes derived from a non-DMD (healthy) donor (KM155) in combination with the endosomal escape promoter SO1861-EMCH (4 μM). Surprisingly, this revealed a strong promotion of exon skipping of exon 51 after 72 h only in combination with SO1861 (Table A3): exposure to 20 μM DMD-ASO alone showed no skipping activity, whereas 0.08 μM DMD-ASO+SO1861-EMCH already revealed 40% exon skipping, pointing to a greater than 250-fold improvement (Figure 1A). Similarly, exposure to 20 μM DMD-PMO resulted in 4% skipping, whereas 1.25 μM DMD-ASO+SO1861-EMCH already achieved 5% skipping and 20 μM achieved 34%, pointing to a 16-fold increase in potency for the non-targeted oligo in conjunction with the non-targeted SO1861-EMCH (Figure 1B).

[0245] 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.

[0246] [Table 14]

[0247] 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 363 nM hCD71-DMD-ASO (Figure 4A, left panel), whereas, surprisingly, a comparable skipping rate (15%) already occurred at a 720-fold lower concentration of 0.50 nM hCD71-DMD-ASO + 4 μM SO1861-EMCH. The skipping efficiency was significantly increased up to 64–68% with hCD71-DMD-ASO at exposure concentrations of only 18.2–109 nM (Figure 4A, right panel).Similarly, 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.

[0248] 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.

[0249] [Table 15]

[0250] [Table 16]

[0251] 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).

[0252] 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).

[0253] Example 3. mCD71-SO1861+M23D or IGF-1-SO1861+M23D Two different conjugates, mCD71-SO1861(DAR3.8), were generated by conjugating either SO1861-EMCH or SO1861-SC-maleimide (Figure 6A) to a TCEP-treated anti-CD71 monoclonal antibody targeting mouse CD71 (mCD71-SH, Figure 6B), respectively (Figure 6C). Each of these conjugates was co-administered with a constant concentration of 500 nM M23D in the concentration range of 0-720 nM into C2C12 differentiated myotubes. This revealed that the combinations mCD71-SO1861 (SO1861-EMCH) + 500 nM M23D (12% skip rate at 710 nM and 8% skip rate at 142 nM) (Figure 7A, left panel) and mCD71-SO1861 (SO1861-SC-Mal) + 500 nM M23D (19% skip rate at 710 nM and 10% skip rate at 142 nM) potently promoted M23D efficacy in exon 23 skipping to the same extent, whereas 500 nM M23D alone (0 nM conjugate) only showed a 5% skip rate (Figure 7A, right panel). The data indicate that the addition of the mCD71-SO1861-Mal conjugate significantly increases the on-target effect mediated by M23D, i.e. exon skipping, independent of the conjugation and linker.

[0254] Next, IGF-1 ligand was conjugated to SO1861-hydrazone-NHS via lysine to generate IGF-1-SO1861 (DAR2.7), as shown in Figure 6D. IGF-1-SO1861 was tested in C2C12 differentiated myotubes at concentrations ranging from 0 to 3333 nM and co-administered with 500 nM M23D. This revealed that the combination of IGF-1-SO1861 + 500 nM M23D promoted M23D cytoplasmic delivery, i.e., dystrophin exon 23 skipping (19% skip rate at 3333 nM, 11% skip rate at 667 nM, and 8% skip rate at 133 nM) (Figure 7B).

[0255] Taken together, these data indicate that antibody and non-antibody ligand conjugated, i.e. targeted, SO1861 in a co-administration setting leads to a significant efficacy enhancement, i.e. on-target delivery of the PMO payload in muscle cells.

[0256] Example 4. hCD71-DMD-ASO-SO1861 or hCD71-DMD-PMO-SO1861 TCEP-treated anti-CD71 monoclonal antibody targeting human CD71 (hCD71-SH, FIG. 8A) was conjugated with SO1861-SC-Mal via cysteine ​​(FIG. 8B) and PEG4-SPDP was conjugated to lysine to generate hCD71-SO1861-PEG4-SPDP (FIG. 8C), to which either DMD-ASO-SH or DMD-PMO-SH was conjugated to generate hCD71-DMD-ASO-SO18 hCD71-DMD-PMO-SO1861 (DAR2.1 / 4.2) (Figure 8D) and hCD71-DMD-PMO-SO1861 (DAR2.6 / 4.2) (Figure 8E) were generated; DMD-ASO-SH or DMD-PMO-SH were also 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, ranging from 0 to 2.4%, 72 hours after treatment, at concentrations ranging from 0.084 nM to 651 nM for hCD71-DMD-ASO (Figure 9A, left panel; Table A6) and 0.078 nM to 610 nM hCD71-DMD-PMO (Figure 9B, left panel; Table A6). However, when 4 μM SO1861-SC-Mal was co-administered with either hCD71-DMD-ASO (Figure 9A, right panel; Table A7) or hCD71-DMD-PMO (Figure 9B, right panel; Table A7), a strong promotion of exon skipping was observed in differentiated human myotubes: already at about 0.5 nM, 0-5% and 0-4% exon skipping was observed for hCD71-DMD-ASO and hCD71-DMD-PMO, respectively, which increased to 29-40% for 109 nM hCD71-DMD-ASO and 4-5% for 102 nM hCD71-DMD-PMO, respectively, making the improvement of potency by several orders of magnitude compared to conditions without SO1861-SC-Mal.

[0257] Most strikingly, incubation with hCD71-DMD-ASO-SO1861, a conjugate incorporating both ASO and SO1861 conjugated to hCD71, revealed exon 51 skipping (8.5-8.6%) already at 10.0 nM conjugate, which increased to 36-53% at 361 nM, thus achieving a significant improvement in efficacy of the same order of magnitude compared to conditions without SO1861 and compared to the improvement achieved by co-administration of SO1861-SC-Mal (Figure 9C; Table A8).

[0258] More relevantly, in differentiated myotubes from DMD-affected donors, treatment with the targeted conjugates hCD71-DMD-ASO and hCD71-DMD-PMO also revealed skip rates up to 7-11% at 72 h post-treatment with 651 nM hCD71-DMD-ASO (Figure 9A, left panel; Table A7) and 610 nM hCD71-DMD-PMO (Figure 9B, 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 10A, right panel) and 37-57% skipping rates with 102 nM hCD71-DMD-PMO+SO1861-SC-Mal (Figure 10B, 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. Again, most strikingly, incubation with the ASO conjugated to hCD71 and the conjugate incorporating SO1861, hCD71-DMD-ASO-SO1861, revealed strong promotion of exon 51 skipping (94-96%) at 361 nM, and still 30-40% at the 10.0 nM conjugate (Figure 9C; Table A8), thus achieving a significant improvement in efficacy of the same order of magnitude compared to conditions without SO1861 and compared to the improvement achieved by co-administration of SO1861-SC-Mal.

[0259] [Table 17]

[0260] [Table 18]

[0261] [Table 19]

[0262] Example 5. mCD71-M23D-SO1861 (in vitro) Anti-CD71 monoclonal antibody targeting mouse CD71 (mCD71) was conjugated with SO1861-EMCH via cysteine ​​and M23D-thiol via lysine to generate mCD71-M23D-SO1861(DAR1.6 / 3.8) (Figure 8F, Figure 8G) and mCD71-M23D(DAR1.2) (Figure 2D), which were tested for dystrophin exon 23 skipping in differentiated mouse C2C12 myotubes. Treatment did not affect cell viability as determined by CTG assay, but enhanced exon 23 skipping was evident at 336 nM (40% skip rate), 84 nM (28% skip rate), 21 nM (21% skip rate), 5 nM (12% skip rate), and 1 nM (6% skip rate) (Figure ​(Figure111),11), whereas, as shown previously, no appreciable skipping was observed for mCD71-M23D at any of the concentrations tested (Figure 5, left panel). These data indicate that mCD71-M23D-SO1861 improves on-target delivery of M23D in myotubes by at least 1-2 orders of magnitude.

[0263] Example 6. mCD71-M23D-SO1861 (in vivo efficacy) CD-1 male mice were given a single injection of mCD71-M23D-SO1861 (DAR1.6 / 3.8) (Figure 8C, Figure 8D) or mCD71-M23D (DAR1.2) (Figure 2D) standardized with a similar nominal dose of the exon skipping active compound M23D (2.91 and 2.80 mg / kg, respectively). In addition, two dose groups with lower doses of mCD71-M23D-SO1861 were also included, as well as a vehicle control group.

[0264] As shown in Figures 12(A-C) and 13, animals receiving mCD71-M23D (2.80 mg / kg PMO; group 2) did not show exon 23 skipping in any of the tissues examined or at any time point (day 4, day 14, or day 28). Importantly, however, significant skipping was observed in the group receiving mCD71-M23D-SO1861 (2.91 mg / kg PMO, group 3) in all animals and in all muscle tissues examined: on day 4, 8.8% skipping rate in gastrocnemius, 6.4% in diaphragm, and even 17.1% in myocardium; on day 14, skipping increased to 19.3% skipping rate in gastrocnemius, 10.2% in diaphragm, and 18.4% in myocardium. The effect of a single dose was long-lasting with a skip rate of 8.5% in the gastrocnemius and 9.0% in the diaphragm even at day 28, and a skip rate of 7.6% was measured in the myocardium despite the administration of only an absolute PMO payload of 2.91 mg / kg (all percentages are mean ± standard error of the mean for n=2 / 3 tissues). Notably, low levels of skipping were observed for the mid and even low doses in some tissues and time points, notably 3.8% was observed in the myocardium at day 14 for the mid dose, despite containing an absolute PMO payload of only 0.42 mg / kg.

[0265] The data show that the conjugate containing the targeting ligand, oligonucleotide payload and most importantly SO1861 is highly effective and already at a single dose of 2.91 mg / kg of DMD targeting PMO, substantial exon skipping can be achieved in a variety of different skeletal muscles, but most importantly also in cardiac muscle, whereas the conjugate without SO1861 does not achieve any skipping at the equivalent dose. Even doses containing as little as 0.42 mg / kg absolute PMO payload can produce measurable skipping. The data support at least 1-2 orders of magnitude increase in potency over in vivo wild-type content.

[0266] In vivo tolerance of mCD71-M23D-SO1861 in CD-1 mice Conjugates mCD71-M23D-SO1861 and mCD71-M23D were prepared as described in Figures 8C, 8D and administered as detailed in Table A2. During the course of the study, the mCD71-M23D-SO1861 conjugate was well tolerated at all doses, with no treatment-specific adverse findings or any specific clinical observations indicating that the conjugate was not tolerated. However, one animal (out of the remaining six in Group 2) treated with mCD71-M23D was found dead on Day 11, and biomarker data were also missing for one of three mice on Day 28. At the time of sacrifice on day 14, two mice (out of three) treated with mCD71-M23D in group 2 showed renal abnormalities and elevated serum creatinine (Figure 14A), whereas importantly, no abnormalities were observed in the groups treated with mCD71-M23D-SO1861. No significant or sustained changes in the renal biomarker ALT were evident, with one at 161 U / L and two at 30 and 34 U / L, respectively, at the highest mCD71-M23D-SO1861 dose (group 3), resulting in a high degree of variability in this small group of N=3 (Figure 14B). Notably, from day 14 and 28 onwards, ALT levels in all treatment groups were comparable to vehicle controls (group 1), especially for mCD71-M23D-SO1861. In conclusion, mCD71-M23D-SO1861 was well tolerated at all doses tested. Collectively, these data indicate that the mCD71-M23D-SO1861 conjugate is highly effective while also being well tolerated in this study, exhibiting a more favorable tolerability profile than mCD71-M23D.

[0267] Methods (as carried out in Examples 7-9): SO1861 SO1861 is derived from Saponaria officinalis L (Extrasynthese, France) and was coupled to the respective handles according to methods known in the art by Symeres (NL).

[0268] Synthesis of DBCO-(M23D)2 The synthesis of DBCO-(M23D)2 was carried out by Symeres (NL).

[0269] Conjugation of SO1861 with antibodies Custom conjugate production of mCD63-SO1861 was carried out by Abzena (UK).

[0270] Conjugation of DBCO-(M23D)2 and antibodies Custom conjugate production of mCD71-M23D and mCD63-M23D was carried out by Abzena (UK).

[0271] Conjugation of DBCO-(M23D)2 and SO1861-antibody Custom conjugate production of mCD63-M23D-SC-SO1861 was carried out by Abzena (UK).

[0272] Conjugation of 3'-disulfide amide-M23D and SO1861-antibody Custom conjugate production of mCD71-M23D-EMCH-SO1861 (2) was carried out by Fleet Bioprocessing (UK).

[0273] 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.

[0274] 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.

[0275] 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.

[0276] 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.

[0277] 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.

[0278] 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.

[0279] 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.

[0280] 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.

[0281] 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.

[0282] mCD71-M23D-EMCH-SO1861(2) To an aliquot of mCD71-EMCH-SO1861 at DAR4.5 (20 mg, 0.126 μmol, 2.53 mg / ml) was added an aliquot of freshly prepared SMCC solution (2.0 mg / ml, 7 molar equivalents, 0.886 μmol), the mixture was vortexed briefly, and then incubated with end-over-end mixing for 60 min at 20° C. After incubation, the reaction was quenched by adding an aliquot of freshly prepared glycine solution (2.0 mg / ml, approximately 20 molar equivalents, 2.5 μmol), the mixture was vortexed briefly, and then incubated with end-over-end mixing for >15 min at 20° C. The conjugate was purified by Superdex 200 column eluted with TBS pH 7.5 and analyzed by UV-Vis to give purified mCD71-SO1861-SMCC (18.6 mg, yield: 93%, 0.942 mg / ml, SMCC to mCD71 ratio=3.9).

[0283] Separately, an aliquot of M23D-SS-amide (17.2 mg, 1.99 μmol, 10.0 mg / ml) reconstituted using TBS pH 7.5 was added to freshly prepared THPP solution (50 mg / ml, 5 molar equivalents, 10 μmol, 41 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).

[0284] To an aliquot of mCD71-SO1861-SMCC (17.2 mg, 0.11 μmol, 0.95 mg / ml), an aliquot of M23D-SH (4.1 mg / ml, 8 molar equivalents, 0.88 μmol, 1.72 ml) was added, 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-EMCH-SO1861(2) 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 the mCD71-M23D-EMCH-SO1861(2) conjugate (total yield=15.1 mg, 79%, ratio of M23D to mCD71-SO1861=2.1).

[0285] DBCO-(M23D)2 synthesis Intermediate 1 (N-[2-(4-{2-azatricyclo[10.4.0.0 4 , 9 ]Hexadeca-1(12),4(9),5,7,13,15-hexaen-10-yn-2-yl}-N-(2-{[(tert-butoxy)carbonyl]amino}ethyl)-4-oxobutanamido)ethyl]carbamate tert-butyl To a solution of DBCO acid (50.0 mg, 0.164 mmol) in DMF (1.00 ml) was added DIPEA (34.0 μL, 0.195 mmol) and HATU (62.3 mg, 0.164 mmol) and the mixture was stirred for 15 min. Then, di-tert-butyl (azanediylbis(ethane-2,1-diyl))dicarbamate (59.6 mg, 0.197 mmol) was added and the reaction mixture was stirred at room temperature. After 30 min, the reaction mixture was added to water (10.0 ml). The resulting thick suspension was centrifuged (5000 RPM, 3 min) to give a clear solution with a solid on top. The solution was removed by pipette and the solid was dissolved in acetonitrile (10.0 ml). The resulting solution was concentrated in vacuo. The residue was purified by flash chromatography (DCM-methanol / DCM (1 / 9, v / v) gradient 100:0 increasing to 0:50) to give the title product (90.0 mg, 93%) as a colorless solidified oil. 100% purity based on LC-MS. LRMS(m / z):591[M+H] 1+ LC-MS rt(min): 2.12 1

[0286] 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)

[0287] 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)

[0288] 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)

[0289] 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)

[0290] 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)

[0291] 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.

[0292] 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.

[0293] 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.

[0294] 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.

[0295] [Table 20]

[0296] Conjugation of mouse anti-CD63 (mCD63) mAb with SO1861 1. Preparation of mCD63 mCD63 was buffer exchanged into DPBS+5 mM EDTA, pH 7.4 using Vivaspin (50 kDa MWCO) to a final concentration of 8.0 mg / ml.

[0297] 2. Conjugation of mCD63 mCD63 was preincubated at 37° C. for about 15 min, followed by the addition of TCEP (3.8 eq.). The reaction mixture was diluted to 6.5 mg / ml and then incubated at 37° C. for 1 h. The reduction of mCD63 was monitored by modified LC-MS. The reaction was equilibrated at 22° C. and SO1861 (8.0 eq.) was added. The reaction was monitored by modified LC-MS, and when the reaction was complete, it was quenched with DTT (100 eq.).

[0298] 3. Purification of mCD63 Conjugates The reaction mixture was directly purified by P2 desalting column using DPBS. The conjugate was characterized by SEC and modified LC-MS (DAR determination, final extinction coefficient), then concentrated to >10 mg / ml using Vivaspin (50 kDa MWCO), sterile filtered through 0.22 μm filter unit and stored at +4° C. until further use.

[0299] [Table 21]

[0300] Conjugation of mouse anti-CD63 (mCD63) mAb-SO1861 with DBCO-(M23D)2 1. Preparation of mCD63 The mCD63-SO1861 conjugate was buffer exchanged into TBS using Vivaspin (50 kDa MWCO) to a final concentration of 10.0 mg / ml.

[0301] 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.

[0302] 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 mCD63. It was stored at 4° C. in the dark until further use in conjugation.

[0303] Step 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 eq, 1 mM in DPBS, pH 7.4) was added to this mCD63 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 a 0.22 μm filter unit and stored at 4°C until further use.

[0304] [Table 22]

[0305] 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.

[0306] 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.

[0307] 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.

[0308] 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.

[0309] [Table 23]

[0310] 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 or 180,000 cells per well (cpw) in 24-well plates or 40,000 or 30,000 cpw in 96-well plates (wp) and incubated at 37°C with 5% CO2. 24 hours after seeding, cells were switched to differentiation medium (2% horse serum in DMEM) and incubated for 3 days before changing the medium. After another 24 hours, the medium was changed again and compounds were added and incubated for 48 or 72 hours. After a total of 48 or 72 hours of treatment, cells at 24wp were harvested for exon skipping analysis and cell viability was assessed at 96wp.

[0311] Exon skipping analysis and quantification (mouse in vitro) Performed as described above in "Methods" (as carried out in Examples 1-6).

[0312] Cell viability (mouse in vitro) Performed as described above in "Methods" (as carried out in Examples 1-6).

[0313] Results (Examples 7 to 9) Example 7. 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 16. 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 17 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. 18A, right panel) and mCD63-M23D (clear band down to 6.0 nM, two orders of magnitude improvement) (FIG. 18B, right panel). mCD71-M23D alone showed no activity at any concentration tested up to 758 nM (Figure 18A, left panel), and mCD63-M23D alone showed only a 2% skip rate at 755 nM (Figure 18B, 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.

[0314] Example 8. mCD63-SC-SO1861+M23D or mCD71-M23D (in vitro) SO1861-SC-Mal was conjugated to an anti-CD63 monoclonal antibody targeting mouse CD63 to generate mCD63-SC-SO1861 (DAR4.1) (see FIG. 19 for conjugation procedure). mCD63-SC-SO1861 was co-administered with a fixed concentration of 500 nM M23D (phosphorodiamidate morpholino oligomer antisense oligonucleotide that induces exon 23 skipping of mouse dystrophin) into differentiated C2C12 mouse myotubes. This revealed a promotion of exon 23 skipping with 28% skipping rate at 662 nM mCD63-SC-SO1861 and 2% skipping rate at 1.6 nM after 48 hours of treatment (FIG. 20A), while no exon skipping activity was observed with 500 nM M23D alone (FIG. 20C). These data demonstrate that mCD63-SC-SO1861 induces on-target enhanced cytoplasmic delivery of M23D, thereby induced enhanced exon 23 skipping.

[0315] Next, mCD63-SC-SO1861 (Figure 19) was co-administered with a fixed concentration of 80 nM mCD71-M23D (Figure 17). Treatment with 80 nM mCD71-M23D alone did not result in exon skipping (Figure 20C). Co-administration of mCD63-SC-SO1861 with 80 nM mCD71-M23D revealed enhanced exon 23 skipping with 10% skipping at 662 nM mCD63-SC-SO1861 and still 4% skipping at 5.3 nM after 48 h of treatment (Figure 20B).

[0316] Taken together, these data indicate that combining SO1861 conjugated with ligand 1 (i.e., targeted SO1861) with a PMO payload conjugated with ligand 2 (i.e., targeted M23D) leads to significant potency enhancement (an example of a targeted two-component system).

[0317] Example 9. mCD71-M23D-EMCH-SO1861(2) or mCD63-M23D-SC-SO1861 (in vitro) Anti-CD71 monoclonal antibody targeting mouse CD71 was conjugated with both SO1861-EMCH and M23D-thiol to generate mCD71-M23D-EMCH-SO1861(2)(DAR2.1 / 4.5) (conjugation procedure previously described in Figure 8F, Figure 8G). Anti-CD63 monoclonal antibody targeting mouse CD63 was conjugated with both SO1861-SC-Mal and DBCO-(M23D)2 to generate mCD63-M23D-SC-SO1861(DAR3.6 / 4.1) (see Figure 21 for conjugation procedure). Treatment of differentiated C2C12 mouse myotubes with these conjugates for 72 hours revealed enhanced exon 23 skipping for mCD71-M23D-EMCH-SO1861(2) at 1200 nM (65% skip rate), 240 nM (27% skip rate), 48 nM (17% skip rate), 9.6 nM (6% skip rate), and 1.9 nM (7% skip rate) (Figure 22A). Enhanced exon 23 skipping was observed for mCD63-M23D-SC-SO1861 at 1200 nM (53% skip rate), 240 nM (22% skip rate), and 48 nM (11% skip rate) (Figure 22B). Treatment did not affect cell viability as determined by CTG assay. These data indicate that mCD71-M23D-EMCH-SO1861 (2) and mCD63-M23D-SC-SO1861 improve on-target delivery of M23D in myotubes by at least 1–3 orders of magnitude (examples of targeted one-component systems).

[0318] Methods (Examples 10 to 12) SO1861-SC-Maleimide SO1861 is derived from Saponaria officinalis L (Extrasynthese, France) and was coupled to the respective handles according to methods known in the art by Symeres (NL).

[0319] Conjugation of SO1861 with antibodies Custom conjugate production of hCD71-SC-SO1861 and hCD63-SC-SO1861 was carried out by Fleet Bioprocessing (UK).

[0320] Conjugation of 5'-thiol-DMD-ASO, 5'-disulfide amide-DMD-PMO (1), and 3'-disulfide amide-DMD-PMO (1, 2, 3, 4, and 5) with antibodies Custom conjugate production of hCD71-5'-SS-DMD-ASO, hCD71-5'-SS-DMD-PMO(1), hCD71-3'-SS-DMD-PMO(1), hCD71-3'-SS-DMD-PMO(2), hCD71-3'-SS-DMD-PMO(3), hCD71-3'-SS-DMD-PMO(4), hCD71-3'-SS-DMD-PMO(5), and hCD63-5'-SS-DMD-ASO was performed by Fleet Bioprocessing (UK).

[0321] Conjugation of 5'-thiol-DMD-ASO and 3'-disulfide amide-DMD-PMO (1, 2, 3, 4, and 5) with SO1861-antibody hCD71-5'-SS-DMD-ASO-SC-SO1861, hCD71-3'-SS-DMD-PMO(1)-SC-SO1861, hCD71-3'-SS-DMD-PMO(2)-SC-SO186 1, hCD71-3'-SS-DMD-PMO(3)-SC-SO1861, hCD71-3'-SS-DMD-PMO(4)-SC-SO1861, hCD71-3'-SS-DMD-PMO(5)-SC-S Custom conjugate production of O1861, hCD63-5'-SS-DMD-ASO-SC-SO1861, hCD63-3'-SS-DMD-PMO(1)-SC-SO1861, hCD63-3'-SS-DMD-PMO(2)-SC-SO1861, hCD63-3'-SS-DMD-PMO(3)-SC-SO1861, and hCD63-3'-SS-DMD-PMO(4)-SC-SO1861 was carried out by Fleet Bioprocessing (UK).

[0322] Analysis and preparation methods Conjugates were characterized by UV-Vis spectrophotometry, BCA colorimetric assay, analytical SEC, SDS-PAGE, Western blotting, and urea-PAGE gel electrophoresis. Prior to conjugation with SO1861-SC-maleimide, reduced mAb-SH (either hCD71-SH or hCD63-SH) was analyzed by UV-Vis spectrophotometry and Ellman's assay.

[0323] 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

[0324] 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.

[0325] 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).

[0326] 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.

[0327] 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).

[0328] hAb-SC-SO1861 A summary of the conjugations of hAb-SO1861, including hCD71-SC-SO1861 and hCD63-SC-SO1861, is given below, where the quantities given in italics in brackets are shown as examples for hCD71-SC-SO1861.

[0329] An aliquot of hAb was buffer exchanged into TBS pH 7.5 and normalized to 5 mg / ml. An aliquot of hAb (95.8 mg, 6.39 x 10 -4mmol, 4.94 mg / ml) with an aliquot of freshly prepared TCEP (3.18 equiv., 2.03 × 10 -3 A 1.0 mg aliquot (0.203 ml) of the reaction mixture was removed after incubation, purified on a Zeba 7K spin desalting column eluted with TBS pH 7.5, and characterized by UV-vis and Ellman assay (3.407 mg / ml, SH to hAb ratio = 4.8). To this bulk reaction, an aliquot of SO1861-SC-maleimide (6 mol equivalents, 3.79 x 10 -3 2 × 0.25 mg of desalted hAb-SH (1.67 × 10 mmol, 8.3 mg, 4.14 ml) was added with gentle stirring, the mixture was vortexed briefly, and then incubated for 120 min at 20 °C. -6 mmol, 0.073 ml) was dispensed and NEM (8 mol equivalents, 1.34 × 10 -5 10 mmol, 6.7 μl, 0.25 mg / ml) or TBS pH 7.5 (6.7 μl) and then incubated in parallel with bulk conjugation as positive and negative reaction controls, respectively. After incubation, 0.5 mg aliquots (approximately 0.100 ml) of the hAb-SC-SO1861 mixture were removed and purified by Zeba 7K spin desalting column eluted with TBS pH 7.5 and characterized in parallel with positive and negative controls by Ellman's assay to obtain SO1861 incorporation. After reaction, the bulk hAb-SC-SO1861 mixture was diluted with an aliquot of freshly prepared NEM solution (5 mol equivalents, 3.16 × 10 -3The reaction was quenched by the addition of 100 mmol of 2.5 mg / ml solution (158 μl of 2.5 mg / ml solution). The quenched reaction mixture was purified using a sanitized 2.6×40 cm Superdex 200 column eluted with DPBS pH 7.5. The purified hAb-SC-SO1861 was collected and analyzed by UV-Vis spectrophotometry. It was then concentrated to >2.5 mg / ml using Vivaspin 20 centrifugal filters, standardized to 2.5 mg / ml, and 0.2 μm filtered under laminar flow. The product was distributed into aliquots for product testing, characterization, and further conjugation work. The results were as follows: hCD71-SC-SO1861 (total yield = 78.4 mg, 82%, ratio of SO1861 to hCD71 = 4.0) hCD63-SC-SO1861 conjugate (total yield = 58.8 mg, SO1861 to hCD63 ratio = 4.8).

[0330] hAb-DMD-oligonucleotide The conjugation of hAb-targeted DMD oligonucleotides, including hCD71-5'-SS-DMD-ASO, hCD71-5'-SS-DMD-PMO(1), hCD71-3'-SS-DMD-PMO(1-5), and hCD63-5'-SS-DMD-ASO, is outlined below, with the amounts shown in italics within the brackets being shown as an example for hCD71-5'-SS-DMD-PMO(1).

[0331] 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.

[0332] 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).

[0333] An aliquot of hAb-SPDP (hCD71-SPDP, 10.2 mg, 6.79×10-5 mmol, 2.04 mg / ml) was added with an aliquot of oligonucleotide-SH (DMD-PMO(1)-SH, 2.73 mg / ml, 8.0 molar equivalents, 5.43×10-4 mmol, 2.01 ml), the mixture was vortexed briefly, and then incubated overnight at 20° C. with end-over-end mixing. After approximately 16 hours, the conjugate mixture was analyzed by UV-Vis to confirm incorporation by transfer of PDT, and then purified using a sanitized 2.6×60 cm Superdex 200PG column eluted with DPBS pH 7.5. The conjugate was analyzed by UV-Vis and BCA colorimetric assay to confirm fresh ε 280 was assigned. The material was concentrated (using Vivacell 100 (30K MWCO) to the highest concentration achievable) and then distributed into aliquots for product testing and characterization. The result, as an example, was hCD71-5'-SS-DMD-PMO(1) conjugate (total yield = 65%; DMD-PMO(1) to hCD71 ratio = 2.2). Other results were as follows: hCD71-3'-SS-DMD-PMO(1) (total yield = 58%, DMD-PMO(1) to hCD71 ratio = 2.1) hCD71-3'-SS-DMD-PMO(2) (total yield = 70%, DMD-PMO(2) to hCD71 ratio = 3.0) hCD71-3'-SS-DMD-PMO(3) (total yield = 65%, DMD-PMO(3) to hCD71 ratio = 2.6) hCD71-3'-SS-DMD-PMO(4) (total yield = 68%, DMD-PMO(4) to hCD71 ratio = 2.3) hCD71-3'-SS-DMD-PMO(5) (total yield = 67%, DMD-PMO(5) to hCD71 ratio = 2.1) hCD71-5'-SS-DMD-ASO (overall yield = 76%, DMD-ASO to hCD71 ratio = 2.1) hCD63-5'-SS-DMD-ASO (total yield = 60%, DMD-ASO to hCD63 ratio = 2.3)

[0334] hAb-DMD-oligonucleotide-SC-SO1861 A summary of the conjugation of hAb-DMD-oligo-SC-SO1861 is shown below, including hCD71-5'-SS-DMD-ASO-SC-SO1861, hCD63-5'-SS-DMD-ASO-SC-SO1861, hCD71-3'-SS-DMD-PMO(1-5)-SC-SO1861 and hCD63-3'-SS-DMD-PMO(1-5)-SC-SO1861.

[0335] hAb-SC-SO1861 (DAR4) was conjugated to a series of DMD oligonucleotides via lysine residues, targeting a DAR value of 4. Bulk activation of hAb-SC-SO1861 was performed, and the resulting SPDP intermediate was divided into aliquots for conjugation with each oligonucleotide. Initially, a scale of about 100 mg hAb was functionalized with PEG4-SPDP, then purified by gel filtration using a sanitized 5×50 cm Superdex 200 column, and characterized by UV-Vis to obtain hAb-SPDP. Prior to coupling of the DMD oligonucleotides, each oligonucleotide was activated, for example, by treatment with THPP, and then analyzed by UV-Vis and Ellman's assay to confirm the presence of thiols.

[0336] Each aliquot of hAb-SPDP was reacted with 8 equivalents of the corresponding oligonucleotide (i.e., 2 equivalents for each hAb-SPDP; 0.37-0.54 mg of oligonucleotide-SH is required per mg of hAb-SPDP) to obtain the hAb-oligonucleotide via a labile disulfide bond. Oligonucleotide incorporation was estimated by the transfer of PDT measured by UV-Vis. The conjugates were purified by gel filtration using a dedicated 2.6 x 40 cm Superdex 200 column eluted with Dulbecco's PBS pH 7.5. The purified conjugates were analyzed by BCA colorimetric assay to confirm hAb concentration and UV-Vis spectrophotometry, then concentrated and standardized before final filtration and distribution under laminar flow. A small aliquot of each (e.g., 0.25 mg) was reserved for characterization by aSEC, SDS-PAGE, Western blotting, and urea Page electrophoresis. The results were as follows: hCD71-3'-SS-DMD-PMO(1)-SC-SO1861 (total yield = 67%, SO1861 to hCD71 ratio = 4.0, DMD-PMO(1) to hCD71 ratio = 2.4) hCD71-3'-SS-DMD-PMO(2)-SC-SO1861 (total yield = 57%, SO1861 to hCD71 ratio = 4.0, DMD-PMO(2) to hCD71 ratio = 3.1) hCD71-3'-SS-DMD-PMO(3)-SC-SO1861 (total yield = 61%, SO1861 to hCD71 ratio = 4.0, DMD-PMO(3) to hCD71 ratio = 2.5) hCD71-3'-SS-DMD-PMO(4)-SC-SO1861 (total yield = 61%, SO1861 to hCD71 ratio = 4.0, DMD-PMO(4) to hCD71 ratio = 2.0) hCD71-3'-SS-DMD-PMO(5)-SC-SO1861 (total yield = 54%, SO1861 to hCD71 ratio = 4.0, DMD-PMO(5) to hCD71 ratio = 2.0) hCD71-5'-SS-DMD-ASO-SC-SO1861 (total yield = 62%, SO1861 to hCD71 ratio = 4.0, DMD-ASO to hCD71 ratio = 2.3) hCD63-3'-SS-DMD-PMO(1)-SC-SO1861 (total yield = 69%, SO1861 to hCD63 ratio = 4.8, DMD-PMO(1) to hCD63 ratio = 2.7) hCD63-3'-SS-DMD-PMO(2)-SC-SO1861 (total yield = 59%, SO1861 to hCD63 ratio = 4.8, DMD-PMO(2) to hCD63 ratio = 2.7) hCD63-3'-SS-DMD-PMO(3)-SC-SO1861 (total yield = 67%, SO1861 to hCD63 ratio = 4.8, DMD-PMO(3) to hCD63 ratio = 2.8) hCD63-3'-SS-DMD-PMO(4)-SC-SO1861 (total yield = 58%, SO1861 to hCD63 ratio = 4.8, DMD-PMO(4) to hCD63 ratio = 2.4) hCD63-5'-SS-DMD-ASO-SC-SO1861 (total yield = 51%, SO1861 to hCD63 ratio = 4.8, DMD-ASO to hCD63 ratio = 2.2)

[0337] Cell culture (human) Immortalized human myoblasts from a non-DMD donor (KM155) were cultured as described above in "Methods" (as carried out in Examples 1-6).

[0338] Human myoblasts from a fixed DMD-affected donor (KM1328) were cultured in homemade growth medium containing 80 ml 199 medium (Thermo Fisher Scientific) and 320 ml DMEM (Thermo Fisher Scientific) supplemented with 20% fetal bovine serum (Gibco, United Kingdom), 50 μg / ml gentamicin (Thermo Fisher Scientific), 25 μg / ml fetuin (Thermo Fisher Scientific), 5 ng / ml human epidermal growth factor (Thermo Fisher Scientific), 0.5 ng / ml basic fibroblast growth factor (Thermo Fisher Scientific), 5 μg / ml insulin (Sigma), and 0.2 μg / ml dexamethasone (Sigma). For differentiation, cells were seeded onto Matrigel-coated surfaces, which were prepared by incubation with 0.1 mg Corning™ Matrigel™ basement membrane matrix (Corning) per ml DMEM (Gibco) for 60 min at 37° C. At 100% confluence, growth medium was replaced with DMEM (Gibco) supplemented with 2% fetal bovine serum (Gibco), 10 μg / ml insulin (Sigma), and 50 μg / ml gentamicin (Thermo Fisher Scientific). After at least 3 days, but no longer than 5 days of differentiation, treatment was initiated based on the presence of differentiated myotubes).

[0339] 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.

[0340] For exon 51 and exon 53 skipping analysis in KM155 myotubes, the priming premix contained 1 μl dNTP mix (10 mM each) and 1 μl specific reverse primer (for KM155, exon 51: h53R 5′-CTCCGGTTCTGAAGGTGTTC-3′ [SEQ ID NO: 5]; exon 53: h55R 5′-ATCCTGTAGGACATTGGCAGTT-3 [SEQ ID NO: 6]). This mixture was heated at 70° C. for 5 min and then cooled on ice for at least 1 min. 0.5 μl rRNasin (Promega), 4.0 μl 5× RT buffer (Promega), 1.0 μl M-MLV RT (Promega), and 4.5 μl Reaction mixtures containing RNase-free water were prepared and added to the cooled mixtures to give a total volume of 20 μl for each reaction. RT-PCR was performed for 60 min at 42°C, then 5 min at 85°C and cooled on ice. For skip analysis, the nested PCR approach was followed. For this purpose, in the first PCR, 3 μl cDNA was added to a mixture of 2.5 μl 10× SuperTaq PCR buffer, 0.5 μl dNTP mix (10 mM each), 0.125 μl Taq DNA polymerase TAQ-RO (5 U / μl; Roche), 16.875 μl RNase-free water and 1 μl (10 pmol / μl) of each primer flanking the targeted exon. The following primers were used: for KM155, exon 51: h48F 5′-AAAAGACCTTGGGCAGCTTG-3′ [SEQ ID NO: 7] and h53R exon 53: h50F 5'-AGGAAGTTAGAAGATCTGAGC-3' [SEQ ID NO:20] and h55R 5'-ATCCTGTAGGACATTGGCAGTT-3' [SEQ ID NO:6]. These samples were subjected to a PCR run at 94°C for 5 minutes, followed by 25 cycles of 94°C for 40 seconds, 60°C for 40 seconds, 72°C for 180 seconds, followed by 72°C for 7 minutes.For the second PCR, 1.5 μl PCR1 sample was added to 5 μl 10× SuperTaq PCR buffer, 1 μl dNTP mix (10 mM each), 0.25 μl Taq DNA polymerase TAQ-RO (5 U / μl; Roche), 38.25 μl RNase-free water and 2 μl (10 pmol / μl) of a mixture of each primer flanking the targeted exon. The following primers were used: for KM155, exon 51: h49F 5′-CCAGCCACTCAGCCAGTG-3′ [SEQ ID NO: 10] and h52R2 5′-TTCTTCCAACTGGGGACGC-3′ [SEQ ID NO: 11], exon 53: h52F 5′-CCCCAGTTGGAAGAACTCATT-3′ [SEQ ID NO: 21] and h54R 5′-CCAAGAGGCATTGATATTCTC-3′ [SEQ ID NO: 9]. These samples were subjected to a PCR run at 94°C for 5 min, followed by 32 cycles of 94°C for 40 s, 60°C for 40 s, and 72°C for 60 s, followed by 72°C for 7 min. Exon skipping levels were quantified using the Femto Pulse system 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).

[0341] For exon 51 and exon 53 skipping analysis in KM1328 myotubes, the priming premix contained 1 μl dNTP mix (10 mM each) and 1 μl specific reverse primer (for KM1328, exons 51 and 53: h57R 5'-TCTGAACTGCTGGAAAGTCG-3' [SEQ ID NO: 22]). This mixture was heated at 70°C for 5 min and then cooled on ice for at least 1 min. Reaction mixtures containing 0.5 μl rRNasin (Promega), 4.0 μl 5x RT buffer (Promega), 1.0 μl M-MLV RT (Promega), and 4.5 μl RNase-free water were prepared and added to the cooled mixtures to yield a total volume of 20 μl for each reaction. RT-PCR was performed at 42°C for 60 min, then 70°C for 10 min and cooled on ice. For skipping analysis, the nested PCR approach was followed. For this purpose, in the first PCR, 3 μl cDNA was added to a mixture of 2.5 μl 10× SuperTaq PCR buffer, 0.5 μl dNTP mix (10 mM each), 0.125 μl Taq DNA polymerase TAQ-RO (5 U / μl; Roche), 16.875 μl RNase-free water and 1 μl (10 pmol / μl) of each primer flanking the targeted exon. The following primers were used: for KM1328, exon 51 and exon 53: h48F 5′-AAAAGACCTTGGGCAGCTTG-3′ [SEQ ID NO: 7] and h57R 5′-TCTGAACTGCTGGAAAGTCG-3′ [SEQ ID NO: 22]. The samples were subjected to a PCR run at 94° C. for 5 min, followed by 25 cycles of 94° C. for 40 s, 60° C. for 40 s, 72° C. for 180 s, followed by 72° C. for 7 min. For the second PCR, 1.5 μl PCR1 sample was added to a mixture of 5 μl 10× SuperTaq PCR buffer, 1 μl dNTP mix (10 mM each), 0.25 μl Taq DNA polymerase TAQ-RO (5 U / μl; Roche), 38.25 μl RNase-free water, and 2 μl (10 pmol / μl) of each primer flanking the targeted exon.The following primers were used: for KM1328, exons 51 and 53: h49F2 5'-AAACTGAAATAGCAGTTCAAGC-3' [SEQ ID NO: 23] and h54R 5'-CCAAGAGGCATTGATATTCTC-3' [SEQ ID NO: 9]. The samples were subjected to a PCR run of 5 min at 94°C, followed by 32 cycles of 40 s at 94°C, 40 s at 60°C, 90 s at 72°C, followed by 7 min at 72°C. Exon skipping levels were quantified by analyzing specific PCR fragments using a Bioanalyzer 2100 with a DNA1000 chip (Lab-on-a-Chip; Agilent). For exon 51 skipping, the expected non-skipped product is 793 bp in size (KM1328) and the skipped product is 560 bp (KM1328). For exon 53 skipping, the predicted non-skipped product is 793 bp in size (KM1328) and the skipped product is 581 bp (KM1328).

[0342] Results (Examples 10 to 12) Example 10. 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 23A-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 24A, left panel; Table A9), 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 24A, right panel; Table A10). Similarly, exposure to hCD71-5'-SS-DMD-PMO(1) induced minimal exon 51 skipping (0.4-1.1%) at 16.6-600 nM (Figure 24B, left panel; Table A9), 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 24B, right panel; Table A10), 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.

[0343] 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 23A-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 25A, left panel; Table A9), 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 25A, right panel; Table A10).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 25B, right panel; Table A10), whereas exposure to hCD71-3'-SS-DMD-PMO(2) alone resulted in only 5.7% exon 51 skipping even at 600 nM conjugate (Figure 25B, left panel; Table A9), 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 25C, right panel; Table A10), 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 25C, left panel; Table A9). 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.

[0344] 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 23A-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 26A, left panel; Table A9), whereas 2.78 nM hCD71-3'-SS-DMD-PMO(4) + SO1861-SC-Mal already resulted in a 5.4% exon 53 skipping rate (Figure 26A, right panel; Table A10). Similarly, exposure to 600 nM hCD71-3'-SS-DMD-PMO(5) alone resulted in an exon 53 skipping rate of 0.3% (Figure 26B, left panel; Table A9), 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 26B, right panel; Table A10), 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.

[0345] 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').

[0346] [Table 24]

[0347] [Table 25]

[0348] Example 11. hCD71-5'-SS-DMD-ASO + hCD63-SC-SO1861 or hCD63-5'-SS-DMD-ASO + hCD71-SC-SO1861 (in vitro) DMD-ASO-SH was conjugated to either an anti-CD71 monoclonal antibody targeting human CD71 or an anti-CD63 monoclonal antibody targeting human CD63 to generate hCD71-5'-SS-DMD-ASO (DAR2.1) and hCD63-5'-SS-DMD-ASO (DAR2.3), respectively (see Figures 23A-D for conjugation procedure). SO1861-SC-Mal was also conjugated to either an anti-CD71 monoclonal antibody targeting human CD71 or an anti-CD63 monoclonal antibody targeting human CD63 to generate hCD71-SC-SO1861 (DAR4.0) and hCD63-SC-SO1861 (DAR4.8), respectively (see Figure 27 for conjugation procedure). To differentiated human myotubes from a non-DMD (healthy) donor (KM155), hCD71-5'-SS-DMD-ASO and hCD63-5'-SS-DMD-PMO were co-administered with a fixed concentration of 100 nM hCD63-SC-SO1861 or hCD71-SC-SO1861, respectively. Strikingly, co-administration of hCD71-5'-SS-DMD-ASO with 100 nM hCD63-SC-SO1861 revealed enhanced exon 51 skipping, with an exon skipping rate of 28.7% already at 0.46 nM hCD71-5'-SS-DMD-ASO, which increased to exon skipping rates of 50.0-68.1% at 16.6-100 nM hCD71-5'-SS-DMD-ASO (Figure 28A; Table A11). Coadministration of hCD63-5'-SS-DMD-ASO with 100 nM hCD71-SC-SO1861 revealed a 29.2% exon skipping rate at 16.6 nM and enhanced exon 51 skipping (Figure 28B; Table A11). These data indicate that hCD63-SC-SO1861 and hCD71-SC-SO1861 induce enhanced on-target cytoplasmic delivery of hCD71-targeted or hCD63-targeted DMD-ASO, thereby enhancing exon 51 skipping.

[0349] Next, we switched between titrated conjugates and co-administered conjugates at a fixed concentration. Differentiated human muscle from a non-DMD (healthy) donor (KM155) was treated with hCD63-SC-SO1861 in combination with a fixed concentration of 55.6 nM hCD71-5'-SS-DMD-ASO. This revealed that exon 51 skipping was promoted with 72.6% exon skipping rate at 16.6 nM hCD63-SC-SO1861, 53.1% exon skipping rate at 2.78 nM, and still 21.0% exon skipping rate at 0.46 nM (Figure 29A; Table A12). More strikingly and relevantly, in differentiated myotubes from a DMD-affected donor (KM1328), co-administration of hCD63-SC-SO1861 with a constant concentration of 55.6 nM hCD71-5'-SS-DMD-ASO already resulted in a 53.8% skip rate at 0.46 nM hCD63-SC-SO1861, which increased to a 93.4% skip rate at 16.6 nM and a 97.0% skip rate at 600 nM hCD63-SC-SO1861 (Figure 29B, Table A12).

[0350] Taken together, these data indicate that combining a ligand 1-conjugated oligonucleotide payload (i.e., either hCD71- or hCD63-targeted DMD-ASO) with a ligand 2-conjugated SO1861 (i.e., either hCD71- or hCD63-targeted SO1861), or vice versa, i.e., combining a ligand 2-conjugated oligonucleotide payload with a ligand 1-conjugated SO1861, leads to a significant enhancement of efficacy in disease-relevant cell lines, such as human myotubes, specifically differentiated myotubes from DMD-affected donors (an example of a two-target, two-component system).

[0351] [Table 26]

[0352] [Table 27]

[0353] Example 12. hCD71-5'-SS-DMD-ASO-SC-SO1861 or hCD63-5'-SS-DMD-ASO-SC-SO1861 or hCD71-3'-SS-DMD-PMO(1, 2, 3, 4, or 5)-SC-SO1861 or hCD63-DMD-PMO(1, 2, 3, or 4)-SC-SO1861 (in vitro) SO1861-SC-Mal was conjugated to either an anti-CD71 monoclonal antibody targeting human CD71 (hCD71) or an anti-CD63 monoclonal antibody targeting human CD63 (hCD63), followed by conjugation of DMD-ASO-SH via disulfide bond formation to the 5' to generate hCD71-5'-SS-DMD-ASO-SC-SO1861 (DAR 2.3 / 4.0) and hCD63-5'-SS-DMD-ASO-SC-SO1861 (DAR 2.2 / 4.8), respectively (see Figures 30A-C for conjugation procedure). Strikingly, treatment of differentiated human myotubes from a DMD-affected donor (KM1328) with these conjugates revealed a strong promotion of exon 51 skipping: hCD71-5'-SS-DMD-ASO-SC-SO1861 showed 99.6% skipping rate at 600 nM conjugate, 96.3% skipping rate at 100 nM, 64.7% skipping rate at 16.6 nM, and still 79.2% skipping rate at 2.78 nM (Figure 31A, left panel; Table A13). Exon skipping was still measurable down to at least 0.46 nM hCD71-5'-SS-DMD-ASO-SC-SO1861. Similarly, hCD63-5'-SS-DMD-ASO-SC-SO1861 showed 100.0% exon skipping rate at 100-600 nM, 74.6% skipping rate at 16.6 nM, and still 62.4% skipping rate at 2.78 nM conjugate (Figure 31A, right panel; Table A13). These data indicate that incubation with DMD-ASO conjugated to either hCD71 or hCD63 and conjugates incorporating SO1861-SC-Mal results in a marked improvement in achieving maximum dystrophin exon 51 skipping rate in human myotubes from DMD-affected donors.

[0354] Next, conjugates incorporating DMD-PMO(1, 2, or 3) conjugated to either hCD71 or hCD63 and SO1861-SC-Mal were tested for dystrophin exon 51 skipping in human myotubes from a DMD-affected donor (KM1328). SO1861-SC-Mal was conjugated to either an anti-CD71 monoclonal antibody targeting human CD71 or an anti-CD63 monoclonal antibody targeting human CD63, followed by conjugation of DMD-PMO(1)-SH, DMD-PMO(2)-SH, or DMD-PMO(3)-SH through disulfide bond formation to the 3' to obtain hCD71-3'-SS-DMD-PMO(1)-SC-SO1861 (DAR2.4 / 4.0), hCD63-3'-SS-DMD-PMO(1)-SC-SO1861 (DAR2.4 / 4.0), hCD63-3'-SS-DMD-PMO(1)-SC-SO1 The following conjugated antibodies were generated: hCD71-3'-SS-DMD-PMO(2)-SC-SO1861 (DAR 2.7 / 4.8), hCD71-3'-SS-DMD-PMO(2)-SC-SO1861 (DAR 3.1 / 4.0), hCD63-3'-SS-DMD-PMO(2)-SC-SO1861 (DAR 2.7 / 4.8), hCD71-3'-SS-DMD-PMO(3)-SC-SO1861 (DAR 2.5 / 4.0), and hCD63-3'-SS-DMD-PMO(3)-SC-SO1861 (DAR 2.8 / 4.8) (see Figures 30A-C for conjugation procedure). Treatment with both of these conjugates was found to significantly promote exon 51 skipping in human myotubes from a DMD-affected donor (KM1328) (Table A13): 75.8% exon skipping rate with 600 nM hCD71-3'-SS-DMD-PMO(1)-SC-SO1861 (Figure 31B, left panel) and 96.5% exon skipping rate with 600 nM hCD63-3'-SS-DMD-PMO(1)-SC-SO1861 (Figure 31B, right panel). Exon 51 skipping was still 76.9% with 2.78 nM hCD63-3'-SS-DMD-PMO(1)-SC-SO1861, with measurable effects down to at least 0.46 nM hCD63-3'-SS-DMD-PMO(1)-SC-SO1861.Even more strikingly, hCD63-3'-SS-DMD-PMO(2)-SC-SO1861 revealed 100.0% exon skipping at 2.78-600 nM, and still 53.6% at 0.46 nM (Figure 31C, right panel).Similarly, hCD71-3'-SS-DMD-PMO(2)-SC-SO1861 revealed strong promotion of exon 51 skipping, with 100.0% exon skipping at 600 nM, 97.6% at 100 nM, 92.1% at 16.6 nM, 76.3% at 2.78 nM, and still 28.3% at 0.46 nM (Figure 31C, left panel). Finally, hCD71-3'-SS-DMD-PMO(3)-SC-SO1861 and hCD63-3'-SS-DMD-PMO(3)-SC-SO1861 also showed strong promotion of exon 51 skipping, with 86.7% skipping rate at 600 nM hCD71-3'-SS-DMD-PMO(3)-SC-SO1861 (Figure 31D, left panel) and 89.6-98.2% exon skipping rate at 2.78-600 nM hCD63-3'-SS-DMD-PMO(3)-SC-SO1861 (Figure 31D, right panel). Significant exon 51 skipping was measurable down to at least 0.46 nM (20.0%) hCD63-3'-SS-DMD-PMO(3)-SC-SO1861. Taken together, these data indicate that incubation with conjugates incorporating both DMD-PMO and SO1861-SC-Mal conjugated to either hCD71 or hCD63 produced a marked improvement in achieving maximal exon 51 skipping rates in human myotubes from DMD-affected donors bearing a variety of different DMD-PMO targeting sequences.

[0355] In addition, conjugates incorporating DMD-PMO (4 or 5) and SO1861-SC-Mal conjugated to either hCD71 or hCD63 were tested for dystrophin exon 53 skipping in human myotubes from a DMD-affected donor (KM1328). SO1861-SC-Mal was conjugated to either an anti-CD71 monoclonal antibody targeting human CD71 or an anti-CD63 monoclonal antibody targeting human CD63, followed by conjugation of DMD-PMO(4)-SH or DMD-PMO(5)-SH via disulfide bond formation to the 3' to generate hCD71-3'-SS-DMD-PMO(4)-SC-SO1861 (DAR 2.0 / 4.0), hCD63-3'-SS-DMD-PMO(4)-SC-SO1861 (DAR 2.4 / 4.8), and hCD71-3'-SS-DMD-PMO(5)-SC-SO1861 (DAR 2.0 / 4.0) (see Figures 30A-C for conjugation procedure). Treatment with both of these conjugates was found to significantly promote exon 53 skipping in human myotubes from a DMD-affected donor (KM1328) (Table A13): 93.6% exon skipping rate with 600 nM hCD71-3'-SS-DMD-PMO(4)-SC-SO1861 (Figure 32A, left panel) and 98.0% skipping rate with 600 nM hCD63-3'-SS-DMD-PMO(4)-SC-SO1861 (Figure 32A, right panel). Exon 53 skipping was still 90.6% with 2.78 nM hCD63-3'-SS-DMD-PMO(4)-SO1861, and the effect was measurable down to at least 0.46 nM hCD63-3'-SS-DMD-PMO(4)-SC-SO1861. Similarly, hCD71-3'-SS-DMD-PMO(5)-SC-SO1861 revealed an exon skipping rate of 94.4% at 600 nM and still 19.1% at 2.78 nM (Figure 32B).These data indicate that incubation with conjugates incorporating both DMD-PMO and SO1861-SC-Mal conjugated to either hCD71 or hCD63 produced a marked improvement in achieving maximal exon 53 skipping rates in human myotubes from DMD-affected donors bearing a variety of different DMD-PMO targeting sequences.

[0356] In conclusion, conjugates incorporating DMD oligonucleotides conjugated to either hCD71 or hCD63 and SO1861-SC-Mal with a variety of different oligonucleotide targeting sequences (various different exons, various different sequences) lead to significant enhancement of efficacy in relevant cell lines such as differentiated myotubes from DMD-affected donors (an example of a targeted one-component system).

[0357] [Table 28]

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Claims

1. 1. A pharmaceutical composition for use in the treatment or prevention of a muscle wasting disorder, comprising: saponin, nucleic acids, and Ligands for endocytic receptors on muscle cells In a composition comprising a covalently linked conjugate comprising The composition, wherein the saponin is a triterpenoid 12,13-dehydrooleanane-type saponin.

2. The muscle wasting disorder is a muscle cell-related genetic disorder or is 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 a muscle cell-related genetic disorder that is familial dilated cardiomyopathy; Or, the muscle wasting disorder is a muscle cell-related genetic disorder that is a dystrophinopathy 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. 3. The composition for use according to claim 1 or 2, wherein the saponin, at least in its unconjugated state, comprises an aldehyde group at the C-23 position of the saponin aglycone core structure.

5. The saponin aglycone core structure is Quila acid; Gypsogenin; 2α-hydroxyoleanolic acid; 16α-hydroxyoleanolic acid; Hederagenin (23-hydroxyoleanolic acid); 16α,23-dihydroxyoleanolic acid; Protoestigenin-21(2-methylbut-2-enoate)-22-acetate; 23-Oxo-vallingtogenol C-21,22-bis(2-methylbut-2-enoate); 23-Oxo-barringtogenol C-21(2-methylbut-2-enoate)-16,22-diacetate; 3,16,28-trihydroxyoleanan-12-ene; Gypsogenic acid; and Its derivatives is selected from one or more of the following:

3. The composition for use according to claim 1 or 2, wherein the saponin aglycone core structure is selected from quillaric acid, gypsogenin, and derivatives thereof, or wherein the saponin aglycone core structure is quillaric acid.

6. The sugar fraction of the saponin is as shown in the following table: Table 1 Table 2 3. The composition for use according to claim 1 or 2, comprising a glycan selected from any one of the glycans as listed in Group A or Group B presented in .

7. the saponin is at least a bidesmosidic saponin containing a first sugar chain selected from Group A and a second sugar chain selected from Group B; or the first glycan comprises a terminal glucuronic acid residue and / or the second glycan comprises at least four saccharide 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.

8. 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. 3. The composition for use according to claim 1 or 2, wherein the conjugate comprises two or more molecules of the saponin, or comprises 2 to 32 molecules of the saponin, or 4 to 16 molecules of the saponin, or 4 to 8 molecules of the saponin.

10. 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 comprising a chiral acid aglycone core structure selected from: d) List D: Escin Ia, escinate, α-hederin, AMA-1, AMR, AS6.2, AS64R, assamsaponin F, dipsacoside B, esculentoside A, macrantoidin A, NP-005236, NP-012672, primulic acid 1, saikosaponin A, saikosaponin D, tea seed saponin I, and tea seed saponin J A saponin having a 12,13-dehydrooleanane-type aglycone core structure without an aldehyde group at the C-23 position of the aglycone, selected from One or more of the following: Or, the saponin is any one or more of a saponin selected from list A, B or C, or a saponin selected from list B or C, or a saponin selected from list C.

11. 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; or the saponin is any one or more of QS-21, SO1832, SO1861, SA1641, and GE1741; or the saponin is QS-21, SO1832, or SO1861; Or the composition for use according to claim 1 or 2, wherein the saponin is SO1861.

12. The saponin is a saponin isolated from soapwort (Saponaria officinalis), or the saponin is any one or more of saponarioside B, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862, and SO1904; or the saponin is any one or more of SO1832, SO1861, and SO1862; or the saponins are SO1832 and SO1861; Or the composition for use according to claim 1 or 2, wherein the saponin is SO1861.

13. 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 in size, or 8-100 nt, or 10-50 nt; or the oligonucleotide is an antisense oligonucleotide, or a mutation-specific antisense oligonucleotide, or an oligonucleotide designed to induce exon skipping.

14. The oligonucleotide may be selected from the group consisting of morpholino phosphorodiamidate oligomers (PMO), 2'-O-methyl (2'-OMe) phosphorothioate RNA, 2'-O-methoxyethyl (2'-O-MOE) RNA {2'-O-methoxyethyl-RNA (MOE)}, locked or bridged nucleic acids (LNA or BNA), 2'-O,4'-aminoethylene bridged nucleic acids (BNANC), peptide nucleic acids (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 DNA, single-stranded RNA or DNA, double-stranded RNA (dsRNA) or DNA. comprising or consisting of any one of: Or the composition for use according to claim 13, wherein the oligonucleotide comprises or consists of a morpholino phosphorodiamidate oligomer (PMO) or a 2'-O-methyl (2'-OMe) phosphorothioate RNA.

15. the oligonucleotide 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 oligonucleotide 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 13, wherein the oligonucleotide is selected from eteplirsen, drisapersen, golodirsen, viltolarsen, and casimersen.

16. the conjugate comprises two or more molecules of the nucleic acid; or said two or more molecules of said nucleic acid are 2 to 16 molecules, or 2 to 8 molecules, or optionally 2, 3, 4, 5, or 6 molecules; Or the two or more molecules of the nucleic acid are two or more oligonucleotides; optionally two or more different oligonucleotides, and at least one of the two or more different oligonucleotides is an antisense oligonucleotide.

17. 3. The composition for use according to claim 1 or 2, wherein the conjugate comprises 1 to 16 molecules of the saponin and 1 to 5 molecules of the nucleic acid per molecule of the ligand.

18. the conjugate comprises 2 to 8 molecules of the saponin per molecule of the ligand; or 3 to 6 molecules of said saponin per molecule of said ligand; or 4 to 5 molecules of said saponin per molecule of said ligand; Or the composition for use according to claim 17, wherein the conjugate comprises an average of 4 to 4.5 molecules of the saponin per molecule of the ligand.

19. the conjugate comprises 2 to 5 molecules of the nucleic acid per molecule of the ligand; or 3 to 4 molecules of the nucleic acid per molecule of the ligand; Or the composition for use according to claim 17, wherein the conjugate comprises an average of four molecules of the nucleic acid per molecule of the ligand.

20. 3. The composition for use according to claim 1 or 2, wherein the endocytic receptor on muscle cells to which the ligand binds is selected from transferrin receptor (CD71), insulin-like growth factor 1 (IGF-I) receptor (IGF1R), insulin-like growth factor 2 (IGF-II) receptor (IGF2R), tetraspanin CD63; muscle-specific kinase (MuSK), glucose transporter GLUT4, cation-independent mannose-6-phosphate receptor (CI-MPR), and LDL receptor.

21. 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 a binding fragment thereof specific for binding to the endocytosis receptor, or the endocytosis receptor is selected from transferrin receptor (CD71), insulin-like growth factor 1 (IGF-I) receptor (IGF1R), insulin-like growth factor 2 (IGF-II) receptor (IGF2R), 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: or the ligand is an antibody or a binding fragment thereof specific for binding to the transferrin receptor, or the ligand is a monoclonal antibody or Fab' fragment or at least one single domain antibody specific for binding to the transferrin receptor, or the composition for use according to claim 1 or 2, wherein the ligand is a monoclonal antibody specific for binding to the transferrin receptor.

22. the covalent linking of the saponin within the conjugate is via a first linker to which the saponin is covalently attached; Or the first linker is containing a covalent bond selected from any one or more of a semicarbazone bond, a hydrazone bond, an imine bond, an acetal bond including a 1,3-dioxolane bond, a ketal bond, an ester bond, an oxime bond, a disulfide bond, a thioether bond, an amide bond, a peptide bond, and an ester bond, or which is a hydrazone bond or a semicarbazone bond; or the saponin, at least in an unconjugated state, is a saponin comprising an aldehyde group at C-23 position of the saponin aglycone core structure, and the aldehyde group participates in the formation of a covalent bond with the first linker.

23. the first linker is a cleavable linker that undergoes cleavage under acidic, reducing, enzymatic and / or photoinduced conditions; Or the first linker is bonds that undergo cleavage under acidic conditions, such as semicarbazone bonds, hydrazone bonds, imine bonds, acetal bonds including 1,3-dioxolane bonds, ketal bonds, ester bonds, and / or oxime bonds; a proteolytically susceptible bond, optionally an amide or peptide bond, or a bond that is subject to proteolysis by cathepsin B; - Bonds that are cleavable by oxidation and reduction, such as disulfide bonds, or bonds that are susceptible to thiol exchange reactions, such as thioether bonds comprising a cleavable bond selected from: or an acid-sensitive bond that undergoes cleavage in vivo under acidic conditions present in the endosomes and / or lysosomes of human cells, or at a pH of 4.0 to 6.5, or at a pH of ≦5.5; or an acid-sensitive bond selected from any one or more of a semicarbazone bond, a hydrazone bond, an imine bond, an acetal bond including a 1,3-dioxolane bond, a ketal bond, an ester bond, and / or an oxime bond, or selected from a semicarbazone bond and a hydrazone bond; or a hydrazone bond.

24. the first linker further comprises any oligomeric or polymeric structure, whether it is a dendron, such as a polyamidoamine (PAMAM) dendrimer, or a polyethylene glycol, such as any of PEG3 to PEG30; 23. The composition for use according to claim 22, wherein the polymer or oligomer structure is any one of PEG4 to PEG12, or any one of G2 dendron, G3 dendron, G4 dendron and G5 dendron, or a G2 dendron or a G3 dendron or PEG3 to PEG30.

25. the covalent linking of the nucleic acid within the conjugate is effected by a second linker to which the nucleic acid is covalently attached; or said second linker comprises or consists of the linker succinimidyl 3-(2-pyridyldithio)propionate (SPDP); 3. The composition for use according to claim 1 or 2, wherein optionally the second linker covalently links the nucleic acid to a lysine residue, or a lysine residue that is a lysine residue contained in the ligand, or to a glycan residue, or a partially trimmed glycan.

26. the second linker is a cleavable linker that undergoes cleavage under acidic, reducing, enzymatic and / or photoinduced conditions; Or the second linker is bonds that undergo cleavage under acidic conditions, such as semicarbazone bonds, hydrazone bonds, imine bonds, acetal bonds including 1,3-dioxolane bonds, ketal bonds, ester bonds, and / or oxime bonds; a proteolytically susceptible bond, optionally an amide or peptide bond, or a bond that is subject to proteolysis by cathepsin B; - Bonds that are cleavable by oxidation and reduction, such as disulfide bonds, or bonds that are susceptible to thiol exchange reactions, such as thioether bonds comprising a cleavable bond selected from: or an acid-sensitive bond that undergoes cleavage in vivo under acidic conditions present in the endosomes and / or lysosomes of human cells, or at a pH of 4.0 to 6.5, or at a pH of ≦5.5; or an acid-sensitive bond selected from any one or more of a semicarbazone bond, a hydrazone bond, an imine bond, an acetal bond including a 1,3-dioxolane bond, a ketal bond, an ester bond, and / or an oxime bond, or selected from a semicarbazone bond and a hydrazone bond; or a hydrazone bond.

27. 23. The composition for use according to claim 22, wherein the first linker and / or the second linker are directly or indirectly covalently linked to the ligand.

28. the covalent linking of the ligand within the conjugate is effected by a third linker to which the ligand is covalently attached; or the ligand comprises a chain of amino acid residues comprising at least one cysteine ​​residue and / or at least one lysine residue, and the third linker comprises a covalent bond made with the at least one cysteine ​​residue or the at least one lysine residue; or wherein the ligand comprises a chain of amino acid residues comprising a multicysteine ​​repeat, which is optionally a tetracysteine ​​repeat represented by the sequence HRWCCPGCCKTF (SEQ ID NO: 4), and the third linker comprises a covalent bond to any one or more of the cysteine ​​residues of the multicysteine ​​repeat; and optionally two or more third linkers are linked to one molecule of the ligand by each of the two or more third linkers binding to a distinct cysteine ​​residue of the multicysteine ​​repeat comprised in the ligand.

29. the conjugate comprises a central linker for achieving a covalent linkage between at least one molecule of the saponin, at least one molecule of the nucleic acid, and at least one molecule of the ligand, the covalent linkage being achieved either directly or via the first linker, the second linker, and / or the third linker, respectively; Or the central linker is a trifunctional linker.

30. The central linker, in its unconjugated form, has the structure A: 【Chemistry 1】 30. The composition for use of claim 29, wherein the trifunctional linker is represented by:

31. 30. The composition for use according to claim 29, wherein the conjugate comprises 1 to 4 of said trifunctional linkers, or 1 to 2 trifunctional linkers, or an average of 1.2 to 1.8 trifunctional linkers, for each molecule of said ligand contained in said conjugate.

32. The trifunctional linker, in its conjugated form, has the structure B: 【Chemistry 2】 (In the formula, S is the at least one molecule of the saponin; L1 is the first linker to which the at least one molecule of the saponin is covalently attached; E is at least one molecule of the nucleic acid; L2 is the second linker to which the at least one molecule of the nucleic acid is covalently attached; A is at least one molecule of said ligand, or an antibody or binding fragment thereof; and L3 is the third linker to which the at least one molecule of the ligand is covalently bound; 30. The composition for use according to claim 29, wherein L1, L2 and L3 are the same or different.

33. 3. The composition for use according to claim 1 or 2, wherein the saponin is or comprises at least one molecule of SO1861, the nucleic acid is drisapersen or eteplirsen or golodirsen or viltolarsen, or drisapersen or eteplirsen, and the ligand is an anti-CD71 antibody or a binding fragment thereof.

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

35. 3. A composition for use according to claim 1 or 2, comprising a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent.

36. 1. A covalently linked conjugate for delivery of a therapeutic nucleic acid into muscle cells, comprising: saponin, nucleic acids, and Ligands for endocytic receptors on muscle cells In a conjugate comprising The saponin is a triterpenoid 12,13-dehydrooleanane-type saponin, and The conjugate comprises 1 to 16 molecules of the saponin and 1 to 5 molecules of the nucleic acid per molecule of the ligand.

37. the conjugate comprises 2 to 8 molecules of the saponin per molecule of the ligand; or 3 to 6 molecules of said saponin per molecule of said ligand; or 4 to 5 molecules of the saponin per molecule of the ligand. Including; Or the conjugate of claim 36, wherein the conjugate comprises an average of 4 to 4.5 molecules of the saponin per molecule of the ligand.

38. the conjugate comprises 2 to 5 molecules of the nucleic acid per molecule of the ligand; or 3 to 4 molecules of the nucleic acid per molecule of the ligand. Including; Or the conjugate of claim 36 or 37, wherein the conjugate comprises an average of four molecules of the nucleic acid per molecule of the ligand.

39. 38. The conjugate of claim 36 or 37, wherein the saponin, at least in its unconjugated state, comprises an aldehyde group at the C-23 position of the saponin aglycone core structure.

40. The saponin aglycone core structure is Quila acid; Gypsogenin; 2α-hydroxyoleanolic acid; 16α-hydroxyoleanolic acid; Hederagenin (23-hydroxyoleanolic acid); 16α,23-dihydroxyoleanolic acid; Protoestigenin-21(2-methylbut-2-enoate)-22-acetate; 23-Oxo-vallingtogenol C-21,22-bis(2-methylbut-2-enoate); 23-Oxo-barringtogenol C-21(2-methylbut-2-enoate)-16,22-diacetate; 3,16,28-trihydroxyoleanan-12-ene; Gypsogenic acid; and Its derivatives is selected from one or more of the following: or the saponin aglycone core structure is selected from quillaric acid, gypsogenin, and derivatives thereof, or the saponin aglycone core structure is quillaric acid.

41. The sugar fraction of the saponin is as shown in the following table: Table 3 Table 4 Table 5 38. The conjugate of claim 36 or 37, comprising a sugar chain selected from any one of the sugar chains as listed in Group A or Group B presented in .

42. the saponin is at least a bidesmosidic saponin containing a first sugar chain selected from Group A and a second sugar chain selected from Group B; or the first glycan comprises a terminal glucuronic acid residue and / or the second glycan comprises at least four saccharide 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.

43. 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.

44. 38. The conjugate of claim 36 or 37, wherein the conjugate comprises two or more molecules of the saponin, or comprises 2 to 32 molecules of the saponin, or 4 to 16 molecules of the saponin, or 4 to 8 molecules of the saponin.

45. 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 comprising a chiral acid aglycone core structure selected from: d) List D: Escin Ia, escinate, α-hederin, AMA-1, AMR, AS6.2, AS64R, assamsaponin F, dipsacoside B, esculentoside A, macrantoidin A, NP-005236, NP-012672, primulic acid 1, saikosaponin A, saikosaponin D, tea seed saponin I, and tea seed saponin J A saponin having a 12,13-dehydrooleanane-type aglycone core structure without an aldehyde group at the C-23 position of the aglycone, selected from One or more of the following: Or the conjugate of claim 36 or 37, wherein the saponin is any one or more of a saponin selected from list A, B or C, or a saponin selected from list B or C, or a saponin selected from list C.

46. 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; or the saponin is any one or more of QS-21, SO1832, SO1861, SA1641, and GE1741; or the saponin is QS-21, SO1832, or SO1861; Or the saponin is SO1861.

47. The saponin is a saponin isolated from soapwort (Saponaria officinalis), or the saponin is any one or more of saponarioside B, SO1542, SO1584, SO1658, SO1674, SO1700, SO1730, SO1772, SO1832, SO1861, SO1862, and SO1904; or the saponin is any one or more of SO1832, SO1832, SO1861, and SO1862; or the saponins are SO1832 and SO1861; Or the saponin is SO1861.

48. the nucleic acid is an oligonucleotide, defined as a nucleic acid not exceeding 150 nt, or the oligonucleotide is 5-150 nt in size, or 8-100 nt, or 10-50 nt; 38. The conjugate of claim 36 or 37, wherein the oligonucleotide is an antisense oligonucleotide, or a mutation-specific antisense oligonucleotide, or an oligonucleotide designed to induce exon skipping.

49. The oligonucleotide may be selected from the group consisting of morpholino phosphorodiamidate oligomers (PMO), 2'-O-methyl (2'-OMe) phosphorothioate RNA, 2'-O-methoxyethyl (2'-O-MOE) RNA {2'-O-methoxyethyl-RNA (MOE)}, locked or bridged nucleic acids (LNA or BNA), 2'-O,4'-aminoethylene bridged nucleic acids (BNANC), peptide nucleic acids (PNA), 2'-deoxy-2'-fluoroarabinonucleic acid (FANC), and 2'-deoxy-2'-fluoroarabinonucleic acid (FANC). NA), 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 DNA; Or the conjugate of claim 48, wherein the oligonucleotide comprises or consists of a morpholino phosphorodiamidate oligomer (PMO) or a 2'-O-methyl (2'-OMe) phosphorothioate RNA.

50. the oligonucleotide 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 oligonucleotide 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 conjugate of claim 48, wherein the oligonucleotide is selected from eteplirsen, drisapersen, golodirsen, viltolarsen, and casimersen.

51. the conjugate comprises two or more molecules of the nucleic acid; or said two or more molecules of said nucleic acid are 2 to 16 molecules, or 2 to 8 molecules, or optionally 2, 3, 4, 5, or 6 molecules; Or the two or more molecules of the nucleic acid are two or more oligonucleotides; optionally two or more different oligonucleotides, and at least one of the two or more different oligonucleotides is an antisense oligonucleotide.

52. 38. The conjugate of claim 36 or 37, wherein the conjugate comprises 1 to 16 molecules of the saponin and 1 to 5 molecules of the nucleic acid per molecule of the ligand.

53. the conjugate comprises 2 to 8 molecules of the saponin per molecule of the ligand; or 3 to 6 molecules of said saponin per molecule of said ligand; or 4 to 5 molecules of said saponin per molecule of said ligand; Or the conjugate of claim 52, wherein the conjugate comprises an average of 4 to 4.5 molecules of the saponin per molecule of the ligand.

54. the conjugate comprises 2 to 5 molecules of the nucleic acid per molecule of the ligand; or 3 to 4 molecules of the nucleic acid per molecule of the ligand; Or the conjugate of claim 52, wherein the conjugate comprises an average of four molecules of the nucleic acid per molecule of the ligand.

55. 38. The conjugate of claim 36 or 37, wherein the endocytic receptor on muscle cells to which the ligand binds is selected from transferrin receptor (CD71), insulin-like growth factor 1 (IGF-I) receptor (IGF1R), insulin-like growth factor 2 (IGF-II) receptor (IGF2R), tetraspanin CD63; muscle-specific kinase (MuSK), glucose transporter GLUT4, and cation-independent mannose-6-phosphate receptor (CI-MPR).

56. 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 a binding fragment thereof specific for binding to the endocytosis receptor, or the endocytosis receptor is selected from transferrin receptor (CD71), insulin-like growth factor 1 (IGF-I) receptor (IGF1R), insulin-like growth factor 2 (IGF-II) receptor (IGF2R), 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: or the ligand is an antibody or a binding fragment thereof specific for binding to the transferrin receptor, or the ligand is a monoclonal antibody or a Fab' fragment or at least one single domain antibody specific for binding to the transferrin receptor, or the ligand is a monoclonal antibody specific for binding to the transferrin receptor.

57. the covalent linking of the saponin within the conjugate is via a first linker to which the saponin is covalently attached; Or the first linker is containing a covalent bond selected from any one or more of a semicarbazone bond, a hydrazone bond, an imine bond, an acetal bond including a 1,3-dioxolane bond, a ketal bond, an ester bond, an oxime bond, a disulfide bond, a thioether bond, an amide bond, a peptide bond, and an ester bond, or containing a covalent bond which is a hydrazone bond or a semicarbazone bond; or the conjugate of claim 36 or 37, wherein the saponin, at least in its unconjugated state, comprises an aldehyde group at C-23 of the saponin aglycone core structure, and the aldehyde group participates in the formation of a covalent bond with the first linker.

58. the first linker is a cleavable linker that undergoes cleavage under acidic, reducing, enzymatic and / or photoinduced conditions; Or the first linker is bonds that undergo cleavage under acidic conditions, such as semicarbazone bonds, hydrazone bonds, imine bonds, acetal bonds including 1,3-dioxolane bonds, ketal bonds, ester bonds, and / or oxime bonds; a proteolytically susceptible bond, optionally an amide or peptide bond, or a bond that is subject to proteolysis by cathepsin B; - Bonds that are cleavable by oxidation and reduction, such as disulfide bonds, or bonds that are susceptible to thiol exchange reactions, such as thioether bonds and a cleavable bond selected from or an acid-sensitive bond that undergoes cleavage in vivo under acidic conditions present in the endosomes and / or lysosomes of human cells, or at a pH of 4.0 to 6.5, or at a pH of ≦5.5; or an acid-sensitive bond selected from any one or more of a semicarbazone bond, a hydrazone bond, an imine bond, an acetal bond including a 1,3-dioxolane bond, a ketal bond, an ester bond, and / or an oxime bond, or selected from a semicarbazone bond and a hydrazone bond; or a hydrazone bond.

59. the first linker further comprises any oligomeric or polymeric structure, whether it is a dendron, such as a polyamidoamine (PAMAM) dendrimer, or a polyethylene glycol, such as any of PEG3 to PEG30; 58. The conjugate of claim 57, wherein the polymer or oligomer structure is any one of PEG4 to PEG12, or any one of G2 dendron, G3 dendron, G4 dendron, and G5 dendron, or a G2 dendron or a G3 dendron or PEG3 to PEG30.

60. the covalent linking of the nucleic acid within the conjugate is effected by a second linker to which the nucleic acid is covalently attached; or said second linker comprises or consists of the linker succinimidyl 3-(2-pyridyldithio)propionate (SPDP); 38. The conjugate of claim 36 or 37, wherein optionally the second linker covalently links the nucleic acid to a lysine residue, or a lysine residue that is a lysine residue contained in the ligand, or to a glycan residue, or a partially trimmed glycan.

61. the second linker is a cleavable linker that undergoes cleavage under acidic, reducing, enzymatic and / or photoinduced conditions; Or the second linker is bonds that undergo cleavage under acidic conditions, such as semicarbazone bonds, hydrazone bonds, imine bonds, acetal bonds including 1,3-dioxolane bonds, ketal bonds, ester bonds, and / or oxime bonds; a proteolytically susceptible bond, optionally an amide or peptide bond, or a bond that is subject to proteolysis by cathepsin B; - Bonds that are cleavable by oxidation and reduction, such as disulfide bonds, or bonds that are susceptible to thiol exchange reactions, such as thioether bonds and a cleavable bond selected from or an acid-sensitive bond that undergoes cleavage in vivo under acidic conditions present in the endosomes and / or lysosomes of human cells, or at a pH of 4.0 to 6.5, or at a pH of ≦5.5; or an acid-sensitive bond selected from any one or more of a semicarbazone bond, a hydrazone bond, an imine bond, an acetal bond including a 1,3-dioxolane bond, a ketal bond, an ester bond, and / or an oxime bond, or selected from a semicarbazone bond and a hydrazone bond; or a hydrazone bond.

62. 58. The conjugate of claim 57, wherein the first linker and / or the second linker are directly or indirectly covalently linked to the ligand.

63. the covalent linking of the ligand within the conjugate is effected by a third linker to which the ligand is covalently attached; or the ligand comprises a chain of amino acid residues comprising at least one cysteine ​​residue and / or at least one lysine residue, and the third linker comprises a covalent bond made with the at least one cysteine ​​residue or the at least one lysine residue; or the ligand comprises a chain of amino acid residues comprising a multi-cysteine ​​repeat, which is optionally a tetracysteine ​​repeat represented by the sequence HRWCCPGCCKTF (SEQ ID NO: 4), and the third linker comprises a covalent bond to any one or more of the cysteine ​​residues of the multi-cysteine ​​repeat; and optionally two or more third linkers are linked to one molecule of the ligand by each of the two or more third linkers binding to a distinct cysteine ​​residue of the multi-cysteine ​​repeat comprised in the ligand.

64. the conjugate comprises a central linker for achieving a covalent linkage between at least one molecule of the saponin, at least one molecule of the nucleic acid, and at least one molecule of the ligand, the covalent linkage being achieved either directly or via the first linker, the second linker, and / or the third linker, respectively; Or the central linker is a trifunctional linker.

65. The central linker, in its unconjugated form, has the structure A: 【Transformation 3】 65. The conjugate of claim 64, wherein the trifunctional linker is represented by:

66. 65. The conjugate of claim 64, wherein the conjugate comprises 1 to 4 of said trifunctional linkers, or 1 to 2 trifunctional linkers, or an average of 1.2 to 1.8 trifunctional linkers for each molecule of said ligand contained in said conjugate.

67. The trifunctional linker, in its conjugated form, has the structure B: 【Chemistry 4】 (In the formula, S is the at least one molecule of the saponin; L1 is the first linker to which the at least one molecule of the saponin is covalently attached; E is at least one molecule of the nucleic acid; L2 is the second linker to which the at least one molecule of the nucleic acid is covalently attached; A is at least one molecule of said ligand, or an antibody or binding fragment thereof; and L3 is the third linker to which the at least one molecule of the ligand is covalently bound; 65. The conjugate of claim 64, wherein L1, L2 and L3 are the same or different.

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