WNT signaling agonist molecules in the treatment of a bone-related disease or disorder

Abstract

The present application relates to agents capable of activating (GPR)124 / RECK / Frizzled / lipoprotein receptor-related protein (LRP)-mediated Wnt signaling, wherein said agents do not activate Frizzled / LRP –mediated Wnt signaling in the absence of RECK or GPR124, for use in the treatment and / or prevention of a bone-related disease or disorder in a subject.

Description

[0001] WNT SIGNALING AGONIST MOLECULES IN THE TREATMENT OF A BONE- RELATED DISEASE OR DISORDER

[0002] FIELD

[0003] The invention is broadly in the medical field of bone-related diseases, and more precisely relates to treatments of bone-related diseases or disorders associated with at least one of low bone formation, low bone mineral density, low bone mineral content, low bone mass, low bone quality, and / or low bone strength.

[0004] BACKGROUND

[0005] Loss of bone mineral content can result from a wide variety of conditions and may lead to significant medical problems. For example, osteoporosis is a bone disorder that increases a subject’s risk of fractures due to low bone mass, deterioration of bone tissue, disruption of bone microarchitecture and optionally decreased bone strength. This asymptomatic condition often remains undiagnosed until fractures occur, which may cause important secondary health problems and even death. As a result, osteoporosis has a significant financial impact on the healthcare system and society and management of osteoporosis and its associated health problems is necessary to improve quality of life of the patients and to reduce the economic burden.

[0006] Preventive measures and treatments are available to combat osteoporosis. As a preventive strategy, lifestyle changes, exercise, intake of calcium and vitamin D, and refraining from alcohol, smoking and excessive intake of salt could be implemented. Medicaments used in osteoporosis may be those that inhibit bone resorption, like bisphosphonates, denosumab, calcitonin, selective estrogen receptor modulators (SERMs), estrogen and progesterone, or that stimulate bone formation, such as parathyroid hormone (PTH) and Teriparatide. Specialist referral should be considered for patients who sustain fractures while undergoing osteoporosis therapy.

[0007] However, there remains a need in the art to find further agents capable of stimulating bone formation.

[0008] SUMMARY

[0009] Present inventors previously developed agonists capable of activating Wnt signaling selectively in cells expressing RECK and GPR124, like cerebral endothelial cells (as described in International patent application W02019180204A1). Present inventors now found to their surprise that these agonists also act as potentiators of bone formation. More particularly, present inventors found to their surprise that the expression of selective Wnt agonists as described in International patent application W02019180204A1, exemplified by Wnt7aK190A, in vascular endothelium led to a systemic increase in bone ossification in mice. As a result thereof, selective Wnt agonists can be used in the treatment and / or prevention of bone diseases or disorders, preferably bone diseases or disorders associated with at least one of low bone formation, low bone mineral density, low bone mineral content, low bone mass, low bone quality and / or low bone strength.

[0010] Accordingly, an aspect provides an agent capable of activating G-protein coupled receptor (GPR)124 / RECK / Frizzled / lipoprotein receptor-related protein (LRP) -mediated Wnt signaling, for use in the treatment and / or prevention of a bone -related disease or disorder in a subject, wherein said agent does not activate Frizzled / LRP -mediated Wnt signaling in the absence of RECK or GPR124.

[0011] A further aspect provides a nucleic acid encoding an agent as disclosed herein for use in the treatment and / or prevention of a bone-related disease or disorder in a subject, wherein said agent is a protein, polypeptide or a peptide.

[0012] A further aspect provides a nucleic acid expression cassette for use in the treatment and / or prevention of a bone-related disease or disorder in a subject, wherein said nucleic acid expression cassette comprises a nucleic acid as disclosed herein operably linked to a promoter and / or transcriptional and translational regulatory signals.

[0013] A further aspect provides a vector for use in the treatment and / or prevention of a bone-related disease or disorder in a subject, wherein said vector comprises a nucleic acid as disclosed herein or a nucleic acid expression cassette as disclosed herein, such as a viral vector. These and further aspects and preferred embodiments of the invention are described in the following sections and in the appended claims. The subject-matter of the appended claims is hereby specifically incorporated in this specification.

[0014] BRIEF DESCRIPTION OF DRAWINGS

[0015] The following description of the figures of specific embodiments of the invention is merely exemplary in nature and is not intended to limit the present teachings, their application or uses.

[0016] Fig. 1. Endothelial Wnt7aK190A expression leads to a systemic increase in bone ossification (A) Micro CT Scan lateral head view of control and Cdh5 CreERT ; Rosa26 Wnt7a K190A / + mice injected with Tamoxifen. Insets in A show a magnified view of the sphenoid bone. (B-D) Measures of volume for indicated bones. In C and D, the “bone volume” measure refers to the ossified portion of the indicated bone. Note the near absence of trabecular space in Wnt7aK190A transgenic mice. Abbreviations : IP - interparietal bone ; S - sphenoid bone ; V - vertebra. DESCRIPTION OF EMBODIMENTS

[0017] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0018] The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass “consisting of’ and “consisting essentially of’, which enjoy well-established meanings in patent terminology.

[0019] The recitation of numerical ranges by endpoints includes all intervening values between the lower and upper endpoints, as well as the recited endpoints. Intervening values may be integers or, where applicable, fractions, i.e., more broadly any real numbers such as any rational numbers. This applies to numerical ranges irrespective of whether they are introduced by the expression “from . . . to... ” or the expression “between... and... ” or another expression. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. For example, each sub-range between any stated value in a stated range and any other stated value in that stated range is also specifically disclosed. Each sub-range between any stated value in a stated range and either the lower endpoint or the upper endpoint of the stated range is also specifically disclosed. The stated value may be an isolated value or an endpoint of a range subsumed by or overlapping with the stated range. For example, for a stated range with lower endpoint LI and upper endpoint U1 (i.e., stated range Ll-Ul) and a stated sub-range nested within the stated range with lower endpoint L2 and upper endpoint U2 (i.e., stated sub-range L2-U2), also specifically disclosed are the subranges L1-L2, L1-U2, L2-U1, and U2-U1. The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and still more preferably + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” refers is itself also specifically, and preferably, disclosed.

[0020] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. Whereas the terms “one or more” or “at least one”, such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members. In another example, “one or more” or “at least one” may refer to 1, 2, 3, 4, 5, 6, 7 or more.

[0021] As used herein, the term “and / or” when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a list is described as comprising group A, B, and / or C, the list can comprise A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.

[0022] The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.

[0023] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.

[0024] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation is meant to apply throughout this specification, i.e., also in the context of other aspects or embodiments of the invention, unless otherwise defined.

[0025] In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0026] Reference throughout this specification to “one embodiment”, “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0027] Similarly, it should be appreciated that in the description of illustrative embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects.

[0028] In the present description of the invention, reference is made to the accompanying drawings that form a part hereof, and in which are shown by way of illustration only of specific embodiments in which the invention may be practiced. Parenthesized or emboldened reference numerals affixed to respective elements merely exemplify the elements by way of example, with which it is not intended to limit the respective elements. Unless otherwise indicated, all figures and drawings in this document are not to scale and are chosen for the purpose of illustrating different embodiments of the invention. In particular the dimensions of the various components are depicted in illustrative terms only, and no relationship between the dimensions of the various components should be inferred from the drawings, unless so indicated.

[0029] The present inventors have previously characterised the first ever “Wnt decoding module” capable of discriminating Wnt ligands that are otherwise largely synonymous in their capacity to bind Frizzled, thereby helping cells interpret the complexity of Wnt signaling inputs in order to orchestrate tissue development and homeostasis. In this Wnt decoding module, selectivity is conferred by RECK, which mediates Wnt7-specific binding in a Frizzled-independent manner. G- protein coupled receptor GPR124, a RECK binding partner, assembles Wnt7 ligand-specific RECK / GPR124 / Frizzled / lipoprotein receptor-related protein (LRP) signalosomes. Following the characterization of said “Wnt decoding module”, the present inventors also designed agonists capable of activating Wnt signaling selectively in cells expressing RECK and GPR124 and demonstrated their usefulness in stimulating Wnt / beta-catenin signaling in cerebral endothelial cells substantially without cross-reactivity with other Frizzled pathways (as described in International patent application W02019180204 Al). Moreover, in International patent applications W02019180204 Al and WO2023094581 Al, present inventors established the use of their agonists capable of activating Wnt signaling selectively in cells expressing RECK and GPR124 as therapeutics for neurovascular disorders or central nervous system (CNS) disorders comprising neurovascular dysfunction, and in glioblastoma multiforme (GBM).

[0030] Present inventors now found to their surprise that their agonists have a positive effect on bone formation, and therefore are also useful as therapeutics for bone-related diseases or disorders, including diseases characterized by abnormal osteoblast and / or osteoclast activity as well as bone injuries, like fractures. More particularly, present inventors demonstrated that the expression of the exemplary Wnt7aK190A(i.e. a selective agonist as described in International patent application W02019180204A1) in vascular endothelium led to a systemic increase in bone ossification in mice.

[0031] Accordingly, an aspect provides an agent capable of activating Wnt signaling mediated by the GPR124 / RECK / Frizzled / LRP receptor complex, wherein said agent does not activate Wnt signaling mediated by the Frizzled / LRP receptor complex in the absence of RECK and / or GPR124, for use in the treatment (e.g. treatment and / or prevention) of a bone-related disease or disorder in a subject.

[0032] In particular embodiments, activating Wnt signaling mediated by the GPR124 / RECK / Frizzled / LRP receptor complex, while not activating Wnt signaling mediated by the Frizzled / LRP receptor complex in the absence of RECK and / or GPR124 allows to increase at least one of bone formation, bone mineral density, bone mineral content, bone mass, bone quality and / or bone strength.

[0033] A further aspect provides a method of treating a bone -related disease or disorder in a subject in need of such a treatment, comprising administering a therapeutically effective amount of an agent capable of activating G-protein coupled receptor (GPR)124 / RECK / Frizzled / lipoprotein receptor- related protein (LRP)-mediated Wnt signaling, wherein said agent does not activate Frizzled / LRP - mediated Wnt signaling in the absence of RECK and / or GPR124, the nucleic acid encoding the agent as described herein, the nucleic acid expression cassette as described herein, the vector as described herein or the pharmaceutical composition as described herein, to the subject.

[0034] A further aspect provides the use of an agent capable of activating G-protein coupled receptor (GPR)124 / RECK / Frizzled / lipoprotein receptor-related protein (LRP) -mediated Wnt signaling, wherein said agent does not activate Frizzled / LRP -mediated Wnt signaling in the absence of RECK and / or GPR124, the nucleic acid encoding the agent as described herein, the nucleic acid expression cassette as described herein, the vector as described herein or the pharmaceutical composition as described herein, for the manufacture of a medicament for the treatment of a bone- related disease or disorder. The terms “treat” or “treatment” encompass both the therapeutic treatment of an already developed disease or condition, such as the therapy of an already developed bone -related disease or disorder, as well as prophylactic or preventive measures, wherein the aim is to prevent or lessen the chances of incidence of an undesired affliction, such as to prevent occurrence, development and progression of a bone-related disease or disorder. Beneficial or desired clinical results may include, without limitation, alleviation of one or more symptoms or one or more biological markers, diminishment of extent of disease, stabilised (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and the like. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0035] The term “bone-related disease or disorder” as used herein refers to any type of disease or disorder that leads to an alteration in bone strength, bone structure and / or flexibility of the bone, such as compared to a healthy bone. These conditions may be caused by aging, trauma, genetics, hormonal changes, and / or nutritional deficiencies. Lifestyle factors such as low levels of physical activity, smoking, and / or alcohol consumption can also increase the risk of a bone-related disease or disorder. The bone-related disease or disorder may be a local or a systemic disease, and / or a primary or secondary disease (i.e. disease resulting after and as a consequence of a primary disease). The bone-related disease or disorder may be diagnosed using any method known in the art, including but not limited to X-ray imaging, MRI scans, blood tests or biopsies.

[0036] In particular embodiments, the bone-related disease or disorder is characterized by defective bone formation and / or mineralisation.

[0037] In particular embodiments, the bone-related disease or disorder may be characterised by, e.g., decreased bone formation and / or excessive bone resorption, by decreased number, viability and / or function of osteoblasts and / or osteocytes present in the bone, decreased bone mass in a subject, thinning of bone, decreased bone quality, decreased bone mineral content, decreased bone mineral density, decreased bone strength, decreased bone elasticity, or combinations thereof, as compared to a healthy subject. Preferably, the bone-related disease or disorder is a disease or disorder associated with or characterized by at least one of decreased bone formation, decreased bone mineral density, decreased bone mineral content, decreased bone mass, decreased bone quality and / or decreased bone strength (e.g. as compared to a healthy subject).

[0038] In particular embodiments, said bone-related disease or disorder is characterized by decreased / reduced bone formation, reduced bone mineral density, reduced bone mineral content, reduced bone mass, reduced bone quality and / or reduced bone strength, so the subject would benefit from stimulation of bone formation and / or bone mineralisation. In particular embodiments, the bone-related disease or disorder is a disease or disorder characterized by at least one of decreased bone formation, decreased bone mineral density, decreased bone mineral content, decreased bone mass, decreased bone quality and / or decreased bone strength (e.g. as compared to a healthy subject), or with a need for stimulation of bone formation and / or bone mineralization (e.g. bone repair and / or regeneration, such as in the case of bone injury or bone lesions).

[0039] In particular embodiments, the bone-related disease or disorder is characterized by defective bone formation and / or bone mineralization as a primary pathological feature. In particular embodiments, the bone-related disease or disorder is characterized by reduced bone formation, reduced bone mineral density, reduced bone mineral content, reduced bone mass, reduced bone quality and / or reduced bone strength, so the subject would benefit from stimulation of bone formation and / or bone mineralisation, as a primary pathological feature.

[0040] In particular embodiments, the bone-related disease or disorder is characterized by an increased or an abnormally high bone resorption.

[0041] In particular embodiments, the bone-related disease or disorder is a disease or disorder characterized by a therapeutic need for stimulation of bone formation and / or bone mineralization.

[0042] In particular embodiments, the bone-related disease or disorder is a disease or disorder characterized by a therapeutic need for stimulation of endochondral and / or intramembranous ossification.

[0043] In particular embodiments, the bone-related disease or disorder is a disease or disorder that may benefit from an increase in the rate of bone formation. By way of example and not limitation the bone-related disease or disorder may be selected from the group consisting of osteopenia, osteoporosis (e.g., primary or secondary osteoporosis, postmenopausal, senile, steroid-induced like corticoid-induced, regional, seizure or depression-induced), osteoporosis pseudoglioma syndrome (OPPG), secondary, mono- or multisite osteonecrosis (e.g., osteonecrosis of the jaw), bone injury (e.g. a non-union fracture, a mal-union fracture, a delayed union fracture, a compression fracture; a maxilla-facial fracture or a condition requiring bone fusion like spinal fusions and rebuilding), bone reconstruction (e.g., after traumatic injury or cancer surgery, cranio-facial bone reconstruction), osteogenesis imperfecta, bone lesions (e.g. benign or malignant, such as bone cancer), bone cancer (e.g., osteolytic bone cancer), Paget's Disease (e.g. juvenile), renal osteodystrophy, osteomalacia, adynamic bone disease, arthritis, rheumatoid arthritis, hyperparathyroidism (e.g., primary or secondary hyperparathyroidism), familial hyperparathyroidism syndromes, hyperthyroidism, thyroid disease, Cushing’s disease, Cushing’s syndrome, periodontal disease, Gorham-Stout disease, McCune-Albright syndrome, fibrious dysplasia, growth plate injuries, achondroplasia, cleidocranial dysostosis, enchondromatosis, fibrous dysplasia (e.g. polyostotic fibrous dysplasia), Gaucher's Disease, Marfan's syndrome, multiple hereditary osteochondromas (HMO), osteopoikilosis, sclerotic lesions, pseudoarthrosis, osteomyelitis, anti-epileptic drug induced bone loss, weightlessness induced bone loss, osteoarthritis, infiltrative disorders of bone, melorheostosis, metabolic bone diseases, mastocytosis-associated bone loss, organ transplant related bone loss, bone loss associated with systemic lupus erythematosus, ankylosing spondylitis, Turner Syndrome, Down Syndrome, Klinefelter Syndrome, Perthes' Disease, adolescent idiopathic scoliosis, Winchester Syndrome, ischemic bone disease (e.g., Legg-Calve-Perthes disease, regional migratory osteoporosis), glucocorticoid-induced bone loss, bone marrow disorders, acromegaly, bone loss associated with joint replacement, human immunodeficiency virus (HIV) associated bone loss, bone loss associated with loss of growth hormone, bone loss associated with cystic fibrosis, chemotherapy associated bone loss, tumor-induced bone loss, cancer-related bone loss, hormone ablative bone loss, multiple myeloma, drug-induced bone loss (e.g., Gonadotropin-releasing hormone agonists induced low bone mass, thyroid medication induced low bone mass, phenytoin, divalproex sodium induced low bone mass, immunosuppressant induced low bone mass, blood thinning agents induced low bone mass, anticonvulsant treatment induced low bone mass, corticosteroid treatment induced low bone mass), bone loss associated with malnutrition (e.g. diet low in calcium or vitamin D), bone loss associated with eating disorder (e.g., anorexia nervosa), bone loss associated with aging, bone loss associated with space travel, bone loss associated with prolonged inactivity, bone loss associated with immobility, decreased bone mass density associated with hyperprolactinemia, bone loss associated with Grave's disease, bone loss associated with malabsorption syndromes, bone loss associated with smoking, bone loss associated with alcohol abuse, Hajdu-Cheney syndrome, Singleton -Merten syndrome, reflex sympathetic osteodystrophy, Hallermann-Streiff syndrome (HSS), bone loss associated with prednisolone use, geroderma osteodysplastica, Torg osteolysis syndrome, Albright's hereditary osteodystrophy and hypochondroplasia.

[0044] In particular embodiments, the bone-related disease or disorder may be selected from the group consisting of osteoporosis, osteoporosis pseudoglioma syndrome (OPPG), osteopenia, osteonecrosis, osteolysis, osteodystrophy, osteomalacia, bone injury, bone reconstruction, osteogenesis imperfecta, bone lesions, bone cancer, Paget's Disease, arthritis, periodontal disease, growth plate injuries, fibrous dysplasia, and pseudoarthrosis.

[0045] In particular embodiments, the bone-related disease or disorder may be selected from the group consisting of osteoporosis, osteopenia, osteonecrosis, osteolysis, osteodystrophy, osteomalacia, bone injury, bone reconstruction, osteogenesis imperfecta, bone lesions, bone cancer, Paget's Disease, arthritis, periodontal disease, growth plate injuries, fibrous dysplasia, and pseudoarthrosis, such as selected from the group consisting of osteoporosis, osteopenia, osteonecrosis, osteolysis, osteodystrophy, osteomalacia, bone injury, bone reconstruction, bone lesions, bone cancer, Paget's Disease, arthritis, periodontal disease, growth plate injuries, fibrous dysplasia, and pseudoarthrosis.

[0046] In particular embodiments, the bone-related disease or disorder is not osteoporosis pseudoglioma syndrome (OPPG).

[0047] In particular embodiments, the bone-related disease or disorder may be selected from the group consisting of osteoporosis, osteopenia, osteonecrosis, osteolysis, osteodystrophy, osteomalacia, bone injury, bone reconstruction, osteogenesis imperfecta, bone lesions, bone cancer, Paget's Disease, arthritis, periodontal disease, growth plate injuries, fibrous dysplasia, and pseudoarthrosis, wherein said bone-related disease or disorder is characterized by reduced bone formation, reduced bone mineral density, reduced bone mineral content, reduced bone mass, reduced bone quality and / or reduced bone strength, so the subject would benefit from stimulation of bone formation and / or bone mineralisation.

[0048] In particular embodiments, the bone-related disease or disorder may be a bone injury, such as a bone fracture. In particular embodiments, the bone fracture is a non-union fracture, a mal -union fracture, a delayed union fracture, a compression fracture; a maxilla-facial fracture or a condition requiring bone fusion (e.g., spinal fusions and rebuilding).

[0049] In particular embodiments, the bone-related disease or disorder arises independently of hereditary factors. In particular embodiments, the bone-related disease or disorder is not of genetic origin (e.g. not caused by an inherited genetic defect).

[0050] In particular embodiments, the bone-related disease or disorder is acquired by the subject, e.g. such as by age.

[0051] Also provided herein is the agent as described herein for use in a method of improving the outcome in a mammal undergoing one or more of an orthopaedic procedure, dental procedure, implant surgery, joint replacement, bone grafting, bone cosmetic surgery, fracture healing, nonunion healing, delayed union healing and facial reconstruction in a subject, wherein said agent as described herein is administered to said subject before, during and / or after said procedure, replacement, graft, surgery or repair. In other words, also provided herein is a method of improving the outcome in a mammal undergoing one or more of an orthopaedic procedure, dental procedure, implant surgery, joint replacement, bone grafting, bone cosmetic surgery, fracture healing, nonunion healing, delayed union healing and facial reconstruction in a subject, comprising administering said agent to said subject before, during and / or after said procedure, replacement, graft, surgery or repair. Except when noted, the terms “subject” or “patient” can be used interchangeably and refer to animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, still more preferably primates, and specifically includes human patients and non-human mammals and primates. Preferred subjects are human subjects. The terms “subject” or “patient” include subjects in need of treatment, more particularly subjects that would benefit from treatment of a given condition, particularly a bone-related disease or disorder. Such subjects may include, without limitation, those that have been diagnosed with said condition, those prone to develop said condition and / or those in who said condition is to be prevented.

[0052] In particular embodiments, such as if the bone -related disease or disorder is associated with aging, like osteoporosis, the subject is a mid-age or an elderly subject. In particular embodiments, the subject may be older than 40, older than 45, older than 50, older than 55, older than 60, older than 65, older than 70, older than 75, older than 80, older than 85, or older than 90 years of age. In particular embodiments, such as if the bone -related disease or disorder is osteoporosis, the subject may be older than 60, older than 65, older than 70, older than 75, older than 80, older than 85, or older than 90 years of age. In particular embodiments, the subject is a woman, such as a postmenopausal woman.

[0053] In particular embodiments, the bone-related disease or disorder is not a juvenile-onset disorder (e.g. OPPG).

[0054] The therapeutic uses or methods as taught herein allow to administer a therapeutically and / or prophylactically effective amount of an agent as described herein, a nucleic acid encoding the agent as described herein, a nucleic acid expression cassette as described herein, a vector as described herein or a pharmaceutical composition as described herein, in subjects having a bone-related disease or disorder which will benefit from such treatment. The term “therapeutically effective amount” as used herein, refers to an amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a subject that is being sought by a surgeon, researcher, veterinarian, medical doctor or other clinician, which may include inter alia alleviation of the symptoms of the disease or condition being treated. The term “prophylactically effective amount” refers to an amount of an active compound or pharmaceutical agent that inhibits or delays in a subject the onset of a disorder as being sought by a researcher, veterinarian, medical doctor or other clinician. Methods are known in the art for determining therapeutically and / or prophylactically effective doses of an agent, a nucleic acid encoding the agent, a nucleic acid expression cassette, or a pharmaceutical composition, as described herein.

[0055] The term “therapeutically effective dose” as used herein refers to an amount of an agent, a nucleic acid encoding the agent, a nucleic acid expression cassette, or a pharmaceutical composition, as described herein, that when administered brings about a positive therapeutic response with respect to treatment of a patient having a bone-related disease or disorder.

[0056] Appropriate therapeutically effective doses of an agent, a nucleic acid encoding the agent, a nucleic acid expression cassette, or a pharmaceutical composition, as described herein, may be determined by a qualified physician with due regard to the nature of the disease condition and severity, and the age, size and condition of the patient.

[0057] In particular embodiments, the therapeutically effective dose is an amount sufficient to increase at least one of bone formation, bone mineral density, bone mineral content, bone mass, bone quality and / or bone strength.

[0058] References to any peptides, polypeptides, proteins or nucleic acids denote the respective peptides, polypeptides, proteins or nucleic acids as commonly known under the respective designations in the art. More particularly, the references to “G-protein coupled receptor 124” (GPR124), “Reversion-inducing cysteine-rich protein with Kazal motifs” (RECK), “Frizzled” (FZD), or “lipoprotein receptor-related protein” (LRP) denote the respective peptides, polypeptides, proteins or nucleic acids, as apparent from the context, as commonly known under said designations in the art.

[0059] The terms encompass the peptides, polypeptides, proteins or nucleic acids when forming a part of a living organism, organ, tissue or cell, when forming a part of a biological sample, as well as when at least partly isolated from such sources. The terms also encompass the peptides, polypeptides, proteins or nucleic acids when produced by recombinant or synthetic means.

[0060] Unless otherwise apparent from the context, reference herein to any peptide, polypeptide, protein or nucleic acid also encompasses modified forms of said peptide, polypeptide, protein or nucleic acid, such as forms bearing post-expression modifications including, for example, phosphorylation, glycosylation, lipidation, methylation, cysteinylation, sulphonation, glutathionylation, acetylation, oxidation of methionine to methionine sulphoxide or methionine sulphone, and the like.

[0061] By means of additional guidance, G protein -coupled receptor 124 is also known in the art as Adhesion G protein-coupled receptor A2 (ADGRA2) or Tumor endothelial marker 5 (TEM5). By means of an example, human GPR124 gene is annotated under NCBI Genbank (http: / / www.ncbi.nlm.nih.gov / ) Gene ID 25960. Human GPR124 mRNA is annotated under NCBI Genbank accession number NM_032777.9. Nucleotides 387 (start codon) to 4403 (stop codon) of NM_032777.9 constitute the GPR124 coding sequence. Human GPR124 protein sequence is annotated under NCBI Genbank accession number NP_116166.9, and Uniprot (www.uniprot.org) accession number Q96PE1-1, and is further reproduced below (SEQ ID NO: 1): >NP_116166.9 adhesion G protein-coupled receptor A2 precursor [Homo sapiens]

[0062] MGAGGRRMRGAPARLLLPLLPWLLLLLAPEARGAPGCPLSIRSCKCSGERPKGLSGGVPGP ARRRVVCSGGDLPEPPEPGLLPNGTVTLLLSNNKITGLRNGSFLGLSLLEKLDLRNNIISTVQ PGAFLGLGELKRLDLSNNRIGCLTSETFQGLPRLLRLNISGNIFSSLQPGVFDELPALKVVDL GTEFLTCDCHLRWLLPWAQNRSLQLSEHTLCAYPSALHAQALGSLQEAQLCCEGALELHT HHLIPSLRQVVFQGDRLPFQCSASYLGNDTRIRWYHNRAPVEGDEQAGILLAESLIHDCTFI TSELTLSHIGVWASGEWECTVSMAQGNASKKVEIVVLETSASYCPAERVANNRGDFRWPR TLAGITAYQSCLQYPFTSVPLGGGAPGTRASRRCDRAGRWEPGDYSHCLYTNDITRVLYTF VLMPINASNALTLAHQLRVYTAEAASFSDMMDWYVAQMIQKFLGYVDQIKELVEVMV DMASNLMLVDEHLLWLAQREDKACSRIVGALERIGGAALSPHAQHISVNARNVALEAYLI KPHSYVGLTCTAFQRREGGVPGTRPGSPGQNPPPEPEPPADQQLRFRCTTGRPNVSLSSFHI KNSVALASIQLPPSLFSSLPAALAPPVPPDCTLQLLVFRNGRLFHSHSNTSRPGAAGPGKRR GVATPVIFAGTSGCGVGNLTEPVAVSLRHWAEGAEPVAAWWSQEGPGEAGGWTSEGCQ LRSSQPNVSALHCQHLGNVAVLMELSAFPREVGGAGAGLHPVVYPCTALLLLCLFATIITY ILNHS SIRVSRKGWHMLLNLCFHIAMTS AVFAGGITLTNYQMVCQAVGITLHYS SLSTLLW MGVKARVLHKELTWRAPPPQEGDPALPTPSPMLRFYLIAGGIPLIICGITAAVNIHNYRDHS PYCWLVWRPSLGAFYIPVALILLITWIYFLCAGLRLRGPLAQNPKAGNSRASLEAGEELRG STRLRGSGPLLSDSGSLLATGSARVGTPGPPEDGDSLYSPGVQLGALVTTHFLYLAMWAC GALAVSQRWLPRVVCSCLYGVAASALGLFVFTHHCARRRDVRASWRACCPPASPAAPHA PPRALPAAAEDGSPVFGEGPPSLKSSPSGSSGHPLALGPCKLTNLQLAQSQVCEAGAAAGG

[0063] EGEPEPAGTRGNLAHRHPNNVHHGRRAHKSRAKGHRAGEACGKNRLKALRGGAAGALE LLSSESGSLHNSPTDSYLGSSRNSPGAGLQLEGEPMLTPSEGSDTSAAPLSEAGRAGQRRSA SRDSLKGGGALEKESHRRSYPLNAASLNGAPKGGKYDDVTLMGAEVASGGCMKTGLWK SETTV

[0064] By means of additional guidance, RECK (Reversion-Inducing Cysteine-Rich Protein With Kazal Motifs) is also known in the art as Suppressor of tumorigenicity 15 protein (ST15). By means of an example, human RECK gene is annotated under NCBI Genbank (http: / / www.ncbi.nlm.nih.gov / ) Gene ID 8434. Human RECK mRNA (transcript variant 1) is annotated under NCBI Genbank accession number NM_021111.2. Nucleotides 87 (start codon) to 3002 (stop codon) of NM_021111.2 constitute the RECK coding sequence. Human RECK protein sequence is annotated under NCBI Genbank accession number NP_066934.1, and Uniprot accession number 095980-1, and is further reproduced below (SEQ ID NO: 2):

[0065] >NP_066934.1 reversion-inducing cysteine-rich protein with Kazal motifs isoform 1 precursor [Homo sapiens] MATVRASLRGALLLLLAVAGVAEVAGGLAPGSAGALCCNHSKDNQMCRDVCEQIFSSKS ESRLKHLLQRAPDYCPETMVEIWNCMNSSLPGVFKKSDGWVGLGCCELAIALECRQACK QASSKNDISKVCRKEYENALFSCISRNEMGSVCCSYAGHHTNCREYCQAIFRTDSSPGPSQI

[0066] KAVENYCASISPQLIHCVNNYTQSYPMRNPTDSLYCCDRAEDHACQNACKRILMSKKTEM EIVDGLIEGCKTQPLPQDPLWQCFLESSQSVHPGVTVHPPPSTGLDGAKLHCCSKANTSTC RELCTKLYSMSWGNTQSWQEFDRFCEYNPVEVSMLTCLADVREPCQLGCRNLTYCTNFN

[0067] NRPTELFRSCNAQSDQGAMNDMKLWEKGSIKMPFINIPVLDIKKCQPEMWKAIACSLQIKP CHSKSRGSIICKSDCVEILKKCGDQNKFPEDHTAESICELLSPTDDLKNCIPLDTYLRPSTLG

[0068] NIVEEVTHPCNPNPCPANELCEVNRKGCPSGDPCLPYFCVQGCKLGEASDFIVRQGTLIQVP SSAGEVGCYKICSCGQSGLLENCMEMHCIDLQKSCIVGGKRKSHGTSFSIDCNVCSCFAGN LVCSTRLCLSEHSSEDDRRTFTGLPCNCADQFVPVCGQNGRTYPSACIARCVGLQDHQFEF GSCMSKDPCNPNPCQKNQRCIPKPQVCLTTFDKFGCSQYECVPRQLACDQVQDPVCDTDH MEHNNLCTLYQRGKSLSYKGPCQPFCRATEPVCGHNGETYSSVCAAYSDRVAVDYYGDC QAVGVLSEHSSVAECASVKCPSLLAAGCKPIIPPGACCPLCAGMLRVLFDKEKLDTIAKVT NKKPITVLEILQKIRMHVSVPQCDVFGYFSIESEIVILIIPVDHYPKALQIEACNKEAEKIESLI NSDSPTLASHVPLSALIISQVQVSSSVPSAGVRARPSCHSLLLPLSLGLALHLLWTYN

[0069] The term “Frizzled” or “FZD” or “FZ” encompasses any and all members of the Frizzled family. By means of additional guidance, Table 1 presents 10 known human Frizzled family members, as annotated in NCBI Genbank and Uniprot.

[0070] Table 1

[0071] The term “lipoprotein receptor-related protein” or “LRP” encompasses any and all lipoprotein receptor-related proteins, also known in the art as low density lipoprotein receptor-related proteins or prolow-density lipoprotein receptor-related proteins, and particularly denote LPR proteins involved in Wnt signaling. In certain particularly preferred embodiments, the terms denote LRP5 (GenelD: 4041), LRP6 (GenelD: 4040), or LRP5 or LRP6 (LRP5 / 6).

[0072] Hence, in particular embodiments, the agent is an agent capable of activating GPR124 / RECK / Frizzled / LRP5 / 6-mediated Wnt signaling, wherein said agent does not activate Frizzled / LRP5 / 6-mediated Wnt signaling in the absence of RECK and / or GPR124.

[0073] By means of additional guidance, human LRP5 mRNA is annotated under NCBI Genbank accession numbers NM_002335.3 (transcript variant 1; nucleotides 107 (start codon) to 4954 (stop codon) of NM_002335.3 constitute the LRP5 coding sequence) or NM_001291902.1 (transcript variant 2; nucleotides 1872 (start codon) to 4976 (stop codon) of NM_001291902.1 constitute the LRP5 coding sequence). Human LRP5 protein sequence is annotated under NCBI Genbank accession numbers NP_002326.2 (isoform 1 precursor) or NP_001278831.1 (isoform 2), and Uniprot accession number 075197-1, the LRP5 protein sequence annotated as NP_002326.2 being reproduced below (SEQ ID NO: 3):

[0074] >NP_002326.2 low-density lipoprotein receptor-related protein 5 isoform 1 precursor [Homo sapiens]

[0075] MEAAPPGPPWPLLLLLLLLLALCGCPAPAAASPLLLFANRRDVRLVDAGGVKLESTIVVSG LEDAAAVDFQFSKGAVYWTDVSEEAIKQTYLNQTGAAVQNVVISGLVSPDGLACDWVGK KLYWTDSETNRIEVANLNGTSRKVLFWQDLDQPRAIALDPAHGYMYWTDWGETPRIERA GMDGSTRKIIVDSDIYWPNGLTIDLEEQKLYWADAKLSFIHRANLDGSFRQKVVEGSLTHP FALTLSGDTLYWTDWQTRSIHACNKRTGGKRKEILSALYSPMDIQVLSQERQPFFHTRCEE DNGGCSHLCLLSPSEPFYTCACPTGVQLQDNGRTCKAGAEEVLLLARRTDLRRISLDTPDF TDIVLQVDDIRHAIAIDYDPLEGYVYWTDDEVRAIRRAYLDGSGAQTLVNTEINDPDGIAV DWVARNLYWTDTGTDRIEVTRLNGTSRKILVSEDLDEPRAIALHPVMGLMYWTDWGENP

[0076] KIECANLDGQERRVLVNASLGWPNGLALDLQEGKLYWGDAKTDKIEVINVDGTKRRTLL EDKLPHIFGFTLLGDFIYWTDWQRRSIERVHKVKASRDVIIDQLPDLMGLKAVNVAKVVG TNPCADRNGGCSHLCFFTPHATRCGCPIGLELLSDMKTCIVPEAFLVFTSRAAIHRISLETNN NDVAIPLTGVKEASALDFDVSNNHIYWTDVSLKTISRAFMNGSSVEHVVEFGLDYPEGMA VDWMGKNLYWADTGTNRIEVARLDGQFRQVLVWRDLDNPRSLALDPTKGYIYWTEWG GKPRIVRAFMDGTNCMTLVDKVGRANDLTIDYADQRLYWTDLDTNMIESSNMLGQERV VIADDLPHPFGLTQYSDYIYWTDWNLHSIERADKTSGRNRTLIQGHLDFVMDILVFHSSRQ DGLNDCMHNNGQCGQLCLAIPGGHRCGCASHYTLDPSSRNCSPPTTFLLFSQKSAISRMIP DDQHSPDLILPLHGLRNVKAIDYDPLDKFIYWVDGRQNIKRAKDDGTQPFVLTSLSQGQNP DRQPHDLSIDIYSRTLFWTCEATNTINVHRLSGEAMGWLRGDRDKPRAIVVNAERGYLYF TNMQDRAAKIERAALDGTEREVLFTTGLIRPVALVVDNTLGKLFWVDADLKRIESCDLSG ANRLTLEDANIVQPLGLTILGKHLYWIDRQQQMIERVEKTTGDKRTRIQGRVAHLTGIHAV EEVSLEEFSAHPCARDNGGCSHICIAKGDGTPRCSCPVHLVLLQNLLTCGEPPTCSPDQFAC ATGEIDCIPGAWRCDGFPECDDQSDEEGCPVCSAAQFPCARGQCVDLRLRCDGEADCQDR SDEADCDAICLPNQFRCASGQCVLIKQQCDSFPDCIDGSDELMCEITKPPSDDSPAHSSAIGP VIGIILSLFVMGGVYFVCQRVVCQRYAGANGPFPHEYVSGTPHVPLNFIAPGGSQHGPFTGI ACGKSMMSSVSLMGGRGGVPLYDRNHVTGASSSSSSSTKATLYPPILNPPPSPATDPSLYN MDMFYSSNIPATARPYRPYIIRGMAPPTTPCSTDVCDSDYSASRWKASKYYLDLNSDSDPY PPPPTPHSQYLSAEDSCPPSPATERSYFHLFPPPPSPCTDSS

[0077] By means of additional guidance, human LRP6 mRNA is annotated under NCBI Genbank accession number NM_002336.2. Nucleotides 143 (start codon) to 4984 (stop codon) of NM_002336.2 constitute the LRP6 coding sequence. Human LRP6 protein sequence is annotated under NCBI Genbank accession number NP_002327.2 and Uniprot accession number 075581-1, and is further reproduced below (SEQ ID NO: 4):

[0078] >NP_002327.2 low-density lipoprotein receptor-related protein 6 precursor [Homo sapiens]

[0079] MGAVLRSLLACSFCVLLRAAPLLLYANRRDLRLVDATNGKENATIVVGGLEDAAAVDFV FSHGLIYWSDVSEEAIKRTEFNKTESVQNWVSGLLSPDGLACDWLGEKLYWTDSETNRIE VSNLDGSLRKVLFWQELDQPRAIALDPSSGFMYWTDWGEVPKIERAGMDGSSRFIIINSEIY WPNGLTLDYEEQKLYWADAKLNFIHKSNLDGTNRQAVVKGSLPHPFALTLFEDILYWTD WSTHSILACNKYTGEGLREIHSDIFSPMDIHAFSQQRQPNATNPCGIDNGGCSHLCLMSPVK PFYQCACPTGVKLLENGKTCKDGATELLLLARRTDLRRISLDTPDFTDIVLQLEDIRHAIAI DYDPVEGYIYWTDDEVRAIRRSFIDGSGSQFVVTAQIAHPDGIAVDWVARNLYWTDTGTD RIEVTRLNGTMRKILISEDLEEPRAIVLDPMVGYMYWTDWGEIPKIERAALDGSDRVVLVN TSLGWPNGLALDYDEGKIYWGDAKTDKIEVMNTDGTGRRVLVEDKIPHIFGFTLLGDYVY WTDWQRRSIERVHKRSAEREVIIDQLPDLMGLKATNVHRVIGSNPCAEENGGCSHLCLYR PQGLRCACPIGFELISDMKTCIVPEAFLLFSRRADIRRISLETNNNNVAIPLTGVKEASALDF DVTDNRIYWTDISLKTISRAFMNGSALEHVVEFGLDYPEGMAVDWLGKNLYWADTGTNR

[0080] IEVSKLDGQHRQVLVWKDLDSPRALALDPAEGFMYWTEWGGKPKIDRAAMDGSERTTL VPNVGRANGLTIDYAKRRLYWTDLDTNLIESSNMLGLNREVIADDLPHPFGLTQYQDYIY WTDWSRRSIERANKTSGQNRTIIQGHLDYVMDILVFHSSRQSGWNECASSNGHCSHLCLA VPVGGFVCGCPAHYSLNADNRTCSAPTTFLLFSQKSAINRMVIDEQQSPDIILPIHSLRNVR AIDYDPLDKQLYWIDSRQNMIRKAQEDGSQGFTVVVSSVPSQNLEIQPYDLSIDIYSRYIYW TCEATNVINVTRLDGRSVGVVLKGEQDRPRAVVVNPEKGYMYFTNLQERSPKIERAALDG TEREVLFFSGLSKPIALALDSRLGKLFWADSDLRRIESSDLSGANRIVLEDSNILQPVGLTVF ENWLYWIDKQQQMIEKIDMTGREGRTKVQARIAQLSDIHAVKELNLQEYRQHPCAQDNG GCSHICLVKGDGTTRCSCPMHLVLLQDELSCGEPPTCSPQQFTCFTGEIDCIPVAWRCDGFT ECEDHSDELNCPVCSESQFQCASGQCIDGALRCNGDANCQDKSDEKNCEVLCLIDQFRCA NGQCIGKHKKCDHNVDCSDKSDELDCYPTEEPAPQATNTVGSVIGVIVTIFVSGTVYFICQ RMLCPRMKGDGETMTNDYVVHGPASVPLGYVPHPSSLSGSLPGMSRGKSMISSLSIMGGS SGPPYDRAHVTGASSSSSSSTKGTYFPAILNPPPSPATERSHYTMEFGYSSNSPSTHRSYSYR PYSYRHFAPPTTPCSTDVCDSDYAPSRRMTSVATAKGYTSDLNYDSEPVPPPPTPRSQYLS AEENYESCPPSPYTERSYSHHLYPPPPSPCTDSS

[0081] A skilled person can appreciate that any sequences represented in sequence databases or in the present specification may be of precursors of the respective peptides, polypeptides, proteins or nucleic acids and may include parts which are processed away from mature molecules.

[0082] The term “protein” as used throughout this specification generally encompasses macromolecules comprising one or more polypeptide chains, i.e., polymeric chains of amino acid residues linked by peptide bonds. The term may encompass naturally, recombinantly, semi -synthetically or synthetically produced proteins. The term also encompasses proteins that carry one or more co- or post-expression-type modifications of the polypeptide chain(s), such as, without limitation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of pro-enzymes or pre-hormones into active forms, etc. The term further also includes protein variants or mutants which carry amino acid sequence variations vis-a-vis a corresponding native proteins, such as, e.g., amino acid deletions, additions and / or substitutions. The term contemplates both full-length proteins and protein parts or fragments, e.g., naturally-occurring protein parts that ensue from processing of such full-length proteins.

[0083] The term “polypeptide” as used throughout this specification generally encompasses polymeric chains of amino acid residues linked by peptide bonds. Hence, especially when a protein is only composed of a single polypeptide chain, the terms “protein” and “polypeptide” may be used interchangeably herein to denote such a protein. The term is not limited to any minimum length of the polypeptide chain. The term may encompass naturally, recombinantly, semi -synthetically or synthetically produced polypeptides. The term also encompasses polypeptides that carry one or more co- or post-expression-type modifications of the polypeptide chain, such as, without limitation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of pro-enzymes or pre-hormones into active forms, etc. The term further also includes polypeptide variants or mutants which carry amino acid sequence variations vis-a-vis a corresponding native polypeptide, such as, e.g., amino acid deletions, additions and / or substitutions. The term contemplates both full-length polypeptides and polypeptide parts or fragments, e.g., naturally -occurring polypeptide parts that ensue from processing of such full-length polypeptides. The term “peptide” as used throughout this specification preferably refers to a polypeptide as used herein consisting essentially of 50 amino acids or less, e.g., 45 amino acids or less, preferably 40 amino acids or less, e.g., 35 amino acids or less, more preferably 30 amino acids or less, e.g., 25 or less, 20 or less, 15 or less, 10 or less or 5 or less amino acids.

[0084] A peptide, polypeptide or protein can be naturally occurring, e.g., present in or isolated from nature, e.g., produced or expressed natively or endogenously by a cell or tissue and optionally isolated therefrom. A peptide, polypeptide or protein can be recombinant, i.e., produced by recombinant DNA technology, and / or can be, partly or entirely, chemically or biochemically synthesised. Without limitation, a peptide, polypeptide or protein can be produced recombinantly by a suitable host or host cell expression system and optionally isolated therefrom (e.g., a suitable bacterial, yeast, fungal, plant or animal host or host cell expression system), or produced recombinantly by cell-free translation or cell-free transcription and translation, or non-biological peptide, polypeptide or protein synthesis.

[0085] The term “nucleic acid” as used throughout this specification typically refers to a polymer (preferably a linear polymer) of any length composed essentially of nucleoside units. A nucleoside unit commonly includes a heterocyclic base and a sugar group. Heterocyclic bases may include inter alia purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) which are widespread in naturally -occurring nucleic acids, other naturally-occurring bases (e.g., xanthine, inosine, hypoxanthine) as well as chemically or biochemically modified (e.g., methylated), non-natural or derivatised bases. Exemplary modified nucleobases include without limitation 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. In particular, 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability and may be preferred base substitutions in for example antisense agents, even more particularly when combined with 2'-O-methoxyethyl sugar modifications. Sugar groups may include inter alia pentose (pentofiiranose) groups such as preferably ribose and / or 2 -deoxyribose common in naturally- occurring nucleic acids, or arabinose, 2-deoxyarabinose, threose or hexose sugar groups, as well as modified or substituted sugar groups (such as without limitation 2'-O-alkylated, e.g., 2'-O- methylated or 2'-O-ethylated sugars such as ribose; 2'-O-alkyloxyalkylated, e.g., 2’-O- methoxyethylated sugars such as ribose; or 2'-O,4'-C-alkylene-linked, e.g., 2'-O,4'-C-methylene- linked or 2'-O,4'-C-ethylene-linked sugars such as ribose; 2 ’-fluoro-arabinose, etc.). Nucleic acid molecules comprising at least one ribonucleoside unit may be typically referred to as ribonucleic acids or RNA. Such ribonucleoside unit(s) comprise a 2'-OH moiety, wherein -H may be substituted as known in the art for ribonucleosides (e.g., by a methyl, ethyl, alkyl, or alkyloxyalkyl). Preferably, ribonucleic acids or RNA may be composed primarily of ribonucleoside units, for example, > 80%, > 85%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99% or even 100% (by number) of nucleoside units constituting the nucleic acid molecule may be ribonucleoside units. Nucleic acid molecules comprising at least one deoxyribonucleoside unit may be typically referred to as deoxyribonucleic acids or DNA. Such deoxyribonucleoside unit(s) comprise 2'-H. Preferably, deoxyribonucleic acids or DNA may be composed primarily of deoxyribonucleoside units, for example, > 80%, > 85%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99% or even 100% (by number) of nucleoside units constituting the nucleic acid molecule may be deoxyribonucleoside units. Nucleoside units may be linked to one another by any one of numerous known inter-nucleoside linkages, including inter alia phosphodiester linkages common in naturally -occurring nucleic acids, and further modified phosphate- or phosphonate-based linkages such as phosphorothioate, alkyl phosphorothioate such as methyl phosphorothioate, phosphorodithioate, alkylphosphonate such as methylphosphonate, alkylphosphonothioate, phosphotriester such as alkylphosphotriester, phosphoramidate, phosphoropiperazidate, phosphoromorpholidate, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate; and further siloxane, carbonate, sulfamate, carboalkoxy, acetamidate, carbamate such as 3’-N-carbamate, morpholino, borano, thioether, 3 ’-thioacetal, and sulfone intemucleoside linkages. Preferably, inter-nucleoside linkages may be phosphate-based linkages including modified phosphate-based linkages, such as more preferably phosphodiester, phosphorothioate or phosphorodithioate linkages or combinations thereof. The term “nucleic acid” also encompasses any other nucleobase containing polymers such as nucleic acid mimetics, including, without limitation, peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA), morpholino phosphorodiamidate-backbone nucleic acids (PMO), cyclohexene nucleic acids (CeNA), tricyclo- DNA (tcDNA), and nucleic acids having backbone sections with alkyl linkers or amino linkers (see, e.g., Kurreck 2003 (Eur J Biochem 270: 1628-1644)). “Alkyl” as used herein particularly encompasses lower hydrocarbon moieties, e.g., C1-C4 linear or branched, saturated or unsaturated hydrocarbon, such as methyl, ethyl, ethenyl, propyl, 1 -propenyl, 2-propenyl, and isopropyl. Nucleic acids as intended herein may include naturally occurring nucleosides, modified nucleosides or mixtures thereof. A modified nucleoside may include a modified heterocyclic base, a modified sugar moiety, a modified inter-nucleoside linkage or a combination thereof.

[0086] The term “nucleic acid” further preferably encompasses DNA, RNA and DNA / RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemically synthesised) DNA, RNA or DNA / RNA hybrids. RNA is inclusive of RNAi (inhibitory RNA), dsRNA (double stranded RNA), siRNA (small interfering RNA), mRNA (messenger RNA), miRNA (micro -RNA), tRNA (transfer RNA, whether charged or discharged with a corresponding acylated amino acid), and cRNA (complementary RNA). A nucleic acid can be naturally occurring, e.g., present in or isolated from nature, e.g., produced natively or endogenously by a cell or a tissue and optionally isolated therefrom. A nucleic acid can be recombinant, i.e., produced by recombinant DNA technology, and / or can be, partly or entirely, chemically or biochemically synthesised. Without limitation, a nucleic acid can be produced recombinantly by a suitable host or host cell expression system and optionally isolated therefrom (e.g., a suitable bacterial, yeast, fungal, plant or animal host or host cell expression system), or produced recombinantly by cell -free transcription, or non -biological nucleic acid synthesis. A nucleic acid can be double -stranded, partly double stranded, or singlestranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.

[0087] The reference to any peptides, polypeptides, proteins or nucleic acids encompass such peptides, polypeptides, proteins or nucleic acids of any organism where found, and particularly of animals, preferably warm-blooded animals, more preferably vertebrates, yet more preferably mammals, including humans and non-human mammals, still more preferably of humans.

[0088] Hence, in certain embodiments, one or more and preferably all of GPR124, RECK, FZD and LRP as employed herein is or are of animal origin, preferably warm-blooded animal origin, more preferably vertebrate origin, yet more preferably mammalian origin, including human origin and non-human mammalian origin, still more preferably human origin.

[0089] The reference to any peptides, polypeptides, proteins or nucleic acids may particularly encompass such peptides, polypeptides, proteins or nucleic acids with a native sequence, i.e., ones of which the primary sequence is the same as that of the peptides, polypeptides, proteins or nucleic acids found in or derived from nature. A skilled person understands that native sequences may differ between different species due to genetic divergence between such species. Moreover, native sequences may differ between or within different individuals of the same species due to normal genetic diversity (variation) within a given species. Also, native sequences may differ between or even within different individuals of the same species due to somatic mutations, or post -transcriptional or post- translational modifications. Any such variants or isoforms of peptides, polypeptides, proteins or nucleic acids are intended herein. Accordingly, all sequences of peptides, polypeptides, proteins or nucleic acids found in or derived from nature are considered “native”.

[0090] In certain embodiments, the peptides, polypeptides, proteins or nucleic acids may be human, i.e., their primary sequence may be the same as a corresponding primary sequence of or present in naturally occurring human peptides, polypeptides, proteins or nucleic acids. In certain embodiments the qualifier “human” relates to the primary sequence of the respective peptides, polypeptides, proteins or nucleic acids, rather than to their origin or source. For example, such peptides, polypeptides, proteins or nucleic acids may be present in or isolated from samples of human subjects or may be obtained by other means (e.g., by recombinant expression, cell -free transcription or translation, or non-biological nucleic acid or peptide synthesis).

[0091] In certain embodiments, the peptides, polypeptides, proteins or nucleic acids may be wild-type. While most native peptides, polypeptides, proteins or nucleic acids may be considered wild-type, those carrying naturally-occurring mutations leading to partial or complete loss of function, which may contribute to or be causative of a disease phenotype, are generally excluded from the scope of the term “wild-type”.

[0092] The reference to any peptides, polypeptides, proteins or nucleic acids may also encompass variants or fragments of such peptides, polypeptides, proteins or nucleic acids, particularly of naturally- occurring, native or wild-type forms thereof.

[0093] The term “fragment” as used throughout this specification with reference to a peptide, polypeptide, or protein generally denotes a portion of the peptide, polypeptide, or protein, such as typically an N- and / or C-terminally truncated form of the peptide, polypeptide, or protein. Preferably, a fragment may comprise at least about 30%, e.g., at least about 50% or at least about 70%, preferably at least about 80%, e.g., at least about 85%, more preferably at least about 90%, and yet more preferably at least about 95% or even about 99% of the amino acid sequence length of said peptide, polypeptide, or protein. For example, insofar not exceeding the length of the full-length peptide, polypeptide, or protein, a fragment may include a sequence of > 5 consecutive amino acids, or > 10 consecutive amino acids, or > 20 consecutive amino acids, or > 30 consecutive amino acids, e.g., > 40 consecutive amino acids, such as for example > 50 consecutive amino acids, e.g., > 60, > 70, > 80, > 90, > 100, > 200, >300, >400, > 500, > 600, > 700, > 800, > 900 or > 1000 consecutive amino acids of the corresponding full-length peptide, polypeptide, or protein.

[0094] The term “fragment” with reference to a nucleic acid (polynucleotide) generally denotes a 5’- and / or 3 ’-truncated form of a nucleic acid. Preferably, a fragment may comprise at least about 30%, e.g., at least about 50% or at least about 70%, preferably at least about 80%, e.g., at least about 85%, more preferably at least about 90%, and yet more preferably at least about 95% or even about 99% of the nucleic acid sequence length of said nucleic acid. For example, insofar not exceeding the length of the full-length nucleic acid, a fragment may include a sequence of > 5 consecutive nucleotides, or > 10 consecutive nucleotides, or > 20 consecutive nucleotides, or > 30 consecutive nucleotides, e.g., > 40 consecutive nucleotides, such as for example > 50 consecutive nucleotides, e.g., > 60, > 70, > 80, > 90, > 100, > 200, > 300, > 400, > 500, > 600, > 700, > 800, > 900, > 1000, > 1500, > 2000, > 2500, > 3000, > 3500 or > 4000 consecutive nucleotides of the corresponding full-length nucleic acid. The terms encompass fragments arising by any mechanism, in vivo and / or in vitro, such as, without limitation, by alternative transcription or translation, exo- and / or endo-proteolysis, exo- and / or endo-nucleolysis, or degradation of the peptide, polypeptide, protein or nucleic acid, such as, for example, by physical, chemical and / or enzymatic proteolysis or nucleolysis.

[0095] The term “variant” of a protein, polypeptide, peptide or nucleic acid generally refers to proteins, polypeptides or peptides the amino acid sequence of which, or nucleic acids the nucleotide sequence of which, is substantially identical (i.e., largely but not wholly identical) to the sequence of the protein, polypeptide, peptide, or nucleic acid, e.g., at least about 80% identical or at least about 85% identical, e.g., preferably at least about 90% identical, e.g., at least 91% identical, 92% identical, more preferably at least about 93% identical, e.g., at least 94% identical, even more preferably at least about 95% identical, e.g., at least 96% identical, yet more preferably at least about 97% identical, e.g., at least 98% identical, and most preferably at least 99% identical to the sequence of the recited protein, polypeptide, peptide, or nucleic acid. Preferably, a variant may display such degrees of identity to a recited protein, polypeptide, peptide or nucleic acid when the whole sequence of the recited protein, polypeptide, peptide or nucleic acid is queried in the sequence alignment (i.e., overall sequence identity). Sequence identity may be determined using suitable algorithms for performing sequence alignments and determination of sequence identity as know per se. Exemplary but non-limiting algorithms include those based on the Basic Local Alignment Search Tool (BLAST) originally described by Altschul et al. 1990 (J Mol Biol 215: 403-10), such as the “Blast 2 sequences” algorithm described by Tatusova and Madden 1999 (LEMS Microbiol Lett 174: 247-250), for example using the published default settings or other suitable settings (such as, e.g., for the BLASTN algorithm: cost to open a gap = 5, cost to extend a gap = 2, penalty for a mismatch = -2, reward for a match = 1, gap x_dropoff = 50, expectation value = 10.0, word size = 28; or for the BLASTP algorithm: matrix = Blosum62 (Henikoff et al., 1992, Proc. Natl. Acad. Sci., 89: 10915-10919), cost to open a gap = 11, cost to extend a gap = 1, expectation value = 10.0, word size = 3).

[0096] An example procedure to determine the percent identity between a particular amino acid sequence and the amino acid sequence of a query polypeptide will entail aligning the two amino acid sequences using the Blast 2 sequences (B12seq) algorithm, available as a web application or as a standalone executable programme (BLAST version 2.2.31+) at the NCBI web site (www.ncbi.nlm.nih.gov), using suitable algorithm parameters. An example of suitable algorithm parameters include: matrix = Blosum62, cost to open a gap = 11, cost to extend a gap = 1, expectation value = 10.0, word size = 3). If the two compared sequences share homology, then the output will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the output will not present aligned sequences. Once aligned, the number of matches will be determined by counting the number of positions where an identical amino acid residue is presented in both sequences. The percent identity is determined by dividing the number of matches by the length of the query polypeptide, followed by multiplying the resulting value by 100. The percent identity value may, but need not, be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 may be rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 may be rounded up to 78.2. It is further noted that the detailed view for each segment of alignment as outputted by B12seq already conveniently includes the percentage of identities.

[0097] A variant of a protein, polypeptide, peptide or nucleic acid may be a homologue (e.g., orthologue or paralogue) of said protein, polypeptide, peptide or nucleic acid. As used herein, the term “homology” generally denotes structural similarity between two macromolecules from same or different taxons, wherein said similarity is due to shared ancestry.

[0098] A variant of a protein, polypeptide, or peptide may comprise one or more amino acid additions, deletions, or substitutions relative to (i.e., compared with) the corresponding protein or polypeptide. For example, a variant (substitution variant) of a protein, polypeptide, or peptide may comprise up to 70 (e.g., not more than one, two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, 50, 60, or 70) conservative amino acid substitutions relative to (i.e., compared with) the corresponding protein or polypeptide; and / or a variant (substitution variant) of a protein, polypeptide, or peptide may comprise up to 20 (e.g., not more than one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, or 19) non -conservative amino acid substitutions relative to (i.e., compared with) the corresponding protein or polypeptide.

[0099] A conservative amino acid substitution is a substitution of one amino acid for another with similar characteristics. Conservative amino acid substitutions include substitutions within the following groups: valine, alanine and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. The nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (i.e., basic) amino acids include arginine, lysine and histidine. The negatively charged (i.e., acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above-mentioned polar, basic, or acidic groups by another member of the same group can be deemed a conservative substitution. By contrast, a non-conservative substitution is a substitution of one amino acid for another with dissimilar characteristics. Alternatively or in addition, for example, a variant (deletion variant) of a protein, polypeptide, or peptide may lack up to 20 amino acid segments (e.g., one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 segments) relative to (i.e., compared with) the corresponding protein or polypeptide. The deletion segment(s) may each independently consist of one amino acid, two contiguous amino acids or three contiguous amino acids. The deletion segments may be non-contiguous, or two or more or all of the deletion segments may be contiguous.

[0100] A variant of a nucleic acid may comprise one or more nucleotide additions, deletions, or substitutions relative to (i.e., compared with) the corresponding nucleic acid.

[0101] Reference to “fragment or variant” or “variant or fragment” of any peptide, polypeptide, protein or nucleic acid, also encompasses fragments of variants of such peptide, polypeptide, protein or nucleic acid, and variants of fragments of such peptide, polypeptide, protein or nucleic acid.

[0102] Particularly envisaged are biologically active fragments and / or variants of the recited peptides, polypeptides or proteins. The term “biologically active” is interchangeable with terms such as “functionally active” or “functional”, denoting that the fragment and / or variant at least partly retains the biological activity or intended functionality of the respective or corresponding peptide, polypeptide or protein. Reference to the “activity” of a peptide, polypeptide or protein may generally encompass any one or more aspects of the biological activity of the peptide, polypeptide or protein, such as without limitation any one or more aspects of its biochemical activity, enzymatic activity, signaling activity, interaction activity, ligand activity, and / or structural activity, e.g., within a cell, tissue, organ or an organism.

[0103] Preferably, a functionally active fragment or variant may retain at least about 20%, e.g., at least about 25%, or at least 30%, or at least about 40%, or at least about 50%, e.g., at least 60%, more preferably at least about 70%, e.g., at least 80%, yet more preferably at least about 85%, still more preferably at least about 90%, and most preferably at least about 95% or even about 100% of the intended biological activity or functionality compared with the corresponding peptide, polypeptide or protein. In certain embodiments, a functionally active fragment or variant may even display higher biological activity or functionality compared with the corresponding peptide, polypeptide or protein, for example may display at least about 100%, or at least about 150%, or at least about 200%, or at least about 300%, or at least about 400%, or at least about 500% of the intended biological activity or functionality compared with the corresponding peptide, polypeptide or protein. By means of an example, where the activity of a given peptide, polypeptide or protein can be readily measured in an assay with a quantitative output, for example an enzymatic assay or a signaling assay or a binding assay producing a quantifiable signal, a functionally active fragment or variant of the peptide, polypeptide or protein may produce a signal which is at least about 20%, or at least about 25%, or at least 30%, or at least about 40%, or at least about 50%, or at least 60%, more preferably at least about 70%, or at least 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 300%, or at least about 400%, or at least about 500% of the signal produced by the corresponding peptide, polypeptide or protein.

[0104] By means of an example and not limitation, a biologically active fragment or variant of GPR124, RECK, FZD or LRP polypeptide or protein will at least partly retain one or more aspects of the biological activity of the corresponding native or wild-type GPR124, RECK, FZD or LRP polypeptide or protein, respectively. For example, reference to the biological activity of the GPR124, RECK, FZD or LRP polypeptide or protein may particularly denote the ability to participate in a GPR124 / RECK / FZD / LRP complex, e.g., the ability to bind to one or more other component(s) of said complex, and / or the ability to mediate Wnt signaling as part of the GPR124 / RECK / FZD / LRP complex.

[0105] Hence, the specification in particular discloses the therapeutic use of an agent capable of activating GPR124 / RECK / FZD / LRP-mediated Wnt signaling, wherein said agent does not activate FZD / LRP -mediated Wnt signaling in the absence of RECK and / or GPR124, wherein one or more or preferably all of GPR124, RECK, FZD or LRP is or are native or wild-type GPR124, RECK, FZD or LRP.

[0106] The specification further in particular discloses the therapeutic use of an agent capable of activating GPR124 / RECK / FZD / LRP-mediated Wnt signaling, wherein said agent does not activate FZD / LRP -mediated Wnt signaling in the absence of RECK and / or GPR124, wherein one or more or preferably all of GPR124, RECK, FZD or LRP is or are native or wild-type human GPR124, RECK, FZD or LRP.

[0107] The specification further in particular discloses the therapeutic use of an agent capable of activating GPR124 / RECK / FZD / LRP-mediated Wnt signaling, wherein said agent does not activate FZD / LRP -mediated Wnt signaling in the absence of RECK and / or GPR124, wherein one or more or preferably all of GPR124, RECK, FZD or LRP is or are native or wild-type human GPR124, RECK, FZD or LRP as annotated under the Genbank or Uniprot entries set forth elsewhere in this specification. The reader is reminded that where Genbank or Uniprot entries provide the sequence of precursor polypeptides or proteins, the corresponding mature forms would be expected to participate in the GPR124 / RECK / FZD / LRP complex.

[0108] The specification further in particular discloses the therapeutic use of an agent capable of activating GPR124 / RECK / FZD1 / LRP5 or LRP6-mediated Wnt signaling, wherein said agent does not activate FZD5 / LRP5 or LRP6-mediated Wnt signaling in the absence of RECK and / or GPR124, wherein one or more or preferably all of GPR124, RECK, FZD1, FZD5, LRP5 or LRP6 is or are native or wild-type human GPR124, RECK, FZD1, FZD5, LRP5 or LRP6 as annotated under the Genbank or Uniprot entries set forth elsewhere in this specification.

[0109] The specification further in particular discloses the therapeutic use of an agent capable of activating GPR124 / RECK / FZD / LRP-mediated Wnt signaling, wherein said agent does not activate FZD / LRP -mediated Wnt signaling in the absence of RECK and / or GPR124, wherein one or more of GPR124, RECK, FZD or LRP is or are a biologically active fragment or variant of native or wild-type GPR124, RECK, FZD or LRP.

[0110] The specification further in particular discloses the therapeutic use of an agent capable of activating GPR124 / RECK / FZD / LRP-mediated Wnt signaling, wherein said agent does not activate FZD / LRP -mediated Wnt signaling in the absence of RECK and / or GPR124, wherein one or more of GPR124, RECK, FZD or LRP is or are a biologically active fragment or variant of native or wild-type human GPR124, RECK, FZD or LRP.

[0111] The terms “complex”, “protein complex” or “polypeptide complex” are well-understood in the art. By means of further guidance and without limitation, the terms broadly denote a cluster comprising two or more proteins or polypeptides. The cluster may be stabilised by non -covalent bonds, more particularly non-covalent protein-protein interactions, wherein all or only part of the polypeptides present within the cluster physically interact. A protein complex can be comprised entirely of peptides, polypeptides or proteins, or it may include other molecules or macromolecules such as carbohydrates, lipids, glycolipids, nucleic acids, oligonucleotides, nucleoproteins, nucleosides, nucleoside phosphates, enzyme co-factors, porphyrins, metal ions and the like. The terms encompass without limitation protein complexes which may be obligate or non-obligate, transient or permanent, and / or homomultimeric or heteromultimeric. The terms encompass without limitation protein complexes which may be located at the cell membrane (plasma membrane), extracellularly, within cytoplasm, within cellular organelles, or at membranes of cellular organelles. Complexes located at the cell membrane typically contain at least one membrane -anchored or transmembrane protein. The terms also encompass membrane microdomains and membrane- associated macromolecular organelle -like structures known as “signalosome” which compartmentalise a given signaling pathway. Such higher-order protein complexes can be at least partly stabilised by intracellular scaffolds. The terms may thus also denote situations in which certain proteins or polypeptides are locally concentrated or accumulated at a given site of a cell membrane, such as to allow signal transduction through the cell membrane at said site mediated by said proteins or polypeptides. A GPR124 / RECK / FZD / LRP receptor complex broadly denotes a protein complex, particularly a membrane-associated protein complex, more particularly a plasma membrane-associated protein complex comprising at least one GPR124 polypeptide, at least one RECK polypeptide, at least one FZD polypeptide and at least one LRP polypeptide. A GPR124 / RECK / FZD / LRP receptor complex, when located at the plasma membrane of a cell containing the downstream members of the Wnt / p- catenin signaling pathway (e.g., Dishevelled (Dvl)), is capable of activating Wnt / p-catenin signaling in said cell in response to extracellularly provided Wnt7 ligand.

[0112] A FZD / LRP receptor complex broadly denotes a protein complex, particularly a membrane- associated protein complex, more particularly a plasma membrane-associated protein complex comprising at least one FZD polypeptide and at least one LRP polypeptide. A FZD / LRP receptor complex, when located at the plasma membrane of a cell containing the downstream members of the Wnt / p-catenin signaling pathway (e.g., Dvl), is capable of activating Wnt / p-catenin signaling in said cell in response to extracellularly provided Wnt ligand, such as, but not limited to Wnt7 ligand.

[0113] The term “Wnt signaling” as used herein refers to the mechanism by which a biologically active Wnt ligand exerts its effect upon a cell to modulate said cell’s activity and / or actions. Biologically active Wnt ligands modulate cell activity and / or action by binding to Wnt receptor(s), such as the FZD / LRP receptor complex. Once activated by binding of the Wnt ligand, the Wnt receptor(s) will activate one or more intracellular signaling pathways. Three Wnt signaling pathways are classically recognized: the canonical (i.e., mediated by P-catenin activation as a transcriptional co-activator) Wnt pathway, the non-canonical planar cell polarity pathway, and the non-canonical Wnt / calcium pathway. All three pathways are typically activated by binding of a Wnt ligand to a FZD receptor. However, the recruitment of the LRP receptor appears to be a prerequisite for inducing canonical (or P-catenin-dependent) Wnt signaling. As known in the art, in the absence of a Wnt / FZD / LRP- complex (“inactive canonical Wnt signaling”), P-catenin is phosphorylated in the cytoplasm by Casein Kinase and glycogen synthase kinase-3 (GSK-3). The interaction between these kinases and P-catenin is facilitated by the scaffolding proteins, Axin and adenomatous polyposis coli (APC). Together, these proteins form a ‘degradation complex’, which allows phosphorylated P-catenin to be recognized by beta-transducin repeat-containing protein (P-TrCP), targeted for ubiquitination, and degraded by the proteasome. Active canonical (or beta-catenin-dependent) Wnt signaling involves binding of Wnt ligands to a receptor complex of FZD and LRP on the cell surface. During signaling, FZD cooperates with LRP in such a way that binding of the Wnt protein leads to dimerization of the two receptors. It is theorized that this dimerization leads to a conformational change of the FZD and LRP receptors. As a consequence, the cytoplasmic tail of LRP recruits and binds to the scaffold protein Axin in a phosphorylation-dependent manner and leads to formation of a complex involving DVL, Axin, and GSK3. Multimers of receptor-bound DVL and Axin molecules might support the formation of the LRP-FZD dimer. As a result of the recruitment of GSK to the cell membrane, P-catenin phosphorylation is inhibited, releasing -catenin from the degradation complex and allowing p-catenin to accumulate in the cytoplasm. The accumulation of P-catenin in the cytoplasm allows to P-catenin to enter the nucleus and to interact with TCF / LEF transcription factors.

[0114] The present inventors previously identified Wnt7-specific RECK / GPR124 / Frizzled / LRP-mediated signaling, in which Wnt7 binds specifically to Reck in a FZD-independent manner and GPR124, a RECK binding partner, bridges RECK-bound Wnt7 to the FZD / LRP complex thereby assembling Wnt7-ligand specific RECK / GPR124 / FZD / LRP signalosomes and activating canonical Wnt signaling (as described in International patent application W02019180204A1).

[0115] In view of the above, the skilled person will understand that, as is typically the case for Wnt signaling mediated by the FZD and LRP polypeptides, an FZD / LRP complex might not be formed on a cell’s membrane in the absence of or independently of an extracellular agent capable of activating Wnt signaling through said FZD / LRP complex. In other words, the agent may play an active role in assembling or organizing FZD and LRP polypeptides into the FZD / LRP complex, such as by means of protein-protein interactions between the agent and each of FZD and LRP. This results in the formation of the FZD / LRP complex and activation of Wnt signaling mediated by the complex.

[0116] Similarly, a GPR124 / RECK / FZD / LRP receptor complex might not be formed on a cell’s membrane in the absence of or independently of an extracellular agent capable of activating Wnt signaling through said GPR124 / RECK / FZD / LRP complex. In other words, the agent may play an active role in assembling or organizing two or more of GPR124, RECK, FZD and LRP polypeptides into the GPR124 / RECK / FZD / LRP complex, such as by means of protein-protein interactions between the agent and each of said two or more of GPR124, RECK, FZD and LRP. This results in the formation of the GPR124 / RECK / FZD / LRP complex and activation of Wnt signaling mediated by the complex.

[0117] Accordingly, the phrase “an agent capable of activating Gprl24 / Reck / Frizzled / LRP -mediated Wnt signaling” or “an agent capable of activating GPR124 / RECK / FZD / LRP -mediated Wnt signaling” refers to the ability of the agent to induce Wnt signaling through the Gprl24, Reck, Frizzled and LRP polypeptides. The phrase “an agent capable of activating Gprl24 / Reck / Frizzled / LRP- mediated Wnt signaling” does not necessarily imply the existence of a Gprl24 / Reck / Frizzled / LRP receptor complex prior to contacting a cell expressing Gprl24, Reck, Frizzled and LRP with the agent. For example and without limitation, the agent may contribute to or facilitate the formation or assembly of a Gprl24 / Reck / Frizzled / LRP signaling complex, thus activating Wnt signaling mediated by said complex. Similarly, a phrase “an agent capable of activating Frizzled / LRP- mediated Wnt signaling” refers to the ability of the agent to induce Wnt signaling through the Frizzled and LRP polypeptides. The phrase “an agent capable of activating Frizzled / LRP -mediated Wnt signaling” would not necessarily imply the existence of a Frizzled / LRP receptor complex prior to contacting a cell expressing Frizzled and LRP with such agent. For example and without limitation, such agent might contribute to or facilitate the formation or assembly of a Frizzled / LRP signaling complex, thus activating Wnt signaling mediated by said complex. The phrase “said agent does not activate Frizzled / LRP-mediated Wnt signaling in the absence of Reck and / or Gprl24” does not necessarily imply the existence of a Frizzled / LRP receptor complex in a cell expressing Frizzled and LRP but not Reck and / or Gprl24. For example and without limitation, the agent may fail to contribute to or facilitate the formation or assembly of a Frizzled / LRP signaling complex in such cell, thus failing to activate Wnt signaling mediated by said complex. The skilled person shall further appreciate that a GPR124 / RECK / FZD / LRP receptor complex as envisaged herein may include further component(s), which may or need not functionally modulate the complex. For example, the complex may include Dishevelled (Dvl), forming intracellular scaffolds capable of bridging GPR124 and Frizzled or any other additional adaptor, such as described in America et al., An integrated model for Gprl24 function in Wnt7a / b signaling among vertebrates, Cell Rep., 2022.

[0118] In particular embodiments, the cell expressing - GPR124, RECK, FZD and LRP polypeptides at its plasma membrane and containing the downstream members of the Wnt / P-catenin signaling pathway is a cell naturally expressing all GPR124, RECK, FZD and LRP polypeptides at the cell surface, such as a cerebral endothelial cell. In further embodiments, the cell expressing the GPR124, RECK, FZD and LRP polypeptides at its plasma membrane and containing the downstream members of the Wnt / P-catenin signaling pathway is a cell modified (e.g., genetically engineered) in such a way that it expresses all GPR124, RECK, FZD and LRP polypeptides at its surface. For example, the cell may be a cultured cell naturally expressing FZD and LRP but not naturally expressing GPR124 and RECK, and genetically engineered to express the GPR124 and RECK polypeptides (e.g., stably or transiently transfected with expressible nucleic acid(s) encoding said GPR124 and RECK polypeptides. Preferably, the cell is a mammalian cell, more preferably a human cell.

[0119] In particular embodiments, the capability of the agent to activate GPR124 / RECK / Frizzled / LRP- mediated Wnt signaling, but not activate Frizzled / LRP-mediated Wnt signaling in the absence of RECK and / or GPR124, denotes the capability of the agent to activate canonical Wnt signaling in the presence of RECK and GPR124, but not in the absence of RECK and / or GPR124.

[0120] The phrase “the presence of RECK and GPR124” refers to the occurrence of the RECK and GPR124 polypeptides at or close to the cell membrane, preferably in close mutual proximity with the Frizzled and LRP polypeptides. The close mutual proximity can facilitate the formation of the GPR124 / RECK / Frizzled / LRP receptor complex under conditions conducive thereto, such as when an agent as taught herein is externally supplied to a cell. On the other hand, the phrase “the absence of RECK and / or GPR124” refers to lack or non-occurrence of the RECK, the GPR124, or the RECK and the GPR124 polypeptides at the cell membrane. Absence of RECK and / or GPR124 at or close to the cell membrane may occur when a cell does not express, translate, or correctly translocate RECK and / or GPR124. The absence of RECK and / or GPR124 need not denote the complete absence of the RECK and / or GPR124 polypeptide at the cell membrane, but may for example refer to an amount of RECK and / or GPR124 polypeptide which is not detectable by, or falls below the sensitivity range of, conventional protein detection or quantification assays known by the person skilled in the art, such as for example immunoblotting, immunocytochemistry or immunofluorescence .

[0121] In particular embodiments, the capability of the agent to activate GPR124 / RECK / Frizzled / LRP- mediated Wnt signaling, but not activate Frizzled / LRP -mediated Wnt signaling in the absence of RECK and / or GPR124, denotes the capability of the agent to activate Wnt signalling in cells capable of mediating Wnt signalling by the GPR124 / RECK / Frizzled / LRP receptor complex, but not in cells capable of Frizzled / LRP -mediated Wnt signaling which do not express GPR124 and / or RECK.

[0122] Cells capable of mediating Wnt signaling by the GPR124 / RECK / Frizzled / LRP receptor complex may be cells naturally expressing all cellular components required for GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling, such as cerebral endothelial cells or cell lines. Cells capable of mediating Wnt signaling by the GPR124 / RECK / Frizzled / LRP receptor complex may also be cells which naturally express none or not all of the cellular components required for GPR124 / RECK / Frizzled / LRP -mediated Wnt signaling, which may be modified such as genetically engineered in order to compensate for the missing cellular components required for GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling. Such modifications are known in the art and are described elsewhere herein.

[0123] Cells capable of mediating Wnt signaling by the GPR124 / RECK / Frizzled / LRP receptor complex may be vertebrate cells or cell lines. Non-limiting examples include human embryonic kidney 293 T (HEK239T), human umbilical vein endothelial cells (HUVEC), human dermal microvascular endothelial cells (HDMVEC), human coronary artery endothelial cells (HCAEC), human aortic endothelial cells (HAEC), human brain microvascular endothelial cells (HBMECs), Bend3 cells, Bend5 cells, or hCMEC / D3 cells. HEK293T cells may be obtained from public collections maintained for example by American Type Culture Collection (ATCC) (10801 University Blvd. Manassas, Virginia 20110-2209, USA), including without limitation, HEK293T with ATCC acc.

[0124] No. CRL-3216.

[0125] In particular embodiments, the capability of the agent to activate GPR124 / RECK / Frizzled / LRP- mediated Wnt signaling, but not activate Frizzled / LRP -mediated Wnt signaling in the absence of RECK and / or GPR124, denotes the capability of the agent to activate Wnt signalling in cells expressing GPR124, RECK, FZD and LRP, but not in cells expressing FZD and LRP but not expressing GPR124 and / or RECK, wherein the cells expressing GPR124, RECK, FZD and LRP and the cells expressing FZD and LRP but not expressing GPR124 and / or RECK are otherwise substantially identical.

[0126] In particular embodiments, the capability of the agent to activate GPR124 / RECK / Frizzled / LRP- mediated Wnt signaling, but not activate Frizzled / LRP -mediated Wnt signaling in the absence of RECK and / or GPR124, denotes the capability of the agent to activate Wnt signalling in mouse or human vascular endothelial cells or cell lines, while not activating Wnt-signalling in mouse or human vascular endothelial cells or cell lines in which the RECK and / or GPR124 gene is knocked- out. Determination of the activation of Wnt signalling is known in the art and can be performed as described elsewhere herein.

[0127] Brain vascular endothelial cells or cell lines serve as a good in vitro model system for identifying these agents capable of activating GPR124 / RECK / Frizzled / LRP -mediated Wnt signaling, while not activating Frizzled / LRP -mediated Wnt signaling in the absence of RECK and / or GPR124, useful for the treatment (e.g. treatment and / or prevention) of a bone-related disease or disorder. Nonlimiting examples for human brain vascular endothelial cells or cell lines include brain microvascular endothelial cells (HBMECs), Bend3 cells, Bend5 cells or hCMEC / D3 cells. Bend3 cells may be obtained from public collections maintained for example by ATCC, including without limitation, bEnd.3 with ATCC acc. No. CRL-2299.

[0128] Furthermore, Wnt7aa / _zebrafish (Danio rerio) when being transgenically expressed in the endothelium of said zebrafish, serve as a good in vivo model system for identifying these agents capable of activating GPR124 / RECK / Frizzled / LRP -mediated Wnt signaling, while not activating Frizzled / LRP-mediated Wnt signaling in the absence of RECK and / or GPR124, useful for the treatment (e.g. treatment and / or prevention) of a bone-related disease or disorder. Accordingly, in particular embodiments, the capability of the agent to activate GPR124 / RECK / Frizzled / LRP- mediated Wnt signaling, but not activate Frizzled / LRP-mediated Wnt signaling in the absence of RECK and / or GPR124, denotes the capability of the agent to restore the brain vasculature and / or blood-brain barrier in Wnt7aa / _zebrafish (Danio rerio) when being transgenically expressed in the endothelium of said zebrafish, while microinjection of mRNA encoding the agent in a wild-type zebrafish embryo does not induce a posteriorization phenotype, preferably wherein mRNA is injected at a dose of 1 pg to 100 pg, or while micro injection of mRNA encoding the agent in a wild-type Xenopus embryo does not induce axis duplication.

[0129] The capability of the agent to restore the brain vasculature and / or blood-brain barrier in Wnt7aa / _zebrafish when being transgenically expressed in the endothelium of said zebrafish can be determined by any method known in the art as described in Martin et al. Engineered Wnt ligands enable blood-brain barrier repair in neurological disorders. Science 2022. For example, restoration of the brain vasculature and / or blood-brain barrier can be evaluated by determining the amount of hindbrain central arteries (QAs), the aggregate cerebral capillary length, the mean vessel size and / or the average capillary branches. Determining the expression of GLUT1 in the brain region of the Wnt7aa / _zebrafish transgenically expressing the agent as taught herein (e.g. using in situ hybridization or immunohistochemistry) can be used to visualize the brain vasculature in said zebrafish. Subsequently, the GLUT1 expression pattern in the brain region of the Wnt7aa / _zebrafish transgenically expressing the agent as taught herein can be compared to the GLUT1 expression pattern in the brain region of a control, such as a wild-type, zebrafish.

[0130] In particular embodiments, the brain vasculature and blood-brain barrier of the Wnt7aa / _zebrafish transgenically expressing the agent as disclosed herein is considered to be restored if the amount of hindbrain CtAs in Wnt7aa / _zebrafish transgenically expressing the agent as taught herein is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the amount of hindbrain CtAs in a wild-type zebrafish.

[0131] Methods of generating Wnt7aa / _zebrafish and transgenically expressing an agent in the endothelium of a zebrafish are well known in the art, for example, using CRISPR / Cas-directed gene editing, such as described in Shankaran et al., CRISPR / Cas9 -directed gene editing for the generation of loss-of-fiinction mutants in high-throughput zebrafish Fo screens, Curr Protoc Mol Biol., 119:31.9.1-31.9.22 (2017).

[0132] Microinjection of mRNA in a zebrafish embryo, preferably a one to four-cell stage zebrafish embryo, as well as the evaluation of the occurrence or absence of a phenotype in zebrafish are well known in the art, for example see Rosen et al., Microinjection of zebrafish embryos to analyse gene function, J Vis Exp. (25): 1115 (2009). The posteriorization phenotype in zebrafish includes posteriorization of the anterior neuroectoderm during gastrulation, loss of forebrain and loss of the eye structure in zebrafish (van de Water et al., Ectopic Wnt signal determines the eyeless phenotype of zebrafish masterblind mutant. Development. 128(20):3877-3888 (2001)). Evaluation of the occurrence or absence axis duplication upon injection of an agent in a Xenopus embryo is well known in the art, for example see Kuhl et al., Dorsal axis duplication as a functional readout for Wnt activity. Methods Mol Biol. 469:467-476 (2008).

[0133] As used herein, the term “agent” broadly refers to any chemical (e.g., inorganic or organic), biochemical or biological substance, molecule or macromolecule (e.g., biological macromolecule), a combination or mixture thereof, a sample of undetermined composition, or an extract made from biological materials such as bacteria, fungi, plants, or animal cells or tissues. Preferred though nonlimiting “agents” include nucleic acids, oligonucleotides, ribozymes, peptides, polypeptides, proteins, peptidomimetics, antibodies, antibody fragments, antibody-like protein scaffolds, aptamers, photoaptamers, spiegelmers, chemical substances, preferably organic molecules, more preferably small organic molecules, lipids, carbohydrates, polysaccharides, etc., and any combinations thereof. Depending on the context, the term “agent” may denote a “therapeutic agent” or “drug”, useful for or used in the treatment, cure, prevention, or diagnosis of a disease.

[0134] In particular embodiments, the agent as disclosed herein comprises or is selected from a group consisting of a chemical substance, an antibody, an antibody fragment, an antibody-like protein scaffold, a protein or polypeptide, a peptide, a peptidomimetic, an aptamer, a photoaptamer, a spiegelmer and a nucleic acid, preferably wherein said agent comprises, essentially consists of or consists of a protein or polypeptide.

[0135] The agent as disclosed herein may comprise a combination of two or more of a chemical substance, an antibody, an antibody fragment, an antibody-like protein scaffold, a protein or polypeptide, a peptide, a peptidomimetic, an aptamer, a photoaptamer, a spiegelmer and a nucleic acid. For example, the agent as disclosed herein may comprise a combination of one or more polypeptide regions and one or more non-polypeptide regions.

[0136] In particular embodiments, the agent as disclosed herein comprises or is selected from a group consisting of a protein, polypeptide, or a peptide.

[0137] As used herein, the term “chemical substance” is used in its broadest sense and generally refers to any substantially pure substance that has a constant chemical composition and characteristic properties. The chemical substance may be an organic molecule, preferably a small organic molecule. The term “small molecule” refers to compounds, preferably organic compounds, with a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, peptides, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, e.g., up to about 4000, preferably up to 3000 Da, more preferably up to 2000 Da, even more preferably up to about 1000 Da, e.g., up to about 900, 800, 700, 600 or up to about 500 Da. The term “antibody” is used herein in its broadest sense and generally refers to any immunologic binding agent, such as a whole antibody, including without limitation a chimeric, humanized, human, recombinant, transgenic, grafted and single chain antibody, and the like, or any fusion proteins, conjugates, fragments, or derivatives thereof that contain one or more domains that selectively bind to an antigen of interest. The term antibody thereby includes a whole immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, or an immunologically effective fragment of any of these. The term thus specifically encompasses intact monoclonal antibodies, polyclonal antibodies, multivalent (e.g., 2-, 3- or more-valent) and / or multi-specific antibodies (e.g., bi- or more-specific antibodies) formed from at least two intact antibodies, and antibody fragments insofar they exhibit the desired biological activity (particularly, ability to specifically bind an antigen of interest), as well as multivalent and / or multi-specific composites of such fragments. The term “antibody” is not only inclusive of antibodies generated by methods comprising immunisation, but also includes any polypeptide, e.g., a recombinantly expressed polypeptide, which is made to encompass at least one complementarity-determining region (CDR) capable of specifically binding to an epitope on an antigen of interest. Hence, the term applies to such molecules regardless whether they are produced in vitro, in cell culture, or in vivo.

[0138] The term “immunoglobulin sequence” - whether used herein to refer to a heavy chain antibody or to a conventional 4-chain antibody - is used as a general term to include both the full-size antibody, the individual chains thereof, as well as all parts, domains or fragments thereof (including but not limited to antigen-binding domains or fragments such as VHH domains or VH / VL domains, respectively). In addition, the term “sequence” as used herein (for example in terms like “immunoglobulin sequence”, “antibody sequence”, “variable domain sequence”, “VHH sequence” or “protein sequence”), should generally be understood to include both the relevant amino acid sequence as well as nucleic acid sequences or nucleotide sequences encoding the same, unless the context requires a more limited interpretation.

[0139] The term “epitope” includes any polypeptide determinant capable of specifically binding to an immunoglobulin or T-cell receptor. Epitope determinants may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and may have specific three dimensional structural characteristics, and / or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. An antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0140] The terms “binding region”, “binding site” or “interaction site” shall herein have the meaning of a particular site, part, domain or stretch of amino acid residues that is responsible for binding to an antigen of interest. Such binding region essentially consists of specific amino acid residues of the antibodies described herein, which residues are in contact with the target molecule.

[0141] The term “specificity” refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding molecule or antigen-binding protein (such as an antibody) molecule can bind. The specificity of an antigen -binding protein can be determined based on affinity and / or avidity. The affinity, represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding protein (KD), is a measure for the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding protein: the lesser the value of the KD, the stronger the binding strength between an antigenic determinant and the antigen-binding molecule (alternatively, the affinity can also be expressed as the affinity constant (KA), which is 1 / KD). As will be clear to the skilled person, affinity can be determined in a manner known per se, depending on the specific antigen of interest. Avidity is the measure of the strength of binding between an antigen-binding molecule (such as an antibody) and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen-binding molecule and the number of pertinent binding sites present on the antigenbinding molecule. Typically, antigen-binding proteins (such as antibodies) will bind with a dissociation constant (KD) of 10'5to 10'12moles / liter (M) or less, and preferably 10'7to 10'12moles / liter (M) or less and more preferably 10'8to 10'12moles / liter, and / or with an association constant(KA) of at least 107M1, preferably at least 108M1, more preferably at least 109M1, such as at least 1012M1. Any KD value greater than 10'4M is generally considered to indicate non-specific binding. Preferably, an antibody will bind to the desired antigen with an KD less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art.

[0142] A full-length antibody as it exists naturally is an immunoglobulin molecule comprising 2 heavy (H) chains and 2 light (L) chains interconnected by disulfide bonds. The amino terminal portion of each chain includes a variable region of about 100-110 amino acids primarily responsible for antigen recognition via the complementarity determining regions (CDRs) contained therein. The carboxy - terminal portion of each chain defines a constant region primarily responsible for effector function.

[0143] The CDRs are interspersed with regions that are more conserved, termed framework regions (FR). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of 3 CDRs and 4 FRs, arranged from amino -terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDRs of the light chain are referred to as “LCDR1, LCDR2, and LCDR3” and the 3 CDRs of the heavy chain are referred to as “HCDR1, HCDR2, and HCDR3.” The CDRs contain most of the residues which form specific interactions with the antigen. The numbering and positioning of CDR amino acid residues within the LCVR and HCVR regions is in accordance with the well-known Kabat numbering convention, which refers to a system of numbering amino acid residues which are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain regions of an antibody (Kabat, et al., Ann. NYAcad. Sci. 190:382-93 (1971 ); Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91- 3242 (1991 )). The positioning of CDRs in the variable region of an antibody follows Kabat numbering or simply, “Kabat.”

[0144] Light chains are classified as kappa or lambda, and are characterized by a particular constant region as known in the art. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the isotype of an antibody as IgG, IgM, IgA, IgD, or IgE, respectively. IgG antibodies can be further divided into subclasses, e.g., IgGl, IgG2, IgG3, IgG4. Each heavy chain type is characterized by a particular constant region with a sequence well known in the art.

[0145] In certain embodiments, an antibody may be any of IgA, IgD, IgE, IgG and IgM classes, and preferably IgG class antibody.

[0146] In certain embodiments, the antibody may be a polyclonal antibody, e.g., an antiserum or immunoglobulins purified there from (e.g., affinity -purified).

[0147] In other embodiments, the antibody may be a monoclonal antibody or a mixture of monoclonal antibodies. Monoclonal antibodies can target a particular antigen or a particular epitope within an antigen with greater selectivity and reproducibility.

[0148] As used herein, the term “monoclonal antibody” refers to an antibody that is derived from a single copy or clone including, for example, any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Monoclonal antibodies preferably exist in a homogeneous or substantially homogeneous population. Monoclonal antibodies and antigen -binding fragments thereof of the present invention can be produced, for example, by recombinant technologies, phage display technologies, synthetic technologies, e.g., CDR-grafting, or combinations of such technologies, or other technologies known in the art.

[0149] By means of example and not limitation, monoclonal antibodies may be made by the hybridoma method first described by Kohler et al. 1975 (Nature 256: 495), or may be made by recombinant DNA methods (e.g., as in US 4,816,567). Monoclonal antibodies may also be made using phage antibody libraries using techniques as described by Clackson et al. 1991 (Nature 352: 624-628) and Marks et al. 1991 (J Mol Biol 222: 581-597). These later techniques are based on the phage display technology as described inter alia in US5837500, US5571698, US5223409, US7118879, US7208293 and US7413537. Briefly, therapeutic candidate molecules, e.g., human antibody fragments (e.g., Fabs), peptides, and small proteins, are displayed on the surface of a small bacterial virus called a bacteriophage (or phage). A collection of displayed molecules is known as a library. Phage display enables to search through these libraries to identify molecules that bind, preferably with high specificity and / or affinity, to targets of interest, e.g. therapeutic targets. Non-limiting examples of phage antibody libraries include HuCAL® (Human Combinatorial Antibody Library, Morphosys), Ylanthia® (Morphosys), the human Fab fragment libraries described in W0200070023, and the macaque antibody library as described in WO 1996040878. The Human Combinatorial Antibody Library (Morphosys) has been prepared as described in WO 199708320 using synthetic consensus sequences which cover the structural repertoire of antibodies encoded in the human genome.

[0150] The term “antibody fragment” or “antigen -binding moiety” comprises a portion or region of a full length antibody, generally the antigen binding or variable domain thereof. Examples of antibody fragments include Fab, Fab', F(ab)2, Fv , scFv fragments, single domain (sd)Fv, such as VH domains , VL domains and VHH domains, diabodies, linear antibodies, single-chain antibody molecules, in particular heavy-chain antibodies; and multivalent and / or multispecific antibodies formed from antibody fragment(s), e.g., dibodies, tribodies, and multibodies. The above designations Fab, Fab', F(ab')2, Fv, scFv etc. are intended to have their art -established meaning.

[0151] The term “antigen-binding portion” or “antigen-binding region” refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. These may also be bispecific, dual specific, or multi -specific formats; specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term “antigenbinding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature, 341 : 544-546 (1989); PCT publication WO 90 / 05144), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv) (Bird et al., Science, 242: 423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci., 85: 5879-5883 (1988)). Such single chain antibodies are also intended to be encompassed within the term “antigen -binding portion” of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed.

[0152] Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (Holliger, et al., Proc. Natl. Acad. Sci., 90: 6444-6448 (1993); Poljak, et al., Structure 2: 1121-1123 (1994)). Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer- Verlag. New York. 790 pp. (ISBN 3-540-41354-5).

[0153] Still further, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S.M., et al., Human Antibodies and Hybridomas, 6: 93-101 (1995)) and use of a cysteine residue, a marker peptide and a C -terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, et al., Mol. Immunol., 31 : 1047-1058 (1994)). Antibody portions, such as Fab and F(ab')2 fragments, may be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules may be obtained using standard recombinant DNA techniques.

[0154] In certain embodiments, the antibody fragment may be a Nanobody®. The terms “Nanobody®” and “Nanobodies®” are trademarks of Ablynx NV (Belgium). The term “Nanobody” is well- known in the art and as used herein in its broadest sense encompasses an immunological binding agent obtained (1) by isolating the VHH domain of a naturally occurring heavy -chain antibody, preferably a heavy-chain antibody derived from camelids; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by "humanization" of a naturally occurring VHH domain or by expression of a nucleic acid encoding a such humanized VHH domain; (4) by "camelization" of a naturally occurring VH domain from any animal species, and in particular from a mammalian species, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (5) by "camelisation" of a "domain antibody" or "dAb" as described in the art, or by expression of a nucleic acid encoding such a camelized dAb; (6) by using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences known per se (7) by preparing a nucleic acid encoding a Nanobody using techniques for nucleic acid synthesis known per se, followed by expression of the nucleic acid thus obtained; and / or (8) by any combination of one or more of the foregoing. “Camelids" as used herein comprise old world camelids (Camelus bactrianus and Camelus dromaderius) and new world camelids (for example Lama paccos, Lama glama and Lama vicugna).

[0155] The amino acid sequence and structure of a Nanobody can be considered - without however being limited thereto - to be comprised of four framework regions or "FR's", which are referred to in the art and herein as "Framework region 1" or "FR1"; as "Framework region 2" or "FR2"; as "Framework region 3" or "FR3"; and as "Framework region 4" or "FR4", respectively; which framework regions are interrupted by three complementary determining regions or "CDR's", which are referred to in the art as "Complementarity Determining Region l"or "CDR1"; as "Complementarity Determining Region 2" or "CDR2"; and as "Complementarity Determining Region 3" or "CDR3", respectively. The total number of amino acid residues in a Nanobody can be in the region of 110-120, and preferably 112-115. It should however be noted that parts, fragments, analogs or derivatives of a Nanobody are not particularly limited as to their length and / or size, as long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are preferably suitable for the purposes described herein.

[0156] For a general description of heavy chain antibodies and the variable domains thereof, reference is inter alia made to the following references, which are mentioned as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103 of the Vrije Universiteit Brussel; WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231 and WO 02 / 48193 of Unilever; WO 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527 of the Vlaams Instituut voor Biotechnologie (VIB); WO 03 / 050531 of Algonomics N.V. and applicant; WO 01 / 90190 by the National Research Council of Canada; WO 03 / 025020 (= EP 1 433 793) by the Institute of Antibodies; as well as WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551 by applicant and the further published patent applications by applicant; Hamers -Casterman et al., Nature 1993 June 3; 363 (6428): 446-8; Davies and Riechmann, FEBS Lett. 1994 Feb 21; 339(3): 285-90; Muyldermans et al., Protein Eng. 1994 Sep; 7(9): 1129-3; Davies and Riechmann, Biotechnology (NY) 1995 May; 13(5): 475-9; Gharoudi et al., 9th Forum of Applied Biotechnology, Med. Fac. Landbouw Univ. Gent. 1995; 60 / 4a part I: 2097-2100; Davies and Riechmann, Protein Eng. 1996 Jun; 9(6): 531-7; Desmyter et al., Nat Struct Biol. 1996 Sep; 3(9): 803-11; Sheriff et al., Nat Struct Biol. 1996 Sep; 3(9): 733-6; Spinelli et al., Nat Struct Biol. 1996 Sep; 3(9): 752-7; Arbabi Ghahroudi et al., FEBS Lett. 1997 Sep 15; 414(3): 521-6; Vu et al., Mol Immunol. 1997 Nov-Dec; 34(16-17): 1121-31; Atarhouch et al., Journal of Camel Practice and Research 1997; 4: 177-182; Nguyen et al., J. Mol. Biol. 1998 Jan 23; 275(3): 413-8; Lauwereys et al., EMBO J. 1998 Jul 1; 17(13): 3512-20; Frenken et al., Res Immunol. 1998 Jul-Aug; 149(6): 589-99; Transue et al., Proteins 1998 Sep 1; 32(4): 515-22; Muyldermans and Lauwereys, J. Mol. Recognit. 1999 Mar- Apr; 12 (2): 131-40; van der Linden et al., Biochim. Biophys. Acta 1999 Apr 12; 1431(1): 37-46.; Decanniere et al., Structure Fold. Des. 1999 Apr 15; 7(4): 361-70; Ngyuen et al., Mol. Immunol. 1999 Jun; 36(8): 515-24; Woolven et al., Immunogenetics 1999 Oct; 50 (1-2): 98- 101; Riechmann and Muyldermans, J. Immunol. Methods 1999 Dec 10; 231 (1-2): 25-38; Spinelli et al., Biochemistry 2000 Feb 15; 39(6): 1217-22; Frenken et al., J. Biotechnol. 2000 Feb 28; 78(1): 11-21; Nguyen et al., EMBO J. 2000 Mar 1; 19(5): 921- 30; van der Linden et al., J. Immunol. Methods 2000 Jun 23; 240 (1-2): 185-95; Decanniere et al., J. Mol. Biol. 2000 Jun 30; 300 (1): 83-91; van der Linden et al., J. Biotechnol. 2000 Jul 14; 80(3): 261-70; Harmsen et al., Mol. Immunol. 2000 Aug; 37(10): 579-90; Perez et al., Biochemistry 2001 Jan 9; 40(1): 74-83; Conrath et al., J. Biol. Chem. 2001 Mar 9; 276 (10): 7346-50; Muyldermans et al., Trends Biochem Sci. 2001 Apr;26(4):230-5; Muyldermans S., J. Biotechnol. 2001 Jun; 74 (4): 277-302; Desmyter et al., J. Biol. Chem. 2001 Jul 13 ;276 (28): 26285-90; Spinelli et al., J. Mol. Biol. 2001 Aug 3; 311 (1): 123-9; Conrath et al., Antimicrob Agents Chemother. 2001 Oct; 45 (10): 2807-12; Decanniere et al., J. Mol. Biol. 2001 Oct 26; 313(3): 473-8; Nguyen et al., Adv Immunol. 2001; 79: 261-96; Muruganandam et al., FASEB J. 2002 Feb; 16 (2): 240-2; Ewert et al., Biochemistry 2002 Mar 19; 41 (11): 3628-36; Dumoulin et al., Protein Sci. 2002 Mar; 11 (3): 500- 15; Cortez-Retamozo et al., Int. J. Cancer. 2002 Mar 20; 98 (3): 456-62; Su et al., Mol. Biol. Evol.

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[0159] In accordance with the terminology used in the above references, the variable domains present in naturally occurring heavy chain antibodies will also be referred to as “VHH domains”, in order to distinguish them from the heavy chain variable domains that are present in conventional 4 -chain antibodies (which will be referred to herein as “VH domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which will be referred to herein as “VL domains”). As mentioned in the prior art referred to above, VHH domains have a number of unique structural characteristics and functional properties which make isolated VHH domains (as well as Nanobodies based thereon, which share these structural characteristics and functional properties with the naturally occurring VHH domains) and proteins containing the same highly advantageous for use as functional antigen-binding domains or proteins. In particular, and without being limited thereto, VHH domains (which have been “designed” by nature to functionally bind to an antigen without the presence of, and without any interaction with, a light chain variable domain) and Nanobodies can function as a single, relatively small, functional antigen -binding structural unit, domain or protein. This distinguishes the VHH domains from the VH and VL domains of conventional 4-chain antibodies, which by themselves are generally not suited for practical application as single antigen-binding proteins or domains, but need to be combined in some form or another to provide a functional antigen-binding unit (as in for example conventional antibody fragments such as Fab fragments; in ScFv's fragments, which consist of a VH domain covalently linked to a VL domain).

[0160] In certain embodiments, the antibody fragment may be a domain antibody (dAb). For the term "dAb", reference is for example made to Ward et al. (Nature 1989 Oct. 12; 341 (6242): 544-6), to Holt et al., Trends Biotechnol., 2003, 21(l l):484-490; as well as to for example WO 06 / 030220, WO 06 / 003388 and other published patent applications of Domantis Ltd. Single domain antibodies or single variable domains can be derived from certain species of shark (for example, the so-called "IgNAR domains", see for example WO 05 / 18629). In further embodiments, the antibody or antibody fragment may be multispecific (such as a bispecific, trispecific, etc. antibody) comprising at least two (such as two, three, etc.) binding sites, each directed against a different antigen or antigenic determinant.

[0161] In some embodiments, the therapeutic agent may be a dual variable domain immunoglobulin (DVD-Ig™).

[0162] The term antibody includes antibodies originating from or comprising one or more portions derived from any animal species, preferably vertebrate species, including, e.g., birds and mammals. Without limitation, the antibodies may be chicken, turkey, goose, duck, guinea fowl, quail or pheasant. Also without limitation, the antibodies may be human, murine (e.g., mouse, rat, etc.), porcine, donkey, rabbit, goat, sheep, guinea pig, monkey (e.g., cynomolus monkeys), camel (e.g., Camelus bactrianus and Camelus dromaderius) also including camel heavy -chain antibodies, llama (e.g., Lama paccos, Lama glama or Lama vicugna) also including llama heavy -chain antibodies, or horse.

[0163] The term antibody as used herein also encompasses “chimeric antibodies” which originate from at least two animal species. More specifically, the term “chimeric antibody” or “chimeric antibodies” refers to antibodies which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as for example antibodies having murine heavy and light chain variable regions linked to human, non-human primate, canine, equine, or feline constant regions. Chimeric antibodies comprise a portion of the heavy and / or light chain that is identical to or homologous with corresponding sequences from antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous with corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, exhibiting the desired biological activity (See e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1984)). Chimeric antibodies are made through merging DNA encoding a portion, such as the Fv region, of a monoclonal antibody from one species, e.g. mouse or monkey, with the antibody-producing DNA from another species, e.g. human.

[0164] In certain embodiments, the therapeutic agent may be a “fully human antibody”. As used herein, the term “fully human antibody” refers to an antibody of which the encoding genetic information is of human origin. Accordingly, the term “fully human antibody” refers to antibodies having variable and constant regions derived only from human germline immunoglobulin sequences. The term “fully human antibody” is thus not to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as a mouse, have been grafted onto human framework sequences. Fully human antibodies may be derived from phage human antibody libraries as described above, or they may be obtained through immunization of transgenic mice which have been engineered to replace the murine immunoglobulin encoding region as described in Lonberg and Husznar 1995 (Int. Rev. Immunol. 13 (1): 65-93). Fully human antibodies that are made using phage display are preferably produced by recombinant expression in a human cell line resulting in antibodies with a human glycosylation pattern. Non-limiting examples of fully human antibodies are HuCAL® antibodies (Morphosys). The genetic information for constructing a HuCAL® antibody is extracted from the HuCAL® antibody library (Morphosys) and introduced into human PER.C6® cells in the form of a vector (i.e., transfection). The transfected cells translate the genetic information into protein. The protein is further modified by glycosylation and the resulting antibody molecule is finally secreted by the cells into the culture medium.

[0165] The term antibody as used herein also encompasses “humanized antibodies”, which are antibodies derived from non-human species whose protein sequence have been modified so as to increase their similarity to antibodies produced naturally in humans. More particularly, the term “humanized antibody” refers to antibodies which comprise heavy and light chain variable region sequences from a non -human species (e.g., a mouse) but in which at least a portion of the VH and / or VL sequence has been altered to be more “human-like”, i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which non-human CDR sequences are introduced into human VH and VL sequences to replace the corresponding human CDR sequences.

[0166] The humanized antibody is an antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody. A humanized antibody comprises substantially all, or at least one, and typically two, variable domains (Fab, Fab', F(ab') 2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. A humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. A humanized antibody may contain both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CHI, hinge, CH2, CH3, and CH4 regions of the heavy chain. Alternatively, a humanized antibody may only contain a humanized light chain, or a humanized heavy chain. An exemplary humanized antibody contains a humanized variable domain of a light chain and a humanized variable domain of a heavy chain. Also, for example, humanized antibodies may be derived from conventional antibodies (i.e. an immunoglobulin molecule comprising 2 heavy (H) chains and 2 light (L) chains interconnected by disulfide bonds) from the family Camelidae, in particular from the llama (e.g., Lama paccos, Lama glama or Lama vicugna), whose variable domains exhibit a high degree of amino acid sequence identity with the variable domains of human antibodies. A suitable platform for the production of such humanized antibodies is the SIMPLE Antibody™ platform (ArGEN-X) as described in WO 2011080350.

[0167] A skilled person will understand that an antibody can include one or more amino acid deletions, additions and / or substitutions (e.g., conservative substitutions), insofar such alterations preserve its binding of the respective antigen. For example, mutations may be introduced into the antibody, in particular in the Fc region, to extend in vivo half-life without compromising immunogenicity as described in US patent 8,323,962.

[0168] An antibody may also include one or more native or artificial modifications of its constituent amino acid residues (e.g., glycosylation, etc.).

[0169] Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art, as are methods to produce recombinant antibodies or fragments thereof (see for example, Harlow and Lane, “Antibodies: A Laboratory Manual”, Cold Spring Harbour Laboratory, New York, 1988; Harlow and Lane, “Using Antibodies: A Laboratory Manual”, Cold Spring Harbour Laboratory, New York, 1999, ISBN 0879695447; “Monoclonal Antibodies: A Manual of Techniques”, by Zola, ed., CRC Press 1987, ISBN 0849364760; “Monoclonal Antibodies: A Practical Approach”, by Dean & Shepherd, eds., Oxford University Press 2000, ISBN 0199637229; Methods in Molecular Biology, vol. 248: “Antibody Engineering: Methods and Protocols”, Lo, ed., Humana Press 2004, ISBN 1588290921).

[0170] Methods for immunising animals, e.g., non-human animals such as laboratory or farm animals, using immunising antigens optionally fused to or covalently or non -covalently linked, bound or adsorbed to a presenting carrier, and preparation of antibody or cell reagents from immune sera is well-known per se and described in documents referred to elsewhere in this specification. The animals to be immunised may include any animal species, preferably warm-blooded species, more preferably vertebrate species, including, e.g. , birds and mammals. Without limitation, the antibodies may be chicken, turkey, goose, duck, guinea fowl, quail or pheasant. Also without limitation, the antibodies may be human, murine (e.g., mouse, rat, etc.), porcine, donkey, rabbit, goat, sheep, guinea pig, camel, llama or horse. The term “presenting carrier” or “carrier” generally denotes an immunogenic molecule which, when bound to a second molecule, augments immune responses to the latter, usually through the provision of additional T cell epitopes. The presenting carrier may be a (poly)peptidic structure or a non-peptidic structure, such as inter alia glycans, polyethylene glycols, peptide mimetics, synthetic polymers, etc. Exemplary non-limiting carriers include human Hepatitis B virus core protein, multiple C3d domains, tetanus toxin fragment C or yeast Ty particles. Following immunization, the antibody -producing cells from the animals may be isolated and used to generate monoclonal antibody-producing hybridoma cells using techniques well-known in the art.

[0171] Methods for producing recombinant antibodies or fragments thereof need a host organism or cell. The terms “host cell” and “host organism” may suitably refer to cells or organisms encompassing both prokaryotes, such as bacteria, and eukaryotes, such as yeast, fungi, protozoan, plants and animals. Contemplated as host organisms or cells for the production of antibodies include inter alia unicellular organisms, such as bacteria (e.g. , E. coll), and (cultured) animal cells (e.g., mammalian cells or human cells). The advantages of producing antibodies in bacteria are amongst other the relatively safe and straightforward handling of bacterial cells and the rapid replication cycles of microorganisms. Bacteria are particularly suitable for the production of antibody fragments with a simple structure. For the production of full-length immunoglobulins or more complex antibody fragments in prokaryotic cells, the bacterial cells may be transformed with at least two nucleic acids each encoding a different portion of the antibody fragment or the immunoglobulin, e.g., the heavy chain or the light chain, as described in WO 2009021548 for full-length immunoglobulins. In the bacterial cell, the genetic information encoding the antibody is read and translated into a protein. The resulting antibodies accumulate in the periplasmic space and can be harvested upon lysis of the bacterial cells. A further separation step may be performed to purify the antibodies. WO 2009021548 describes an E. Coli-\>asQ secretion system wherein the bacteria release the antibodies in the surrounding culture medium due to the introduction of a signal sequence into the antibody encoding construct. This enables the easy and convenient purification of the antibodies from the cell culture medium. An exemplary mammalian cell line that can be used for the production of antibodies is the Chinese hamster ovary (CHO) cell line. An exemplary human cell line suitable for the production of antibodies includes the PER.C6® cell line as deposited under ECAC no. 96022940 and described in WO 2000063403 or a derivative thereof. Human cell lines are particularly suitable for the production of fully human antibodies because they produce antibodies with a human glycosylation pattern.

[0172] Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to standard handbooks as well as to the general background art referred to herein and to the further references cited therein. The capability of activating Wnt signaling refers to the ability of the agent as disclosed herein to mimic, reproduce or approximate the signal transduction effect and / or activity of a natural Wnt ligand binding to FZD and LRP, such as to a FZD / LRP complex.

[0173] Activation of Wnt signaling may be suitably determined and / or quantitated by measuring the expression of one or more Wnt target genes, TCF reporter gene expression, beta-catenin stabilization, LRP phosphorylation, and / or translocation of Axin from cytoplasm to cell membrane as known in the art. For instance, activation of Wnt signaling may be suitably determined and / or quantitated by measuring the expression of TCF gene (e.g., by RT-PCR or any other transcript detection method), a primary output of Wnt signaling (Nature, 1997, vol. 385(6619), 829-33). For example, a TCF reporter assay (also known as TOP / FOP or TOPflash) may be used to assess changes in the transcription of TCF / LEF controlled genes. The TCF reporter assay may be a luciferase reporter assay. Further for example, activation of Wnt signaling may be suitably determined and / or quantitated by measuring the expression of c-myc (He et al., 1998, Science, 281(5382), 1509-12), n-myc (Ten Berge et al., 2008, Development, 135(19), 3247-57), LEF1 (Hovanes et al., 2001, Nat Genet, 28(1), 53-7; Filali et al., 2002, J Biol Chem, 277(36), 33398- 410), or c-jun (Mann et al., 1999, Proc Natl Acad Sci U S A, 96(4), 1603-8).

[0174] Alternatively, activation of Wnt signaling may be determined by measuring the location, level and / or phosphorylation status of P-catenin. A non-limiting example of such an assay is the “P- Catenin Redistribution Assay" (Thermo Scientific) which provides recombinant U20S cells stably expressing human P-catenin fused to the C-terminus of enhanced green fluorescent protein (EGFP). The assay allows visualization and monitoring of the translocation of a GFP- P-catenin fusion protein from the membrane to the nucleus. Another way of determining activation of Wnt signaling is the visualization of Axin translocation, for example with a GFP -Axin fusion protein.

[0175] In particular embodiments, the agent as disclosed herein may be considered capable of activating (canonical) Wnt signaling if the agent enhances Wnt / p-catenin signaling at least 10-fold more, at least 20-fold more, at least 30-fold more, at least 40-fold more, at least 50-fold more, at least 100- fold more, at least 250-fold more, at least 500-fold more, at least 750-fold more, at least 1000-fold more, at least lxl04-fold more, or at least lxl05-fold more compared to Wnt / P-catenin signaling baseline or background induced by a neutral substance or negative control, for example as measured in an assay as described elsewhere herein.

[0176] In particular embodiments, the agent as disclosed herein may be considered to not activate (canonical) Wnt signaling if the agent enhances Wnt / p-catenin signaling less than 10-fold more, such as particularly at most 5-fold more or at most 2.5-fold more, or if the agent does not enhance or even reduces (e.g., 2-fold less or 5-fold less or 10-fold less) Wnt / p-catenin signaling compared to Wnt / P-catenin signaling baseline or background induced by a neutral substance or negative control, for example as measured in an assay as described elsewhere herein.

[0177] In particular embodiments, the agent as disclosed herein may be considered to activate the GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling, but not activate Frizzled / LRP-mediated Wnt in the absence of RECK and / or GPR124, if the GPR124 / RECK / Frizzled / LRP -mediated Wnt signaling activity induced by said agent is at least 3.5-fold more, at least 5-fold more, at least 10- fold more, at least 15-fold more, at least 20-fold more, at least 25-fold more, at least 50-fold more, at least 100-fold more, at least 500-fold more, at least 1000-fold more, at least lxl04-fold more, or at least lxl05-fold more, preferably at least 50-fold more, than the Frizzled / LRP-mediated Wnt signaling activity induced by said agent, in absence of RECK and / or GPR124. Before comparing the GPR124 / RECK / Frizzled / LRP -mediated Wnt signaling activity (denoted “activity 1” in this paragraph) and the Frizzled / LRP-mediated Wnt signaling activity in the absence of RECK and / or GPR124 (denoted “activity 2” in this paragraph), activity 1 and activity 2 induced by the agent may be normalized to activity 1 and activity 2 induced by wild-type Wnt7a, respectively, the latter for example set to represent 100% activity.

[0178] By means of an example, in an in vitro or in vivo cell assay system comprising 1) cells expressing GPR124, RECK, FZD and LRP and separately 2) cells expressing FZD and LRP but not expressing GPR124 and / or RECK, wherein the cells under 1) and 2) are otherwise substantially identical, the agent may be considered to activate GPR124 / RECK / Frizzled / LRP -mediated Wnt signaling, but not activate FZD / LRP -mediated Wnt signaling in the absence of RECK and / or GPR124, when the Wnt signaling activity induced by the same quantity of said agent under substantially identical conditions is at least 3.5-fold more, 5-fold more, at least 10-fold more, at least 15-fold more, at least 20-fold more, at least 25-fold more, at least 50-fold more, at least 100-fold more, at least 500- fold more, at least 1000-fold more, at least lxl04-fold more, or at least lxl05-fold more in cells under 1) than in cells under 2). For example, the cells under 1) and 2) may be from the same primary cell source, or may be of the same cell line, and may be genetically engineered to differ in expression of GPR124 and / or RECK.

[0179] Activation of the Wnt signaling pathway may occur by promoting the close association or mutual proximity of the Frizzled and LRP polypeptides at the cell membrane, thereby forming membrane - associated hetero-oligomers comprising the Frizzled and LRP polypeptides. Upon ligand-driven formation of the Frizzled-LRP hetero-oligomer, the intracellular portion of the LRP polypeptide becomes accessible for phosphorylation, for example by CK1 and GSK-3, which greatly increases its affinity for Axin. Second, when present in a hetero-oligomer with the LRP polypeptide, the intracellular portion of the Frizzled polypeptide is able to induce the phosphorylation and recruitment of DVL. The resulting assembly of an activated LRP-FZD-DVL-Axin complex leads indirectly to the dissociation of the destruction complex of beta-catenin, thereby allowing beta- catenin to accumulate in the cytoplasm and translocate to the cell nucleus where beta-catenin may induce gene transcription.

[0180] Accordingly, in particular embodiments, the agent as disclosed herein is capable of inducing heteromerization (such as, preferably, heterodimerization) of Frizzled and LRP polypeptides at a cell membrane in the presence of RECK and GPR124, but not in the absence of RECK and / or GPR124.

[0181] In order to achieve heteromerization or close association of the Frizzled and LRP polypeptides at the cell membrane, an agent which is capable of binding concurrently both the Frizzled and the LRP polypeptide, for example by binding the extracellular portions of both the Frizzled and the LRP polypeptide, may be used.

[0182] Heteromerization of the Frizzled and LRP polypeptides at the cell membrane may be determined by any method known in the art to determine heteromerization of membrane proteins, such as visualizing the heteromerization of fluorescently labelled membrane proteins by immunofluorescence staining, fluorescence resonance energy transfer (FRET) or determining the occurrence of downstream events of heteromerization of Frizzled and LRP polypeptides, such as detecting the presence and phosphorylation of Dvl.

[0183] Accordingly, in particular embodiments, the agent as disclosed herein is capable of concurrently binding to Frizzled and LRP polypeptides at a cell membrane, in the presence of RECK and GPR124, but not in the absence of RECK and / or GPR124.

[0184] The terms “bind”, “interact”, “specifically bind” or “specifically interact” as used throughout this specification mean that an agent binds to or influences one or more desired molecules or analytes substantially to the exclusion of other molecules which are random or unrelated, and optionally substantially to the exclusion of other molecules that are structurally related. The terms do not necessarily require that an agent binds exclusively to its intended target(s). For example, an agent may be said to specifically bind to target(s) of interest if its affinity for such intended target(s) under the conditions of binding is at least about 2 -fold greater, preferably at least about 5 -fold greater, more preferably at least about 10-fold greater, yet more preferably at least about 25 -fold greater, still more preferably at least about 50 -fold greater, and even more preferably at least about 100-fold or more greater, such as, e.g., at least about 1000-fold or more greater, at least about lxl04-fold or more greater, or at least about lxl05-fold or more greater, than its affinity for a nontarget molecule.

[0185] The binding or interaction between the agent and its intended target(s) may be covalent (i.e., mediated by one or more chemical bonds that involve the sharing of electron pairs between atoms) or, more typically, non-covalent (i.e., mediated by non-covalent forces, such as for example, hydrogen bridges, dipolar interactions, van der Waals interactions, and the like). Preferably, the agent may bind to or interact with its intended target(s) with affinity constant (KA) of such binding KA > IxlO6M1, more preferably KA > I xlO7M1, yet more preferably KA > IxlO8M1, even more preferably KA > IxlO9M1, and still more preferably KA > IxlO10M1or KA > IxlO11M1, wherein KA = [A_T] / [A][T], A denotes the agent, T denotes the intended target. Determination of KA can be carried out by methods known in the art, such as for example, using equilibrium dialysis and Scatchard plot analysis.

[0186] The binding of an agent as described herein to a target and the affinity and specificity of said binding may be determined by any methods known in the art. Non-limiting examples thereof include co-immunoprecipitation, bimolecular fluorescence complementation, affinity electrophoresis, label transfer, phage display, proximity ligation assay (PLA), Tandem affinity purification (TAP), in-silico docking and calculation of the predicted Gibbs binding energy and competition binding assays.

[0187] In particular embodiments, the agent as disclosed herein is capable of binding to one or more, such as two or more, of the GPR124 polypeptide, the RECK polypeptide, the Frizzled polypeptide and the LRP polypeptide.

[0188] In particular embodiments, the agent as disclosed herein is capable of binding to one or more different Frizzled polypeptides, such as one or more Frizzled polypeptides selected from the group consisting of Fzd 1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, and FzdlO. Preferably, the agent as disclosed herein may be capable of specifically binding to one or more Fzd that are believed to be dominant in bone cells and / or endothelial cells. In particular embodiments, the agent as disclosed herein may be capable of specifically binding to at least Fzd4 and / or Fzd9, and optionally to one or more other Fzd; or may be capable of specifically binding to Fzd4 substantially to the exclusion of other Fzd. More preferably, the agent as disclosed herein is capable of specifically binding to human Fzd4 and / or Fzd9. The agent as disclosed herein may be selective for the one or more preferred Frizzled polypeptides, for example having a specificity for the one or more preferred Frizzled polypeptides of at least 5-fold, at least 10-fold, at least 25-fold, at least 50- fold, at least 100-fold, at least 1000-fold, at least lxl04-fold, or at least lxl05-fold, compared to other non-preferred Frizzled polypeptides.

[0189] In particular embodiments, the agent as disclosed herein is capable of binding to one or more different LRP polypeptides involved in Wnt signaling. Preferably, the agent as disclosed herein is capable of binding to LRP5 and / or LRP6, e.g., any one or each of LRP5 and LRP6. More preferably, the agent as disclosed herein is capable of binding to human LRP5 and / or LRP6, e.g., any one or each of human LRP5 and human LRP6. The agent as disclosed herein may be selective for the one or more preferred LRP polypeptides, for example having a specificity for the one or more preferred LRP polypeptides of at least 5-fold, at least 10-fold, at least 25-fold, at least 50- fold, at least 100-fold, at least 1000-fold, at least lxl04-fold, or at least lxl05-fold, compared to other non-preferred LRP polypeptides.

[0190] In particular embodiments, the agent as disclosed herein is capable of binding to the GPR124 and / or the RECK polypeptide. In other words, in particular embodiments, the agent as disclosed herein is capable of binding to the GPR124 polypeptide, the RECK polypeptide, or to both the GPR124 polypeptide and the RECK polypeptide.

[0191] GPR124 is composed of an intracellular domain (ICD) comprising a Dvl binding motif and a C- terminal PDZ -binding motif or ETTV motif, a 7-transmembrane domain (7TM) and an extracellular domain composed of a leucine -rich repeat (LRR), an immunogloblulin (Ig) domain, a hormone receptor (HormR) domain and a putative GPCR autoproteolysis -inducing (GAIN) domain with GPCR proteolysis site (GPS).

[0192] RECK is composed of five N-terminal cysteine-knot (CK) motifs or regions (i.e. CK1, CK2, CK3, CK4 and CK5), a cysteine-rich domain (CRD) and three Kazal motifs preceding a Glycosylphosphatidylinositol (GPI)-anchor site. The CK motifs, the CRD and the Kazal motifs are located extracellularly. Accordingly, the agent capable of binding to the RECK polypeptide as disclosed herein may bind the CK1 motif, CK2 motif, CK3 motif, CK4 motif, CK5 motif, CRD, and / or one or more of the Kazal motifs of the RECK polypeptide.

[0193] Accordingly, in particular embodiments, the agent as disclosed herein is capable of binding to the CK4 and / or CK5 regions of RECK polypeptide.

[0194] The CK4 motif spans from amino acid C216 to C263, and the CK5 motif spans from amino acid C292 to C338 of the amino acid sequence of the human RECK protein annotated under NCBI Genbank accession number NP_066934.1 as disclosed elsewhere herein. Accordingly, the CK4 motif of human RECK comprises, consists essentially of or consists of the amino acid sequence CCDRAEDHACQNACKRILMSKKTEMEIVDGLIEGCKTQPLPQDPLWQC (SEQ ID NO: 5) and the CK5 motif of human RECK comprises, consists essentially of or consists of the amino acid sequence CCSKANTSTCRELCTKLYSMSWGNTQSWQEFDRFCEYNPVEVSMLTC (SEQ ID NO: 6).

[0195] In particular embodiments, the agent as disclosed herein is capable of binding to the amino acid sequence of SEQ ID NO: 5 and / or the amino acid sequence of SEQ ID NO: 6.

[0196] The Frizzled receptor is a G protein-coupled receptor protein and ranges in length from about 500 to about 700 amino acids. The N-terminus is predicted to be extracellular and comprises a cysteine rich domain (CRD) of approximately 120 amino acids followed by a hydrophilic linker region of approximately 40-100 amino acids. The Frizzled receptor also comprises seven hydrophobic domains that are predicted to form transmembrane alpha-helices. The intracellular C-terminal domain has a variable length and, though the intracellular domain is overall not well conserved among different family members, it comprises a proximal KTXXXW amino acid motif, wherein X can be any amino acid, which is highly conserved in Frizzled polypeptides and which is required for canonical Wnt signaling.

[0197] The Frizzled CRD domain comprises a motif of 10 invariantly spaced cysteines and is largely conserved between the known Frizzled family members, but also in several other proteins, such as RECK, secreted frizzled related proteins (SFRPs), receptor tyrosine kinases (RTKs), and collagen al XVIII. The CRD domain is important for ligand (e.g. Wnt) binding to the Frizzled polypeptide, for example by recognition and / or binding of cis -unsaturated fatty acyl groups present in the ligand contact site 1 or the residues located ate the Wnt ligand “index” contact site 2.

[0198] In particular embodiments, the agent as disclosed herein is capable of binding to the cysteine -rich domain (CRD) of the Frizzled polypeptide. In certain embodiments, the agent as disclosed herein is capable of binding to the cis- un saturated fatty acyl group-binding domain located within the CRD domain of the Frizzled polypeptide or the “index” contact site within the CRD.

[0199] The N-terminal extracellular domain of LRP is composed of four YWTD (SEQ ID NO: 8) repeat domains (or beta-propeller domains), four epidermal growth factor (EGF)-like domains and an LDLR-like domain (LDLRLD). A single YWTD repeat domain comprises six tandem YWTD sequences. The first two, most N-terminal YWPD repeat domains are predicted to bind to Wnt ligands, and the second two sets of YWPD repeat domains are predicted to bind to Dickkopf (DKK). The N-terminal extracellular domain is followed by a single membrane -spanning segment and a cytoplasmic tail harboring between one and three NPXY motifs (, wherein X can be any amino acid (e g. in LRP1, LRP2, LRP4, APOER2, LDLR, LRP9), or between one and five PPPSP motifs (SEQ ID NO: 10) (e.g. in LRP5 and LRP6).

[0200] The term “Dickkopf’ or “DKK” encompasses any and all members of the DKK family, such as without limitation the known human DKK proteins including DKK1 (RefSeq Protein: NP 036374.1; GenelD: 22943), DKK2 (RefSeq Protein: NP 055236.1; GenelD: 27123), DKK3 (RefSeq Protein: NP 001317149.1 ;GeneID: 27122), and DKK4 (RefSeq Protein: NP 055235.1; GenelD: 27121). In certain embodiments, the terms may particularly denote DKK1.

[0201] In particular embodiments, the agent as disclosed herein is capable of binding to the extracellular domain of the LRP polypeptide.

[0202] In particular embodiments, the agent as disclosed herein is capable of binding to the DKK -binding site of the LRP polypeptide. In more particular embodiments, the agent as disclosed herein is capable of binding to the DKK-binding site, preferably the DKKl-binding site, of the LRP5 and / or LRP6 polypeptide.

[0203] In particular embodiments, the agent as disclosed herein is capable of binding to the Wnt -binding site of the LRP polypeptide. In more particular embodiments, the agent as disclosed herein is capable of binding to the Wnt-binding site of the LRP5 and / or LRP6 polypeptide.

[0204] In further particular embodiments, the agent as disclosed herein is capable of binding to the DKK- binding site and the Wnt-binding site of the LRP polypeptide.

[0205] In particular embodiments, the agent as disclosed herein is capable of binding to P-propeller-EGF- like domains 1 and 2 (P1E1P2E2) and / or P-propeller-EGF-like domains 3 and 4 (P3E3P4E4) of the LRP polypeptide.

[0206] In particular embodiments, the agent as disclosed herein is capable of concurrently binding to the GPR124 and / or the RECK polypeptide, preferably to the cysteine knot 4 (CK4) region, to the CK5 region, or to the CK4 and CK5 regions, of the RECK polypeptide; the cysteine-rich domain (CRD) of the Frizzled polypeptide; and / or the DKK-binding site and / or the Wnt-binding site of the LRP polypeptide.

[0207] In particular embodiments, the agent as disclosed herein is capable of concurrently binding to Frizzled and LRP polypeptides, and in addition to the GPR124 and / or the RECK polypeptide. In particular embodiments, the agent as disclosed herein is capable of concurrently binding to Frizzled, LRP and RECK polypeptides.

[0208] In particular embodiments, the agent as disclosed herein comprises one or more, such as two or more of a GPR124-binding domain, a RECK-binding domain, a Frizzled-binding domain, and / or a LRP -binding domain.

[0209] In particular embodiments, the agent as disclosed herein comprises a RECK-binding domain and a Frizzled-binding domain, and optionally a LRP -binding domain. In more particular embodiments, the agent as disclosed herein comprises a RECK-binding domain, a Frizzled-binding domain, and a LRP -binding domain.

[0210] The GPR124-binding domain of the agent as disclosed herein may be any domain that is capable of binding to a GPR124 polypeptide at high affinity; such as binding to a GPR124 polypeptide with a dissociation constant (KD) of at least about 1 x 10'6M, l x 10'7M, at least 1 x 10'8M, at least 1 x 10'9M, at least 1 x IO-10M, at least 1 x 1011M, or at least 1 x 10'12M. For example, KD of at least about 5 x 10'6M. In particular embodiments, said GPR124-binding domain comprises a GPR124 specific antibody or comprises the GPR124-binding region, preferably the variable region sequence or CDRs, of one or more GPR124 specific antibody or antibodies. Preferably, the GPR124 specific antibody is directed to the extracellular portion of the GPR124 polypeptide, such as directed to the leucine-rich repeat (LRR), immunogloblulin (Ig) domain, hormone receptor (HormR) domain or putative GPCR autoproteolysis-inducing (GAIN) domain with GPCR proteolysis site (GPS) of the GPR124 polypeptide. Non-limiting examples of GPR124 antibodies include antibodies which are known in the art and commercially available, such as from Abeam (e.g. abl98817 directed towards the GPCR), or Sigma-Aldrich (e.g. SAB2900508).

[0211] In particular embodiments, the GPR 124 -binding domain comprises at least one, at least two, at least three, at least four, at least five or at least six CDR(s), each independently having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, preferably 100%, sequence identity with the respective CDR(s) of a GPR124 specific antibody.

[0212] In particular embodiments, the GPR 124 -binding domain is GPR124 specific scFv, such as scFv comprising one or more (preferably all 6) CDR(s) of a GPR124 specific antibody.

[0213] In particular embodiments, the GPR 124 -binding domain is GPR124 specific VHH, such as VHH comprising one or more (preferably all 3) CDR(s) of a GPR124 specific heavy chain antibody.

[0214] In particular embodiments, the GPR 124 -binding domain of the agent as disclosed herein may comprise two or more CDR(s) each independently having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, preferably 100%, sequence identity with the respective CDR(s) of two or more different GPR124 specific antibodies. The RECK-binding domain of the agent as disclosed herein may be any domain that is capable of binding to a RECK polypeptide at high affinity; such as binding to a RECK polypeptide with a dissociation constant (KD) of at least about 1 x 10'6M, l x 10'7M, at least 1 x 10'8M, at least 1 x 10'9M, at least 1 x 10"10M, at least 1 x 10'11M, or at least 1 x 10'12M. For example, KD of at least about 5 x 10'6M.

[0215] In particular embodiments, said RECK-binding domain comprises a RECK specific antibody or comprises the RECK-binding region, preferably the variable region sequence or CDRs, of one or more RECK specific antibody or antibodies. Preferably, the RECK specific antibody is directed to the extracellular portion of the RECK polypeptide, such as directed to the CK4 and / or CK5 region(s) of the RECK polypeptide. Non-limiting examples of RECK antibodies include antibodies which are known in the art and commercially available, such as from Abeam (e.g. ab88249 and ab89915 specific for human full-length RECK), Cell Signaling Technology (e.g. #3433 specific for human, mouse, rat, monkey RECK (D8C7)), Santa Cruz (e.g. sc-373929 specific for C-terminus of human RECK). In particular embodiments, the RECK-binding domain comprises at least one, at least two, at least three, at least four, at least five or at least six CDR(s), each independently having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, preferably 100%, sequence identity with the respective CDR(s) of a RECK specific antibody.

[0216] In particular embodiments, the RECK-binding domain is RECK specific scFv, such as scFv comprising one or more (preferably all 6) CDR(s) of a RECK specific antibody.

[0217] In particular embodiments, the RECK-binding domain is RECK specific VHH, such as VHH comprising one or more (preferably all 3) CDR(s) of a RECK specific heavy chain antibody.

[0218] In particular embodiments, the RECK-binding domain of the agent as disclosed herein may comprise two or more CDR(s) each independently having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, preferably 100%, sequence identity with the respective CDR(s) of two or more different RECK specific antibodies.

[0219] In particular embodiments, said RECK-binding domain of the agent as disclosed herein comprises, consists essentially of, consists of or is derived from (e.g., is a biologically active fragment and / or variant of) a RECK-binding polypeptide, such as a Wnt ligand or Wnt polypeptide. Preferably, the RECK-binding domain is derived from a Wnt7 polypeptide (e.g., Wnt7a or Wnt7b) as described elsewhere herein, more preferably from a human or murine Wnt7 polypeptide, even more preferably from a human Wnt7 polypeptide (e.g., human Wnt7a or human Wnt7b). For example, in certain embodiments, the RECK-binding domain may comprise, consist essentially of or consist of a RECK-binding fragment of Wnt7 (e.g., Wnt7a or Wnt7b), preferably of human or mouse Wnt7, more preferably of human Wnt7 (e.g., human Wnt7a or human Wnt7b) or variant thereof. In a further example, in certain embodiments, the RECK-binding domain may comprise, consist essentially of or consist of the an amino acid sequence having at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, preferably 100%, sequence identity to the amino acid sequence VEPVRASRNKRPTFLKIKKPLSYRKPMDT (SEQ ID NO: 18) or VEVVRASRLRQPTFLRIKQLRSYQKPMET (SEQ ID NO: 19).

[0220] The term “Wnt”, “Wnt ligand” or “Wnt polypeptide” encompasses any and all members of the Wnt family. By means of additional guidance, Table 2 presents 19 known human Wnt family members, as annotated in NCBI Genbank and Uniprot.

[0221] Table 2

[0222] In certain embodiments, the Wnt polypeptide as employed herein is of animal origin, preferably warm-blooded animal origin, more preferably vertebrate origin, yet more preferably mammalian origin, including human origin and non-human mammalian origin, still more preferably human origin.

[0223] The Frizzled-binding domain of the agent as disclosed herein may be any domain that is capable of binding to one or more Frizzled polypeptides . Without being limited to any mechanism or theory, the affinity of the Frizzled-binding domain of the agent may be sufficiently high to activate GPR124 / RECK / Frizzled / LRP -mediated Wnt signaling (e.g., when binding of the agent to RECK, of RECK to GPR124, and of GPR124 to Frizzled via DVL network ensures the proximity or presentation of the agent and more particularly its Frizzled-binding domain to Frizzled), but sufficiently low to avoid activating (for example, lack of activation or at most minimal activation, such as physiologically inconsequential degree or extent of activation) Frizzled / LRP -mediated Wnt signaling in the absence of RECK and / or GPR124. Without limitation, in certain embodiments the Frizzled-binding domain may be capable of binding to one or more Frizzled polypeptides with a KD lower than about 1 x 10'9M, lower than about 1 x IO-8M, or lower than about 1 x 10'7M, or lower than about 1 x 10'6M, such as for example between about 1 x 10'6M and about 1 x 10'7M, or between about 1 x 10'6M and about 5 x 10'6M.

[0224] In particular embodiments, the Frizzled-binding domain comprises a Frizzled specific antibody or comprises the Frizzled-binding region, preferably the variable region sequence or CDR, of one or more Frizzled specific antibody or antibodies. Preferably, the Frizzled specific antibody is directed to the extracellular portion of the Frizzled receptor, such as directed to the CRD of the Frizzled receptor. Non-limiting examples of Frizzled antibodies include antibodies which are known in the art and commercially available, such as from Biolegend (e.g. clone CH3A4a7 specific for human Frizzled 4, clone W3C4E11 specific for human Frizzled 9) and antibodies available from Abeam (e.g. ab64636 specific for Frizzled 7, ab83042 specific for human Frizzled 4, ab77379 specific for human Frizzled 7, ab75235 specific for human Frizzled 8, abl02956 specific for human Frizzled 9). For example, the Frizzled-binding domain may comprise the six CDR regions of the pan specific frizzled antibody OMP-18R5 (vantictumab).

[0225] In particular embodiments, the Frizzled-binding domain comprises at least one, at least two, at least three, at least four, at least five or at least six CDR(s), each independently having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, preferably 100%, sequence identity with the respective CDR(s) of a Frizzled specific antibody.

[0226] In particular embodiments, the Frizzled-binding domain is Frizzled specific scFv, such as scFv comprising one or more (preferably all 6) CDR(s) of a Frizzled specific antibody.

[0227] In particular embodiments, the Frizzled-binding domain is Frizzled specific VHH, such as VHH comprising one or more (preferably all 3) CDR(s) of a Frizzled specific heavy chain antibody.

[0228] In particular embodiments, the Frizzled-binding domain may comprise two or more CDR(s) each independently having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, preferably 100%, sequence identity with the respective CDR(s) of two or more different Frizzled specific antibodies.

[0229] In particular embodiments, the Frizzled-binding domain of the agent as disclosed herein comprises, consists essentially of, consists of or is derived from (e.g., is a biologically active fragment and / or variant of) a Frizzled-binding polypeptide, such as a Wnt ligand or Wnt polypeptide. Preferably, the Frizzled-binding domain is derived from a Wnt7 polypeptide (e.g., Wnt7a or Wnt7b) as described elsewhere herein, more preferably from a human Wnt7 polypeptide (e.g., human Wnt7a or human Wnt7b).

[0230] The Frizzled binding region of a Wnt polypeptide, can be determined by modelling a three- dimensional structure of the Wnt polypeptide based on Xenopus Wnt8a crystallographic analysis (C. Y. Janda, D. Waghray, A. M. Levin, C. Thomas, K. C. Garcia, Science. 337, 59-64 (2012)).

[0231] XWnt8 was shown to bind Frizzled to two discontinued sites. One is called the thumb (site 1) and the other the index (site2). Critical residues of the XWnt8 thumb appear to be K182, 1186, S187, G188 and W196. S187 harbors a palmitoleic acid which is critical for binding at site 1. Site 2 contacts appear to be dominated by F317, W319, C321, T322 and V323. In addition, XWnt8 could form higher-order Wnt / Fz multimers though pseudo site 3 composed of: P34, Y37,L38,S41,A42,A45,V46,T70,L71,A74,F147, G150, L151 and T153. It appears that critical amino acid residues in site 1 (especially KI 82, SI 87, G188 and W196) and site 2 (especially W319, C321 and V323) are highly conserved between the different Wnt polypeptides, while pseudo-site 3 residues are more divergent. In view thereof, the skilled person will understand that the Frizzled binding region of a Wnt polypeptide can be determined using sequence alignment with XWnt8. In particular embodiments, the Frizzled-binding domain of the agent as disclosed herein comprises, consists essentially of or consists of the ‘thumb’ (site 1) of a Wnt ligand, preferably, the Frizzled- binding domain comprises an amino acid sequence XKXXXXSGXXXXXXXW (SEQ ID NO: 11 ), wherein X can be any amino acid, preferably wherein said amino acid sequence shows at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, preferably 100%, sequence identity to CKCHGVSGSCTTKTCW (SEQ ID NO: 12).

[0232] In preferred embodiments, the serine at amino acid position 7 of SEQ ID NO: 11 or SEQ ID NO: 12 is linked to a fatty-acid residue, preferably wherein said serine at amino acid position 7 of SEQ ID NO: 11 or SEQ ID NO: 12 is palmitoylated.

[0233] In preferred embodiments, the Frizzled-binding domain of the agent as disclosed herein does not comprise the ‘index’ (site 2) of a Wnt ligand. More particularly, the Frizzled-binding domain of the agent as disclosed herein preferably does not comprise an amino acid sequence WXCXV, wherein X can be any amino acid.

[0234] LRP -binding domain of the agent as disclosed herein may be any domain that is capable of binding to one or more LRP polypeptides. Without being limited to any mechanism or theory, the affinity of the LRP-binding domain of the agent may be sufficiently high to activate GPR124 / RECK / Frizzled / LRP -mediated Wnt signaling (e.g. when binding of the agent to RECK, of RECK to GPR124, and of GPR124 to Frizzled via DVL network ensures the proximity or presentation of the agent and more particularly its LRP-binding domain to LRP), but sufficiently low to avoid activating (for example, lack of activation or at most minimal activation, such as physiologically inconsequential degree or extent of activation) Frizzled / LRP -mediated Wnt signaling in the absence of RECK and / or GPR124. Without limitation, in certain embodiments the LRP-binding domain may be capable of binding to one or more LRP polypeptides with a KD lower than about 1 x 10'9M, or lower than about 1 x 10'8M, or lower than about 1 x 10'7M, such as for example between about 1 x 10'3M and about 1 x 10'9M, or between about 1 x 10'3M and about 1 x 10'8M, or between about 1 x 10'3M and about 1 x 10'7M.

[0235] In particular embodiments, said LRP-binding domain comprises a LRP specific antibody or the LRP-binding region, preferably the variable region sequence or CDR, of one or more LRP specific antibody or antibodies. Preferably, the LRP specific antibodies are LRP5 and / or LRP6 specific antibodies. Preferably, the LRP specific antibody is directed to the extracellular portion of the LRP polypeptide, such as directed to the DKK-binding domain of LRP5 and / or LRP6. Non-limiting examples of LRP5 specific antibodies include antibodies which are known in the art and commercially available, such as from Abeam (e.g. ab36121 specific for rabbit and human LRP5), or Santa Cruz (e.g. sc-21390 specific for human LRP5). Non-limiting examples of LRP6 specific antibodies include antibodies available from Abeam (e.g. abl34146 specific for mouse, rat and human LRP6), or Santa Cruz (e.g. sc -25317 specific for human LRP6).

[0236] In particular embodiments, the LRP -binding domain comprises at least one, at least two, at least three, at least four, at least five or at least six CDR(s), each independently having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, preferably 100%, sequence identity with the respective CDR(s) of a LRP specific antibody, preferably a LRP5 or LRP6 specific antibody.

[0237] In particular embodiments, the LRP -binding domain is LRP5 or LRP6 specific scFv, such as scFv comprising one or more (preferably all 6) CDR(s) of a LRP5 or LRP6 specific antibody.

[0238] In particular embodiments, the LRP -binding domain is LRP5 or LRP6 specific VHH, such as VHH comprising one or more (preferably all 3) CDR(s) of a LRP5 or LRP6 specific heavy chain antibody.

[0239] In particular embodiments, the LRP -binding domain of the agent as disclosed herein may comprise two or more CDR(s) each independently having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, preferably 100%, sequence identity with the respective CDR(s) of two or more different LRP specific antibodies, preferably LRP5 or LRP6 specific antibodies.

[0240] In particular embodiments, said LRP -binding domain of the agent as disclosed herein comprises, consists essentially of, consists of or is derived from (e.g., is a biologically active fragment and / or variant of) a LRP -binding polypeptide, such as a Wnt ligand or Wnt polypeptide (e.g., Wnt7a or Wnt7b), a DKK polypeptide (e.g. DKK1, DKK2, DKK3, or DKK4), Sclerostin (or SOST), or Wise (or SOSTDC1), or a variant thereof.

[0241] By means of an example, human SOST gene is annotated under NCBI Genbank (http: / / www.ncbi.nlm.nih.gov / ) Gene ID 50964. Human SOST mRNA is annotated under NCBI Genbank accession number NM_025237.2. Nucleotides 48 (start codon) to 689 (stop codon) of NM_025237.2 constitute the SOST coding sequence. Human SOST protein sequence is annotated under NCBI Genbank accession number NP_079513.1, and Uniprot (www.uniprot.org) accession number Q9BQB4. 1.

[0242] By means of an example, human WISE (or sclerostin domain containing 1 (SOSTDC1)) gene is annotated under NCBI Genbank (http: / / www.ncbi.nlm.nih.gov / ) Gene ID 25928. Human WISE mRNA is annotated under NCBI Genbank accession number NM_015464.2. Nucleotides 182 (start codon) to 802 (stop codon) of NM_015464.2 constitute the WISE coding sequence. Human WISE protein sequence is annotated under NCBI Genbank accession number NP_056279.1, and Uniprot (www.uniprot.org) accession number Q6X4U4.2. Preferably, the LRP -binding domain of the agent as disclosed herein is derived from a Wnt7 (e.g., Wnt7a or Wnt7b) polypeptide, more preferably from a human Wnt7 polypeptide (e.g., human Wnt7a or human Wnt7b) or a variant thereof.

[0243] In particular embodiments, said LRP -binding domain of the agent as disclosed herein comprises, consists essentially of, consists of or is derived from a DKK polypeptide, preferably a DKK1 polypeptide, or a Wnt polypeptide, preferably a Wnt7 polypeptide. In preferred embodiments, said LRP -binding domain of the agent as disclosed herein is derived from a Wnt7 polypeptide.

[0244] Ahn V.E. et al. (Ahn V.E. et al., Dev Cell. 2011 Nov 15;21(5):862-873) discloses that the C- terminal region of DKK1 is responsible for binding to LRP6. Accordingly, in particular embodiments, said LRP-binding domain of the agent as disclosed herein comprises, consists essentially of, consists of or is derived from the C-terminal domain of DKK1. For example, the C- terminal domain of human DKK1 (human DKK1 is annotated under NCBI Genbank accession number NP_036374.1 and Uniprot accession number 094907.1) comprises amino acid sequence MYHTKGQEGSVCLRSSDCASGLCCARHFWSKICKPVLKEGQVCTKHRRKGSHGLEIFQRC YCGEGLSCRIQKDHHQASNSSRLHTCQRH (SEQ ID NO: 14).

[0245] On the other hand, Bourhis E. et al. (Bourhis, Eric et al., Structure , Volume 19 , Issue 10 , 1433 - 1442) identified a LRP5 / 6 interaction motif NXI / V. wherein X can be any amino acid, at the N- terminal end of DKK1, but also in DKK2, DKK4, WISE, and SOST. In view hereof, LRP-binding domain may comprise NXI or NXV, wherein X can be any amino acid.

[0246] Without being limited to any mechanism or theory, the affinity of the Frizzled-binding domain and the LRP-binding domain of the agent may be collectively sufficiently high to activate GPR124 / RECK / Frizzled / LRP -mediated Wnt signaling (e.g., when binding of the agent to RECK, of RECK to GPR124, and of GPR124 to Frizzled via DVL network ensures the proximity or presentation of the agent and more particularly its LRP-binding domain to LRP), but collectively sufficiently low to avoid activating (for example, lack of activation or at most minimal activation, such as physiologically inconsequential degree or extent of activation) Frizzled / LRP -mediated Wnt signaling in the absence of RECK and / or GPR124.

[0247] In particular embodiments, the agent as disclosed herein is a protein conjugate.

[0248] The terms “fusion protein” or “fusion polypeptide” and “protein conjugate” or “polypeptide conjugate” denote hybrid or chimeric molecules comprising at least two proteins or polypeptides linked, connected or joined together in a manner not normally found in nature. The molecules may be suitably denoted as amino acid-based compounds, i.e., as substances or molecules as including primarily but not necessarily exclusively amino acid residues. Any recombinantly, semi- synthetically or synthetically produced fusions or conjugates are encompassed. The fusions or conjugates may be if desired modified by glycosylation, phosphorylation, sulfonation, methylation, acetylation, lipidation, pegylation or the like.

[0249] More particularly, the terms “fusion protein” or “fusion polypeptide” denote genetic fusions, whereby two or more proteins, polypeptides or variants or fragments thereof are joined by a colinear, covalent linkage via their individual polypeptide backbones, through genetic expression of a single contiguous polynucleotide molecule encoding the fusion product. Typically, to produce the contiguous polynucleotide molecule encoding the fusion product, two or more open reading frames (ORFs) each encoding a given polypeptide segment are joined to form a continuous longer ORF in a manner that maintains the correct reading frame for each original ORF. In the resulting recombinant fusion polypeptide the two or more polypeptide segments encoded by the original ORFs are joined in the same polypeptide molecule, whereas they are not normally so joined in nature. While the reading frame is thus made continuous throughout the fused genetic segments, the so fused polypeptide segments may be physically or spatially separated by, for example, an inframe polypeptide or peptide linker.

[0250] More particularly, the terms “protein conjugate” or “polypeptide conjugate” denote substances or molecules in which two or more proteins, polypeptides or variants or fragments thereof are joined by non-genetic means, whereas they are not normally so joined in nature. The polypeptide segments may be joined via their individual polypeptide backbones or via one or more of their respective amino acid side chains, or one protein segment may be joined via its polypeptide backbone to an amino acid side chain of another polypeptide segment.

[0251] In particular embodiments, the GPR124-binding domain, the RECK-binding domain, the Frizzled- binding domain and / or the LRP -binding domain of the agent as disclosed herein may be coupled by one or more linkers.

[0252] In the context of present invention, the term “coupled” as used herein is synonymous with “connected”, “bound”, “fused”, “joined” and refers to a physical link between at least two elements or components.

[0253] As used herein, the term “linker” refers to a connecting element that serves to link other elements. The linker may be a rigid linker or a flexible linker. In particular embodiments, the linker is a covalent linker, achieving a covalent bond. The terms “covalent” or “covalent bond” refer to a chemical bond that involves the sharing of one or more electron pairs between two atoms. For many molecules, the sharing of electrons allows each atom to attain the equivalent of a full outer electron shell, corresponding to a stable electronic configuration. Covalent bonds include different types of interactions, including o-bonds, 7i-bonds, metal-to-metal bonds, agostic interactions, bent bonds and three-center two-electron bonds. In particular embodiments, the linker is a (poly) peptide linker or a non-peptide linker, such as a non-peptide polymer, such as a non-biological polymer. Preferably, the linkage(s) between the RECK-binding domain, the Frizzled-binding domain and / or the LRP-binding domain may be hydrolytically stable linkage(s), i.e., substantially stable in water at useful pH values, including in particular under physiological conditions, for an extended period of time, e.g., for days. In particular embodiments, the linker is a peptide linker of one or more amino acids.

[0254] The term “amino acid” encompasses naturally occurring amino acids, naturally encoded amino acids, non-naturally encoded amino acids, non-naturally occurring amino acids, amino acid analogues and amino acid mimetics that function in a manner similar to the naturally occurring amino acids, all in their D and L stereoisomers, provided their structure allows such stereo -isomeric forms. Amino acids are referred to herein by either their name, their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. The term “naturally occurring” generally refers to materials which are found in nature and are not manipulated by man. The terms “non-naturally occurring”, “un-natural” and the like generally refer to a material that is not found in nature or that has been structurally modified, semi- synthesised or synthesised by man. A “naturally encoded amino acid” refers to an amino acid that is one of the 20 common amino acids or pyrrolysine, pyrroline -carboxy-lysine or selenocysteine. The 20 common amino acids are: Alanine (A or Ala), Cysteine (C or Cys), Aspartic acid (D or Asp), Glutamic acid (E or Glu), Phenylalanine (F or Phe), Glycine (G or Gly), Histidine (H or His), Isoleucine (I or He), Lysine (K or Lys), Leucine (L or Leu), Methionine (M or Met), Asparagine (N or Asn), Proline (P or Pro), Glutamine (Q or Gin), Arginine (R or Arg), Serine (S or Ser), Threonine (T or Thr), Valine (V or Vai), Tryptophan (W or Trp), and Tyrosine (Y or Tyr). A “non- naturally encoded amino acid” refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine, pyrroline-carboxy -lysine or selenocysteine. The term includes without limitation amino acids that occur by a modification (such as a post -translational modification) of a naturally encoded amino acid, but are not themselves naturally incorporated into a growing polypeptide chain by the translation complex, as exemplified without limitation by N- acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine. Further examples of non-naturally encoded, un-natural or modified amino acids include 2-Aminoadipic acid, 3-Aminoadipic acid, beta-Alanine, beta-Aminopropionic acid, 2 -Aminobutyric acid, 4- Aminobutyric acid, piperidinic acid, 6 -Aminocaproic acid, 2-Aminoheptanoic acid, 2- Aminoisobutyric acid, 3 -Aminoisobutyric acid, 2-Aminopimelic acid, 2,4 Diaminobutyric acid, Desmosine, 2,2’-Diaminopimelic acid, 2,3 -Diaminopropionic acid, N-Ethylglycine, N- Ethylasparagine, homoserine, homocysteine, Hydroxylysine, allo -Hydroxy lysine, 3- Hydroxyproline, 4-Hydroxyproline, Isodesmosine, allo-Isoleucine, N-Methylglycine, N- Methylisoleucine, 6-N-Methyllysine, N-Methylvaline, Norvaline, Norleucine, or Ornithine. Also included are amino acid analogues, in which one or more individual atoms have been replaced either with a different atom, an isotope of the same atom, or with a different functional group. Also included are un-natural amino acids and amino acid analogues described in Ellman et al. Methods Enzymol. 1991, vol. 202, 301-36. The incorporation of non-natural amino acids into proteins or polypeptides may be advantageous in a number of different ways. For example, D -amino acidcontaining polypeptides exhibit increased stability in vitro or in vivo compared to L-amino acidcontaining counterparts. More specifically, D-amino acid-containing polypeptides may be more resistant to endogenous peptidases and proteases, thereby providing improved bioavailability of the agent and prolonged lifetimes in vivo.

[0255] More particularly, the peptide linker may be 1 to 50 amino acids long or 2 to 50 amino acids long or 1 to 45 amino acids long or 2 to 45 amino acids long, preferably 1 to 40 amino acids long or 2 to 40 amino acids long or 1 to 35 amino acids long or 2 to 35 amino acids long, more preferably 1 to 30 amino acids long or 2 to 30 amino acids long. Further preferably, the linker may be 5 to 25 amino acids long or 5 to 20 amino acids long. Particularly preferably, the linker may be 5 to 15 amino acids long or 7 to 15 amino acids long. Hence, in certain embodiments, the linker may be 1, 2, 3 or 4 amino acids long. In other embodiments, the linker may be 5, 6, 7, 8 or 9 amino acids long. In further embodiments, the linker may be 10, 11, 12, 13 or 14 amino acids long. In still other embodiments, the linker may be 15, 16, 17, 18 or 19 amino acids long. In further embodiments, the linker may be 20, 21, 22, 23, 24 or 25 amino acids long. In certain embodiments, the linker is 4-10 or 5-9 or 6-8 or 7 amino acids long. In other embodiments, the linker is 12-18 or 13-17 or 14-16 or 15 amino acids long.

[0256] The nature of amino acids constituting the linker is not of particular relevance so long as the biological activity of the polypeptide segments linked thereby is not substantially impaired and the linker provides for the intended spatial separation of the GPR124-binding domain, the RECK- binding domain, the Frizzled-binding domain and / or the LRP-binding domain of the agent. Preferred linkers are essentially non-immunogenic and / or not prone to proteolytic cleavage.

[0257] In certain preferred embodiments, the peptide linker may comprise, consist essentially of or consist of amino acids selected from the group consisting of Glycine, Serine, Alanine, Threonine, and combinations thereof. In even more preferred embodiments, the linker may comprise, consist essentially of or consist of amino acids selected from the group consisting of Glycine, Serine, and combinations thereof. Such linkers provide for particularly good flexibility. In certain embodiments, the linker may consist of only Glycine residues. In certain embodiments, the linker may consist of only Serine residues. In particular embodiments, the linker is a non-peptide linker. In preferred embodiments, the nonpeptide linker may comprise, consist essentially of or consist of a non-peptide polymer. The term “non-peptide polymer” as used herein refers to a biocompatible polymer including two or more repeating units linked to each other by a covalent bond excluding the peptide bond. For example, the non-peptide polymer may be 2 to 200 units long or 2 to 100 units long or 2 to 50 units long or 2 to 45 units long or 2 to 40 units long or 2 to 35 units long or 2 to 30 units long or 5 to 25 units long or 5 to 20 units long or 5 to 15 units long. The non-peptide polymer may be selected from the group consisting of polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers such as PL A (poly (lactic acid) and PLGA (polylactic -glycolic acid), lipid polymers, chitins, hyaluronic acid, and combinations thereof. Particularly preferred is poly (ethylene glycol) (PEG). The molecular weight of the non-peptide polymer preferably may range from 1 to 100 kDa, and preferably 1 to 20 kDa. The non-peptide polymer may be one polymer or a combination of different types of polymers. The non-peptide polymer has reactive groups capable of binding to the RECK-binding domain, the Frizzled-binding domain and / or the LRP -binding domain which are to form the conjugate. Preferably, the non-peptide polymer has a reactive group at each end. Preferably, the reactive group is selected from the group consisting of a reactive aldehyde group, a propione aldehyde group, a butyl aldehyde group, a maleimide group and a succinimide derivative. The succinimide derivative may be succinimidyl propionate, hydroxy succinimidyl, succinimidyl carboxymethyl or succinimidyl carbonate. The reactive groups at both ends of the non-peptide polymer may be the same or different. In certain embodiments, the non- peptide polymer has a reactive aldehyde group at both ends. For example, the non-peptide polymer may possess a maleimide group at one end and, at the other end, an aldehyde group, a propionic aldehyde group or a butyl aldehyde group. When a polyethylene glycol (PEG) having a reactive hydroxy group at both ends thereof is used as the non-peptide polymer, the hydroxy group may be activated to various reactive groups by known chemical reactions, or a PEG having a commercially-available modified reactive group may be used so as to prepare the protein conjugate.

[0258] The inclusion of one or more linkers increases the spatial separation between the GPR124-binding domain, the RECK-binding domain, the Frizzled-binding domain and / or the LRP -binding domain of the agent, which can advantageously affect the Wnt signaling activity of the agent.

[0259] In particular embodiments, the agent as disclosed herein is provided as a single chain or a multimer. To achieve multimeric forms, GPR 124 -binding domain, RECK-binding domain, Frizzled-binding domain and / or LRP -binding domain may be attached to a scaffold or backbone composed of, for example, dendrimers, antibodies or poly-L-lysine. In particular embodiments, the agent as disclosed herein is conjugated to a bone cell or tissuespecific targeting agent, such as an aptamer binding to human osteogenic-induced progenitor cells (Ardjomandi, N., et al. (2013). Identification of an aptamer binding to human osteogenic-induced progenitor cells. Nucleic Acid Ther. 23, 44-61. Doi: 10.1089 / nat.2012.0349).

[0260] In particular embodiments, the agent as disclosed herein comprises a RECK-binding domain, a Frizzled-binding domain and a LRP -binding domain, wherein said RECK-binding domain, said Frizzled-binding domain, and said LRP -binding domain are derived from a Wnt7 polypeptide, such as from a Wnt7a or Wnt7b polypeptide, preferably the human Wnt polypeptide, such as from human Wnt7a or Wnt7b polypeptide.

[0261] In particular embodiments, the agent as disclosed herein comprises a RECK-binding domain, a Frizzled-binding domain and a LRP -binding domain, wherein at least one, at least two or all three binding domains are derived from a Wnt7 polypeptide, such as from a Wnt7a or Wnt7b polypeptide, preferably the human Wnt polypeptide, such as from human Wnt7a or Wnt7b polypeptide.

[0262] By means of an example, human WNT7A gene is annotated under NCBI Genbank (http: / / www.ncbi.nlm.nih.gov / ) Gene ID 7476. Human WNT7A mRNA (transcript variant 1) is annotated under NCBI Genbank accession number NM_004625.3. Nucleotides 306 (start codon) to 1355 (stop codon) of NM_004625.3 constitute the WNT7A coding sequence. Human WNT7A protein sequence is annotated under NCBI Genbank accession number NP_004616.2, and Uniprot accession number 000755.2, and is further reproduced below (SEQ ID NO: 15):

[0263] > NP_004616.2 protein Wnt-7a precursor [Homo sapiens]

[0264] MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIG EGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAAC TQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMN LHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEP VRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQ ASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK.

[0265] By means of an example, the mature form of human Wnt7a protein (not comprising the signal peptide) comprises an amino sequence as further reproduced below (SEQ ID NO: 51): LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVF GKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGG CSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTT KTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYI EKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFH WCCYVKCNTCSERTEMYTCK. By means of an example, human WNT7B gene is annotated under NCBI Genbank Gene ID 7477. Human WNT7B mRNA (transcript variant 1) is annotated under NCBI Genbank accession number NM_058238.2. Nucleotides 375 (start codon) to 1424 (stop codon) of NM_058238.2 constitute the WNT7B coding sequence. Human WNT7B protein sequence is annotated under NCBI Genbank accession number NP 478679.1, and Uniprot accession number P56706.2, and is further reproduced below (SEQ ID NO: 16):

[0266] > NP_478679. 1 protein Wnt-7b precursor [Homo sapiens]

[0267] MHRNFRKWIFYVFLCFGVLYVKLGALSSVVALGANIICNKIPGLAPRQRAICQSRPDAIIVI GEGAQMGINECQYQFRFGRWNCSALGEKTVFGQELRVGSREAAFTYAITAAGVAHAVTA ACSQGNLSNCGCDREKQGYYNQAEGWKWGGCSADVRYGIDFSRRFVDAREIKKNARRL MNLHNNEAGRKVLEDRMQLECKCHGVSGSCTTKTCWTTLPKFREVGHLLKEKYNAAVQ VEVVRASRLRQPTFLRIKQLRSYQKPMETDLVYIEKSPNYCEEDAATGSVGTQGRLCNRTS PGADGCDTMCCGRGYNTHQYTKVWQCNCKFHWCCFVKCNTCSERTEVFTCK

[0268] In particular embodiments, said RECK -binding domain comprises, consists essentially of or consists of an amino acid sequence as set forth in HVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 17), VEPVRASRNKRPTFLKIKKPLSYRKPMDT (SEQ ID NO: 18) or

[0269] VEVVRASRLRQPTFLRIKQLRSYQKPMET (SEQ ID NO: 19), or an amino acid sequence having at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence SEQ ID NO: 17, SEQ ID NO: 18; or SEQ ID NO: 19. In particular embodiments, said RECK-binding domain derived from a Wnt7 polypeptide comprises, consists essentially of or consists of the amino acid sequence SEQ ID NO: 17, SEQ ID NO: 18; or SEQ ID NO: 19.

[0270] In particular embodiments, said RECK-binding domain comprises, consists essentially of or consists of the amino acid sequence XXXVXAXRXXXXXFLXIXXXXXYXKXXXX (SEQ ID NO: 20), VXAXRXXXXXFLXIXXXXXYXK (SEQ ID NO: 21),

[0271] XXXVXAXRXXXXXFLXXXXXXXXXKXXXX (SEQ ID NO: 22) or

[0272] VXAXRXXXXXFLXXXXXXXXXK (SEQ ID NO: 23), wherein X is any amino acid, preferably wherein the amino acid sequence shows at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, preferably 100%, sequence identity to any one of SEQ ID NO: 18 or SEQ ID NO: 19. In particular embodiments, said RECK-binding domain is derived from a Wnt7 polypeptide, and the Frizzled-binding domain and LRP -binding domain are not derived from a Wnt7 polypeptide.

[0273] In particular embodiments, said RECK-binding domain is derived from a Wnt7 polypeptide, and the Frizzled-binding domain and LRP -binding domain are derived from a Wnt polypeptide selected from the list consisting of Wntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a Wnt5b, Wnt6, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt 10a, Wnt 10b, Wnt 11 and Wnt 16.

[0274] In particular embodiments, the agent as disclosed herein is or consists essentially of a fragment of a Wnt7 polypeptide, such as a fragment of a Wnt7a or Wnt7b polypeptide, preferably a fragment of a human Wnt7 polypeptide, such as a fragment of a human Wnt7a or human Wnt7b polypeptide.

[0275] In particular embodiments, the fragment of the Wnt7 polypeptide has at least 30%, and preferably at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, preferably 100%, of the GPR124 / RECK / Frizzled / LRP -mediated Wnt signaling activity of the full-length Wnt7 polypeptide.

[0276] In particular embodiments, the fragment of the Wnt7 polypeptide has a GPR124 / RECK / Frizzled / LRP -mediated Wnt signaling activity which is higher (e.g., 1.1-fold, 1.2- fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold or 2-fold higher or even higher) than the GPR124 / RECK / Frizzled / LRP -mediated Wnt signaling activity of the full-length Wnt7 polypeptide.

[0277] The N-terminal domain of Wnt7a is capable of binding to RECK and activating Wnt signaling mediated by the GPR124 / RECK / Frizzled / LRP receptor complex, while not activating Wnt signaling mediated by the Frizzled / LRP receptor complex in the absence of RECK and / or GPR124.

[0278] Hence, in particular embodiments, the fragment of the Wnt7 polypeptide has Frizzled / LRP- mediated Wnt signaling activity in the absence of RECK and / or GPR124 which is at least 10-fold less, or at least 100-fold less, or at least 1000-fold less, or at least 1 x 104-fold less, or at least 1 x 105-fold less, or at least 1 x 106-fold less than the Frizzled / LRP -mediated Wnt signaling activity of the full-length Wnt7 polypeptide in the absence of RECK and / or GPR124.

[0279] In particular embodiments, said fragment of the Wnt7 polypeptide is or consists essentially of the N-terminal domain (NTD) of the Wnt7 polypeptide, such as the NTD of a Wnt7a or Wnt7b polypeptide, preferably the NTD of human Wnt7 polypeptide, such as human Wnt7a or Wnt7b. The N-terminal domain of the human Wnt7a or Wnt7b polypeptide typically ranges from the Leucine (L) residue at the position corresponding to position 1 in SEQ ID NO: 24 (Wnt7a) or SEQ ID NO: 25 (Wnt7b) to the cysteine (C) residue at the position corresponding to position 247 in SEQ ID NO: 24 (Wnt7a) or SEQ ID NO: 25 (Wnt7b). In particular embodiments, said fragment of the Wnt7 polypeptide comprises at least the NTD of the Wnt7 polypeptide and does not comprise the C-terminal domain (CTD) of the Wnt7 polypeptide.

[0280] In particular embodiments, the agent as disclosed herein comprises, consists essentially of, or consists of at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, contiguous amino acids of the NTD of human Wnt7a polypeptide, more particularly, the agent as disclosed herein comprises, consists essentially of or consists of at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, contiguous amino acids of the amino acid sequence

[0281] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVF GKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGG CSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTT KTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYI EKSPNYC (SEQ ID NO: 24) or

[0282] LGANIICNKIPGLAPRQRAICQSRPDAIIVIGEGAQMGINECQYQFRFGRWNCSALGEKTVF GQELRVGSREAAFTYAITAAGVAHAVTAACSQGNLSNCGCDREKQGYYNQAEGWKWGG CSADVRYGIDFSRRFVDAREIKKNARRLMNLHNNEAGRKVLEDRMQLECKCHGVSGSCT TKTCWTTLPKFREVGHLLKEKYNAAVQVEVVRASRLRQPTFLRIKQLRSYQKPMETDLVY

[0283] IEKSPNYC (SEQ ID NO: 25), preferably SEQ ID NO:24.

[0284] In particular embodiments, the agent as disclosed herein comprises, consists essentially of, or consists of an amino acid sequence as set forth in SEQ ID NO: 24 or SEQ ID NO: 25, preferably SEQ ID NO: 24, or an amino acid sequence having at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, sequence identity to the amino acid sequence SEQ ID NO: 24 or SEQ ID NO: 25, preferably SEQ ID NO: 24.

[0285] The skilled person will understand that if it is envisaged to express and secrete the agent as taught herein by a host cell, the nucleic acid encoding the agent as taught herein preferably encodes a precursor form of the agent including an N-terminal signal peptide sequence. Accordingly, the nucleic acid may encode a fragment of the precursor polypeptide of Wnt7 (i.e. including the signal peptide), such as the precursor polypeptide of human Wnt7a or Wnt7b. In particular embodiments, the nucleic acid encodes an agent comprising, consisting essentially of, or consists of an amino acid sequence as set forth in SEQ ID NO: 24 or SEQ ID NO: 25 or an amino acid sequence having having at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 24 or SEQ ID NO: 25, wherein said amino acid sequence is preceded N-terminally by a signal peptide having an amino acid sequence MNRKARRCLGHLFLSLGMVYLRIGGFSSVVA (SEQ ID NO: 26) or

[0286] MHRNFRKWIFYVFLCFGVLYVKLGALSSVVA (SEQ ID NO: 27), respectively. Alternatively, the nucleic acid encoding the agent as taught herein may be comprised within a vector providing for a signal peptide. The signal peptide may be a homologous or heterologous signal peptide, depending on the host cell used for production of the agent as taught herein. Furthermore, for prokaryotic expression of the agent as taught herein, a protease cleavage site motif may be present C-terminally of said signal peptide and N-terminally of the agent as taught herein.

[0287] Certain variants of the human or murine Wnt7a polypeptide are as effective or more effective in activating Wnt signaling mediated by the GPR124 / RECK / Frizzled / LRP receptor complex, while not activating Wnt signaling mediated by the Frizzled / LRP receptor complex in the absence of RECK and / or GPR124, compared to the wild-type human or murine Wnt7a polypeptide, respectively.

[0288] Accordingly, in particular embodiments, the agent as disclosed herein is a variant of a Wnt7 polypeptide, such as a variant of a Wnt7a or Wnt7b polypeptide . In further particular embodiments, the agent as disclosed herein is the NTD or a variant of the NTD of a Wnt7a or Wnt7b polypeptide.

[0289] In certain embodiments, in such variant, the glutamine (Q) residue at the position corresponding to position 17 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the isoleucine (I) residue at the position corresponding to position 20 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the proline (P) residue at the position corresponding to position 25 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the alanine (A) residue at the position corresponding to position 27 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the isoleucine (I) residue at position corresponding to position 28 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the glutamate (E) residue at the position corresponding to position 33 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the methionine (M) residue at the position corresponding to position 37 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the leucine (L) residue at the position corresponding to position 39 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the glutamate (E) residue at the position corresponding to position 41 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the phenylalanine (F) residue at the position corresponding to position 44 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the arginine (R) residue at the position corresponding to position 50 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the asparagine (N) residue at the position corresponding to position 52 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the valine (V) residue at the position corresponding to position 68 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the isoleucine (I) residue at the position corresponding to position 129 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the phenylalanine (F) residue at the position corresponding to position 131 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51 ; the lysine (K) residue at the position corresponding to position 133 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the phenylalanine (F) residue at the position corresponding to position 135 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the isoleucine (I) residue at the position corresponding to position 141 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the arginine (R) residue at the position corresponding to position 146 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the arginine (R) residue at the position corresponding to position 158 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the lysine (K) residue at the position corresponding to position 159 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the lysine (K) residue at the position corresponding to position 181 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the arginine (R) residue at the position corresponding to position 191 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the lysine (K) residue at the position corresponding to position 198 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the lysine (K) residue at the position corresponding to position 200 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the valine (V) residue at the position corresponding to position 205 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the glutamate (E) residue at the position corresponding to position 208 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the glutamate (E) residue at the position corresponding to position 208 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the arginine (R) residue at the position corresponding to position 214 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the lysine (K) residue at the position corresponding to position 216 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the proline (P) residue at the position corresponding to position 218 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the lysine (K) residue at the position corresponding to position 222 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the isoleucine (I) residue at the position corresponding to position 223 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the tyrosine (Y) residue at the position corresponding to position 229 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the proline (P) residue at the position corresponding to position 232 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the threonine (T) residue at the position corresponding to position 235 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the glutamate (E) residue at the position corresponding to position 248 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the arginine (R) residue at the position corresponding to position 289 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the tryptophan (W) residue at the position corresponding to position 291 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the threonine (T) residue at the position corresponding to position 307 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; and / or the lysine (K) residue at the position corresponding to position 318 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51, is substituted by one or more (preferably not more than three, preferably not more than two, more preferably one) other amino acid residue, such as preferably but without limitation by an alanine (A) residue, an arginine (R) residue or a glutamine (Q) residue, more preferably by an alanine (A) residue . In certain embodiments, in such variant, the glutamine (Q) residue at the position corresponding to position 17 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the isoleucine (I) residue at the position corresponding to position 20 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the proline (P) residue at the position corresponding to position 25 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the isoleucine (I) residue at position corresponding to position 28 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the glutamate (E) residue at the position corresponding to position 33 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the methionine (M) residue at the position corresponding to position 37 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the leucine (L) residue at the position corresponding to position 39 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the glutamate (E) residue at the position corresponding to position 41 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the phenylalanine (F) residue at the position corresponding to position 44 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the arginine (R) residue at the position corresponding to position 50 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the valine (V) residue at the position corresponding to position 68 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the isoleucine (I) residue at the position corresponding to position 129 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the phenylalanine (F) residue at the position corresponding to position 131 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the lysine (K) residue at the position corresponding to position 133 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the phenylalanine (F) residue at the position corresponding to position 135 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the isoleucine (I) residue at the position corresponding to position 141 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the arginine (R) residue at the position corresponding to position 146 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the arginine (R) residue at the position corresponding to position 158 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the lysine (K) residue at the position corresponding to position 181 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the arginine (R) residue at the position corresponding to position 191 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the lysine (K) residue at the position corresponding to position 198 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the lysine (K) residue at the position corresponding to position 200 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51 ; the valine (V) residue at the position corresponding to position 205 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the glutamate (E) residue at the position corresponding to position 208 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the glutamate (E) residue at the position corresponding to position 208 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the arginine (R) residue at the position corresponding to position 214 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the lysine (K) residue at the position corresponding to position 216 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the proline (P) residue at the position corresponding to position 218 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the lysine (K) residue at the position corresponding to position 222 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the isoleucine (I) residue at the position corresponding to position 223 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the tyrosine (Y) residue at the position corresponding to position 229 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the proline (P) residue at the position corresponding to position 232 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the threonine (T) residue at the position corresponding to position 235 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the glutamate (E) residue at the position corresponding to position 248 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the arginine (R) residue at the position corresponding to position 289 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the tryptophan (W) residue at the position corresponding to position 291 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; the threonine (T) residue at the position corresponding to position 307 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51; and / or the lysine (K) residue at the position corresponding to position 318 in SEQ ID NO: 24 or SEQ ID NO: 51 , preferably SEQ ID NO: 51, is substituted by an alanine (A) residue; and / or the alanine (A) residue at the position corresponding to position 27 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51, is substituted by an arginine (R) residue; and / or the asparagine (N) residue at the position corresponding to position 52 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51, is substituted by a glutamine (Q) residue; and / or the lysine (K) residue at the position corresponding to position 159 in SEQ ID NO: 24 or SEQ ID NO: 51, preferably SEQ ID NO: 51, is substituted by an alanine (A), a serine (S) or a leucine (L) residue residue .

[0290] In certain embodiments, the variant of the Wnt7 polypeptide comprises two or more (e.g., preferably two, preferably three, more preferably four) of the amino acid substitutions listed above. In more particular embodiments, the NTD of the Wnt7a or Wnt7b polypeptide comprises two or more (e.g., preferably two, preferably three, more preferably four) of the amino acid substitutions listed above.

[0291] Hence, in particular embodiments, the agent as disclosed herein comprises, consists essentially of or consists of an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 24 or SEQ ID NO: 51, wherein the residue at position 17 is not glutamine, the residue at position 20 is not isoleucine, the residue at position 25 is not proline, the residue at position 27 is not alanine, the residue at position 28 is not isoleucine, the residue at position 33 is not glutamate, the residue at position 37 is not methionine, the residue at position 39 is not leucine, the residue at position 41 is not glutamate, the residue at position 44 is not phenylalanine, the residue at position 50 is not arginine, the residue at position 52 is not asparagine, the residue at position 68 is not valine, the residue at position 129 is not isoleucine, the residue at position 131 is not phenylalanine, the residue at position 133 is not lysine, the residue at position 135 is not phenylalanine, the residue at position 141 is not isoleucine, the residue at position 146 is not arginine, the residue at position 158 is not arginine, the residue at position 159 is not lysine, the residue at position 181 is not lysine, the residue at position 191 is not arginine, the residue at position 198 is not lysine, the residue at position 200 is not lysine, the residue at position 205 is not valine, the residue at position 208 is not glutamate, the residue at position 214 is not arginine, the residue at position 216 is not lysine, the residue at position 218 is not proline, the residue at position 222 is not lysine, the residue at position 223 is not isoleucine, the residue at position 229 is not tyrosine, the residue at position 232 is not proline, the residue at position 235 is not threonine, the residue at position 248 is not glutamate, the residue at position 289 is not arginine, the residue at position 291 is not tryptophan, the residue at position 307 is not threonine, and / or the residue at position 318 is not lysine; preferably wherein the residue at position 27 is arginine, the residue at position 28 is alanine, the residue at position 33 is alanine, the residue at position 41 is alanine, the residue at position 44 is alanine, the residue at position 50 is alanine, the residue at position 52 is glutamine, the residue at position 68 is alanine, the residue at position 129 is alanine, the residue at position 131 is alanine, the residue at position 133 is alanine, the residue at position 135 is alanine, the residue at position 141 is alanine, the residue at position 146 is alanine, the residue at position 158 is alanine, the residue at position 159 is alanine, the residue at position 181 is alanine, the residue at position 191 is alanine, the residue at position 198 is alanine, the residue at position 200 is alanine, the residue at position 205 is alanine, the residue at position 208 is alanine, , the residue at position 214 is alanine, the residue at position 216 is alanine, the residue at position 218 is alanine, the residue at position 222 is alanine, the residue at position 223 is alanine, the residue at position 229 is alanine, the residue at position 232 is alanine, the residue at position 235 is alanine, the residue at position 248 is alanine, the residue at position 289 is alanine, the residue at position 291 is alanine, the residue at position 307 is alanine, and / or the residue at position 318 is alanine.

[0292] In preferred embodiments, the agent as disclosed herein is a variant of the NTD of human Wnt7a comprising, consisting essentially of or consisting of an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 24, wherein the residue at position 17 is not glutamine, the residue at position 20 is not isoleucine, the residue at position 25 is not proline, the residue at position 27 is not alanine, the residue at position 28 is not isoleucine, the residue at position 33 is not glutamate, the residue at position 37 is not methionine, the residue at position 39 is not leucine, the residue at position 41 is not glutamate, the residue at position 44 is not phenylalanine, the residue at position 50 is not arginine, the residue at position 52 is not asparagine, the residue at position 68 is not valine, the residue at position 129 is not isoleucine, the residue at position 131 is not phenylalanine, the residue at position 133 is not lysine, the residue at position 135 is not phenylalanine, the residue at position 141 is not isoleucine, the residue at position 146 is not arginine, the residue at position 158 is not arginine, the residue at position 159 is not lysine, the residue at position 181 is not lysine, the residue at position 191 is not arginine, the residue at position 198 is not lysine, the residue at position 200 is not lysine, the residue at position 205 is not valine, the residue at position 208 is not glutamate, the residue at position 214 is not arginine, the residue at position 216 is not lysine, the residue at position 218 is not proline, the residue at position 222 is not lysine, the residue at position 223 is not isoleucine, the residue at position 229 is not tyrosine, the residue at position 232 is not proline, and / or the residue at position 235 is not threonine.

[0293] In particular embodiments, the agent as disclosed herein comprises, consists essentially of or consists of an amino acid sequence as set forth in SEQ ID NO: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,

[0294] 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,

[0295] 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 7, 9, or 13 , preferably SEQ ID NO: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 47, 48, 49, 53, 54, 55, 56, 57, 58, 59,

[0296] 60, 62, 66, 67, 69, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 90, 91, 92, 93, 94,

[0297] 95, 96, 97, 98, 100, 104, 7 or 13, more preferably SEQ ID NO: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 47, 48, 49, 53, 54, 55, 56, 57, 58, 59, 60, 62, 66, 67 or 69.

[0298] Sequences below are mature Wnt7a polypeptide variants or NTD fragments thereof, wherein one amino acid residue has been substituted by another residue (in bold). hWnt7aNTD-K159A:

[0299] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRAILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 28) hWnt7aK159A:

[0300] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRAILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0301] 71) hWnt7aNTD-A27R:

[0302] LGASIICNKIPGLAPRQRAICQSRPDRIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 29) hWnt7aA27R:

[0303] LGASIICNKIPGLAPRQRAICQSRPDRIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0304] 72) hWnt7aNTD'E33A:

[0305] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGAGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 30) hWnt7aE33A:

[0306] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGAGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN

[0307] KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 73) hWnt7aNTD'E41A:

[0308] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDACQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 31) hWnt7aE41A:

[0309] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDACQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0310] 74) hWnt7aNTD-F44A:

[0311] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQAQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 32) hWnt7aF44A:

[0312] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQAQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0313] 75) hWnt7aNTD-N52Q:

[0314] LGASIICNI<IPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWQCSALGERTVFGI<ELI<V GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 33) hWnt7aN52Q:

[0315] LGASIICNI<IPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRW2CSALGERTVFGI<ELI<V GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0316] 76) hWnt7aNTD-I129A:

[0317] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGAGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 34) hWnt7aI129A:

[0318] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGAGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0319] 77) hWnt7aNTD'F131A:

[0320] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGAAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 35) hWnt7aF131A:

[0321] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGAAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0322] 78) hWnt7aNTD-K133A:

[0323] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAA VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK

[0324] DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 36) hWnt7aK133A:

[0325] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAA VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0326] 79) hWnt7aNTD-I141A:

[0327] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREAKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 37) hWnt7aI141A:

[0328] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREAKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0329] 80) hWnt7aNTD-R158A:

[0330] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGAKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 38) hWnt7aR158A:

[0331] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGAKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0332] 81) hWnt7aNTD-K181A:

[0333] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTATCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 39) hWnt7aK181A:

[0334] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTATCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0335] 82) hWnt7aNTD-R191A:

[0336] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFAELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 40) hWnt7aR191A:

[0337] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFAELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0338] 83) hWnt7aNTD-K198A:

[0339] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLA DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 41) hWnt7aK198A:

[0340] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLA DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 84) hWnt7aNTD-V205A:

[0341] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAAHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 42) hWnt7aV205A:

[0342] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAAHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYEEDPVTGSVGTQGRACNK TAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0343] 85) hWnt7aNTD-E208A:

[0344] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVAPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 43) hWnt7aE208A:

[0345] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVAPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0346] 86) hWnt7aNTD-K216A:

[0347] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNARPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 44) hWnt7aK216A:

[0348] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNARPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0349] 87) hWnt7aNTD-K222A:

[0350] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLAIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 45) hWnt7aK222A:

[0351] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLAIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0352] 88) hWnt7aNTD-I223A:

[0353] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKAKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 46) hWnt7aI223A:

[0354] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKAKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0355] 89) hWnt7aNTD-Y229A:

[0356] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK

[0357] DKYNEAVHVEPVRASRNKRPTFLKIKKPLSARKPMDTDLVYIEKSPNYC (SEQ ID NO: 47) hWnt7aY229A:

[0358] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSARKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0359] 90) hWnt7aNTD-P232A:

[0360] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKAMDTDLVYIEKSPNYC (SEQ ID NO: 48) hWnt7aP232A:

[0361] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKAMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0362] 91) hWnt7aNTD'T235A:

[0363] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDADLVYIEKSPNYC (SEQ ID NO: 49) hWnt7aT235A:

[0364] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDADLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0365] 92) hWnt7aE248A:

[0366] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCAEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 50) hWnt7al<2X9:

[0367] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYAAVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0368] 52) hWnt7aW291A:

[0369] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVAQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0370] 53) hWnt7aT307A:

[0371] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNACSERTEMYTCK (SEQ ID NO:

[0372] 54) hWnt7aNTD-Q17A:

[0373] LGASIICNKIPGLAPRARAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 55) hWnt7aQ17A:

[0374] LGASIICNKIPGLAPRARAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0375] 93) hWnt7aNTD-I20A:

[0376] LGASIICNKIPGLAPRQRAACQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 56) hWnt7aI20A:

[0377] LGASIICNKIPGLAPRQRAACQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0378] 94) hWnt7aNTD-p25A:

[0379] LGASIICNKIPGLAPRQRAICQSRADAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 57) hWnt7aP25A:

[0380] LGASIICNKIPGLAPRQRAICQSRADAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0381] 95) hWnt7aNTD-M37A:

[0382] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQAGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 58) li\Viit7a'13':

[0383] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQAGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0384] 96) hWnt7aNTD'L39A:

[0385] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGADECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 59) hWnt7aL39A:

[0386] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGADECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0387] 97) hWnt7aNTD-I28A:

[0388] LGASIICNKIPGLAPRQRAICQSRPDAAIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 60) hWnt7aI28A:

[0389] LGASIICNKIPGLAPRQRAICQSRPDAAIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0390] 98) hWnt7aNTD-R50A:

[0391] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGAWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK

[0392] DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 61) h\Vnt7al<"'':

[0393] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGAWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0394] 99) hWnt7aNTD-V68A:

[0395] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKA GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 62) hWnt7aV68A:

[0396] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKA GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0397] 100) hWnt7aNTD-F135A:

[0398] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VAVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 63) hWnt7aF135A:

[0399] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VAVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0400] 101) hWnt7aNTD'R146A:

[0401] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNAATLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 64) hWnt7aR146A:

[0402] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNAATLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0403] 102) hWnt7aNTD-K159S:

[0404] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRSILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKD KYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 65) hWnt7aK159S:

[0405] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRSILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKD KYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNK TAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO:

[0406] 103) hWnt7aNTD-K159L:

[0407] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRLILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 66) hWnt7aK159L:

[0408] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRLILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 104) hWnt7aNTD-K200A:

[0409] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DAYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 67) hWnt7aK200A:

[0410] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DAYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC

[0411] EEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSE RTEMYTCK (SEQ ID NO: 7) hWnt7aNTD-R214A:

[0412] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASANKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 68) hWnt7aR214A:

[0413] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASANKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 9) hWnt7aNTD-p218A:

[0414] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 69) hWnt7aP218A: LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 13) hWnt7aK318A:

[0415] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKV GSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAK VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCA (SEQ ID NO: 70)

[0416] In preferred embodiments, in such variant, the lysine (K) residue at the position corresponding to position 159 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by one or more (preferably not more than three, preferably not more than two, more preferably one) other amino acid residue, such as preferably but without limitation by an alanine (A) residue, a serine (S) or a leucine (L) residue. In more preferred embodiments, the agent as disclosed herein comprises, consists essentially of or consists of an amino acid sequence as set forth in SEQ ID NO: 28, 66, 67, 71, 104 or 7, preferably SEQ ID NO: 28 or 71, more preferably SEQ ID NO: 28.

[0417] In particular embodiments, the variant of the Wnt7 polypeptide has at least 35%, preferably at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, preferably 100%, of the GPR124 / RECK / Frizzled / LRP -mediated Wnt signaling activity of the full- length wild-type Wnt7 polypeptide. In preferred embodiments, the variant of the Wnt7 polypeptide has at least 70% of the GPR124 / RECK / Frizzled / LRP -mediated Wnt signaling activity of the full- length wild-type Wnt7 polypeptide. In particular embodiments, the variant of the Wnt7 polypeptide has a GPR124 / RECK / Frizzled / LRP -mediated Wnt signaling activity which is higher (e.g. 1.1 -fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold or 2-fold higher or even higher) than the GPR124 / RECK / Frizzled / LRP -mediated Wnt signaling activity of the full-length wild-type Wnt7 polypeptide.

[0418] In particular embodiments, the agent as disclosed herein is soluble in water. For example, the solubility of the agent can be increased by using a Frizzled-specific antibody or fragment thereof, instead of a palmitoylated Frizzled-binding domain of a Wnt polypeptide. A further aspect relates to a nucleic acid encoding the agent as disclosed herein, wherein the agent is a protein, polypeptide or a peptide, for use in the treatment (e.g. treatment and / or prevention) of a bone-related disease or disorder in a subject.

[0419] By “encoding” is meant that a nucleic acid sequence or part(s) thereof corresponds, by virtue of the genetic code of an organism in question to a particular amino acid sequence, e.g., the amino acid sequence of one or more desired proteins or polypeptides, or to another nucleic acid sequence in a template-transcription product (e.g. RNA or RNA analogue) relationship.

[0420] To allow expression of the nucleic acid encoding the agent as disclosed herein, wherein the agent is a protein, polypeptide or a peptide, the nucleic acid may be inserted into a nucleic acid expression cassette and / or vector, as is well-known in the art.

[0421] Accordingly, a further aspect relates to a nucleic acid expression cassette, for use in the treatment (e.g. treatment and / or prevention) of a bone-related disease or disorder in a subject, wherein said nucleic acid expression cassette comprises the nucleic acid encoding the agent as disclosed herein, operably linked to a promoter and / or transcriptional and translational regulatory signals.

[0422] The term “nucleic acid expression cassettes” as used herein refers to nucleic acid molecules, typically DNA, to which nucleic acid fragments, preferably the recombinant nucleic acid molecule as defined herein, may be inserted to be expressed, wherein said nucleic acid molecules comprise one or more nucleic acid sequences controlling the expression of the nucleic acid fragments. Nonlimiting examples of such more nucleic acid sequences controlling the expression of the nucleic acid fragments include promoter sequences, open reading frames and transcription terminators.

[0423] Preferably, the nucleic acid expression cassette may comprise one or more open reading frames (ORF) encoding said one or more proteins, polypeptides or peptides. An “open reading frame” or “ORF” refers to a succession of coding nucleotide triplets (codons) starting with a translation initiation codon and closing with a translation termination codon known per se, and not containing any internal in-frame translation termination codon, and potentially capable of encoding a protein, polypeptide or peptide. Hence, the term may be synonymous with “coding sequence” as used in the art.

[0424] An “operable linkage” is a linkage in which regulatory sequences and sequences sought to be expressed are connected in such a way as to permit said expression. For example, sequences, such as, e.g., a promoter and an ORF, may be said to be operably linked if the nature of the linkage between said sequences does not: (1) result in the introduction of a frame -shift mutation, (2) interfere with the ability of the promoter to direct the transcription of the ORF, (3) interfere with the ability of the ORF to be transcribed from the promoter sequence. Hence, “operably linked” may mean incorporated into a genetic construct so that expression control sequences, such as a promoter, effectively control transcription / expression of a sequence of interest.

[0425] The precise nature of transcriptional and translational regulatory sequences or elements required for expression may vary between expression environments, but typically include a transcription terminator, and optionally an enhancer.

[0426] Reference to a “promoter” is to be taken in its broadest context and includes transcriptional regulatory sequences required for accurate transcription initiation and where applicable accurate spatial and / or temporal control of gene expression or its response to, e.g., internal or external (e.g., exogenous) stimuli. More particularly, “promoter” may depict a region on a nucleic acid molecule, preferably DNA molecule, to which an RNA polymerase binds and initiates transcription. A promoter is preferably, but not necessarily, positioned upstream, z.e., 5’, of the sequence the transcription of which it controls. Typically, in prokaryotes a promoter region may contain both the promoter per se and sequences which, when transcribed into RNA, will signal the initiation of protein synthesis (e.g., Shine-Dalgamo sequence). A promoter sequence can also include “enhancer regions”, which are one or more regions of DNA that can be bound with proteins (namely the transacting factors) to enhance transcription levels of genes in a gene -cluster. The enhancer, while typically at the 5’ end of a coding region, can also be separate from a promoter sequence, e.g., can be within an intronic region of a gene or 3’ to the coding region of the gene.

[0427] In embodiments, promoters contemplated herein may be constitutive or inducible. A constitutive promoter is understood to be a promoter whose expression is constant under the standard culturing conditions. Inducible promoters are promoters that are responsive to one or more induction cues. For example, an inducible promoter can be chemically regulated (e.g., a promoter whose transcriptional activity is regulated by the presence or absence of a chemical inducing agent such as an alcohol, tetracycline, a steroid, a metal, or other small molecule) or physically regulated (e.g., a promoter whose transcriptional activity is regulated by the presence or absence of a physical inducer such as light or high or low temperatures). An inducible promoter can also be indirectly regulated by one or more transcription factors that are themselves directly regulated by chemical or physical cues. Non-limiting examples of promoters include T7, U6, Hl, retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter, the P-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter.

[0428] In particular embodiments, the promoter is a bone tissue-specific or cartilage -specific promoter, such as an osteoblast-, chondrocyte-, osteoclast-, or osteocyte-specific promoter. For example, osteoblast-tissue specific promoters include osteocalcin gene promoters, such as the OCN-Cre promoter as described in Zhang M, et al. Osteoblast-specific knockout of the insulin -like growth factor (IGF) receptor gene reveals an essential role of IGF signaling in bone matrix mineralization. J Biol Chem. 2002;277:44005-12.

[0429] In particular embodiments, the promoter is an endothelium -specific promoter, such as a bone endothelium-specific promoter. Non-limiting examples of endothelium -specific promoters include the cadherin 5 (Cdh5) promoter.

[0430] The terms “terminator” or “transcription terminator” refer generally to a sequence element at the end of a transcriptional unit which signals termination of transcription. For example, a terminator is usually positioned downstream of, z.e., 3’ of ORF(s) encoding a polypeptide of interest. For instance, where a recombinant nucleic acid contains two or more ORFs, e.g., successively ordered and forming together a multi-cistronic transcription unit, a transcription terminator may be advantageously positioned 3’ to the most downstream ORF.

[0431] In particular embodiments, the nucleic acid expression cassette comprises the nucleic acid encoding the agent as disclosed herein, operably linked to one or more promoters, enhancers, ORFs and / or transcription terminators.

[0432] A further aspect relates to vector comprising the nucleic acid encoding the agent as disclosed herein, or the nucleic acid expression cassette as disclosed herein, such as a viral vector, for use in the treatment (e.g. treatment and / or prevention) of a bone-related disease or disorder in a subject.

[0433] The terms “expression vector” or “vector” as used herein refers to nucleic acid molecules, typically DNA, to which nucleic acid fragments, preferably the recombinant nucleic acid molecule as defined herein, may be inserted and cloned, i.e., propagated. Hence, a vector will typically contain one or more unique restriction sites, and may be capable of autonomous replication in a defined cell or vehicle organism such that the cloned sequence is reproducible. A vector may also preferably contain a selection marker, such as, e.g., an antibiotic resistance gene, to allow selection of recipient cells that contain the vector. Vectors may include, without limitation, plasmids, phagemids, bacteriophages, bacteriophage-derived vectors, PAC, BAC, linear nucleic acids, e.g., linear DNA, transposons, viral vectors, etc., as appropriate (see, e.g., Sambrook et al., 1989; Ausubel 1992). Viral vectors may include inter alia retroviral vectors, lentiviral vectors, adenoviral vectors, or adeno-associated viral vectors, for example, vectors based on HIV, SV40, EBV, HSV or BPV. Expression vectors are generally configured to allow for and / or effect the expression of nucleic acids or open reading frames introduced thereto in a desired expression system, e.g., in vitro, in a cell, organ and / or organism. For example, expression vectors may advantageously comprise suitable regulatory sequences.

[0434] Factors of importance in selecting a particular vector include inter alia: choice of recipient cell, ease with which recipient cells that contain the vector may be recognised and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in particular recipient cells; whether it is desired for the vector to integrate into the chromosome or to remain extra-chromosomal in the recipient cells; and whether it is desirable to be able to “shuttle” the vector between recipient cells of different species.

[0435] Expression vectors can be autonomous or integrative. A nucleic acid can be in introduced into a cell in the form of an expression vector such as a plasmid, phage, transposon, cosmid or virus particle. The recombinant nucleic acid can be maintained extrachromosomally or it can be integrated into the cell chromosomal DNA. Expression vectors can contain selection marker genes encoding proteins required for cell viability under selected conditions (e.g., URA3, which encodes an enzyme necessary for uracil biosynthesis, or LEU2, which encodes an enzyme required for leucine biosynthesis, or TRP1, which encodes an enzyme required for tryptophan biosynthesis) to permit detection and / or selection of those cells transformed with the desired nucleic acids. Expression vectors can also include an autonomous replication sequence (ARS). The ARS may comprise a centromere (CEN) and an origin of replication (ORI). For example, the ARS may be ARS 18 or ARS68.

[0436] Integrative vectors generally include a serially arranged sequence of at least a first insertable DNA fragment, a selectable marker gene, and a second insertable DNA fragment. The first and second insertable DNA fragments are each about 200 (e.g., about 250, about 300, about 350, about 400, about 450, about 500, or about 1000 or more) nucleotides in length and have nucleotide sequences which are homologous to portions of the genomic DNA of the cell species to be transformed. A nucleotide sequence containing a nucleic acid of interest for expression is inserted in this vector between the first and second insertable DNA fragments, whether before or after the marker gene. Integrative vectors can be linearized prior to transformation to facilitate the integration of the nucleotide sequence of interest into the cell genome.

[0437] Prior to introducing the vectors into a cell of interest, the vectors can be grown (e.g., amplified) in bacterial cells such as Escherichia coli (E. coli). The vector DNA can be isolated from bacterial cells by any of the methods known in the art, which result in the purification of vector DNA from the bacterial milieu. The purified vector DNA can be extracted extensively with phenol, chloroform, and ether, to ensure that no E. coli proteins are present in the plasmid DNA preparation, since these proteins can be toxic to mammalian cells.

[0438] As noted elsewhere, an agent may comprise a protein, polypeptide or peptide. Such may be suitably obtained through expression by host cells or host organisms (e.g. a bacterial cell, a yeast cell, or an animal cell), transformed with an expression construct encoding and configured for expression of said protein, polypeptide or peptide in said host cells or host organisms, followed by purification of the protein, polypeptide or peptide.

[0439] Such protein, polypeptide or peptide may be suitably isolated. The term “isolated” with reference to a particular component (such as for instance a nucleic acid, protein, polypeptide or peptide) generally denotes that such component exists in separation from - for example, has been separated from or prepared and / or maintained in separation from - one or more other components of its natural environment. For instance, an isolated human or animal protein or complex may exist in separation from a human or animal body where it naturally occurs. The term “isolated” as used herein may preferably also encompass the qualifier “purified”. As used herein, the term “purified” with reference to peptides, polypeptides, proteins, or nucleic acids does not require absolute purity. Instead, it denotes that such peptides, polypeptides, proteins, or nucleic acids are in a discrete environment in which their abundance (conveniently expressed in terms of mass or weight or concentration) relative to other analytes is greater than in the starting composition or sample. A discrete environment denotes a single medium, such as for example a single solution, gel, precipitate, lyophilisate, etc. Purified nucleic acids, proteins, polypeptides or peptides may be obtained by known methods including, for example, laboratory or recombinant synthesis, chromatography, preparative electrophoresis, centrifugation, precipitation, affinity purification, etc. Purified peptides, polypeptides or proteins may preferably constitute by weight > 10%, more preferably > 50%, such as > 60%, yet more preferably > 70%, such as > 80%, and still more preferably > 90%, such as > 95%, > 96%, > 97%, > 98%, > 99% or even 100%, of the protein content of the discrete environment. Protein content may be determined, e.g., by the Lowry method (Lowry et al. 1951. J Biol Chem 193: 265), optionally as described by Hartree 1972 (Anal Biochem 48: 422-427). Purity of peptides, polypeptides, or proteins may be determined by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Quantity of nucleic acids may be determined by measuring absorbance A260. Purity of nucleic acids may be determined by measuring absorbance A260 / A280, or by agarose- or polyacrylamide-gel electrophoresis and ethidium bromide or similar staining.

[0440] Further, there are several other well-known methods of introducing nucleic acids into animal cells, any of which may be used herein. At the simplest, the nucleic acid can be directly injected into the target cell / target tissue. Other methods include fusion of the recipient cell with bacterial protoplasts containing the nucleic acid, the use of compositions like calcium chloride, rubidium chloride, lithium chloride, calcium phosphate, DEAE dextran, cationic lipids or liposomes or methods like receptor-mediated endocytosis, biolistic particle bombardment ("gene gun" method), infection with viral vectors (i.e. derived from lentivirus, adeno -associated virus (AAV), adenovirus, retrovirus or antiviruses), electroporation, and the like. Other techniques or methods which are suitable for delivering nucleic acid molecules to target cells include the continuous delivery of an NA molecule from poly (lactic-Co-Glycolic Acid) polymeric microspheres or the direct injection of protected (stabilized) NA molecule(s) into micropumps delivering the product. Another possibility is the use of implantable drug-releasing biodegradable microspheres. Also envisaged is encapsulation of NA in various types of liposomes (immunoliposomes, PEGylated (immuno) liposomes), cationic lipids and polymers, nanoparticles or dendrimers, poly (lactic-Co-Glycolic Acid) polymeric microspheres, implantable drug -releasing biodegradable microspheres, etc.; and co-injection of NA with protective agent like the nuclease inhibitor aurintricarboxylic acid. It shall be clear that also a combination of different above-mentioned delivery modes or methods may be used.

[0441] In particular embodiments, the vector comprising the nucleic acid as described herein is a viral vector, preferably a viral vector specifically directed towards bone tissue, such as towards boneforming cells, like osteogenic progenitor cells, or towards endothelial cells in the bone tissue.

[0442] In further particular embodiments, the viral vector is an AAV vector, such as an adeno-associated virus serotype 9 (AAV9) vector, or bone-specific mutant thereof.

[0443] In particular embodiments, the agent as described herein, the nucleic acid encoding the agent as described herein, the nucleic acid expression cassette as described herein or the vector as described herein, is comprised in a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0444] In line therewith, a further aspect relates to a pharmaceutical composition comprising the agent as described herein, the nucleic acid encoding the agent as described herein, the nucleic acid expression cassette as described herein or the vector as described herein, and a pharmaceutically acceptable carrier for use in the treatment and / or prevention of a bone-related disease or disorder in a subject. Such pharmaceutical formulations or compositions may be comprised in a kit of parts.

[0445] The term “pharmaceutically acceptable” as used herein is consistent with the art and means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof.

[0446] As used herein, “carrier” or “excipient” includes any and all solvents, diluents, buffers (such as, e.g., neutral buffered saline or phosphate buffered saline), solubilisers, colloids, dispersion media, vehicles, fillers, chelating agents (such as, e.g., EDTA or glutathione), amino acids (such as, e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavourings, aromatisers, thickeners, agents for achieving a depot effect, coatings, antifungal agents, preservatives, antioxidants, tonicity controlling agents, absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active substance, its use in the therapeutic compositions may be contemplated.

[0447] Illustrative, non-limiting carriers for use in formulating the pharmaceutical compositions include, for example, oil-in-water or water-in-oil emulsions, aqueous compositions with or without inclusion of organic co-solvents suitable for intravenous (IV) use, liposomes or surfactantcontaining vesicles, microspheres, microbeads and microsomes, powders, tablets, capsules, suppositories, aqueous suspensions, aerosols, and other carriers apparent to one of ordinary skill in the art.

[0448] Pharmaceutical compositions as intended herein may be formulated for essentially any route of administration, such as without limitation, oral administration (such as, e.g., oral ingestion or inhalation), intranasal administration (such as, e.g., intranasal inhalation or intranasal mucosal application), parenteral administration (such as, e.g., subcutaneous, intravenous (I.V.), intramuscular, intraperitoneal or intrastemal injection or infusion), transdermal or transmucosal (such as, e.g., oral, sublingual, intranasal) administration, topical administration, rectal, vaginal or intra-tracheal instillation, and the like. In this way, the therapeutic effects attainable by the methods and compositions can be, for example, systemic, local, tissue -specific, etc., depending of the specific needs of a given application.

[0449] For example, for oral administration, pharmaceutical compositions may be formulated in the form of pills, tablets, lacquered tablets, coated (e.g., sugar-coated) tablets, granules, hard and soft gelatin capsules, aqueous, alcoholic or oily solutions, syrups, emulsions or suspensions. In an example, without limitation, preparation of oral dosage forms may be is suitably accomplished by uniformly and intimately blending together a suitable amount of the agent as disclosed herein in the form of a powder, optionally also including finely divided one or more solid carrier, and formulating the blend in a pill, tablet or a capsule. Exemplary but non-limiting solid carriers include calcium phosphate, magnesium stearate, talc, sugars (such as, e.g., glucose, mannose, lactose or sucrose), sugar alcohols (such as, e.g., mannitol), dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. Compressed tablets containing the pharmaceutical composition can be prepared by uniformly and intimately mixing the agent as disclosed herein with a solid carrier such as described above to provide a mixture having the necessary compression properties, and then compacting the mixture in a suitable machine to the shape and size desired. Moulded tablets maybe made by moulding in a suitable machine, a mixture of powdered compound moistened with an inert liquid diluent. Suitable carriers for soft gelatin capsules and suppositories are, for example, fats, waxes, semisolid and liquid polyols, natural or hardened oils, etc.

[0450] For example, for oral or nasal aerosol or inhalation administration, pharmaceutical compositions may be formulated with illustrative carriers, such as, e.g., as in solution with saline, polyethylene glycol or glycols, DPPC, methylcellulose, or in mixture with powdered dispersing agents, further employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other solubilising or dispersing agents known in the art. Suitable pharmaceutical formulations for administration in the form of aerosols or sprays are, for example, solutions, suspensions or emulsions of the agents as taught herein or their physiologically tolerable salts in a pharmaceutically acceptable solvent, such as ethanol or water, or a mixture of such solvents. If required, the formulation can also additionally contain other pharmaceutical auxiliaries such as surfactants, emulsifiers and stabilizers as well as a propellant. Illustratively, delivery may be by use of a single-use delivery device, a mist nebuliser, a breath-activated powder inhaler, an aerosol metered-dose inhaler (MDI) or any other of the numerous nebuliser delivery devices available in the art. Additionally, mist tents or direct administration through endotracheal tubes may also be used.

[0451] Examples of carriers for administration via mucosal surfaces depend upon the particular route, e.g., oral, sublingual, intranasal, etc. When administered orally, illustrative examples include pharmaceutical grades of mannitol, starch, lactose, magnesium stearate, sodium saccharide, cellulose, magnesium carbonate and the like, with mannitol being preferred. When administered intranasally, illustrative examples include polyethylene glycol, phospholipids, glycols and glycolipids, sucrose, and / or methylcellulose, powder suspensions with or without bulking agents such as lactose and preservatives such as benzalkonium chloride, EDTA. In a particularly illustrative embodiment, the phospholipid 1,2 dipalmitoyl -sn-glycero-3 -phosphocholine (DPPC) is used as an isotonic aqueous carrier at about 0.01-0.2% for intranasal administration of the compound of the subject invention at a concentration of about 0. 1 to 3.0 mg / ml.

[0452] For example, for parenteral administration, pharmaceutical compositions may be advantageously formulated as solutions, suspensions or emulsions with suitable solvents, diluents, solubilisers or emulsifiers, etc. Suitable solvents are, without limitation, water, physiological saline solution or alcohols, e.g. ethanol, propanol, glycerol, in addition also sugar solutions such as glucose, invert sugar, sucrose or mannitol solutions, or alternatively mixtures of the various solvents mentioned. The injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid. The agents and pharmaceutically acceptable salts thereof of the invention can also be lyophilised and the lyophilisates obtained used, for example, for the production of injection or infusion preparations. For example, one illustrative example of a carrier for intravenous use includes a mixture of 10% USP ethanol, 40% USP propylene glycol or polyethylene glycol 600 and the balance USP Water for Injection (WFI). Other illustrative carriers for intravenous use include 10% USP ethanol and USP WFI; 0.01-0.1% triethanolamine in USP WFI; or 0.01-0.2% dipalmitoyl diphosphatidylcholine in USP WFI; and 1-10% squalene or parenteral vegetable oil-in-water emulsion. Illustrative examples of carriers for subcutaneous or intramuscular use include phosphate buffered saline (PBS) solution, 5% dextrose in WFI and 0.01- 0.1% triethanolamine in 5% dextrose or 0.9% sodium chloride in USP WFI, or a 1 to 2 or 1 to 4 mixture of 10% USP ethanol, 40% propylene glycol and the balance an acceptable isotonic solution such as 5% dextrose or 0.9% sodium chloride; or 0.01-0.2% dipalmitoyl diphosphatidylcholine in USP WFI and 1 to 10% squalene or parenteral vegetable oil-in-water emulsions.

[0453] Where aqueous formulations are preferred, such may comprise one or more surfactants. For example, the composition can be in the form of a micellar dispersion comprising at least one suitable surfactant, e.g., a phospholipid surfactant. Illustrative examples of phospholipids include diacyl phosphatidyl glycerols, such as dimyristoyl phosphatidyl glycerol (DPMG), dipalmitoyl phosphatidyl glycerol (DPPG), and distearoyl phosphatidyl glycerol (DSPG), diacyl phosphatidyl cholines, such as dimyristoyl phosphatidylcholine (DPMC), dipalmitoyl phosphatidylcholine (DPPC), and distearoyl phosphatidylcholine (DSPC); diacyl phosphatidic acids, such as dimyristoyl phosphatidic acid (DPMA), dipahnitoyl phosphatidic acid (DPPA), and distearoyl phosphatidic acid (DSPA); and diacyl phosphatidyl ethanolamines such as dimyristoyl phosphatidyl ethanolamine (DPME), dipalmitoyl phosphatidyl ethanolamine (DPPE) and distearoyl phosphatidyl ethanolamine (DSPE). Typically, a surfactant: active substance molar ratio in an aqueous formulation will be from about 10: 1 to about 1: 10, more typically from about 5: 1 to about 1:5, however any effective amount of surfactant may be used in an aqueous formulation to best suit the specific objectives of interest.

[0454] When rectally administered in the form of suppositories, these formulations may be prepared by mixing the compounds according to the invention with a suitable non -irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidity and / or dissolve in the rectal cavity to release the drug.

[0455] Suitable carriers for microcapsules, implants or rods are, for example, copolymers of glycolic acid and lactic acid.

[0456] One skilled in this art will recognize that the above description is illustrative rather than exhaustive. Indeed, many additional formulations techniques and pharmaceutically -acceptable excipients and carrier solutions are well-known to those skilled in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens.

[0457] In preferred embodiments, the agent, the nucleic acid encoding the agent, the nucleic acid expression cassette comprising the nucleic acid, the vector comprising the nucleic acid or the nucleic acid expression cassette, the host cell or the pharmaceutical composition as described herein is administered parenterally. More preferably, the agent, the nucleic acid encoding the agent, the nucleic acid expression cassette comprising the nucleic acid or the vector comprising the nucleic acid, the nucleic acid expression cassette , the host cell or the pharmaceutical composition as described herein is administered intravenously, for example by infusion. In particular embodiments, the agent, the nucleic acid encoding the agent, or the nucleic acid expression cassette comprising the nucleic acid as described herein is used in gene therapy. In further particular embodiments, the agent, the nucleic acid encoding the agent, or the nucleic acid expression cassette comprising the nucleic acid as taught herein is used in bone-directed gene therapy, such as in bone-endothelial cell-directed gene therapy.

[0458] Accordingly, also provided herein is a method for gene therapy, in particular bone or bone- endothelial cell-directed gene therapy, in a subject in need of said gene therapy comprising: introducing in the subject, in particular in the bone tissue, such as the endothelial cells in the bone tissue, of the subject, a nucleic acid expression cassette or a vector as described herein; and expressing a therapeutically effective amount of the agent encoded by the nucleic acid as taught herein in the subject, in particular the bone tissue of the subject.

[0459] The term “gene therapy” as used herein refers to the introduction of an exogenous polynucleotide into a host cell for therapeutic or prophylactic purposes, irrespective of the method used for the introduction. Such methods include a variety of well-known techniques such as vector-mediated gene transfer (by, e.g., viral infection / transfection, or various other protein-based or lipid-based gene delivery complexes) as described elsewhere herein. The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. A number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art. For example, the vector- mediated gene transfer of the agent, the nucleic acid encoding the agent, the nucleic acid expression cassette comprising the nucleic acid as described herein may be performed using a viral vector specifically directed towards bone or a viral vector specific for bone endothelial cells.

[0460] In particular embodiments, the nucleic acid encoding the agent, the nucleic acid expression cassette comprising the nucleic acid, the vector comprising the nucleic acid or the nucleic acid expression cassette or the pharmaceutical composition as taught herein is administered to the subject by the injection (e.g., intravenously) or transplantation of allogeneic cells transformed with the vector comprising the nucleic acid or the nucleic acid expression cassette as taught herein. When administered, the injected or transplanted allogenic cells will transcribe and translate the nucleic acid encoding the agent as taught herein in vivo. The dosage or amount of the agent as taught herein, optionally in combination with one or more other active compounds to be administered, depends on the individual case and is, as is customary, to be adapted to the individual circumstances to achieve an optimum effect. Thus, the unit dose and regimen depend on the nature and the severity of the disorder to be treated, and also on factors such as the species of the subject, the sex, age, body weight, general health, diet, mode and time of administration, immune status, and individual responsiveness of the human or animal to be treated, efficacy, metabolic stability and duration of action of the compounds used, on whether the therapy is acute or chronic or prophylactic, or on whether other active compounds are administered in addition to the agent of the invention. In order to optimize therapeutic efficacy, the agent as taught herein can be first administered at different dosing regimens. Typically, levels of the agent in a tissue can be monitored using appropriate screening assays as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. The frequency of dosing is within the skills and clinical judgement of medical practitioners (e.g., doctors, veterinarians or nurses). Typically, the administration regime is established by clinical trials which may establish optimal administration parameters. However, the practitioner may vary such administration regimes according to the one or more of the aforementioned factors, e.g., subject’s age, health, weight, sex and medical status. The frequency of dosing can be varied depending on whether the treatment is prophylactic or therapeutic.

[0461] Toxicity and therapeutic efficacy of the agent as described herein or pharmaceutical compositions comprising the same can be determined by known pharmaceutical procedures in, for example, cell cultures or experimental animals. These procedures can be used, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Pharmaceutical compositions that exhibit high therapeutic indices are preferred. While pharmaceutical compositions that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to normal cells (e.g., non -target cells) and, thereby, reduce side effects.

[0462] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in appropriate subjects. The dosage of such pharmaceutical compositions lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For a pharmaceutical composition used as described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the pharmaceutical composition which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography. Without limitation, depending on the type and severity of the disease, a typical dosage (e.g., a typical daily dosage or a typical intermittent dosage, e.g., a typical dosage for every two days, every three days, every four days, every five days, every six days, every week, every 1.5 weeks, every two weeks, every three weeks, every month, or other) of the agent as taught herein may range from about 10 pg / kg to about 500 pg / kg body weight of the subject, per dose, depending on the factors mentioned above, e.g., such as 20-400 pg / kg or 20-200 pg / kg or 20-100 pg / kg or 40-80 pg / kg body weight of the subject.

[0463] By means of example and without limitation, the agent as taught herein may be administered at about 5 pg / kg, or at about 10 pg / kg, or at about 15 pg / kg, or at about 20 pg / kg, or at about 25 pg / kg, or at about 30 pg / kg, or at about 35 pg / kg, or at about 40 pg / kg, or at about 45 pg / kg, or at about 50 mg / kg, or at about 60 pg / kg, or at about 70 pg / kg, or at about 80 pg / kg, or at about 90 pg / kg, or at about 100 pg / kg, or at about 200 pg / kg, or at about 300 pg / kg, or at about 400 pg / kg, or at about 500 pg / kg per dose.

[0464] In particular embodiments, the agent as taught herein is administered using a sustained delivery system, such as a (partly) implanted sustained delivery system. Skilled person will understand that such a sustained delivery system may comprise a reservoir for holding the agent as taught herein, a pump and infusion means (e.g., a tubing system).

[0465] In particular embodiment, the agent as disclosed herein is the main or only active ingredient of the pharmaceutical composition.

[0466] In particular embodiments, pharmaceutical composition further comprises one or more agents in addition to the agent capable of activating G-protein coupled receptor (GPR)124 / RECK / Frizzled / lipoprotein receptor-related protein (LRP) -mediated Wnt signaling as described herein, wherein said one or more agents are agents known to inhibit bone resorption, such as bisphosphonates, denosumab, calcitonin, SERMs, estrogen and progesterone, and / or agents known to stimulate bone formation, such as PTH and Teriparatide.

[0467] Determining the activation or lack of activation of Wnt signaling mediated by the GPR124 / RECK / Frizzled / LRP receptor complex and Wnt signaling mediated by the Frizzled / LRP receptor complex may be determined as described elsewhere herein. For example, cells transiently transfected with a Super TOPFlash plasmid (e.g. Super 8x TOPFlash plasmid, Addgene plasmid #12456) or a stably transfected Super Top Flash reporter cell line (e.g. HEK293 STF cell line) could be (co-)transfected with plasmids encoding Frizzled, LRP, GPR124 and / or Reck polypeptides, and be used to determine the luciferase activity of the cells in response to addition of the test agent as an indication of the Wnt signaling activity in said cells.

[0468] In particular embodiments, the nucleic acid encoding the agent as disclosed herein, the nucleic acid expression cassette as disclosed herein or the vector as disclosed herein, may be delivered to a cell, such as a bone-targeting cell and / or a bone reparative cell, ex vivo prior to delivering said cell encompassing said nucleic acid, nucleic acid expression cassette or vector to the subject.

[0469] Accordingly, in particular embodiments, the agent as disclosed herein, the nucleic acid encoding the agent as disclosed herein, the nucleic acid expression cassette as disclosed herein or the vector as disclosed herein, may be encompassed by a cell. In particular embodiments, the cell is cell capable to differentiate into bone-forming cells. In particular embodiments, the cell is a MSC, a chondrocyte or an osteoblast.

[0470] In particular embodiments, the cell expresses on its surface a protein capable of homing the cell to bone, such as alpha4 integrin (CD49d) or another cell surface protein as described in Lin W. et al., Mesenchymal stem cells homing to improve bone healing, Journal of Orthopaedic Translation, 2017, Volume 9: 19-27.

[0471] In particular embodiments, the agent as disclosed herein (e.g. an agent comprising a protein or polypeptide), the nucleic acid encoding the agent as disclosed herein, the nucleic acid expression cassette as disclosed herein or the vector as disclosed herein, may be incorporated into a bone graft, scaffold (e.g. a hydrogel scaffold) or a gene activated matrix (GAM). For example, viral gene delivery from scaffolds is as described in Jang JH et al., Engineering biomaterial systems to enhance viral vector gene delivery, Mol Ther. Oi l Aug;19(8): 1407-15. doi: 10.1038 / mt.2011.111. Epub 2011 May 31 or Laird NZ et al., Gene- and RNAi -Activated Scaffolds for Bone Tissue Engineering: Current Progress and Future Directions, Adv Drug Deliv Rev. 2021 Jul; 174: 613— 627. The GAM may release the nucleic acid encoding the agent as disclosed herein, the nucleic acid expression cassette as disclosed herein or the vector as disclosed herein, into the surrounding tissue in vivo, for example, after implantation into an osseous defect.

[0472] In particular embodiments, the nucleic acid encoding the agent as disclosed herein, the nucleic acid expression cassette as disclosed herein or the vector as disclosed herein may be administered to the subject by direct gene therapy, such as by direct injection into an osseous lesion. For example, direct gene therapy for bone regeneration is described in Pelled G et al., Direct gene therapy for bone regeneration: gene delivery, animal models, and outcome measures, Tissue Eng Part B Rev. 2010 Feb;16(l): 13-20. doi: 10.1089 / ten.teb.2009.0156.

[0473] While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and variations as follows in the spirit and broad scope of the appended claims.

[0474] The herein disclosed aspects and embodiments of the invention are further supported by the following non-limiting examples. EXAMPLES

[0475] Example 1. Endothelial Wnt7aK190Aexpression leads to a systemic increase in bone ossification

[0476] Present inventors previously found that specific single amino acid substitutions in Wnt7a can lead to ligand agonists with highly-specific signaling properties through the Gprl24 / Reck / Fzd / Lrp5 / 6 complex and with limited to no activities in the absence of Gprl24 and / or Reck. Present inventors now generated a transgenic mouse line allowing conditional gain-of-function (GOF) after activation of Cre recombination system. More specifically, the mWnt7aK190A(as described in International patent application W02019180204A1) sequence was cloned in the pR26 CAG vector in order to generate transgenic mice using CRISPR / Cas9 mediated knock-in in the Rosa26 locus. Transgenesis was successful and animals positive for the transgene were crossed with Cdh5 CreERT mice for functional validation of the GOF system.

[0477] In each litter from crossings with Cdh5 CreERT mice, smaller individuals with curled incisive teeth were observed. Genotyping results showed that these individuals were systematically positives for the Cdh5 CreERT and the Rosa26 Wnt7a K190A alleles. During brain dissection, Cdh5 CreERT+; Rosa26 Wnt7a K190A / + mice exhibited a thicker and more ossified skull than control mice (wild type or the single transgenics Rosa26 Wnt7a K190A / +). Furthermore, analysis of their skeleton using pCT scan showed an increased volume in their interparietal bone as well as of their femur (trabecular) (Fig. 1A-D), compared to control mice. In addition, the trabecular space in Wnt7aK190Atransgenic mice was nearly absent.

[0478] It is noted that “K190A” indicates the amino acid position of the substitution in the mWnt7a precursor polypeptide (and the substitution “K159A” indicates the amino acid position in mWnt7a mature polypeptide).

[0479] It is further noted that Wnt7aK190Awas selected as a prototype. It should however be clear to a skilled person that also the other agonists listed herein, such as the other Wnt7a singe-residue variants as described in International patent application W02019180204A1, behave similar to the Wnt7aK190A.

Claims

1. CLAIMS1. An agent capable of activating G-protein coupled receptor (GPR)124 / RECK / Frizzled / lipoprotein receptor-related protein (LRP)-mediated Wnt signaling, for use in the treatment and / or prevention of a bone-related disease or disorder in a subject, wherein said agent does not activate Frizzled / LRP -mediated Wnt signaling in the absence of RECK or GPR124.

2. The agent for use according to claim 1, wherein said bone -related disease or disorder is characterized by decreased bone formation, decreased bone mineral density, decreased bone mineral content, decreased bone mass, decreased bone quality and / or decreased bone strength, wherein said bone formation, bone mineral density, bone mineral content, bone mass, bone quality and / or bone strength is decreased as compared to a healthy subject.

3. The agent for use according to claim 1 or 2, wherein said bone-related disease or disorder is not caused by an inherited genetic defect.

4. The agent for use according to any one of claims 1 to 3, wherein said bone-related disease or disorder is not osteoporosis-pseudoglioma syndrome (OPPG).

5. The agent for use according to claim 2, wherein said bone-related disease or disorder is selected from the group consisting of osteoporosis, osteopenia, osteonecrosis, osteolysis, osteodystrophy, osteomalacia, bone injury, bone reconstruction, osteogenesis imperfecta, bone lesions, bone cancer, Paget's Disease, arthritis, periodontal disease, growth plate injuries, fibrous dysplasia, and pseudoarthrosis.

6. The agent for use according to claim 1, wherein said bone-related disease or disorder is selected from the group consisting of osteoporosis, osteopenia, osteonecrosis, osteolysis, osteodystrophy, osteomalacia, bone injury, bone reconstruction, osteogenesis imperfecta, bone lesions, bone cancer, Paget's Disease, arthritis, periodontal disease, growth plate injuries, fibrous dysplasia, and pseudoarthrosis, and wherein said bone-related disease or disorder is characterized by reduced bone formation, reduced bone mineral density, reduced bone mineral content, reduced bone mass, reduced bone quality and / or reduced bone strength, so the subject would benefit from stimulation of bone formation and / or bone mineralisation.

7. The agent for use according to any one of claims 1 to 6, wherein the subject is older than 50 years of age.

8. The agent for use according to any one of claims 1 to 7, wherein said agent is capable of inducing heteromerization of Frizzled and LRP polypeptides at a cell membrane in the presence of RECK and GPR124, but not in the absence of RECK or GPR124.

9. The agent for use according to any one of claims 1 to 8, wherein said agent is capable of concurrently binding to the GPR124 and / or the RECK polypeptide, preferably to the cysteine knot 4 (CK4) region, to the CK5 region, or to the CK4 and CK5 regions, of the RECK polypeptide; the cysteine-rich domain (CRD) of the Frizzled polypeptide; and / or the DKK-binding site and / or the Wnt-binding site of the LRP polypeptide.

10. The agent for use according to any one of claims 1 to 9, wherein the agent comprises or is selected from a group consisting of a chemical substance, an antibody, an antibody fragment, an antibody-like protein scaffold, a protein or polypeptide, a peptide, a peptidomimetic, an aptamer, a photoaptamer, a spiegelmer and a nucleic acid, preferably wherein said agent comprises a protein or polypeptide.

11. The agent for use according to any one of claims 1 to 10, wherein said agent comprises a GPR124-binding domain, a RECK-binding domain, a Frizzled-binding domain and / or a LRP- binding domain, preferably wherein said agent comprises a RECK-binding domain, a Frizzled- binding domain, and a LRP -binding domain derived from a Wnt7 polypeptide, such as from a Wnt7a or Wnt7b polypeptide.

12. The agent for use according to claim 11, wherein said RECK-binding domain comprises:- an amino acid sequence having at least 25% sequence identity to the amino acid sequence HVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 17); or- an amino acid sequence having at least 25% sequence identity to the amino acid sequence VEPVRASRNKRPTFLKIKKPLSYRKPMDT (SEQ ID NO: 18); or- an amino acid sequence having at least 25% sequence identity to the amino acid sequence VEVVRASRLRQPTFLRIKQLRSYQKPMET (SEQ ID NO: 19); or- an amino acid sequence XXXVXAXRXXXXXFLXIXXXXXYXKXXXX (SEQ ID NO:20), VXAXRXXXXXFLXIXXXXXYXK (SEQ ID NO: 21),XXXVXAXRXXXXXFLXXXXXXXXXKXXXX (SEQ ID NO: 22) orVXAXRXXXXXFLXXXXXXXXXK (SEQ ID NO: 23) wherein X is any amino acid, preferably wherein the amino acid sequence shows at least 50% sequence identity to any one of SEQ ID NO: 18 or SEQ ID NO: 19.

13. The agent for use according to any one of claims 1 to 12, wherein said agent is or consists essentially of a fragment of a Wnt7 polypeptide, such as a fragment of a Wnt7a or Wnt7b105 polypeptide, optionally wherein said fragment is or consists essentially of the N-terminal domain (NTD) of the Wnt7 polypeptide.

14. The agent for use according to any one of claims 1 to 13, wherein said agent is a variant of a Wnt7 polypeptide, such as a variant of a Wnt7a or Wnt7b polypeptide.

15. The agent for use according to claim 14, wherein- the glutamine (Q) residue at the position corresponding to position 17 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than glutamine (Q), preferably by an alanine (A) residue;- the isoleucine (I) residue at the position corresponding to position 20 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than isoleucine (I), preferably by an alanine (A) residue;- the proline (P) residue at the position corresponding to position 25 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than proline (P), preferably by an alanine (A) residue;- the alanine (A) residue at the position corresponding to position 27 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than alanine (A), preferably by an arginine (R) residue;- the isoleucine (I) residue at position corresponding to position 28 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than isoleucine (I), preferably by an alanine (A) residue;- the glutamate (E) residue at the position corresponding to position 33 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than glutamate (E), preferably by an alanine (A) residue;- the methionine (M) residue at the position corresponding to position 37 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than methionine (M), preferably by an alanine (A) residue;- the leucine (L) residue at the position corresponding to position 39 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than leucine (L), preferably by an alanine (A) residue;- the glutamate (E) residue at the position corresponding to position 41 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than glutamate (E), preferably by an alanine (A) residue;106- the phenylalanine (F) residue at the position corresponding to position 44 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than phenylalanine (F), preferably by an alanine (A) residue;- the arginine (R) residue at the position corresponding to position 50 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than arginine (R), preferably by an alanine (A) residue;- the asparagine (N) residue at the position corresponding to position 52 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than asparagine (N), preferably by an glutamine (Q) residue;- the valine (V) residue at the position corresponding to position 68 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than valine (V), preferably by an alanine (A) residue;- the isoleucine (I) residue at the position corresponding to position 129 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than isoleucine (I), preferably by an alanine (A) residue;- the phenylalanine (F) residue at the position corresponding to position 131 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than phenylalanine (F), preferably by an alanine (A) residue;- the lysine (K) residue at the position corresponding to position 133 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than lysine (K), preferably by an alanine (A) residue;- the phenylalanine (F) residue at the position corresponding to position 135 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than phenylalanine (F), preferably by an alanine (A) residue;- the isoleucine (I) residue at the position corresponding to position 141 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than isoleucine (I), preferably by an alanine (A) residue;- the arginine (R) residue at the position corresponding to position 146 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than arginine (R), preferably by an alanine (A) residue;107- the arginine (R) residue at the position corresponding to position 158 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than arginine (R), preferably by an alanine (A) residue;- the lysine (K) residue at the position corresponding to position 159 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than lysine (K), preferably by an alanine (A), serine (S) or leucine (L) residue;- the lysine (K) residue at the position corresponding to position 181 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than lysine (K), preferably by an alanine (A) residue;- the arginine (R) residue at the position corresponding to position 191 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than arginine (R), preferably by an alanine (A) residue;- the lysine (K) residue at the position corresponding to position 198 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than lysine (K), preferably by an alanine (A) residue;- the lysine (K) residue at the position corresponding to position 200 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than lysine (K), preferably by an alanine (A) residue;- the lysine (K) residue at the position corresponding to position 205 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than lysine (K), preferably by an alanine (A) residue;- the glutamate (E) residue at the position corresponding to position 208 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than glutamate (E), preferably by an alanine (A) residue;- the arginine (R) residue at the position corresponding to position 214 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than arginine (R), preferably by an alanine (A) residue;- the lysine (K) residue at the position corresponding to position 216 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than lysine (K), preferably by an alanine (A) residue;108- the proline (P) residue at the position corresponding to position 218 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than proline (P), preferably by an alanine (A) residue;- the lysine (K) residue at the position corresponding to position 222 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than lysine (K), preferably by an alanine (A) residue;- the isoleucine (I) residue at the position corresponding to position 223 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by n amino acid residue other than isoleucine (I), preferably by an alanine (A) residue;- the tyrosine (Y) residue at the position corresponding to position 229 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than tyrosine (Y), preferably by an alanine (A) residue;- the proline (P) residue at the position corresponding to position 232 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than proline (P), preferably by an alanine (A) residue;- the threonine (T) residue at the position corresponding to position 235 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than threonine (T), preferably by an alanine (A) residue;- the glutamate (E) residue at the position corresponding to position 248 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than glutamate (E), preferably by an alanine (A) residue;- the arginine (R) residue at the position corresponding to position 289 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than arginine (R), preferably by an alanine (A) residue;- the tryptophan (W) residue at the position corresponding to position 291 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than tryptophan (W), preferably by an alanine (A) residue;- the threonine (T) residue at the position corresponding to position 307 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than threonine (T), preferably by an alanine (A) residue; or109- the lysine (K) residue at the position corresponding to position 318 in SEQ ID NO: 24 or SEQ ID NO: 51 is substituted by an amino acid residue other than lysine (K), preferably by an alanine (A) residue.

16. A nucleic acid encoding an agent as defined in any one of claims 1 to 15 for use in the treatment and / or prevention of a bone-related disease or disorder in a subject, wherein said agent is a protein, polypeptide or a peptide.

17. A nucleic acid expression cassette for use in the treatment and / or prevention of a bone-related disease or disorder in a subject, wherein said nucleic acid expression cassette comprises a nucleic acid as defined in claim 16 operably linked to a promoter and / or transcriptional and translational regulatory signals.

18. A vector for use in the treatment and / or prevention of a bone-related disease or disorder in a subject, wherein said vector comprises a nucleic acid as defined in claim 16 or a nucleic acid expression cassette as defined in claim 17, such as a viral vector.

19. The agent for use according to any one of claims 1 to 15, the nucleic acid for use according to claim 16, the nucleic acid expression cassette for use according to claim 17 or the vector for use according to claim 18, wherein said agent, nucleic acid, nucleic acid expression cassette or vector is comprised in a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

20. The agent for use according to any one of claims 1 to 15, the nucleic acid for use according to claim 16, the nucleic acid expression cassette for use according to claim 17 or the vector for use according to claim 18, wherein said agent, nucleic acid, nucleic acid expression cassette or vector is comprised in a cell, such as a mesenchymal stem cell (MSC).

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